Mutations that confer genetic stability to additional genes in influenza viruses.

Abstract

To provide a recombinant influenza virus.SOLUTION: Provided is an isolated recombinant influenza virus in which, as one aspect, at least one of the viral segments is: a PB2 viral segment encoding PB2 with residue at position 540 that is not asparagine; a PA viral segment encoding PA with a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine; or a PB1 viral segment encoding PB1 with a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid; or any combination thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Application No. 62 / 795,821, filed January 23, 2019, the disclosure of which is incorporated herein by reference.

[0002] Declaration of Government Rights This invention was made with government support under HHSN272201400008C awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Influenza A viruses are respiratory pathogens that cause annual epidemics and sporadic pandemics (Wright et al., 2013). Furthermore, highly pathogenic avian H5N1 and recently emerged H7N9 influenza viruses have caused numerous high-mortality human infections (Watanabe et al., 2013; Zhang et al., 2013). Influenza viruses infect respiratory epithelial cells and alveolar macrophages in mammalian hosts (Yu et al., 2010). The host immune system recognizes the influenza virus RNA genome through cytosolic sensors (Diebold et al., 2004; Pichlmair et al., 2006), triggering an innate immune response that results in the production of type I interferons (IFNs), proinflammatory cytokines, and proinflammatory chemokines (Honda and Taniguchi, 2006). Type I IFNs upregulate the production of antiviral proteins, including myxovirus resistance (Mx), oligoadenylate synthetase (OAS), and interferon-stimulated gene 15 (ISG15) (Garcia-Sastre et al., 2011). Dysregulation of the innate immune response to influenza virus infection leads to lung pathology mediated by infiltrating immune cells, including macrophages and neutrophils (Heron et al., 2008; Perrone et al., 2008). Although several studies have addressed the host response to influenza virus infection (Fakuyama and Kawaoka, 2011), the mechanisms of influenza virus-induced pathology remain incompletely understood.

[0004] To analyze immune responses to influenza virus infection in vivo, viruses expressing fluorescent reporter proteins have been generated (Kittel et al., 2004; Shinya et al., 2004). However, these viruses were significantly attenuated (Kittel et al., 2004; Shinya et al., 2004) and could not accurately reflect natural infection. For example, Manicassamy et al. (2010) generated a GFP-expressing influenza virus that was used to evaluate the antigen presentation pathway during influenza virus infection (Helft et al., 2012). However, the GFP gene was not stably maintained during replication in mouse lung cells or cultured cells (Manicassamy et al., 2010).

[0005] Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype continue to evolve in nature, threatening animal health and public health. These viruses were first identified in Guangdong Province, China, in 1996 (Li et al., 2006) and have since been found in multiple avian species in over 63 countries, repeatedly infecting mammals such as pigs and humans (Li et al., 2010; Neumann et al., 2010). As of December 2013, the World Health Organization (WHO) had confirmed 648 human cases of H5N1 virus infection, 384 of which were fatal, with a mortality rate of approximately 60% (http: / / www.who.int). In addition, novel subtypes of influenza viruses, such as H7N9 and H10N8 viruses, have emerged naturally and sporadically infected humans, resulting in fatal outcomes (Chen et al., 2014; Li et al., 2013) (http: / / www.who.int). Therefore, the current threat posed by influenza viruses highlights the urgent need to gain a thorough understanding of their pathogenic mechanisms in order to develop more effective strategies for controlling influenza virus infection, including the dynamic process of infection, although the virus target cells in vivo remain unknown. Summary of the Invention

[0006] The present disclosure relates, by way of example, to mutations in one or more viral segments of an influenza virus that increase the genetic stability of additional non-influenza viral genes, such as "heterologous" gene sequences inserted into one of the viral segments or present on additional viral segments fused to intact or modified (e.g., truncated or internally deleted) viral protein coding regions. In one embodiment, one or more of these mutations may be used to enhance the stability of an influenza virus that is not enhanced by the heterologous gene sequence. In one embodiment, the heterologous gene sequence is a marker gene, e.g., a fluorescent protein gene such as for GFP, BFP, RFP, or YFP, a luciferase gene, a β-glucuronidase gene, or a β-lactamase gene. In one embodiment, the heterologous sequence is for a prophylactic gene product. In one embodiment, the heterologous sequence encodes a therapeutic gene product.

[0007] As disclosed herein, influenza viruses expressing different colored fluorescent proteins (color-flu viruses) were generated. Viruses containing foreign genes were passaged. When adapted to mice, stable expression of the fluorescent proteins in infected animals enabled their detection by different types of microscopy and flow cytometry. The use of fluorescent influenza viruses, each stably expressing one of four different fluorescent proteins, enabled simultaneous monitoring and live imaging. Several studies were conducted using these viruses to demonstrate their versatility. For example, this system was used for live imaging of virus-infected cells, as well as for differential gene expression studies in viral antigen-positive and viral antigen-negative live cells in the lungs of color-flu-infected mice, to analyze the progression of viral spread in mouse lungs. Therefore, color-flu viruses are a powerful tool for analyzing viral infection at the cellular level in vivo to better understand influenza pathogenesis. Furthermore, different stabilizing mutations in the resulting viruses were identified. These mutations include T380A in the HA protein (numbering is for H1) and E712D in the PB2 protein of the A / PR / 8 / 34 (H1N1) virus, and V25A, R443K, K737R, and P167S amino acid substitutions in the PB2, PA, PB1, and NS1 proteins, respectively, of the A / Vietnam / 1203 / 2004 (H5N1) virus. Individual mutations in the H5 virus alone resulted in viruses containing foreign genes that were more stable in vitro, and all combinations of them provided even greater stability in vivo. These mutations are useful for any HA / NA combination.

[0008] In one embodiment, the recombinant virus has one or more stabilizing mutations, e.g., one or more substitutions in one or more influenza virus proteins that enhance the stability or replication (e.g., enhance the titer) of the recombinant virus having the one or more substitutions compared to a corresponding virus (parent virus) that does not have the one or more substitutions, and / or one or more substitutions in one or more influenza virus proteins that enhance the stability or replication of a heterologous gene sequence present in one of the viral segments in the recombinant virus compared to a corresponding virus that does not have the one or more substitutions with the heterologous gene sequence in the respective viral segment, and / or one or more substitutions in one or more influenza virus proteins that enhance the stability or replication of a heterologous gene sequence present in an additional viral segment in the recombinant virus and having the heterologous gene sequence compared to a corresponding virus that does not have the one or more substitutions. The one or more substitutions include, but are not limited to, substitutions in any of influenza PA, PA-X, PB1, PB1-F2, PB2, NP, NS1, NS2, M1, M2, NA, and / or HA (e.g., HA of influenza A virus), substitutions encoded in the corresponding viral segments (PA, PB1, PB2, NP, NS, M, NA, and / or HA), or combinations of substitutions in any one of these influenza virus proteins or genes, or combinations of one or more substitutions in two or more of these proteins or genes. In one embodiment, the one or more substitutions that enhance influenza virus stability or replication are in the PA protein, for example, a substitution for glutamine at position 180, threonine at position 200, or arginine at position 443 in PA (located on the protein surface), which enhances, for example, RNA replication, PA proteolytic activity, and / or interaction with one or more viral or cellular proteins. In one embodiment, the substitution for arginine at position 443 in PA is a conservative substitution. In one embodiment, the substitution for arginine at position 443 in PA is a non-conservative substitution.In one embodiment, the substitution for glutamine at position 180 in PA is a conservative substitution. In one embodiment, the substitution for glutamine at position 180 in PA is a non-conservative substitution. In one embodiment, the substitution for threonine at position 200 in PA is a conservative substitution. In one embodiment, the substitution for threonine at position 200 in PA is a non-conservative substitution. In one embodiment, one or more substitutions that enhance influenza virus stability or replication are in the PB2 protein, for example, a substitution for valine at position 25, a substitution for asparagine at position 540, and / or a substitution for glutamic acid at position 712 in PB2 that enhance, for example, polymerase activity, interaction with MAVS (at position 25), and / or protein folding or stability (at position 712). In one embodiment, the substitution for valine at position 25 in PB2 is a conservative substitution. In one embodiment, the substitution for valine at position 25 in PB2 is a non-conservative substitution. In one embodiment, the substitution for asparagine at position 540 in PB2 is a conservative substitution. In one embodiment, the substitution for asparagine at position 540 in PB2 is a non-conservative substitution. In one embodiment, the substitution for glutamic acid at position 712 in PB2 is a conservative substitution. In one embodiment, the substitution for glutamic acid at position 712 in PB2 is a non-conservative substitution. In one embodiment, the one or more substitutions that enhance the stability or replication of influenza viruses are in the PB1 protein, for example, a substitution for valine at position 149, a substitution for lysine at position 737, a substitution for glutamic acid at position 684, and / or a substitution for aspartic acid at position 685 in PB1 (which may be located on the protein surface), which alter, for example, polymerase activity or endonuclease activity. In one embodiment, the substitution for lysine at position 737 in PB1 is a conservative substitution. In one embodiment, the substitution for lysine at position 737 in PB1 is a non-conservative substitution. In one embodiment, the substitution for valine at position 149 in PB1 is a conservative substitution.In one embodiment, the substitution for valine at position 149 in PB1 is a non-conservative substitution. In one embodiment, the substitution for glutamic acid at position 684 in PB1 is a conservative substitution. In one embodiment, the substitution for glutamic acid at position 684 in PB1 is a non-conservative substitution. In one embodiment, the substitution for aspartic acid at position 685 in PB1 is a conservative substitution. In one embodiment, the substitution for aspartic acid at position 685 in PB1 is a non-conservative substitution.

[0009] In one embodiment, the one or more substitutions that enhance stability or replication, for example, by altering the interferon interference activity or transcriptional regulatory activity of influenza virus NS1, are in the NS1 protein, for example, a substitution for proline at position 167 in NS1, which may alter interaction with cellular proteins. In one embodiment, the substitution for proline at position 167 in NS1 is a conservative substitution. In one embodiment, the substitution for proline at position 167 in NS1 is a non-conservative substitution. In one embodiment, the one or more substitutions that enhance influenza virus stability or replication are in the HA protein, for example, a substitution for threonine at position 380 in HA (located in the alpha helix of HA-2). In one embodiment, the substitution for threonine at position 380 in HA is a conservative substitution. In one embodiment, the substitution for threonine at position 380 in HA is a non-conservative substitution. In one embodiment, the residue at position 443 in PA is K or H. In one embodiment, the residue at position 737 in PB1 is H or R. In one embodiment, the residue at position 25 in PB2 is A, L, T, I, or G. In one embodiment, the residue at position 712 in PB2 is D. In one embodiment, the residue at position 167 in NS1 is C, M, A, L, I, G, or T.

[0010] The virus may be used as a vaccine or as a gene delivery vector.

[0011] The vector comprises influenza cDNA, e.g., influenza A (e.g., any influenza A gene containing either the 18 HA subtype or the 11 NA subtype), B, or C DNA (see Fields Virology (Fields et al. (eds.), Lippincott, Williams, and Wickens (2006)), specifically incorporated herein by reference).

[0012] In one embodiment, the disclosed positions and substitutions in viral proteins can occur in viral segments from any influenza virus isolate or can be used to select viral segments having particular residues at one or more of the disclosed positions, but PB1, PB2, PA, NP, M, and NS encode proteins that have at least 80%, e.g., 90%, 92%, 95%, 97%, 98%, or 99%, contiguous amino acid sequence identity, inclusive, of any integer between 80 and 99, to a polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15. In one embodiment, PB1, PB2, PA, NP, M, and NS encode proteins that have at least 80%, e.g., 90%, 92%, 95%, 97%, 98%, or 99%, contiguous amino acid sequence identity, inclusive, of any integer between 80 and 99, to a polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15. In one embodiment, the influenza virus polypeptide has one or more, e.g., 2, 5, 10, 15, 20 or more conservative amino acid substitutions, e.g., up to 10% or 20% conservative substitutions of a combination of 2, 5, 10, 15, 20 or more conservative and non-conservative amino acid substitutions, e.g., up to 10% or 20% conservative substitutions of residues, compared to a polypeptide encoded by one of SEQ ID NOs: 1-6 or 10-15, and has hallmark residues described herein that provide stability.

[0013] Recombinant influenza viruses of the present disclosure may be prepared by selecting viral segments for inclusion in a recombinant virus, such as a reassortant virus, that has one or more stabilizing mutations in one or more influenza viral proteins. For example, an HA viral segment encoding an HA having a residue at position 380 that is not threonine may be selected, a PA viral segment encoding a PA having a residue at position 443 that is not arginine may be selected, a PA viral segment encoding a PA having a residue at position 180 that is not glutamine may be selected, a PA viral segment encoding a PA having a residue at position 200 that is not threonine may be selected, a PB1 viral segment encoding a PB1 having a residue at position 737 that is not lysine may be selected, a PB1 viral segment encoding a PB1 having a residue at position 149 that is not valine may be selected, a PB1 viral segment encoding a PB1 having a residue at position 684 that is not glutamic acid may be selected, a PB1 viral segment encoding a PB1 having a residue at position 685 that is not aspartic acid may be selected, a PB2 viral segment encoding a PB2 having a residue at position 25 that is not valine, a residue at position 540 that is not asparagine, or a residue at position 712 that is not glutamic acid may be selected, an NS viral segment encoding an NS1 having a residue at position 167 that is not proline may be selected, or any combination thereof. In one embodiment, the residue at position 443 in PA is K or H. In one embodiment, the residue at position 180 in PA is R, K, or H. In one embodiment, the residue at position 200 in PA is A, G, I, L, or V. In one embodiment, the residue at position 737 in PB1 is H or R. In one embodiment, the residue at position 149 in PB1 is A, T, G, I, or L. In one embodiment, the residue at position 684 in PB1 is D or N. In one embodiment, the residue at position 685 in PB1 is E or Q.In one embodiment, the residue at position 25 in PB2 is A, L, T, I, or G. In one embodiment, the residue at position 712 in PB2 is D. In one embodiment, the residue at position 540 in PB2 is K, R, or H. In one embodiment, the residue at position 167 in NS1 is S, C, M, A, L, I, G, or T. In one embodiment, the residue at position 380 in HA is A, I, V, L, or G.

[0014] In one embodiment, the influenza viruses of the disclosure are recombinant influenza viruses having two or more selected amino acid residues at specific positions in one or more of PA, PB1, PB2, HA, and / or NS1. In one embodiment, the recombinant reassortant influenza virus has a lysine or histidine at position 443 in PA, a histidine or arginine at position 737 in PB1, a leucine, isoleucine, threonine, alanine, or glycine at position 25 in PB2, and / or an aspartic acid, histidine, arginine, lysine, or asparagine at position 712 in PB2; a leucine, alanine, valine, isoleucine, or glycine at position 380 in HA, or a serine, cysteine, methionine, alanine, valine, glycine, isoleucine, or leucine at position 167 in NS1.

[0015] For recombinant influenza viruses of the present disclosure having additional viral segments with heterologous gene sequences ("9-segment" viruses), the viruses may be prepared with enhanced stability and / or replication by selecting viral segments for inclusion in the recombinant virus that have one or more stabilizing mutations in influenza viral proteins. For example, an HA viral segment encoding an HA with a residue at position 380 that is not threonine may be selected; a PA viral segment encoding a PA with a residue at position 443 that is not arginine may be selected; a PB1 viral segment encoding a PB1 with a residue at position 737 that is not lysine may be selected; a PB2 viral segment encoding a PB2 with a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid may be selected; an NS viral segment encapsulating an NS1 with a residue at position 167 that is not proline may be selected; or any combination thereof. The additional viral segments may be derived from any naturally occurring viral segment. In one embodiment, the residue at position 443 in PA is K or H. In one embodiment, the residue at position 737 in PB1 is H or R. In one embodiment, the residue at position 25 in PB2 is A, L, T, I, or G. In one embodiment, the residue at position 180 in PA is R, K, or H. In one embodiment, the residue at position 200 in PA is A, G, I, L, or V. In one embodiment, the residue at position 149 in PB1 is A, T, G, I, or L. In one embodiment, the residue at position 684 in PB1 is D or N. In one embodiment, the residue at position 685 in PB1 is E or Q. In one embodiment, the residue at position 540 in PB2 is K, R, or H. In one embodiment, the residue at position 712 in PB2 is D. In one embodiment, the residue at position 167 in NS1 is C, M, A, L, I, G, or T.The heterologous gene sequence may be of a length such that the viral segment bearing the heterologous gene sequence has a length of up to 4 kb, 4.2 kb, 4.5 kb, 4.7 kb, 5 kb, 5.2 kb, 5.5 kb, 5.7 kb, or 6 kb. In one embodiment, the heterologous gene in the additional viral segment replaces the influenza virus protein coding sequence (e.g., there is a deletion of the influenza virus coding sequence without deleting the encapsidation (integration) sequence in the coding sequence that connects to the encapsidation sequence in the non-coding sequence at one or both ends of the viral segment). In one embodiment, the heterologous gene sequence in the additional viral segment is genome-directed. In one embodiment, the heterologous gene sequence in the additional viral segment is fused in-frame to the N-terminal influenza virus protein coding sequence. In one embodiment, the heterologous gene sequence in the additional viral segment is fused in-frame to the C-terminal influenza virus protein coding sequence. The heterologous gene may encode an RNA, e.g., a microRNA, or a protein, e.g., a prophylactic or therapeutic gene product. In one embodiment, the gene product is an antigen derived from a different influenza virus isolate, or an antigen derived from a bacterium, a virus other than influenza virus, a parasite, or a fungus.

[0016] Recombinant influenza viruses of the present disclosure having a heterologous gene sequence in one of eight viral segments with enhanced stability and / or replication ("eight-segment" viruses) may be prepared by selecting viral segments for inclusion in the recombinant virus that have one or more stabilizing mutations in influenza viral proteins. For example, an HA viral segment encoding an HA with a residue at position 380 that is not threonine may be selected; a PA viral segment encoding a PA with a residue at position 443 that is not arginine may be selected; a PB1 viral segment encoding a PB1 with a residue at position 737 that is not lysine may be selected; a PB2 viral segment encoding a PB2 with a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid may be selected; an NS viral segment encapsulating an NS1 with a residue at position 167 that is not proline may be selected; or any combination thereof. In one embodiment, the residue at position 443 in PA is K or H. In one embodiment, the residue at position 737 in PB1 is H or R. In one embodiment, the residue at position 25 in PB2 is A, L, T, I, or G. In one embodiment, the residue at position 712 in PB2 is D. In one embodiment, the residue at position 180 in PA is R, K, or H. In one embodiment, the residue at position 200 in PA is A, G, I, L, or V. In one embodiment, the residue at position 149 in PB1 is A, T, G, I, or L. In one embodiment, the residue at position 684 in PB1 is D or N. In one embodiment, the residue at position 685 in PB1 is E or Q. In one embodiment, the residue at position 540 in PB2 is K, R, or H. In one embodiment, the residue at position 167 in NS1 is C, M, A, L, I, G, or T.

[0017] For recombinant influenza viruses of the present disclosure having a heterologous gene sequence in one of the influenza virus viral segments that also lacks a viral segment ("7-segment" virus), the virus may be prepared by selecting viral segments for inclusion in the recombinant virus that have enhanced stability and / or replication but have one or more stabilizing mutations in influenza viral proteins. For example, an HA viral segment encoding an HA with a residue at position 380 that is not threonine may be selected; a PA viral segment encoding a PA with a residue at position 443 that is not arginine may be selected; a PB1 viral segment encoding a PB1 with a residue at position 737 that is not lysine may be selected; a PB2 viral segment encoding a PB2 with a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid may be selected; an NS viral segment encapsulating an NS1 with a residue at position 167 that is not proline may be selected; or any combination thereof. The omitted viral segment may be any one of the naturally occurring viral segments, and optionally the encoded protein is provided in trans. In one embodiment, the 7 segment virus comprises a PA viral segment, or the PA protein is provided in trans, and the residue at position 443 in PA is K or H. In one embodiment, the 7 segment virus comprises a PB1 viral segment, or the PB1 protein is provided in trans, and the residue at position 737 in PB1 is H or R. In one embodiment, the 7 segment virus comprises a PB2 viral segment, or the PB2 protein is provided in trans, and the residue at position 25 in PB2 is A, L, T, I, or G. In one embodiment, the 7 segment virus comprises a PB2 viral segment, or the PB2 protein is provided in trans, and the residue at position 712 in PB2 is D. In one embodiment, the 7 segment virus comprises a PA viral segment, or the PA protein is provided in trans, and the residue at position 180 in PA is R, K, or H.In one embodiment, the 7-segment virus comprises a PA viral segment, or the PA protein is provided in trans, and residue 200 in PA is A, G, I, L, or V. In one embodiment, the 7-segment virus comprises a PB1 viral segment, or the PB1 protein is provided in trans, and residue 149 in PB1 is A, T, G, I, or L. In one embodiment, the 7-segment virus comprises a PB1 viral segment, or the PB1 protein is provided in trans, and residue 684 in PB1 is D or N. In one embodiment, the 7-segment virus comprises a PB1 viral segment, or the PB1 protein is provided in trans, and residue 685 in PB1 is E or Q. In one embodiment, the 7-segment virus comprises a PB2 viral segment, or the PB2 protein is provided in trans, and residue 540 in PB2 is K, R, or H. In one embodiment, the seven-segment virus comprises an NS viral segment, or the NS1 protein is provided in trans, and residue 167 in NS1 is C, M, A, L, I, G, or T. The heterologous gene sequence may be of a length such that the viral segment bearing the heterologous gene sequence has a length of up to 4 kb, 4.2 kb, 4.5 kb, 4.7 kb, 5 kb, 5.2 kb, 5.5 kb, 5.7 kb, or 6 kb. In one embodiment, the heterologous gene replaces an influenza virus protein-coding sequence (e.g., a deletion of the influenza virus coding sequence without a deletion of the encapsidation (integration) sequence in the coding sequence linked to the encapsidation sequence in the non-coding sequence at one or both ends of the viral segment). In one embodiment, the heterologous gene sequence in the additional viral segment is genome-directed. In one embodiment, the heterologous gene sequence is fused in-frame to the N-terminal influenza virus protein-coding sequence. In one embodiment, the heterologous gene sequence in the additional viral segment is fused in-frame to the C-terminal influenza virus protein coding sequence.The heterologous gene may encode an RNA, e.g., a microRNA, or a protein, e.g., a gene product that is prophylactic or therapeutic. In one embodiment, the gene product is an antigen from a different influenza virus isolate, or an antigen from a bacterium, a virus other than influenza virus, a parasite, or a fungus.

[0018] A heterologous gene sequence may be inserted into any viral segment. The heterologous gene sequence may be of a length such that the viral segment carrying the heterologous gene sequence is at most 4 kb, 4.2 kb, 4.5 kb, 4.7 kb, 5 kb, 5.2 kb, 5.5 kb, 5.7 kb, or 6 kb in length. In one embodiment, the heterologous gene replaces an internal influenza virus sequence in the viral segment. In one embodiment, insertion of the heterologous gene sequence may result in a "knockout" of the respective influenza virus gene product; to prepare such a virus, an influenza virus protein(s) may be provided in trans to complement the type mutation. In one embodiment, the heterologous gene sequence is in addition to an influenza virus coding sequence in the viral segment. In one embodiment, the heterologous gene sequence is fused in-frame to the N-terminal influenza virus protein coding sequence. In one embodiment, the heterologous gene is fused in-frame to the C-terminal influenza virus protein coding sequence. The heterologous gene may encode an RNA or protein, e.g., a prophylactic or therapeutic gene product. In one embodiment, the gene product is an antigen from a different influenza virus isolate, an antigen from a bacterium, a virus other than influenza virus, a parasite, or a fungus. In one embodiment, the heterologous gene sequence is in the NA viral segment. In one embodiment, the heterologous gene sequence is in the HA viral segment. In one embodiment, the heterologous gene sequence is in the M viral segment. In one embodiment, the heterologous gene sequence is in the NS viral segment. In one embodiment, the heterologous gene sequence is in the NP viral segment (see, e.g., Liu et al., 2012; Wang et al., 2010; Arilor et al., 2010; Dos Santos Afonso et al., 2005). In one embodiment, the heterologous gene sequence is in the PA viral segment. In one embodiment, the heterologous gene sequence is in the PB1 viral segment. In one embodiment, the heterologous gene sequence is in the PB2 viral segment.In one embodiment, the heterologous gene sequence is 5' or 3' to the PA coding sequence in the PA viral segment, replaces at least a portion of the PA coding sequence, or is inserted into the PA coding sequence. In one embodiment, the heterologous gene sequence is 5' or 3' to the PB1 coding sequence in the PB1 viral segment, replaces at least a portion of the PB1 coding sequence, or is inserted into the PB1 coding sequence. In one embodiment, the heterologous gene sequence is 5' or 3' to the PB2 coding sequence in the PB2 viral segment, replaces at least a portion of the PB2 coding sequence, or is inserted into the PB2 coding sequence (see, e.g., Avilov et al., 2012). In one embodiment, the heterologous gene sequence is 5' or 3' to the NS coding sequence in the NS viral segment, replaces at least a portion of the NS coding sequence, or is inserted into the NS coding sequence (Manicassamy et al., 2010). In one embodiment, the heterologous gene sequence is 5' or 3' to the NS1 coding sequence in the NS viral segment, replaces at least a portion of the NS1 coding sequence, or is inserted within the NS1 coding sequence. In one embodiment, the heterologous gene sequence is 5' or 3' to the NS2 coding sequence in the NS viral segment, replaces at least a portion of the NS2 coding sequence, or is inserted within the NS2 coding sequence. In one embodiment, the heterologous gene sequence is 5' or 3' to the HA coding sequence in the HA viral segment, replaces at least a portion of the HA coding sequence, or is inserted within the HA coding sequence. In one embodiment, the heterologous gene sequence is 5' or 3' to the NA coding sequence in the NA viral segment, replaces at least a portion of the NA coding sequence, or is inserted within the NA coding sequence (see, e.g., Perez et al., 2004). In one embodiment, the heterologous gene sequence is 5' or 3' to the M1 coding sequence in the M viral segment, replaces at least a portion of the M1 coding sequence, or is inserted within the M1 coding sequence.In one embodiment, the heterologous gene sequence is 5' or 3' to the M2 coding sequence in the M viral segment, replaces at least a portion of the M2 coding sequence, or is inserted into the M2 coding sequence (see, e.g., Wei et al., 2011).

[0019] Also provided are vaccines comprising the recombinant viruses of the present disclosure, e.g., live attenuated vaccines, or vaccines in which the recombinant viruses have been cold-adapted, one or more vectors comprising one or more viral segments with one or more of the disclosed substitutions, and methods of making and using the recombinant viruses. In one embodiment, the vector for vRNA production comprises a promoter such as an RNA polymerase I promoter, an RNA polymerase II promoter, an RNA polymerase III promoter, a T3 promoter, or a T7 promoter.

[0020] Also provided is a method for producing influenza viruses with modified properties, such as enhanced replication or stability, in a selected avian or mammalian host, comprising serially passaging isolates of influenza virus in distinct host organisms, identifying the distinct viruses with the modified properties, and optionally molecularly characterizing the distinct viruses.

[0021] Further provided is a set of recombinant influenza viruses, wherein each element of the set encodes a distinct optically detectable marker, e.g., an open reading frame fused to an open reading frame of an influenza viral protein, an open reading frame on viral segment 9 for influenza A virus or influenza B virus, or an open reading frame replacing at least a portion of one of the viral protein coding regions. For example, one of the elements includes a photoprotein gene, e.g., a luciferase gene, a fluorescent protein gene, e.g., a green fluorescent protein gene, a yellow fluorescent protein gene, or a red fluorescent protein gene, aequorin photoprotein gene, or an obelin photoprotein gene, a chloramphenicol acetyltransferase gene, a phosphatase gene, e.g., an alkaline phosphatase gene, a peroxidase gene, e.g., a horseradish peroxidase gene, a beta-galactosidase gene, a beta-lactamase gene, or a beta-glucuronidase gene. [Brief explanation of the drawings]

[0022] [Figure 1A] Characterization of mouse-adapted Venus-PR8 in mice. Four B6 mice per group were inoculated intranasally with WT-PR8, WT-Venus-PR8, or MA-Venus-PR8. Mouse weight and survival were monitored for 14 days. [Figure 1B] Characterization of mouse-adapted Venus-PR8 in mice. Four B6 mice per group were inoculated intranasally with WT-PR8, WT-Venus-PR8, or MA-Venus-PR8. Mouse weight and survival were monitored for 14 days. [Figure 1C]Characterization of mouse-adapted Venus-PR8 in mice. Four B6 mice per group were inoculated intranasally with WT-PR8, WT-Venus-PR8, or MA-Venus-PR8. Mouse weight and survival were monitored for 14 days. [Figure 1D] Characterization of mouse-adapted Venus-PR8 in mice. Four B6 mice per group were inoculated intranasally with WT-PR8, WT-Venus-PR8, or MA-Venus-PR8. Mouse weight and survival were monitored for 14 days. [Figure 1E] Characterization of mouse-adapted Venus-PR8 in mice. Four B6 mice per group were inoculated intranasally with WT-PR8, WT-Venus-PR8, or MA-Venus-PR8. Mouse weight and survival were monitored for 14 days. [Figure 1F] Characterization of mouse-adapted Venus-PR8 in mice. Four B6 mice per group were inoculated intranasally with WT-PR8, WT-Venus-PR8, or MA-Venus-PR8. Mouse weight and survival were monitored for 14 days. [Figure 1G] Characterization of mouse-adapted Venus-PR8 in mice. Lungs from animals (3 mice per group) infected with 104 PFU of PR8 or MA-Venus PR8 were harvested on days 3, 5, and 7 post-infection. Viral titers were analyzed by plaque assay in MDCK cells. [Figure 2]Distribution of influenza A virus in the lung. (A) Lung tissue was collected from B6 mice on days 3 and 5 after infection with influenza A virus (105 PFU of MA-eCFP, eGFP, Venus, and mCherry-PR8). The open reading frame (ORF) of the NS1 gene without a stop codon was fused to the N-terminus of the fluorescent reporter genes (Venus, eCFP, eGFP, and mCherry) via a sequence encoding the protein linker GSGG. The fluorescent gene was followed by a sequence encoding a GSG linker, a 57-nucleotide foot-and-mouth disease virus protease 2A autoproteolytic site derived from porcine teschovirus-1, and the NEP ORF. In addition, a silent mutation was introduced into the endogenous splice acceptor site of the NS1 ORF to prevent splicing. Whole-mount images of the cleared lung tissue were obtained using a fluorescent stereomicroscope. (B, C) B6 mice were inoculated intranasally with a mixture of MA-eCFP, eGFP, Venus, and mCherry-PR8 (2.5 × 10 PFU per strain). Scale bar = 5 mm. (B) Lung sections were analyzed on days 2 and 5 postinfection using an inverted fluorescence microscope equipped with a Nuance FX multispectral imaging system with InForm software. Scale bar = 100 μm. (C) Magnified images of the indicated areas in (B) were unmixed and separated into autofluorescence (AF), eCFP, eGFP, Venus, and mCherry fluorescence. Arrows in the merged images indicate cells infected with different influenza virus variants. [Figure 3A] Analysis of macrophage infiltration. Lung tissues were harvested from PBS-inoculated mice (mock) or mice infected with 10 PFU of MA-Venus-PR8 on day 2 postinfection, fixed, and processed for histological analysis. Sections were incubated with PE-anti-Mac3 antibody to detect macrophages (red), and nuclei were visualized by counterstaining with Hoechst dye (blue). Fluorescent signals of Venus protein are shown in green. The scale bar indicates 200 μm. [Figure 3B] Analysis of macrophage infiltration. Lung tissues were harvested from PBS-inoculated mice (mock) or mice infected with 10 PFU of MA-Venus-PR8 on day 2 postinfection, fixed, and processed for histological analysis. Sections were incubated with PE-anti-Mac3 antibody to detect macrophages (red), and nuclei were visualized by counterstaining with Hoechst dye (blue). Fluorescent signals of Venus protein are shown in green. The scale bar indicates 200 μm. [Figure 3C] Analysis of macrophage infiltration. Lung tissues were harvested from PBS-inoculated mice (mock) or mice infected with 10 PFU of MA-Venus-PR8 on day 2 postinfection, fixed, and processed for histological analysis. Sections were incubated with PE-anti-Mac3 antibody to detect macrophages (red), and nuclei were visualized by counterstaining with Hoechst dye (blue). Fluorescent signals of Venus protein are shown in green. The scale bar indicates 200 μm. [Figure 3D] Analysis of macrophage infiltration. Lung tissues were harvested from PBS-inoculated mice (mock) or mice infected with 10 PFU of MA-Venus-PR8 on day 2 postinfection, fixed, and processed for histological analysis. Sections were incubated with PE-anti-Mac3 antibody to detect macrophages (red), and nuclei were visualized by counterstaining with Hoechst dye (blue). Fluorescent signals of Venus protein are shown in green. The scale bar indicates 200 μm. [Figure 3E]Analysis of macrophage infiltration. Kinetics of the interaction between virus-infected cells and lung macrophages. Two-photon microscopy was used to image eGFP-positive cells (green) and CD11b+ macrophages (red) in lung tissue from naive B6 mice (upper left panel) and B6 mice on day 3 after infection with 105 PFU of MA-eGFP-PR8 (upper right panel). Sequential images in the lower panels (1–4) show enlargements of the boxes in the upper right panel. Arrowheads indicate blebbing of eGFP-positive cells. Scale bar = 40 μm. [Figure 3F] Analysis of macrophage infiltration. Kinetics of the interaction between virus-infected cells and lung macrophages. Two-photon microscopy was used to image eGFP-positive cells (green) and CD11b+ macrophages (red) in lung tissue from naive B6 mice (upper left panel) and B6 mice on day 3 after infection with 105 PFU of MA-eGFP-PR8 (upper right panel). Sequential images in the lower panels (1–4) show enlargements of the boxes in the upper right panel. Arrowheads indicate blebbing of eGFP-positive cells. Scale bar = 40 μm. [Figure 3G] Analysis of macrophage infiltration. Kinetics of the interaction between virus-infected cells and lung macrophages. Two-photon microscopy was used to image eGFP-positive cells (green) and CD11b+ macrophages (red) in lung tissue from naive B6 mice (upper left panel) and B6 mice on day 3 after infection with 105 PFU of MA-eGFP-PR8 (upper right panel). Sequential images in the lower panels (1–4) show enlargements of the boxes in the upper right panel. Arrowheads indicate blebbing of eGFP-positive cells. Scale bar = 40 μm. [Figure 3H]Analysis of macrophage infiltration. Kinetics of the interaction between virus-infected cells and lung macrophages. Two-photon microscopy was used to image eGFP-positive cells (green) and CD11b+ macrophages (red) in lung tissue from naive B6 mice (upper left panel) and B6 mice on day 3 after infection with 105 PFU of MA-eGFP-PR8 (upper right panel). Sequential images in the lower panels (1–4) show enlargements of the boxes in the upper right panel. Arrowheads indicate blebbing of eGFP-positive cells. Scale bar = 40 μm. [Figure 3I] Analysis of macrophage infiltration. Infection of macrophages with influenza virus. Single-cell suspensions were obtained from the lungs of PBS-inoculated (mock) mice or mice infected with 10 PFU of MA-Venus-PR8 on day 3 postinfection, stained with antibodies against CD45, CD11b, and F4 / 80, and analyzed by flow cytometry. The panels show Venus expression relative to the CD11b staining profile from cells gated on F4 / 80 and CD45 expression levels. [Figure 3J]Analysis of macrophage infiltration. Gene expression analysis. Total RNA was isolated from sorted macrophages from PBS-inoculated (naive) mice and from sorted Venus-positive (Venus(+)) and Venus-negative (Venus(-)) macrophages from mice inoculated with 105 PFU of MA-Venus-PR8 (nine mice per treatment) on day 3 postinfection and subjected to microarray analysis. Differentially expressed (DE) transcripts were identified by comparing gene expression levels in naive macrophages with those in Venus(+) macrophages derived from infected mice. Similarly, gene expression levels were compared between naive and infected Venus(-) macrophages. DE transcripts were organized by hierarchical clustering, and each cluster was analyzed for enhanced biological function. Heatmaps of clustered transcripts for each condition are shown (color key indicated at the top of the panel), and distinct clusters are indicated by color bars on the left of the heatmap. The enrichment annotations for each cluster are listed to the left of each cluster, with the enrichment score for each annotation in parentheses. The blue line in the heatmap indicates the fold change of DE transcripts when comparing Venus(+) and Venus(-) macrophages. A shift in the blue line to the left indicates higher expression of DE transcripts in Venus(+) macrophages, and a shift to the right indicates higher expression of DE transcripts in Venus(-) macrophages. [Figure 3K] Analysis of macrophage infiltration. This panel shows a heat map comparing the expression levels of type I interferon (IFN) between Venus(+) and Venus(-) macrophages. The color key is shown at the bottom of the panel. NS indicates a statistically insignificant comparison between Venus(-) cells from infected animals and naive macrophages from uninfected animals. [Figure 4]Characterization of MA-Venus-HPAI virus. (A) Four B6 mice per group were inoculated intranasally with MA-Venus-HPAI virus. Mouse weight and survival were monitored for 14 days. (B) Lungs, spleens, kidneys, and brains were collected from B6 mice on day 3 after infection with 10 PFU of MA-Venus-HPAI virus. Viral titers in tissue homogenates were determined using plaque assays in MDCK cells. Each data point represents the mean ± standard deviation (n = 3). (C, D) Lung tissues were collected from B6 mice on days 1 and 2 after infection with 10 PFU of MA-Venus-HPAI virus and PR8. Images of cleared lung tissue (bronchi (red); alveoli (green)) were obtained by two-photon microscopy. Each data point represents the mean ± standard deviation (n = 3). Statistical significance was calculated using Student's t-test. (D) The distribution of Venus-positive cells was assessed by volumetric analysis of Venus-positive bronchial and alveolar areas using 3D images of cleared lung tissue. (E) Cells were collected from the lungs of B6 mice on days 1, 2, 3, and 4 after infection with 105 PFU of MA-Venus-PR8 or MA-Venus-HPAI virus and stained for CD45, CD11b, and F4 / 80. Venus expression in CD45-negative cells and the Venus vs. F4 / 80 staining profile gated on CD45-positive cells were analyzed by flow cytometry. A representative data plot is shown on day 2 after infection, along with the percentage of Venus-positive cells. [Figure 5] Viral yields for various viruses. [Figure 6] Virulence of WT-Venus-H5N1 and RG-MA viruses in mice. Groups of four mice were intranasally infected with WT-Venus-H5N1 virus at doses of 101–105 PFU or RG-MA virus at doses of 100–105 PFU, and their body weight change (AC) and survival rate (BD) were monitored for 2 weeks. [Figure 7]Venus expression in various H5N1 viruses in MDCK cells. MDCK cells were infected with Venus-H5N1 related viruses, and the Venus expression in each virus plaque was observed at 24 hpi using fluorescence microscopy (Axio Observer.Z1, Zeiss). Representative images of each virus are shown. [Figure 8] Venus expression in mouse lungs of various H5N1 viruses. Groups of three mice were intranasally infected with 105 PFU (50 μL) of virus. On day 2 post-infection, the mice were euthanized, and their lungs were collected, fixed in 4% PFA, and then embedded in OCT Compound. Frozen tissues were cut into 5 μm slices and stained with Hoechst 33342. Venus signals were detected using a Nikon A1+ confocal microscope system. Blue represents nuclei stained with Hoechst 33342, and green represents Venus expression. [Figure 9] Genotypes of Venus-H5N1-related reassortant viruses and their virulence in mice. Color indicates the origin of the viral segments (blue: WT-Venus-H5N1 virus; red: MA-Venus-H5N1 virus). MLD values ​​were determined by inoculating groups of four mice with 10-fold serial dilutions containing 100–10 PFU of virus in a volume of 50 μL and calculated using the method of Reed and Muench (30). [Figure 10] Growth kinetics of reassortants in MDCK cells. MDCK cells were infected with the virus at an MOI of 0.0001, and culture supernatants were collected at the indicated times and then titrated in MDCK cells. Reported values ​​are the mean ± standard deviation (SD) from two independent experiments. **, P < 0.01 compared to WT-Venus-H5N1 virus-infected cells. [Figure 11]Polymerase activity of different RNP combinations derived from WT-Venus-H5N1 and MA-Venus-H5N1 viruses. 293 cells were transfected in triplicate with plasmids expressing PB1, PB2, PA, and NP from either WT-Venus-H5N1 or MA-Venus-H5N1 viruses, along with a luciferase reporter plasmid and an internal control plasmid. Segments derived from WT-Venus-H5N1 virus are shown in white, while segments derived from MA-Venus-H5N1 virus are shown in green. Cells were incubated at 37°C for 24 hours, and cell lysates were analyzed to measure firefly luciferase and Renilla luciferase activities. Values ​​shown are the mean ± SD of three independent experiments and are normalized to the activity of WT-Venus-H5N1 (100%). *, P < 0.05 compared to WT-Venus-H5N1 virus. **, P < 0.01 compared with WT-Venus-H5N1 virus. [Figure 12A] Venus-NS and deleted NS segments of Venus-H5N1-related reassortants. Viruses were passaged five times in MDCK cells, and vRNA from the fifth passage was extracted using the QIAamp® Viral RNA Mini Kit (QIAGEN). Each NS segment was then amplified by PCR with NS-specific primers and run on an agarose gel. Lane 1, WT+MA-NS; lane 2, WT+MA-M; lane 3, WT+MA-NA; lane 4, WT+MA-PA; lane 5, WT+MA-PB1; lane 6, WT+MA-PB2; lane 7, WT+MA-(PB2+PA); lane 8, WT-Venus-H5N1; lane 9, RG-MA; lane 10, PR8; lane 11, 1-kb DNA marker. [Figure 12B] Schematic diagram of the deletion viruses. [Figure 13] High expression of Venus reassortants in mouse lungs. [Figure 14]Comparison of the growth ability of mutant viruses in MDCK cells. MDCK cells were infected with PR8 viruses carrying the amino acid substitutions found in the NS1-Venus PR8 MA virus, NS1-Venus PR8 WT virus, NS1-Venus PR8 MA virus, and mutant NS1-Venus PR8 viruses at an MOI of 0.001. Viral titers were determined every 12 hours by plaque assay. Results are expressed as the mean titer (log10 [PFU / mL]) ± standard deviation. [Figure 15] Body weight change and survival rate for mice infected with NS1-Venus PR8 virus. Four mice per group were intranasally infected with 10 PFU, 10 PFU, and 10 PFU of NS1-Venus PR8 virus. Body weight was measured, and survival rate was monitored for 14 days post-infection. [Figure 16A] Viral titers in mouse lungs. Nine mice per group were intranasally infected with 103 PFU of PR8. Three mice per group were euthanized on days 3, 5, and 7 post-infection, and their lungs were harvested to determine viral titers. Viral titers were determined by plaque assay. Results are expressed as the mean titer (log10 PFU / g) ± standard deviation. Statistical significance was calculated using the Tukey-Kramer method. Asterisks indicate significant differences from titers obtained from mice infected with PR8 virus or NS1-Venus WT virus (P<0.05). ND: Not detected (detection limit, 5 PFU / lung). [Figure 16B]Viral titers in mouse lungs. Nine mice per group were intranasally infected with each NS1-Venus PR8 virus. Three mice per group were euthanized on days 3, 5, and 7 post-infection, and their lungs were harvested to determine viral titers. Viral titers were determined by plaque assay. Results are expressed as the mean titer (log10 PFU / g) ± standard deviation. Statistical significance was calculated using the Tukey-Kramer method. Asterisks indicate significant differences from titers obtained from mice infected with PR8 virus or NS1-Venus WT virus (P<0.05). ND: Not detected (detection limit, 5 PFU / lung). [Figure 17] Stability of Venus expression by NS1-Venus PR8 MA virus in vitro and in vivo. The positive rate of Venus expression was examined in MDCK cells and mouse lungs. Left panel: MDCK cells were infected with NS1-Venus PR8 MA virus at an MOI of 0.001, and supernatants were collected every 24 hours. The positive rate of Venus expression was estimated by dividing the number of Venus-expressing plaques by the total number of plaques. Middle panel: NS1-Venus PR8 MA virus was serially passaged five times in MDCK cells, and the positive rate of Venus expression was estimated. Right panel: Nine mice were infected with 103 PFU of NS1-Venus PR8 MA virus. Three mice were euthanized at each time point, and plaque assays were performed using lung homogenates. The positive rate of Venus expression was estimated as described above. [Figure 18A] Comparison of Venus expression in cells infected with each NS1-Venus PR8 virus. Venus protein expression in cells infected with each NS1-Venus PR8 virus was detected by Western blotting. MDCK cells were infected with each virus at an MOI of 1. 12 hours after infection, virus-infected cells were lysed and Western blotting was performed. Venus protein was detected using an anti-GFP antibody, and M1 protein was detected as a control. A band observed at approximately 27 kDa is shown in the M1 panel. Representative results from two independent experiments are shown. [Figure 18B] Comparison of Venus expression in cells infected with each NS1-Venus PR8 virus. Venus protein expression in cells infected with each NS1-Venus PR8 virus was detected by Western blotting. MDCK cells were infected with each virus at an MOI of 1. 12 hours after infection, virus-infected cells were lysed and Western blotting was performed. Venus protein was detected using an anti-GFP antibody, and M1 protein was detected as a control. A band observed at approximately 27 kDa is shown in the M1 panel. Representative results from two independent experiments are shown. [Figure 18C] Comparison of Venus expression in cells infected with each NS1-Venus PR8 virus. Observation of Venus expression using confocal microscopy. MDCK cells were infected with each virus at an MOI of 1. 12 hours after infection, the cells were fixed and Venus expression was observed. Representative results from two independent experiments are shown. MDCK cells were infected with the indicated viruses (MOI of 1) and subjected to confocal microscopy 12 hours later. [Figure 18D] Comparison of Venus expression in cells infected with each NS1-Venus PR8 virus. Observation of Venus expression using confocal microscopy. MDCK cells were infected with each virus at an MOI of 1. 12 hours after infection, the cells were fixed and Venus expression was observed. Representative results from two independent experiments are shown. MDCK cells were infected with the indicated viruses (MOI of 1) and subjected to confocal microscopy 12 hours later. [Figure 18E] Comparison of Venus expression in cells infected with each NS1-Venus PR8 virus. Observation of Venus expression using confocal microscopy. MDCK cells were infected with each virus at an MOI of 1. 12 hours after infection, the cells were fixed and Venus expression was observed. Representative results from two independent experiments are shown. MDCK cells were infected with the indicated viruses (MOI of 1) and subjected to confocal microscopy 12 hours later. [Figure 18F]Comparison of Venus expression in cells infected with each NS1-Venus PR8 virus. Observation of Venus expression using confocal microscopy. MDCK cells were infected with each virus at an MOI of 1. 12 hours after infection, the cells were fixed and Venus expression was observed. Representative results from two independent experiments are shown. MDCK cells were infected with the indicated viruses (MOI of 1) and subjected to confocal microscopy 12 hours later. [Figure 18G] Comparison of Venus expression in cells infected with each NS1-Venus PR8 virus. HEK293 cells were infected with viral protein expression plasmids for NP, PA, PB1, and PB2 or PB-2-E712D together with a plasmid expressing vRNA-encoding firefly luciferase. [Figure 19] Multinucleate cell formation by HEK293 cells infected with wild-type PR8 or PR8 carrying the HA-T380A mutation after exposure to low pH buffers. The threshold for membrane fusion was examined over a pH range of 5.5 to 5.9. HEK293 cells were infected with PR8 or PR8 carrying the HA-T380A mutation. 18 hours after infection, HA on the cell surface was digested with TPCK-trypsin and exposed to the indicated pH buffers. After fixation with methanol, the cells were stained with Giemsa solution. Representative photographs are shown. [Figure 20]Time-lapse observation of Venus-expressing cells in cleared lungs. Venus-expressing cells were observed in all lung lobes. Three mice per group were intranasally infected with NS1-Venus PR8 MA (A–F), NS1-Venus PR8 WT (G, H), or PR8 (I, J) viruses, and lungs were harvested on the indicated days. Mock-treated lungs served as negative controls (K, L). To image Venus-expressing cells more deeply, lung samples were treated with SCALEVIEW A2, which clears the samples, separated into individual lobes, and observed using a stereofluorescence microscope. After imaging the entire lung lobe (untreated), the samples were dissected to expose the bronchi (sections). Samples from mice infected with PR8 or NS1-Venus PR8 WT viruses were prepared on day 3 postinfection and compared with NS1-Venus PR8 MA virus-infected lungs, in which the Venus signal was brightest during infection. Representative images are shown. [Figure 21] Analysis of Venus expression in CC10+ and SP-C+ cells in the lung. Lung sections from mice infected with NS1-Venus PR8 MA virus were stained with several antibodies specific for epithelial cells in the lung. Mice were infected with 104 PFU of NS1-Venus PR8 MA virus, and lungs were harvested 3 and 5 days postinfection. (A) Lung sections from mice infected with NS1-Venus PR8 MA virus were prepared 3 days postinfection and stained with anti-CC10 polyclonal antibody (red). Scale bar: 100 μm. (B) Lung sections from mice infected with NS1-Venus PR8 MA virus were prepared 5 days postinfection and stained with anti-SP-C polyclonal antibody (cyan) and anti-podoplanin (Pdpn) polyclonal antibody (red). Venus-positive cells in the alveolar region included SP-C-positive cells (white arrowheads) and podoplanin-positive cells (white arrows). Scale bar: 50 μm. [Figure 22]Flow cytometry analysis of Venus-positive cells in the lungs of specific cell types. Venus-positive cells in the indicated cell types were analyzed by flow cytometry. Mice were infected with 105 PFU of PR8 virus or NS1-Venus PR8 MA virus, and lungs were harvested 3 and 5 days post-infection. Single-cell suspensions were stained with antibodies. (A) Representative dot plots of live CD45+ cells from the lungs of PBS-inoculated mice are shown. (B, C) The total numbers of each specific cell type are shown at the indicated time points. Results are expressed as the mean cell number per lung ± standard deviation. CD45+ and Via-probe cells were analyzed for monocytes and alveolar macrophages. (D, E) The number of Venus-positive cells in the cells defined in A and B at the indicated time points are shown. Results are expressed as the mean cell number ± standard deviation. AM: alveolar macrophages. [Figure 23] Sorting strategy for recovering Venus-positive and Venus-negative cells in the F4 / 80+ population. Mice were infected with 105 PFU of NS1-Venus PR8 MA virus, and lungs were harvested 3 days postinfection. Single-cell suspensions were stained with a set of antibodies. Lungs from mice inoculated with PBS were similarly stained to confirm autofluorescence of alveolar macrophages. (A) Representative dot plots showing the gating strategy for recovering Venus-positive and Venus-negative cells in the CD45+, Via-Probe-F4 / 80+ cell population. The Venus-positive gate did not contain alveolar macrophages. (B) Venus-positive and Venus-negative cells recovered from the lungs of mice infected with NS1-Venus PR8 MA virus were observed using an immunofluorescence assay. [Figure 24]Genes differentially expressed between Venus-positive F4 / 80+ cells and Venus-negative F4 / 80+ cells. Mice were infected with 105 PFU of NS1-Venus PR8 MA virus, and lungs were harvested 3 days postinfection. Single-cell suspensions were stained using the same method as described in Figure 10. Venus-positive and Venus-negative cells were separately collected using a FACSAria II and subjected to microarray analysis. F4 / 80+ cells isolated from the lungs of mice inoculated with PBS served as a control. (A) A total of 633 genes were selected by Student's t-test (P<0.05) and by sorting genes whose expression changed by at least 4.0-fold between the Venus-positive and Venus-negative groups from genes whose expression changed by at least 2.0-fold from the PBS group level. (B) These selected genes were functionally annotated using Gene Ontology (GO) grouping. Statistical significance was determined by using Fisher's exact test (P<0.01). (C) Hierarchical analysis of genes annotated in "cytokine activity" enriched by genes that were significantly differentially expressed between Venus-positive F4 / 80+ cells and Venus-negative F4 / 80+ cells. (D) Hierarchical analysis of genes annotated in "response to wounding" enriched by genes that were significantly differentially expressed between Venus-positive F4 / 80+ cells and Venus-negative F4 / 80+ cells. [Figure 25] Exemplary parental sequences for PR8HG and Cambridge strains of PR8 (SEQ ID NOs: 1-19). [Figure 26] Schematic diagram of a fusion protein containing a heterologous protein. [Figure 27] Schematic of mutations in polymerase complex proteins that stabilize heterologous gene products mapped onto the structure of the complex (PDB ID: 4WSB). [Figure 28]A) Schematic structure of the eight viral RNA segments contained in WT-Venus-PR8. 2A: Protease 2A autoproteolysis site. (B) Each virus was passaged in MDCK cells. The percentage of Venus-expressing plaques in virus stocks derived from different passages was determined in MDCK cells using fluorescence microscopy. (C) Virus stocks derived from different passages were titrated using plaque assays in MDCK cells. [Figure 29] Effect of PB2-E712D on mutation rate. (A) Each virus was passaged five times in MDCK cells, and the mutations introduced into each segment during passage were counted. (B) The number of mutations per nucleotide in each segment was calculated, and the average value for all eight segments is shown. [Figure 30] RNA and protein expression in infected cells. (A–C) MDCK cells were infected with each virus at an MOI of 1. The relative expression levels of IFN-β mRNA (A), NS vRNA (B), and NP vRNA (C) were determined by quantitative real-time PCR 9 hours postinfection. (D) The NS vRNA / NP vRNA ratio was calculated. (E) MDCK cells were infected with WT-Venus-PR8 (WT) or Venus-PR8-PB2-E712D (712) at an MOI of 1. At the indicated time points, cells were lysed, and the expression of NS1, NP, and β-actin was detected by Western blotting. (F) The NS1 / NP ratio was determined based on the band intensity in Western blotting. Panels A, B, C, D, and F show the mean ± standard deviation of triplicate experiments, with each value representing 1 for Venus-PR8-PB2-E712D-infected cells. **, P<0.01; ns, not significant (Student's t test); hpi, hours post-infection. [Figure 31]An internal deletion occurred in the NS segment of WT-Venus-PR8. (A) Schematic sequence of the NS segment of the WT-Venus-PR8 virus that lost Venus expression after serial passage in MDCK cells. Selected examples are shown. (B) Methodology for coinfection experiments is shown. Synonymous mutations were introduced into the 3' or 5' region of the NS segment of WT-Venus-PR8. MDCK cells were then coinfected with the virus. Viruses not expressing Venus were plaque purified, and the sequences of their NS segments were analyzed. (C) Example sequences of the NS segment of Venus-negative viruses obtained after coinfection experiments. Red "X"s indicate introduced synonymous mutations. [Figure 32] Additional mutations that stabilize the Venus gene inserted into the NS segment. (A) The identified amino acid mutations were mapped onto the influenza polymerase complex (PDB ID 4WSB). (B) The polymerase internal tunnel (shown as a yellow tube). The vRNA promoter (vRNA promotewr) binds to the polymerase, and the template vRNA enters the polymerase complex. The template vRNA passes through the active site where RNA synthesis occurs and then exits through the template exit. The RNA product synthesized at the active site exits through the product exit. (C) Each mutant Venus-PR8 virus was passaged four times in MDCK cells, and the percentage of Venus-expressing plaques after passage was determined in MDCK cells using fluorescence microscopy. (D) The percentage of influenza A virus strains containing mutations that stabilize the Venus gene in Venus-PR8 (i.e., the number of strains containing the indicated amino acids / total number of strains available in the Influenza Research Database). DETAILED DESCRIPTION OF THE INVENTION

[0023] definition As used herein, the term "isolated" refers to the in vitro preparation and / or separation of a nucleic acid molecule, e.g., a vector or plasmid, peptide or polypeptide (protein), or virus of the present disclosure, such that it is not associated with materials in vivo or is substantially purified from materials in vitro. Isolated virus preparations are generally obtained by in vitro culture and propagation and / or through passage in eggs, and are substantially free of other infectious agents.

[0024] As used herein, "substantially purified" means that the species of interest is the predominant species, e.g., on a molar basis, more abundant than any other individual species in a composition, e.g., at least about 80% of the species present, and optionally greater than 90%, e.g., 95%, 98%, 99% or greater of the species present in said composition.

[0025] As used herein, "substantially free" means below the level of detection for a particular infectious agent using standard detection methods for that agent.

[0026] A "recombinant" virus is one that has been manipulated in vitro, for example, using recombinant DNA techniques, to introduce alterations into the viral genome. Reassortant viruses can be prepared by recombinant or non-recombinant techniques.

[0027] As used herein, the term "recombinant nucleic acid" or "recombinant DNA sequence or segment" refers to a nucleic acid, e.g., DNA, that has been derived or isolated from a source and may subsequently be chemically modified in vitro, such that the sequence is not naturally occurring or corresponds to a naturally occurring sequence that is not located where it is located in a natural genome. An example of DNA "derived" from a source is a DNA sequence that is identified as a useful fragment and then chemically synthesized in substantially pure form. An example of such DNA that is "isolated" from a source is a useful DNA sequence that has been excised or removed from the source by chemical means, e.g., by using restriction endonucleases, so that it can be further manipulated, e.g., amplified, for use in the present invention, by genetic engineering methods.

[0028] As used herein, a "heterologous" influenza virus gene or viral segment is derived from a different influenza virus source than the majority of other influenza virus genes or viral segments in the recombinant (eg, reassortant) influenza virus.

[0029] The terms "isolated polypeptide," "isolated peptide," or "isolated protein" include polypeptides, peptides, or proteins encoded by cDNA or recombinant RNA, including those of synthetic origin, or some combination thereof.

[0030] As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule expressed from a recombinant DNA molecule. In contrast, the term "native protein" is used herein to refer to a protein that has been isolated from a naturally occurring (i.e., non-recombinant) source. Molecular biology techniques may be used to produce a recombinant form of a protein that has the same properties as the native form of the protein.

[0031] Methods for aligning sequences for comparison are well known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm.

[0032] Computer implementations of these mathematical algorithms can be used to compare sequences to determine sequence identity. Alignments using these programs can be performed using default parameters. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm may first identify high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that meet or satisfy some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score decreases by the amount X from its maximum achieved value, the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.

[0033] In addition to calculating percent sequence identity, the BLAST algorithm may also perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm may be the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered to be similar to a reference nucleic acid sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, less than about 0.01, or less than about 0.001.

[0034] The BLASTN program (for nucleotide sequences) may use as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program may use as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See http: / / www.ncbi.nlm.nih.gov. Alignments may also be performed manually by inspection.

[0035] For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0036] "Conservative" amino acid substitutions refer to the interchangeability of residues with similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. In one embodiment, conservative amino acid substitution groups are threonine-valine-leucine-isoleucine-alanine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0037] Encapsidation sequence Viral segments for incorporation of heterologous gene sequences into recombinant influenza viruses contain non-coding sequences at each end that provide for encapsidation (integration or packaging) into virions. The viral segment also contains flanking coding sequences from one or both ends that contribute to encapsidation, e.g., enhance encapsidation compared to viral segments lacking flanking coding sequences, but that contain heterologous gene sequences. Thus, vectors containing viral segments bearing heterologous gene sequences contain encapsidation sequences at the 3' end of the vRNA, which may include flanking 5' coding sequences, at the 5' end of the vRNA, which may include 3' coding sequences, or at the 3' end of the vRNA, which may include flanking 5' coding sequences, and at the 5' end of the vRNA, which may include 3' coding sequences. For example, the HA encapsidation sequence comprises a sequence at the 3' end of the HA vRNA that contains 33-nt noncoding sequence and at least 3, 6, 9, or 15, or up to about 216, nt of HA coding sequence, and / or a sequence at the 5' end of the HA vRNA that contains about 45 nt of noncoding sequence and up to about 75, 80, 268, or 291, nt of HA coding sequence (Watanabe et al., 2003).The HS encapsidation sequence comprises a sequence at the 3' end of the NS vRNA that contains at least 30, 60, 90, or 150 nt of coding sequence, and a sequence at the 5' end of the NS vRNA that contains at least 30, 60, 90, or 100 nt of coding sequence (Fujii et al., 2005).

[0038] In one embodiment, the 3' NA incorporation sequence corresponds to any integer between nucleotides 1-183, nucleotides 1-90, nucleotides 1-45, nucleotides 1-21, nucleotides 1-19, or 19-183 of the N-terminal NA coding region, and may include a mutation in the NA start codon. In another embodiment, the 5' NA incorporation sequence corresponds to a sequence in the C-terminal coding region of NA, i.e., 39, 78, or 157, or any integer between 1 and 157, of the 3' terminal nucleotide of the C-terminal NA coding region.

[0039] In one embodiment, the 5' HA incorporation sequence corresponds to a sequence in the C-terminal coding region of HA, i.e., a sequence corresponding to the 3'-terminal nucleotide 75, 80, 268, 291, or 518, or any integer between 1 and 518, of the C-terminal HA coding region. The 3' HA incorporation sequence corresponds to nucleotides 1-3, 1-6, 1-9, 1-15, 1-216, 1-468, or any integer between 1 and 468, of the N-terminal HA coding region.

[0040] In one embodiment, the 3' PB1 or PB2 incorporation sequence corresponds to nucleotides 1-250, nucleotides 1-200, nucleotides 1-150, nucleotides 1-160, or 1-130 of the N-terminal PB1 or PB2 coding region, or any integer between 1 and 250. In one embodiment, the 5' PB1 or PB2 incorporation sequence corresponds to the 3'-terminal nucleotides of the C-terminal PB1 or PB2 coding region, e.g., 1-250 nucleotides, 1-200 nucleotides, nucleotides 1-150, nucleotides 1-160, 1-170, or 1-190, or any integer between 1 and 250.

[0041] In one embodiment, the 3' PA incorporation sequence corresponds to nucleotides 1 to 250, nucleotides 1 to 200, nucleotides 1 to 150, or any integer between 1 and 250 of the N-terminal PA coding region. In one embodiment, the 5' PA incorporation sequence corresponds to the 3' terminal nucleotides of the C-terminal PA coding region, e.g., the 3' 1 to 250 nucleotides, 1 to 200 nucleotides, nucleotides 1 to 150, nucleotides 1 to 160, 1 to 170, or 1 to 190, or any integer between 1 and 250.

[0042] In one embodiment, the 3' M incorporation sequence corresponds to nucleotides 1-250, nucleotides 1-242, nucleotides 1-240, or any integer between 1 and 250 of the N-terminal M coding region, and optionally includes a mutation in the M start codon. In another embodiment, the 5' M incorporation sequence corresponds to a sequence in the C-terminal coding region of M, i.e., a sequence corresponding to 50, 100, or 220, or any integer between 1 and 250, 3'-terminal nucleotides for the C-terminal M coding region.

[0043] In one embodiment, the 3' NS or NP incorporation sequence corresponds to nucleotides 1-250, nucleotides 1-200, nucleotides 1-150, nucleotides 1-30, or any integer between 1 and 250, e.g., 1-60, 1-70, 1-80, or 1-90, of the N-terminal NS or NP coding region, and may include a mutation in the NS or NP start codon. In another embodiment, the 5' NS or NP incorporation sequence corresponds to a sequence in the C-terminal coding region of NS or NP, i.e., 10, 30, 150, 200, or 250, or any integer between 1 and 250, for the C-terminal NS or NP coding region, e.g., a sequence corresponding to nucleotides 1-250, nucleotides 1-200, nucleotides 1-150, nucleotides 1-30, or any integer between 1 and 250, e.g., 1-60, 1-70, 1-80, or 1-90, of the C-terminal NS or NP codon region.

[0044] Thus, the present disclosure provides influenza virus vectors comprising sequences corresponding to the 3' and 5' non-coding regions of a particular vRNA, an incorporation sequence of the corresponding vRNA, and a heterologous nucleic acid segment. Thus, in one embodiment, the vector comprises the 3' non-coding region of an NA vRNA, a 3' NA vRNA incorporation sequence or a 5' NA vRNA incorporation sequence, optionally both the 3' and 5' NA incorporation sequences, a heterologous nucleic acid segment, and the 5' non-coding region of an NA vRNA. In another embodiment, the vector comprises the 3' non-coding region of an HA vRNA, a 5' HA vRNA incorporation sequence or a 3' HA vRNA incorporation sequence, or both the 5' and 3' HA incorporation sequences, a heterologous nucleic acid segment, and the 5' non-coding region of an HA vRNA. In another embodiment, the vector comprises the 3' non-coding region of an NS vRNA, an NS incorporation sequence, a heterologous nucleic acid segment, and the 5' non-coding region of an NS vRNA. In another embodiment, the vector comprises the 3' non-coding region of the M vRNA, a 5' M incorporation sequence, or a 3' M incorporation sequence, or both a 5' M incorporation sequence and a 3' M incorporation sequence, a heterologous nucleic acid segment, and a 5' non-coding region of the M vRNA. In yet another embodiment, the vector comprises the 3' non-coding region of the PB2 vRNA, a heterologous nucleic acid segment, a PB2 incorporation sequence, and a 5' non-coding region of the PB2 vRNA. When two incorporation sequences are used in a vector, they may be separated by the heterologous nucleic acid segment. Each vector may be used to prepare vRNA for introduction into cells or to express vRNA in cells in the presence of other influenza virus vRNAs and proteins necessary for virus production.

[0045] In another embodiment, the heterologous gene sequence comprises a sequence corresponding to an open reading frame for a therapeutic gene. In yet a further embodiment, the heterologous gene sequence comprises a sequence corresponding to an open reading frame for an immunogenic peptide or protein of a pathogen or tumor cell, e.g., one useful for inducing a protective immune response. For example, the heterologous nucleic acid segment may encode an immunogenic epitope useful in cancer therapy or a vaccine. A vector containing a heterologous nucleic acid segment may be prepared such that transcription of the vector vRNA generates an mRNA encoding a fusion protein with an influenza protein such as NA. Thus, it is contemplated that the heterologous nucleic acid segment may be fused to a viral integration sequence to encode a fusion protein, e.g., a fusion with the 21 N-terminal residues of NA. The fusion protein may comprise sequences from two different influenza virus proteins, including sequences from two different NA or HA proteins. In another embodiment, the heterologous nucleic acid segment may comprise a sequence corresponding to an IRES linked to an open reading frame.

[0046] In one embodiment of the present disclosure, the heterologous gene sequence may encode a heterologous protein (such as a glycoprotein or a non-influenza viral protein, such as a cytosolic protein, a nucleoprotein, or a mitochondrial-specific protein) that may confer a detectable phenotype. In one embodiment, the heterologous gene sequence may be fused to a truncated portion of the PB2 coding sequence, e.g., one corresponding to a 5' or 3' PB2-encoding integration sequence, optionally forming a chimeric protein. In one embodiment, the heterologous nucleotide sequence replaces or is introduced into a viral viral segment corresponding to the coding region for that segment without disrupting the integration sequence in the coding region of the viral segment. For example, the heterologous nucleotide sequence may be flanked by about 3 to about 400 nucleotides of the 5' and / or 3' PB2 coding region with adjacent non-coding sequence. In one embodiment, the 3' PB2 integration sequence corresponds to any integer between nucleotides 3-400, 3-300, 3-100, 3-50, or 3-400 of the N-terminal and / or C-terminal PB2 coding region. In one embodiment, after infection of host cells with the biologically contained PB2-KO virus, a heterologous protein is produced that is a fusion with the N-terminus and / or C-terminus of the remaining residues of the deleted PB2 protein.

[0047] The vRNA for the additional viral segment or viral segment carrying the heterologous gene sequence may be incorporated into virions with an efficiency of at least 1%, 5%, 10%, or 30%, or at least 50% of the efficiency of the corresponding wild-type vRNA.

[0048] Structure and proliferation of influenza viruses Influenza A viruses have a genome of eight single-stranded negative-sense viral RNAs (vRNAs) that encode at least 10 proteins. The influenza virus life cycle begins with the binding of hemagglutinin (HA) to sialic acid-containing receptors on the surface of host cells, followed by receptor-mediated endocytosis. The low pH in late endosomes causes a conformational shift in HA, exposing the N-terminus of the HA2 subunit (the so-called fusion peptide). The fusion peptide initiates fusion of the viral and endosomal membranes, releasing the matrix protein (M1) and RNP complex into the cytoplasm. The RNP consists of the nucleoprotein (NP), which encapsidates the vRNA, and the viral polymerase complex, formed by the PA, PB1, and PB2 proteins. The RNP is transported into the nucleus, where transcription and replication occur. The RNA polymerase complex catalyzes three distinct reactions: the synthesis of mRNA with a 5' cap and 3' poly(A) structure, the synthesis of full-length complementary RNA (cRNA), and the synthesis of genomic vRNA using cRNA as a template. The newly synthesized vRNAs, NP, and polymerase proteins are then assembled into RNPs, exported from the nucleus, and transported to the plasma membrane, where progeny virus particles budding occurs. The neuraminidase (NA) protein plays a critical role late in infection by removing sialic acid from sialyloligosaccharides, thus releasing newly assembled virions from the cell surface and preventing virus particle self-aggregation. Viral assembly involves protein-protein and protein-vRNA interactions, but the nature of these interactions remains largely unknown.

[0049] Influenza B virus and influenza C virus are structurally and functionally similar to influenza A virus, but there are some differences. For example, influenza B virus does not have the M2 protein with ion channel activity, but has BM2 and has viral segments with both NA and NB sequences. Influenza C virus has only seven viral segments.

[0050] Cell lines that can be used To isolate and / or propagate influenza virus, any cells, including mutant cells that support efficient influenza virus replication, can be used, such as avian cells or mammalian cells, such as human cells, e.g., 293T cells or PER.C6® cells, or canine cells, e.g., MDCK, bovine cells, equine cells, feline cells, porcine cells, ovine cells, rodent cells, e.g., mink cells, e.g., MvLu1 cells, or hamster cells, e.g., CHO cells, or non-human primate cells, e.g., Vero cells. Isolated viruses can be used to prepare reassortant viruses. In one embodiment, the host cells for vaccine production are mammalian or avian continuous cell lines or continuous cell strains. The cells used may be fully characterized, allowing for appropriate testing of the purity of the final product. Data that can be used to characterize a cell include (a) information about its origin, derivation, and passage history, (b) information about its growth and morphological characteristics, (c) results of tests for adventitious factors, (d) distinctive features such as biochemical, immunological, and cytogenetic patterns that allow the cell to be clearly recognized among other cell lines, and (e) results of tests for tumorigenicity. In one embodiment, the passage level or population doubling of the host cells used is as low as possible.

[0051] In one embodiment, the cells are a continuous cell line certified or certifiable by the WHO. Requirements for certifying such a cell line include characterization of at least one of the lineage, growth characteristics, immunological markers, viral susceptibility, tumorigenicity, and storage conditions, as well as testing in animals, eggs, and cell cultures. Such characterization is used to confirm that the cells are free of detectable adventitious agents. In some countries, karyology may be required. Additionally, tumorigenicity may be tested in cells at the same passage level as those used for vaccine production. Viruses may be purified prior to vaccine production by a process shown to provide consistent results (see, e.g., World Health Organization, 1982).

[0052] Viruses produced by host cells may be highly purified prior to vaccine or gene therapy formulation. Generally, purification procedures result in the extensive removal of cellular DNA and other cellular components and adventitious agents. Procedures that extensively degrade or denature DNA may also be used.

[0053] Influenza vaccine Vaccines of the present disclosure comprise an isolated recombinant influenza virus of the present disclosure, and optionally one or more other isolated viruses, including other isolated influenza viruses, one or more immunogenic proteins or glycoproteins of one or more isolated influenza viruses or one or more other pathogens, e.g., immunogenic proteins from one or more bacteria, non-influenza viruses, yeast, or fungi, or isolated nucleic acids (e.g., DNA vaccines) encoding one or more viral proteins, including one or more immunogenic proteins of an isolated influenza virus of the present disclosure. In one embodiment, the influenza virus of the present disclosure can be a vaccine vector for influenza virus or other pathogens.

[0054] Whole virion vaccines may be concentrated by ultrafiltration and then purified by zonal centrifugation or chromatography. Viruses other than those of the invention, such as those included in multivalent vaccines, may be inactivated before or after purification, for example, using formalin or β-propiolactone.

[0055] Subunit vaccines contain purified glycoproteins. Such vaccines may be prepared as follows: surface antigens are purified, for example, by ultracentrifugation, using a virus suspension fragmented by treatment with a detergent. Thus, subunit vaccines contain primarily HA proteins and also NA. The detergent used may be, for example, a cationic detergent such as hexadecyltrimethylammonium bromide (Bachmeyer, 1975), an anionic detergent such as ammonium deoxycholate (Laver & Webster, 1976), or a non-ionic detergent such as that commercialized under the name TRITON X100. Hemagglutinin may be isolated and then purified after treatment of virions with a protease such as bromelain. Subunit vaccines may also be combined with the attenuated viruses of the present disclosure in multivalent vaccines.

[0056] Disassembled vaccines contain virions that have been treated with a lipid-dissolving agent. Disassembled vaccines can be prepared as follows: an aqueous suspension of purified virus, inactivated or not, obtained as described above, is treated with a lipid solvent, such as ethyl ether or chloroform, associated with a detergent, under agitation. Dissolution of the viral envelope lipids results in fragmentation of the virus particles. An aqueous phase containing the disassembled vaccine, primarily composed of hemagglutinin and neuraminidase, freed from their original lipid environment, and the core or its degradation products, is recovered. Remaining infectious particles are then inactivated, if not already present. Disassembled vaccines may also be combined with the attenuated viruses of the present disclosure in a multivalent vaccine.

[0057] Inactivated Vaccines. Inactivated influenza virus vaccines are provided by inactivating replicated viruses using known methods, such as, but not limited to, treatment with formalin or β-propiolactone. Types of inactivated vaccines that can be used in the present invention include whole virus (WV) vaccines or subvirion (SV) (split) vaccines. WV vaccines contain intact inactivated viruses, while SV vaccines contain purified viruses that are disrupted by detergents that solubilize the lipid-containing viral envelope followed by chemical inactivation of remaining viruses.

[0058] Additionally, vaccines that can be used include those that contain the separated HA and NA surface proteins, referred to as surface antigen or subunit vaccines.

[0059] Live attenuated virus vaccines. Live attenuated influenza virus vaccines, such as those comprising the recombinant viruses of the present disclosure, can be used to prevent or treat influenza virus infection. Attenuation may be achieved in a single step by transfer of an attenuation gene from an attenuated donor virus to a replicative isolate or reassortant virus according to known methods. Because resistance to influenza A viruses is primarily mediated by the development of an immune response against the HA and / or NA glycoproteins, the genes encoding these surface antigens originate from the reassortant virus or clinical isolate. The attenuation gene is derived from the attenuated parent. In this approach, the gene conferring attenuation generally does not encode the HA and NA glycoproteins.

[0060] Viruses (donor influenza viruses) capable of reproducibly attenuating influenza viruses are available; for example, cold-adapted (ca) donor viruses can be used to generate attenuated vaccines. See, for example, Isakova-Sivall et al., 2014. Live attenuated reassortant virus vaccines can be generated by combining a ca donor virus with a virulent replicating virus. Reassortant progeny are then selected at 25°C (restrictive for virulent virus replication) in the presence of an appropriate antiserum that inhibits replication of viruses bearing the surface antigens of the attenuated ca donor virus. Useful reassortant viruses are (a) infectious, (b) attenuated for seronegative non-adult mammals and immunologically primed adult mammals, (c) immunogenic, and (d) genetically stable. The immunogenicity of ca reassortant viruses is comparable to their level of replication. Thus, acquisition of the six transferable genes of the ca donor virus by new wild-type viruses reproducibly attenuated these viruses for use in vaccinating susceptible mammals, both adult and non-adult.

[0061] Other attenuating mutations can be introduced into influenza virus genes by site-directed mutagenesis to rescue infectious viruses harboring these mutated genes. Attenuating mutations can be introduced into non-coding regions of the genome as well as into coding regions. Such attenuating mutations can also be introduced into genes other than HA or NA, such as the PB2 polymerase gene. Thus, new donor viruses can be generated with attenuating mutations introduced by site-directed mutagenesis, and such new donor viruses can be used in the generation of live attenuated reassortant vaccine candidates in a manner similar to that described above for the ca donor virus. Similarly, other known, appropriately attenuated donor strains can be reassortant with influenza viruses to obtain attenuated vaccines suitable for use in vaccinating mammals.

[0062] In one embodiment, such an attenuated virus maintains genes from the virus that encode antigenic determinants substantially similar to those of the original clinical isolate, since the goal of an attenuated vaccine is to provide substantially the same antigenicity as the original clinical isolate of the virus, while at the same time lacking pathogenicity to minimize the likelihood that the vaccine will induce a severe disease state in the vaccinated mammal.

[0063] Thus, the viruses in a multivalent vaccine can be attenuated or inactivated, formulated, and administered according to known methods as a vaccine to induce an immune response in an animal, e.g., a mammal. Methods for determining whether such attenuated or inactivated vaccines maintain similar antigenicity to clinical isolates or high-growth strains derived therefrom are well known. Such known methods include the use of antisera or antibodies to eliminate viruses expressing antigenic determinants of the donor virus, chemical selection (e.g., amantadine or rimantidine), HA and NA activity and inhibition, and nucleic acid screening (e.g., probe hybridization or PCR) to confirm that the donor genes encoding antigenic determinants (e.g., HA or NA genes) are not present in the attenuated virus.

[0064] Illustrative Embodiments By inserting the gene for the Venus fluorescent protein into the NS segment of the influenza A / Puerto Rico / 8 / 34 (PR8, H1N1) virus to generate WT-Venus-PR8, we prepared a reporter influenza virus that allows visualization of virus-infected cells, for example, to understand influenza virus-induced pathology. Although the inserted Venus gene was deleted during serial passage of WT-Venus-PR8, WT-Venus-PR8 was significantly attenuated, and the PB2-E712D mutation was found to stabilize the Venus gene. As disclosed herein, we investigated the mechanism by which the Venus gene deletion occurs and how the polymerase mutation stabilizes the Venus gene. Large-scale sequencing analysis revealed that PB2-E712D did not significantly alter the mutation rate, suggesting that Venus gene stability is not affected by polymerase fidelity. Using quantitative real-time PCR, we found that WT-Venus-PR8 induced high levels of interferon-β (IFN-β) expression. The induction of IFN-β expression appeared to be due to reduced transcription / replication efficiency of the modified NS segment in WT-Venus-PR8. In contrast, the transcription / replication efficiency of the modified NS segment was enhanced by the PB2-E712D mutation. The loss of the Venus gene in WT-Venus-PR8 appeared to be caused by an internal deletion in the NS segment. Furthermore, to further understand the mechanism of Venus stabilization, we identified additional amino acid mutations in the viral polymerase complex that stabilize the Venus gene. Some of these amino acids were found to be located near the template or product exit of the viral polymerase, suggesting that these amino acids contribute to Venus gene stability by affecting the binding affinity between the polymerase complex and the RNA template and product.

[0065] The present disclosure relates to an isolated recombinant influenza virus having a PA viral segment, a PB1 viral segment, a PB2 viral segment, an NP viral segment, an NS viral segment, an M viral segment, an NA viral segment, and an HA viral segment, wherein at least one of the viral segments is a PB2 viral segment encoding a PB2 having a residue at position 540 that is not asparagine, a PA viral segment encoding a PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or an asparagine and a PB1 viral segment encoding PB1 having a residue at position 685 that is not an acid, or any combination thereof, wherein the recombinant influenza virus has enhanced genetic stability or enhanced replication compared to a corresponding recombinant influenza virus having a residue at position 540 in PB2 that is asparagine, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid, or a residue at position 685 in PB1 that is aspartic acid. In one embodiment, the residue at position 540 of PB2 is K, R, D, E, Q, or H; the residue at position 712 of PB2 is D, N, S, H, T, Y, or C; the residue at position 180 in PA is R, K, D, E, N, or H; the residue at position 200 in PA is A, I, L, C, S, M, F, P, G, or V; the residue at position 149 in PB1 is A, T, I, L, C, S, M, F, P, or G; the residue at position 684 is D, Q, S, H, T, Y, C, K, R, or N; or the residue at position 685 in PB1 is E, N, R, H, K, S, T, Y, C, or Q.In one embodiment, the residue at position 540 of PB2 is K, R, H, D, S, H, T, Y, or C; the residue at position 712 of PB2 is D, K, H, R, Q, or N; the residue at position 180 in PA is R, K, D, N, S, H, T, Y, or H; the residue at position 200 in PA is A, I, L, G, S, M, or V; the residue at position 149 in PB1 is A, T, I, L, S, M, or G; the residue at position 684 is D, Q, H, L, R, or N; or the residue at position 685 in PB1 is E, N, R, H, K, or Q. In one embodiment, the residue at position 540 of PB2 is K, R, or H, the residue at position 712 of PB2 is D or N, the residue at position 180 in PA is R, K, or H, the residue at position 200 in PA is A, I, L, G, or V, the residue at position 149 in PB1 is A, T, I, L, or G, the residue at position 684 is D or N, or the residue at position 685 in PB1 is E or Q. In one embodiment, the PA further comprises a residue at position 443 that is not arginine, the PB1 further comprises a residue at position 737 that is not lysine, the PB2 further comprises a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid, the NS viral segment encodes an NS1 having a residue at position 167 that is not proline, and the HA viral segment encodes an HA having a residue at position 380 that is not threonine, or any combination thereof. In one embodiment, the residue at position 443 of PA is K or H, the residue at position 737 of PB1 is H or R, the residue at position 25 of PB2 is A, L, T, I or G, the residue at position 712 of PB2 is D, and the residue at position 167 of NS1 is S, C, M, A, L, I, G or T, or any combination thereof. In one embodiment, at least one of the viral segments comprises a heterologous gene sequence encoding a gene product.In one embodiment, the heterologous sequence is in the NS, M, NP, PA, PB1, or PB2 viral segment. In one embodiment, the heterologous sequence is 5' or 3' to the PA coding sequence in the PA viral segment and 5' or 3' to the PB1 coding sequence in the PB1 viral segment. In one embodiment, the heterologous sequence is 5' or 3' to the PB2 coding sequence in the PB2 viral segment. In one embodiment, the heterologous sequence is 5' or 3' to the NS1 coding sequence in the NS viral segment. In one embodiment, the recombinant virus comprises an additional viral segment comprising a heterologous gene sequence encoding a gene product. In one embodiment, the additional viral segment is an NS, M, NP, PA, PB1, or PB2 viral segment. In one embodiment, the virus has an HA that is H1, H2, H3, H5, H7, H9, or H10. In one embodiment, the virus is an influenza B virus.

[0066] 1. An isolated recombinant influenza virus having a PA viral segment, a PB1 viral segment, a PB2 viral segment, an NP viral segment, an NS viral segment, an M viral segment, an NA viral segment, and an HA viral segment, wherein at least one of said viral segments is a PB2 viral segment encoding PB2 having a residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, and at least one of said other viral segments is a PA viral segment encoding PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, Also provided is an isolated recombinant influenza virus, wherein the PB1 viral segment encodes a PB1 having a residue at position 540 in PB2 that is asparagine, a residue at position 712 in PB2 that is glutamic acid, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid, or a residue at position 685 in PB1 that is aspartic acid, or any combination thereof, wherein the recombinant influenza virus has enhanced genetic stability or replication compared to a corresponding recombinant influenza virus having a residue at position 540 in PB2 that is asparagine, a residue at position 712 in PB2 that is glutamic acid, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid, or a residue at position 685 in PB1 that is aspartic acid. In one embodiment, the residue at position 540 of PB2 is K, R, D, E, Q, or H; the residue at position 712 of PB2 is D, N, S, H, T, Y, or C; the residue at position 180 in PA is R, K, D, E, N, or H; the residue at position 200 in PA is A, I, L, C, S, M, F, P, G, or V; the residue at position 149 in PB1 is A, T, I, L, C, S, M, F, P, or G; the residue at position 684 is D, Q, S, H, T, Y, C, K, R, or N; or the residue at position 685 in PB1 is E, N, R, H, K, S, T, Y, C, or Q.In one embodiment, the residue at position 540 of PB2 is K, R, H, D, S, H, T, Y, or C; the residue at position 712 of PB2 is D, K, H, R, Q, or N; the residue at position 180 in PA is R, K, D, N, S, H, T, Y, or H; the residue at position 200 in PA is A, I, L, G, S, M, or V; the residue at position 149 in PB1 is A, T, I, L, S, M, or G; the residue at position 684 is D, Q, H, L, R, or N; or the residue at position 685 in PB1 is E, N, R, H, K, or Q. In one embodiment, the residue at position 540 of PB2 is K, R, or H, the residue at position 712 of PB2 is D or N, the residue at position 180 in PA is R, K, or H, the residue at position 200 in PA is A, I, L, G, or V, the residue at position 149 in PB1 is A, T, I, L, or G, the residue at position 684 is D or N, or the residue at position 685 in PB1 is E or Q. In one embodiment, the PA further comprises a residue at position 443 that is not arginine, the PB1 further comprises a residue at position 737 that is not lysine, the PB2 further comprises a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid, the NS viral segment encodes an NS1 having a residue at position 167 that is not proline, and the HA viral segment encodes an HA having a residue at position 380 that is not threonine, or any combination thereof. In one embodiment, the residue at position 443 of PA is K or H, the residue at position 737 of PB1 is H or R, the residue at position 25 of PB2 is A, L, T, I or G, the residue at position 712 of PB2 is D, and the residue at position 167 of NS1 is S, C, M, A, L, I, G or T, or any combination thereof. In one embodiment, at least one of the viral segments comprises a heterologous gene sequence encoding a gene product.In one embodiment, the heterologous sequence is in the NS, M, NP, PA, PB1, or PB2 viral segment. In one embodiment, the heterologous sequence is 5' or 3' to the PA coding sequence in the PA viral segment and 5' or 3' to the PB1 coding sequence in the PB1 viral segment. In one embodiment, the heterologous sequence is 5' or 3' to the PB2 coding sequence in the PB2 viral segment. In one embodiment, the heterologous sequence is 5' or 3' to the NS1 coding sequence in the NS viral segment. In one embodiment, the recombinant virus comprises an additional viral segment comprising a heterologous gene sequence encoding a gene product. In one embodiment, the additional viral segment is an NS, M, NP, PA, PB1, or PB2 viral segment. In one embodiment, the virus has an HA that is H1, H2, H3, H5, H7, H9, or H10. In one embodiment, the virus is an influenza B virus.

[0067] 1. An isolated recombinant influenza virus having a PA viral segment, a PB1 viral segment, a PB2 viral segment, an NP viral segment, an NS viral segment, an M viral segment, an NA viral segment, and an HA viral segment, wherein the recombinant virus comprises a PB2 viral segment encoding a PB2 having a residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, a PA viral segment encoding a PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid. or any combination thereof, wherein the recombinant influenza virus has enhanced genetic stability or replication compared to a corresponding recombinant influenza virus having a residue at position 540 in PB2 that is asparagine, a residue at position 712 in PB2 that is glutamic acid, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid, or a residue at position 685 in PB1 that is aspartic acid. In one embodiment, the residue at position 540 of PB2 is K, R, D, E, Q, or H; the residue at position 712 of PB2 is D, N, S, H, T, Y, or C; the residue at position 180 in PA is R, K, D, E, N, or H; the residue at position 200 in PA is A, I, L, C, S, M, F, P, G, or V; the residue at position 149 in PB1 is A, T, I, L, C, S, M, F, P, or G; the residue at position 684 is D, Q, S, H, T, Y, C, K, R, or N; or the residue at position 685 in PB1 is E, N, R, H, K, S, T, Y, C, or Q.In one embodiment, the residue at position 540 of PB2 is K, R, H, D, S, H, T, Y, or C; the residue at position 712 of PB2 is D, K, H, R, Q, or N; the residue at position 180 in PA is R, K, D, N, S, H, T, Y, or H; the residue at position 200 in PA is A, I, L, G, S, M, or V; the residue at position 149 in PB1 is A, T, I, L, S, M, or G; the residue at position 684 is D, Q, H, L, R, or N; or the residue at position 685 in PB1 is E, N, R, H, K, or Q. In one embodiment, the residue at position 540 of PB2 is K, R, or H, the residue at position 712 of PB2 is D or N, the residue at position 180 in PA is R, K, or H, the residue at position 200 in PA is A, I, L, G, or V, the residue at position 149 in PB1 is A, T, I, L, or G, the residue at position 684 is D or N, or the residue at position 685 in PB1 is E or Q. In one embodiment, the PA further comprises a residue at position 443 that is not arginine, the PB1 further comprises a residue at position 737 that is not lysine, the PB2 further comprises a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid, the NS viral segment encodes an NS1 having a residue at position 167 that is not proline, and the HA viral segment encodes an HA having a residue at position 380 that is not threonine, or any combination thereof. In one embodiment, the residue at position 443 of PA is K or H, the residue at position 737 of PB1 is H or R, the residue at position 25 of PB2 is A, L, T, I, or G, the residue at position 712 of PB2 is D, and the residue at position 167 of NS1 is S, C, M, A, L, I, G, or T, or any combination thereof. In one embodiment, at least one of the viral segments comprises a heterologous gene sequence encoding a gene product.In one embodiment, the heterologous sequence is in the NS, M, NP, PA, PB1, or PB2 viral segment. In one embodiment, the heterologous sequence is 5' or 3' to the PA coding sequence in the PA viral segment and 5' or 3' to the PB1 coding sequence in the PB1 viral segment. In one embodiment, the heterologous sequence is 5' or 3' to the PB2 coding sequence in the PB2 viral segment. In one embodiment, the heterologous sequence is 5' or 3' to the NS1 coding sequence in the NS viral segment. In one embodiment, the recombinant virus comprises an additional viral segment comprising a heterologous gene sequence encoding a gene product. In one embodiment, the additional viral segment is an NS, M, NP, PA, PB1, or PB2 viral segment. In one embodiment, the virus has an HA that is H1, H2, H3, H5, H7, H9, or H10. In one embodiment, the virus is an influenza B virus.

[0068] The present disclosure also provides a vaccine comprising the isolated recombinant virus.

[0069] The present disclosure provides a plurality of influenza virus vectors for preparing a reassortant, comprising: A vector for producing vRNA comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector for producing vRNA comprising a promoter operably linked to influenza virus NS cDNA linked to a transcription termination sequence, wherein the PB1 DNA, the PB2 DNA, or the PA DNA in the vector for vRNA productionthe vector for vRNA production, wherein the DNA encodes at least one of a PB2 viral segment encoding PB2 having a residue at position 540 that is not asparagine, a PA viral segment encoding PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 viral segment encoding PB1 having a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid, or a combination thereof; and optionally a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a DNA segment encoding influenza virus PB2, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. The present invention provides multiple influenza virus vectors for preparing the reassortants. In one embodiment, the PB1 DNA, the PB2 DNA, the PA DNA, the NP DNA, the NS DNA, and the M DNA in the vector for vRNA production have sequences corresponding to those encoding polypeptides having at least 95% amino acid sequence identity to the corresponding polypeptides encoded by SEQ ID NOS: 1-6 or 10-15. In one embodiment, the residue at position 540 of PB2 is K, R, or H; the residue at position 180 in PA is R, K, or H; the residue at position 200 in PA is A, I, L, G, or V; the residue at position 149 in PB1 is A, T, I, L, or G; the residue at position 684 is D or N; or the residue at position 685 in PB1 is E or Q. In one embodiment, at least one of the viral segments comprises a heterologous gene sequence encoding a gene product. In one embodiment, the vector comprises an additional vector having a viral segment comprising a heterologous gene sequence encoding a gene product.

[0070] a vector for vRNA production comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence; a vector for vRNA production comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence; a vector for vRNA production comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence; a vector for vRNA production comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence; a vector for vRNA production comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence; a vector for vRNA production comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence; a vector for vRNA production comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence; and a vector for vRNA production comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence, in an amount effective to produce infectious influenza virus; the DNA is at least one of: i) PB2 having a residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, and PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or PB1 having a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid, or any combination thereof; or ii) PB2 having a residue at position 540 that is not asparagine, PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or PB1 having a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid, or any combination thereof;or iii) a vector for producing vRNA encoding PB2 having a residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or PB1 having a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid, or any combination thereof, and optionally a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and and a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NS2. In one embodiment, the cell is an avian cell or a mammalian cell. In one embodiment, the cell is a Vero cell, a human cell, or an MDCK cell. In one embodiment, the PB1 DNA, the PB2 DNA, the PA DNA, the NP DNA, the NS DNA, and the M DNA in the vector for vRNA production areIt has a sequence corresponding to one that encodes a polypeptide having at least 95% amino acid sequence identity to the corresponding polypeptide encoded by SEQ ID NOs: 1 to 6 or 10 to 15. In one embodiment, the residue at position 540 of PB2 is K, R, or H, the residue at position 712 of PB2 is D or N, the residue at position 180 in PA is R, K, or H, the residue at position 200 in PA is A, I, L, G, or V, the residue at position 149 in PB1 is A, T, I, L, or G, the residue at position 684 is D or N, or the residue at position 685 in PB1 is E or Q.

[0071] The heterologous sequence for a therapeutic or prophylactic gene of interest, which may be in, for example, an additional influenza segment, for example, in one of the segments of a 9-segment influenza A or B virus, in one of the 8 viral segments, or in one of the segments of a 7-segment virus, may also be a cancer-associated antigen or an immunogen for a pathogen such as a bacterium, non-influenza virus, fungus, etc. In one embodiment, the influenza virus of the present disclosure may be a vaccine vector for influenza virus and for at least one other pathogen, such as a viral or bacterial pathogen, or for a pathogen other than influenza virus, including, but not limited to, a lentivirus, such as HIV, hepatitis B virus, hepatitis C virus, herpesvirus, such as CMV or HSV, foot and mouth disease virus, measles virus, rubella virus, mumps virus, human rhinovirus, parainfluenza virus, such as respiratory syncytial virus, human parainfluenza virus type 1, coronavirus, Nipah virus, hantavirus, Japanese encephalitis virus, rotavirus, dengue virus, West Nile virus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Bordetella pertussis, or Haemophilus influenzae. For example, an influenza virus encompassed by a biological of the present disclosure may include sequences for the H protein of measles virus, the viral envelope protein E1 of rubella virus, the HN protein of mumps virus, the RV capsid protein VP1 of human rhinovirus, the G protein of respiratory syncytial virus, the S protein of a coronavirus, the G protein or F protein of Nipah virus, the G protein of a hantavirus, the E protein of Japanese encephalitis virus, VP6 of rotavirus, the E protein of dengue virus, the E protein of West Nile virus, PspA of Streptococcus pneumoniae, HSP65 from Mycobacterium tuberculosis, IRP1-3 of Bordetella pertussis, or the heme utilization protein, protective surface antigen D15, heme-binding protein A, or envelope protein P1, P2, P5, or P6 of Haemophilus influenzae.Gene therapy vectors may contain heterologous sequences useful for inhibiting or treating, for example, cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamylase deficiency, and central nervous system tumors or pathogens, or may encode antibodies or fragments thereof, such as scFvs or single chain antibodies.

[0072] Pharmaceutical Composition Pharmaceutical compositions of the present disclosure suitable for inoculation, e.g., intranasal, parenteral, or oral administration, comprise one or more influenza virus isolates, e.g., one or more attenuated or inactivated influenza viruses, their subunits, their isolated protein(s), and / or isolated nucleic acids encoding one or more of these proteins, and optionally further comprise sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions may further comprise adjuvants or excipients as known in the art. The compositions of the present disclosure are generally presented in the form of individual doses (unit doses).

[0073] Typically, conventional vaccines contain about 0.1 to 200 μg, e.g., 30 to 100 μg, 0.1 to 2 μg, 0.5 to 5 μg, 1 to 10 μg, 10 to 20 μg, 15 to 30 μg, or 10 to 30 μg, of HA from each of the strains that go into their composition. The vaccines that form the major component of the vaccine compositions of the present disclosure may comprise a single influenza virus or a combination of influenza viruses, e.g., at least two or three influenza viruses, including one or more reassortant(s).

[0074] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions, which may contain auxiliary substances or excipients known in the art. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Generally, liquid dosage forms for oral administration may contain liposome solutions containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solvents, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. In addition to the inert diluents, such compositions may also contain adjuvants, wetting agents, emulsifying and suspending agents, or sweeteners, flavoring agents, or perfuming agents.

[0075] When the compositions of the present disclosure are used for administration to an individual, they can further contain salts, buffers, adjuvants, or other substances that are desirable to improve the effectiveness of the compositions. For vaccines, adjuvants, substances that can enhance a specific immune response, can be used. Typically, the adjuvant and the composition are mixed before presentation to the immune system, or presented separately but to the same site in the organism to be immunized.

[0076] Vaccine heterogeneity may be provided by mixing replicated influenza viruses of at least two influenza virus strains, for example, 2 to 20 strains, or any range or value therein. Vaccines can be provided for variations in a single strain of influenza virus using techniques known in the art.

[0077] Pharmaceutical compositions according to the present disclosure may further or additionally comprise at least one chemotherapeutic compound, such as, but not limited to, immunosuppressants, anti-inflammatory agents, or immune-enhancing agents for gene therapy, and gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumor necrosis factor-alpha, thiosemicarbazones, methisazone, rifampin, ribavirin, pyrimidine analogs, purine analogs, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, protease inhibitors, or ganciclovir for vaccines.

[0078] The compositions may also contain variable but small amounts of endotoxin-free formaldehyde and preservatives that are known to be safe and do not contribute to undesirable effects in the organism to which the composition is administered.

[0079] Medical purposes Administration of the composition (or the antisera it elicits) may be for either "prophylactic" or "therapeutic" purposes. When provided prophylactically, the compositions of the present disclosure that are vaccines are provided before any symptom or clinical sign of pathogen infection becomes apparent. Prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. When provided prophylactically, the gene therapy compositions of the present disclosure are provided before any symptom or clinical sign of disease becomes apparent. Prophylactic administration of the composition serves to prevent or attenuate one or more symptom or clinical sign associated with the disease.

[0080] A viral vaccine, when provided therapeutically, is provided upon detection of a symptom or clinical sign of an actual infection. The therapeutic administration of the compound(s) serves to attenuate any actual infection. A gene therapy composition, when provided therapeutically, is provided upon detection of a symptom or clinical sign of the disease. The therapeutic administration of the compound(s) serves to attenuate the symptom or clinical sign of the disease.

[0081] Thus, the vaccine compositions of the present disclosure may be provided prior to the onset of infection (to prevent or attenuate anticipated infection) or after the onset of actual infection. Similarly, for gene therapy, the compositions may be provided before any symptom or clinical sign of a disorder or disease becomes evident or after one or more symptoms have been detected.

[0082] A composition is considered to be "pharmacologically acceptable" if its administration can be tolerated by a recipient mammal. Such an agent is considered to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. A composition of the present disclosure is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, for example, enhancing at least one primary or secondary humoral or cellular immune response against at least one strain of infectious influenza virus.

[0083] The "protection" provided need not be absolute, i.e., influenza infection need not be prevented or eradicated altogether, provided there is a statistically significant improvement compared to a control population or set of mammals. Protection may also be limited to reducing the severity or rapidity of onset of symptoms or clinical signs of influenza virus infection.

[0084] Medication administration The compositions of the present disclosure may confer resistance to one or more pathogens, e.g., one or more influenza virus strains, through passive or active immunization. In active immunization, the live attenuated vaccine composition is administered prophylactically to a host (e.g., a mammal), and the host's immune response to the administration provides protection against infection and / or disease. For passive immunization, the elicited antisera can be collected and administered to a recipient suspected of having an infection caused by at least one influenza virus strain. The gene therapy compositions of the present disclosure may produce prophylactic or therapeutic levels of a desired gene product through active immunization.

[0085] In one embodiment, the vaccine is provided to a female mammal (at or before pregnancy or birth) under conditions for a time and in an amount sufficient to generate an immune response that serves to protect the female and her fetus or newborn (either across the placenta or by passive incorporation of antibodies in breast milk).

[0086] Thus, the present disclosure includes methods of preventing or attenuating a disorder or disease, e.g., infection by at least one strain of a pathogen. As used herein, a vaccine is considered to prevent or attenuate a disease if its administration results in a total or partial attenuation (i.e., suppression) of clinical signs or symptoms of the disease, or results in total or partial immunity in an individual against the disease. As used herein, a gene therapy composition is considered to prevent or attenuate a disease if its administration results in a total or partial attenuation (i.e., suppression) of clinical signs or symptoms of the disease, or results in total or partial immunity in an individual against the disease.

[0087] Compositions comprising at least one influenza virus of the present disclosure, including attenuated ones, one or more other isolated viruses, one or more isolated viral proteins thereof, one or more isolated nucleic acid molecules encoding one or more viral proteins thereof, or combinations thereof, may be administered by any means that achieves its intended purpose.

[0088] For example, administration of such compositions may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes. Parenteral administration can be accomplished by bolus injection or by gradual perfusion over time.

[0089] A typical regimen for preventing, suppressing, or treating influenza virus-associated pathology involves the administration of an effective amount of a vaccine composition as described herein, administered as a single treatment or repeated as booster or booster doses, for a period of up to 1 week to about 24 months, or any range or value therein.

[0090] According to the present disclosure, an "effective amount" of a composition is one sufficient to achieve the desired effect. It is understood that the effective dosage may depend on the species, age, sex, health, and weight of the recipient, type of concurrent treatment, if any, frequency of treatment, and the nature of the desired effect. The ranges of effective dosages provided below are not intended to limit the invention, but are intended to indicate dosage ranges.

[0091] A dose of a live attenuated or killed virus vaccine for an animal, such as an adult mammalian organism, is about 10 2 ~10 20 , e.g., 10 3 ~10 12 , 10 2 ~10 10 , 10 5 ~10 11 , 10 6 ~10 15 , 10 2 ~10 10 or 10 15 ~10 20 The dose may be plaque-forming units (PFU) / kg, or any range or value therein. For example, the dose of one virus isolate vaccine in an inactivated vaccine may be in the range of about 0.1 to 1000, e.g., 0.1 to 10 μg, 1 to 20 μg, 30 to 100 μg, 10 to 50 μg, 50 to 200 μg, or 150 to 300 μg of HA protein. However, the dose should be a safe and effective amount as determined by conventional methods using existing vaccines as a starting point.

[0092] The dose of immunoactive HA in each dose of a replicating virus vaccine may be standardized to contain an appropriate amount, e.g., 0.1 μg to 1 μg, 0.5 μg to 5 μg, 1 μg to 10 μg, 10 μg to 20 μg, 15 μg to 30 μg, or 30 μg to 100 μg, or any range or value therein, or an amount recommended by a government agency or recognized professional organization. However, the amount of NA may also be standardized, as this glycoprotein may be unstable during purification and storage.

[0093] The dose of immunoactive HA in each dose of replicating virus vaccine can be standardized to contain an appropriate amount, e.g., 1-50 μg, or any range or value therein, or the amount recommended by the United States Public Health Service (PHS), which is typically 15 μg per component for children older than 3 years and 7.5 μg per component for children younger than 3 years. However, while the amount of NA can be standardized, this glycoprotein may be unstable during processing equipment purification and storage (Kendal et al., 1980; Kerr et al., 1975). Each 0.5 mL dose of vaccine may contain about 100,000,000 to 500,000,000 virus particles, 500,000,000 to 2,000,000,000 virus particles, 1,000,000 to 50,000,000,000 virus particles, 1,000,000,000 to 10,000,000,000 virus particles, 20,000,000,000 to 40,000,000,000 virus particles, 1,000,000,000 to 5,000,000,000 virus particles, or 40,000,000,000 to 80,000,000,000 virus particles.

[0094] The present invention is further illustrated by the following non-limiting examples. [Example]

[0095] Example I method Preparation of Color-Influenza The PR8 NS segment fused to different fluorescent reporter genes, including eCFP, eGFP, Venus, and mCherry, was constructed by overlap fusion PCR as described by Manicassamy et al. (2010). Briefly, the open reading frame (ORF) of the NS1 gene without a stop codon was fused to the N-terminus of the fluorescent reporter gene via a sequence encoding the amino acid linker GSGG. The fluorescent reporter ORF was followed by a sequence encoding a GSG linker, a 57-nucleotide foot-and-mouth disease virus protease 2A autoproteolysis site derived from porcine teschovirus-1 (Manicassamy et al. (2010)), and an ORF for the nuclear export protein (NEP) (Figure 5). In addition, silent mutations were introduced into the endogenous splice acceptor site of the NS1 gene to prevent splicing (Basler et al., 2001). The constructed NS segments (termed eCFP-NS, eGFP-NS, Venus-NS, and mCherry-NS) were then cloned into the pPolI vector for reverse genetics, as described by Newmann et al. (1999). Plasmids encoding the Venus reporter protein were a gift from Dr. A. Miyawaki (Laboratory for Cell Function Dynamics, RIKEN Brain Science Institute, Wako, Japan) (Nagai et al., 2002). WT-Venus-PR8 was generated using the reverse genetics system as described by Newmann et al. (1999). Because the virulence and Venus expression levels of WT-Venus-PR8 were significantly weakened in mice, WT-Venus-PR8 was serially passaged in mice. After six passages, a mutant with increased virulence and enhanced Venus expression (MA-Venus-PR8) was obtained. MA-Venus-PR8 stocks were generated in MDCK cells. Because serial passage in animals typically generates a virus population composed of genetic variants, MA-Venus-PR8 was regenerated using reverse genetics.Similarly, MA-eCFP-PR8, -eGFP-PR8, and -mCherry-PR8, which have the same genetic backbone as MA-Venus-PR8, were generated.

[0096] To generate Venus-HPAI viruses by reverse genetics, the NS segment of A / Vietnam / 1203 / 2004 (H5N1; VN1203) was replaced with the Venus-NS of PR8, and the virus was mouse-adapted as described for MA-Venus-PR8. MA-Venus-HPAI virus stocks were generated in MDCK cells. This set of influenza viruses carrying various fluorescent proteins was collectively referred to as "color-influenza."

[0097] Mouse experiments Six-week-old female C57BL / 6 ('B6') mice were purchased from Japan SLC Co., Ltd. (Shizuoka, Japan). Under sevoflurane anesthesia, mice were intranasally inoculated with influenza A virus in 50 μL of PBS at the doses indicated in the figure panels. Body weight and survival rates were monitored for 14 days. At the time points indicated in the figure panels, lungs were collected from PBS-inoculated or influenza A virus-infected mice for virus titration, flow cytometry analysis, and histological examination. All animal experiments were conducted in accordance with the regulations of the Animal Care and Use Committee of the University of Tokyo and approved by the Animal Care and Use Committee of the Institute of Medical Science, the University of Tokyo.

[0098] Histological and cytological examination Lungs were fixed in 4% paraformaldehyde (PFA) phosphate-buffered saline. Fixed tissues were embedded in OCT compound (Sakura Finetech, Tokyo, Japan), frozen in liquid N2, and stored at -80°C. Cryostat 6 μm sections were treated with PBS containing 1% BSA (PBS-BSA) for 30 minutes to block nonspecific binding, and then incubated with phycoerythrin (PE)-Mac3 (M3 / 84, BD Biosciences, San Jose, CA). To examine cytology of MDCK cells, cells were infected with influenza virus and then fixed in 4% PFA phosphate-buffered saline. Nuclei were stained with Hoechst 33342 (Invitrogen, Carlsbad, CA). Sections and cells were visualized using a confocal microscope (Nikon A1, Nikon, Tokyo, Japan) controlled by NIS-Elements software. For quantitative multicolor imaging analysis, slides were visualized by using an inverted fluorescence microscope (Nikon Eclipse TS100) with a Nuance FX multispectral imaging system with InForm software (PerkinElmer, Waltham, MA).

[0099] Whole-mount imaging of lung tissue Mice were euthanized and perfused intracardially with PBS to remove blood cells from the lungs. Lungs were isolated after intratracheal perfusion with 4% PFA phosphate-buffered saline. Lung tissue was cleared with SCALEVIEW-A2 solution (Olympus, Tokyo, Japan) according to the manufacturer's instructions. Images were acquired using a stereofluorescence microscope (M205FA, Leica Microsystems, Wetzlar, Germany) equipped with a digital camera (DFC365FX, Leica Microsystems).

[0100] Two-photon laser microscopy 10 in total 5B6 mice were inoculated intranasally with PFU of MA-eGFP-PR8. To label lung macrophages, mice were intravenously injected with 50 μL of PE-CD11b (M1 / 70, BioLegend, San Diego, CA) on day 3 postinfection. Thirty minutes after antibody injection, mouse lungs were harvested. The kinetics of eGFP- and PE-positive cells in the lungs were imaged using a multiphoton microscope (LSM 710 NLO, Carl Zeiss, Oberkochen, Germany). During analysis, lungs were maintained in complete medium (RPMI 1640 with 10% fetal bovine serum) in a humidified chamber (37°C, 5% CO). Data were processed using the LSM software Zen2009 (Carl Zeiss). For three-dimensional imaging of HPAI virus-infected lung tissue, B6 mice were inoculated with 10 μL of PE-CD11b (M1 / 70, BioLegend, San Diego, CA). 5 PFU of MA-Venus-HPAI virus was inoculated intranasally. Lung tissues were harvested from mice on day 2 postinfection and treated with SCALEVIEW-A2 solution (Olympus) to clear the tissue as described above. Three-dimensional images of the lung tissue were obtained using a multiphoton microscope (Nikon A1R MP).

[0101] Flow cytometry analysis and cell sorting To obtain single-cell suspensions, lungs were dissociated with Collagenase D (Roche Diagnostics, Mannheim, Germany; final concentration: 2 μg / mL) and DNase I (Worthington Biochemical, Lakewood, NJ; final concentration: 40 U / mL) for 30 minutes at 37°C by crushing the tissue through a nylon filter (BD Biosciences). Red blood cells (RBCs) were lysed by treatment with RBC lysis buffer (Sigma-Aldrich, St. Louis, MO). To block nonspecific antibody binding, cells were incubated with purified anti-mouse CD16 / 32 (Fc Block, BD Biosciences, San Diego, CA). Cells were stained with appropriate combinations of fluorescent antibodies to analyze the population of each immune cell subset. The following antibodies were used: anti-CD45 (30-F11: eBioscience, San Diego, CA), anti-CD11b (M1 / 70: BioLegend), anti-F4 / 80 (BM8: eBioscience), and anti-CD11c (HL3: BD Biosciences). All samples were incubated with 7-aminoactinomycin D (Via-Probe, BD Biosciences) to exclude dead cells. Data from labeled cells were acquired on a FACSAria II (BD Biosciences) and analyzed using FlowJo software version 9.3.1 (Tree Star, San Carlos, CA). To isolate Venus-positive and Venus-negative macrophages from the lung, stained cells were sorted using a FACSAria II (BD Biosciences).

[0102] Microarray analysis Total RNA from sorted macrophages was extracted using TRIzol Reagent (Life Technologies, Carlsbad, CA) and precipitated with isopropanol. RNA amplification was performed using the Arcturus Riboamp Plus RNA Amplification Kit (Life Technologies) according to the manufacturer's instructions. RNA was labeled using the Agilent Low Input Quick Amp Labeling Kit (1 color) (Agilent Technologies, Santa Clara, CA) and hybridized to SurePrint G3 Mouse GE 8X60K microarrays (Agilent Technologies). Arrays were scanned using a DNA Microarray Scanner with SureScan High-Resolution Technology (G2565CA; Agilent Technologies), and data were acquired using Agilent Feature Extraction software version 10.7.3.1 (Agilent Technologies). Probe annotation was provided by Agilent Technologies (AMADID 028005). Probe intensities were background corrected and normalized using the standard exponential and quantile methods, respectively. The log2 of the intensities was then fitted to a linear model comparing groups of interest. 34 All reported p-values ​​were adjusted for multiple hypothesis comparisons using the Benjamini-Hochberg method. Transcripts were considered differentially expressed if there was at least a two-fold change in mean probe intensity between contrasts with adjusted p<0.01. Hierarchical clustering was performed in R. The resulting gene clusters were then analyzed with ToppCluster (Kaimal et al., 2010) to identify enriched gene annotations in each cluster. Reported scores are the -log of the Benjamini-Hochberg adjusted p-value. 10 is.

[0103] Western blot analysis Total lysates from MDCK cells were electrophoresed on an SDS-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and transferred to a PVDF membrane (Milipore, Billerica, MA). The membrane was blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) and incubated with rabbit anti-GFP polyclonal antibody (MBL, Nagoya, Japan), mouse anti-NS1 antibody (188 / 5), rabbit antiserum against A / WSN / 33 (H1N1) (R309), or mouse anti-actin antibody (A2228; Sigma-Aldrich), followed by incubation with HR-conjugated anti-mouse or anti-rabbit IgG antibody (GE Healthcare, Waukesha, WI). After washing the membrane with PBS-Tween, specific proteins were detected using the ECL Plus Western Blotting Detection System (GE Healthcare). Specific protein bands were visualized using a VersaDoc Imaging System (Bio-Rad).

[0104] result To generate fluorescent influenza viruses expressing a reporter protein fused to the NS1 open reading frame, we chose Venus (a GFP variant with eight mutations, including F46L, which improves chromophore formation and increases brightness compared to GFP) (Wagai et al., 2002). As expected based on previous findings of attenuation for influenza viruses expressing reporter proteins (Kittel et al., 2004; Shinhya et al., 2004), the mouse pathogenicity of A / Puerto Rico / 8 / 34 (PR8; H1N1) virus expressing Venus (WT-Venus-PR8) was substantially lower than that of wild-type PR8 (WT-PR8), with a minimum dose required to kill 50% of infected mice (MLD). 50 ) is 10 for WT-PR8 2.5 10 for WT-Venus-PR8 compared with PFU 4.5The WT-Venus-PR8 strain was serially passaged in C57BL / 6 (B6) mice. After six serial passages, the mutant (designated MA-Venus-PR8; harboring a T-to-A mutation at position 380 of the hemagglutinin protein and an E-to-D mutation at position 712 of the polymerase subunit PB2) exhibited significantly higher pathogenicity (MLD) than WT-Venus-PR8. 50 =10 3.5 PFU), but was still less pathogenic than the original PR8 virus (Figure 1A). 4 B6 mice were infected intranasally with PFU of MA-Venus-PR8 or PR8 virus. At all time points tested, lung virus titers were similar in MA-Venus-PR8- and PR8-infected mice (Figure 1B). To test the stability of Venus expression, plaque assays were performed using lung homogenates from infected mice. Only 1 out of 150 plaques was found to be Venus-negative at each of days 3, 5, and 7 postinfection (pi), demonstrating the high genetic stability of Venus expression in this recombinant virus. In contrast, only 70% of NS1-GFP viruses expressed the reporter protein (Manicassamy et al., 2010). The robust toxicity and genetic stability of MA-Venus-PR8 indicate that this virus represents a highly attractive reporter system for visualizing influenza virus-infected cells in vivo. [Table 1]

[0105] To increase the versatility of fluorescent influenza viruses as imaging tools, additional MA-PR8 mutants were generated that expressed different spectral GFP variants, namely, eCFP (ex. 434 nm, em. 477 nm) and eGFP (ex. 489 nm, em. 508 nm) (Patterson, 2001). An mCherry mutant (ex. 587 nm, em. 610 nm) that fluoresces at longer wavelengths than Venus (ex. 515 nm, em. 528 nm) was also generated (Nagai et al., 2002; Shaner et al., 2004). These influenza viruses encoding multispectral fluorescent reporter proteins were collectively referred to as "color-influenza." To determine the pathogenicity of color-influenza viruses, the viral titers and MLDs of MA-eCFP, eGFP, and mCherry-PR8 were measured in mouse lung tissue. 50 The values ​​were compared with MA-Venus-PR8 and MA-PR8. All of the virus strains showed relatively high replication in the lungs, with MLD values ​​of 0.01 and 0.02. 50The values ​​were similar among the color-influenza viruses (Table 1). The stability of fluorescent expression of the color-influenza viruses was tested in vivo and in vitro by plaque assay. When viruses were recovered from mouse lungs on day 7 postinfection, the percentages of fluorescent-positive plaques were 98.0% (MA-eCFP-PR8), 100.0% (MA-eGFP-PR8), and 96.4% (MA-mCherry-PR8). At 72 hours postinfection, the percentages of fluorescent-positive plaques in samples from MDCK cell culture medium were 100.0% (MA-eCFP-PR8), 99.2% (MA-eGFP-PR8), and 98.2% (MA-mCherry-PR8). In addition, the stability of the NS1-fluorescent protein chimera in virus-infected cells was examined by infecting MDCK cells with MA-Venus-PR8 virus and detecting the NS1-Venus chimera protein using anti-GFP and anti-NS1 antibodies. The NS1-Venus chimeric protein was not degraded until 12 hours after infection, indicating that the fluorescent signal in cells infected with influenza virus was primarily generated from the NS1-fluorescent protein chimera, rather than from degradation products. These findings indicate that the pathogenicity and stability of influenza virus were not affected by the different fluorescent reporter genes.

[0106] To evaluate the expression of influenza virus in mouse lungs, we harvested lungs from B6 mice infected with each of the influenza viruses and processed them for visualization as described in the Methods section. All four viruses were clearly visible in the entire cleared lung tissue when analyzed by fluorescent stereomicroscopy (Figure 2A). The fluorescent signal was observed primarily within the bronchial epithelial layer on day 3 postinfection. On day 5 postinfection, the fluorescent signal spread to the peripheral alveolar region. These data indicated that all four influenza viruses are useful for analyzing the distribution of influenza virus-infected cells in mouse lungs. To evaluate the expression of influenza virus in mouse lungs, we harvested lungs from B6 mice infected with each of the influenza viruses and processed them for visualization as described in the Methods section. All four viruses were clearly visible in the entire cleared lung tissue when analyzed by fluorescent stereomicroscopy (Figure 2A). The fluorescent signal was observed primarily within the bronchial epithelial layer on day 3 postinfection. On day 5 postinfection, the fluorescent signal spread to the peripheral alveolar region. These data demonstrate that all four color-infected influenza viruses are useful for analyzing the distribution of influenza virus-infected cells in mouse lungs.

[0107] Next, we used the Nuance™ Spectral Imaging System to test whether the fluorescent signals of all four influenza viruses could be detected simultaneously. A mixture of the four strains (2.5 x 10 in a total volume of 50 μL each) was collected. 4Lung tissue was collected from B6 mice intranasally inoculated with 1000 PFU (1000 ng / mL). Analysis of lung sections obtained on days 2 and 5 postinfection showed that the fluorescent signals of all four influenza viruses were distinguishable from each other (Figure 2B). At day 2 postinfection, clusters of the same fluorescent color were observed in bronchial epithelial cells, suggesting the local spread of individual viruses. At this time point, a limited number of alveolar cells were infected. At day 5 postinfection, we detected clusters of alveolar cells expressing a single fluorescent protein, indicating the initiation of infection and its local spread by a single virus (Figure 2B). Interestingly, epithelial cells expressing two or three fluorescent proteins simultaneously were detected, albeit at low frequency, suggesting coinfection of these cells (Figure 2C). The ability to visualize cells coinfected with different influenza viruses in vivo is a major advancement of the technology and will enable insight into influenza coinfection and the reassortment process.

[0108] Next, we tested the usefulness of color-influenza virus for analyzing host responses to infection. Because macrophages are involved in innate immunity and acute inflammation in influenza virus-infected lungs, we examined lung sections stained with an antibody against macrophages (PE-Mac3) using confocal microscopy. At day 2 postinfection, mice with MA-Venus-PR8 showed macrophage-infiltrated areas containing Venus-positive bronchial epithelial cells (Figure 3A). In contrast, only a few Mac3-positive cells were detected in the alveoli of lungs from mock-infected animals. Based on this finding, we used live imaging to further examine the interaction between influenza virus-infected epithelial cells and macrophages in mouse lungs. Using two-photon laser microscopy, we detected CD11b+ alveolar macrophages in lung tissue from untreated B6 mice. During the observation period (49 min; data not shown), most of these macrophages did not migrate (i.e., showed little migration). In mice infected with the MA-eGFP-PR8 virus, many CD11b macrophages appeared to "attach" to eGFP-positive epithelial cells (data not shown), and some of these eGFP-positive epithelial cells exhibited blebbing similar to apoptotic cells. Interestingly, many CD11b macrophages rapidly migrated to the periphery of eGFP-positive epithelial cells, suggesting a possible macrophage response to inflammatory signals such as IFN or chemokines. Therefore, this system can be used to monitor the in vivo interaction between virus-infected cells and immune cells.

[0109] Many studies have evaluated the transcriptomic and proteomic profiles of influenza virus-infected mice (Go et al., 2012; Zhao et al., 2012). Because these studies used whole lung samples, the results represent the sum of virus-infected and virus-uninfected cells, which results in a dilution of the host response and makes it impossible to distinguish the profile of infected cells from that of uninfected bystander cells. As a first step to overcome this drawback, macrophages derived from the lungs of mice infected with MA-Venus-PR8 (which are known to be infected by influenza viruses (Figure 3B)) were selected based on their fluorescent protein expression and microarray analysis was performed. Macrophages isolated from the lungs of mice inoculated with PBS (naive macrophages) served as a control. In fluorescence-positive macrophages, 6,199 transcripts were differentially expressed compared to naive macrophages. In contrast, in fluorescence-negative macrophages obtained from infected mice, only 4,252 transcripts were differentially expressed compared to naive macrophages. This difference may reflect differences in gene transcription induced by active influenza virus infection. However, it should be noted that the fluorescence-negative cell population obtained from infected animals may have included infected cells in which fluorescent signals had not yet been detected, as expected at the early stages of viral infection. Indeed, confocal microscopy revealed that it took 9 hours to detect fluorescent protein expression in the majority of MDCK cells. Hierarchical clustering of differentially expressed transcripts followed by functional enrichment analysis of each cluster indicated that fluorescence-positive and fluorescence-negative macrophages obtained from infected animals exhibited activation of pathways related to immune responses, cytokine production, and inflammation (Figure 3D, green cluster). The upregulation of these pathways in fluorescence-negative cells could have resulted from cellular activation by IFNs and cytokines released from infected cells and / or cells in the early stages of viral infection (as discussed above).Furthermore, a subset of enriched annotations, e.g., type I IFN-mediated signaling (Figure 3D, light blue cluster), included transcripts that were more highly expressed in fluorescence-positive macrophages. Additionally, type I IFN genes were found to be among the most upregulated transcripts in fluorescence-positive macrophages (Figure 3E). Collectively, this enhanced type I IFN activity is consistent with the suggestion that fluorescence-positive cells were infected, whereas fluorescence-negative cells included both uninfected (but potentially "primed") cells and cells in the early stages of influenza virus infection. Indeed, although it took at least 5 hours to detect fluorescent protein expression after infection with influenza virus, all of the fluorescent proteins (i.e., eCFP, eGFP, Venus, and mCherry) were detectable in the majority of cells by 9 hours postinfection. These findings open up a new avenue in infectious disease research: comparing gene expression (or other types of expression) patterns in reporter protein-positive cells with those in reporter protein-negative cells (potentially primed by released cytokines and / or in the early stages of infection).

[0110] Finally, as discussed in more detail in Example II, we tested whether the mouse-adapted fluorescent influenza virus concept could be applied to other influenza virus strains, such as highly pathogenic avian influenza A (H5N1) (HPAI) viruses, which are a research priority due to the threat they pose to humans. The same strategy used to generate MA-Venus-PR8 was used to generate a MA-Venus-HPAI virus based on A / Vietnam / 1203 / 2004 (VN1203; H5N1), but because Venus viruses carrying the VN1203 NS gene did not contribute to pathogenicity in mice, the PR8 NS gene was used to express the NS1-Venus chimeric protein. The pathogenicity of the MA-Venus-HPAI virus in B6 mice was comparable to that of VN1203, and the MLD for both viruses was significantly higher. 50The values ​​were less than 5 PFU (Figure 4A and Hatta et al., 2007). MA-Venus-HPAI virus also shares with other HPAI viruses the ability to spread systemically, replicating in various organs, including the spleen, kidney, and brain (Figure 4B and Hatta et al., 2007). Furthermore, we successfully constructed a three-dimensional image of HPAI virus-infected bronchi deep within lung tissue using the strong fluorescent signal emitted by MA-Venus-HPAI virus-infected cells (Figure 4C and data not shown). This type of three-dimensional imaging analysis will improve our understanding of the spatial distribution of influenza virus-infected bronchi. When comparing the distribution of virus-infected cells between HPAI viruses and PR8-infected lungs, we found that HPAI viruses spread from the bronchial epithelium to the alveolar region more rapidly than PR8 (Figures 4C and 4D). Using flow cytometry analysis, we found that CD45-negative nonhematopoietic cells and F4 / 80-positive macrophages expressed Venus more frequently in the lungs of mice infected with MA-Venus-HPAI virus than in the lungs of animals inoculated with MA-Venus-PR8 (Figures 4E and 4F), supporting the finding that H5N1 HPAI virus induces a more severe inflammatory response in the lung than PR8, demonstrating the utility of H5N1-influenza viruses for comparative testing of influenza pathogenicity.

[0111] Consideration In this study, we generated color-influenza viruses to study influenza virus infection at the cellular level. Color-influenza viruses combine several improvements over existing systems, including robust viral replication, virulence, stable fluorescent protein expression, and a set of four different colors that can be visualized simultaneously. Color-influenza viruses are applicable to all influenza virus strains. These improvements enable comprehensive transcriptomic analysis of infected and bystander cells and, for the first time, live imaging of influenza virus-infected cells in mouse lungs.

[0112] Previous versions of fluorescent influenza viruses, including our original construct (i.e., WT-Venus-PR8) (Kittel et al., 2004; Shinya et al., 2004), were significantly attenuated in mice. These attenuated fluorescent viruses may still be useful for identifying primary target cells. However, the immune response elicited by these highly attenuated, non-lethal viruses is most likely significantly different from that of the mouse-lethal parent virus, making their use for pathogenicity studies problematic. This problem was resolved by passaging the viruses in mice. This strategy was found to be successful with two different influenza virus strains, suggesting its broad applicability. A second drawback of previously tested fluorescent influenza viruses is the genetic instability of the attached reporter protein (Manicassamy et al., 2010). However, approximately 100% of viral plaques examined in mouse lung samples at day 7 postinfection expressed the reporter protein.

[0113] Because fluorescent reporter proteins must be within the "biological optical window (650-900 nm)" to be detected for imaging tissues in living animals using fluorescent probes (Weisslander, 2001; Jobsis, 1977), influenza virus cannot currently be monitored noninvasively in living animals. None of the fluorescent reporter proteins, including mCherry, which has the longest emission wavelength of any influenza reporter protein, is within this biological optical window. Heaton et al. (2013) generated a luciferase reporter-expressing influenza virus that could be used to monitor viral replication in living animals, but this system requires systemic inoculation of the substrate into the animal at every observation point. Additionally, the resolution of their imaging system (based on the IVIS® system) is not adequate for the in vivo analysis of cellular immune mechanisms that we are able to achieve with this system.

[0114] New imaging analysis techniques (Ghoznari et al., 2013) allowed the development of a set of four distinct influenza color variants that could be distinguished from one another using Nuance™, thus enabling their simultaneous detection. Indeed, our preliminary studies identified lung epithelial cells expressing two or three different fluorescent proteins (Figure 2C). This may be the first visualization of mouse lung cells infected with more than one influenza virus strain. In future studies, these color variants could be used to address long-standing questions in influenza virus research, such as the frequency of in vivo viral coinfections, which may be important for better understanding influenza virus reassortment and, therefore, the generation of novel influenza viruses, such as the 1957 (Schaltissek et al., 1978; Kanaoka et al., 1989), 1968 (Schaltissek et al., 1978; Kanaoka et al., 1989), and 2009 (Smith et al., 2009; Itoh et al., 2009) pandemic viruses.

[0115] Using the described toolset, influenza virus-infected cells were detected in whole lung tissue of mice, allowing observation of the location and distribution of influenza virus in the lung. Furthermore, the interaction of virus-infected epithelial cells with immune cells was observed. Such studies allow direct monitoring of influenza disease progression from acute bronchitis to severe viral pneumonia, which causes considerable morbidity and mortality in highly pathogenic influenza virus infections (Gambotto et al., 2008; Shieh et al., 2009).

[0116] In conclusion, color-influenza virus combined with advanced imaging techniques allows detection at the cellular level in animals.

[0117] Example II As disclosed in Example I, reverse genetics was used to prepare an H5N1 virus (called wild-type Venus-H5N1 virus, abbreviated as WT-Venus-H5N1 virus) carrying a Venus (Nagai et al., 2002) (a mutant of eGFP) reporter gene, which showed moderate virulence and low Venus expression in mice. After six passages in mice, a mouse-adapted Venus-H5N1 virus (abbreviated as MA-Venus-H5N1 virus) was obtained that stably expressed high levels of Venus in vivo and was lethal to mice, with the dose required to kill 50% of infected mice (MLD ). 50 ) was 3.2 plaque-forming units (PFU), while its parent WT-Venus-H5N1 virus was 10 3 However, the mechanism behind this difference in toxicity and Venus stability was unclear.

[0118] In this study, we explored the molecular mechanisms determining the virulence of Venus-H5N1 virus and Venus stability in mice. Using reverse genetics, we rescued various reassortant strains between WT-Venus-H5N1 and MA-Venus-H5N1 viruses and examined their virulence in mice to identify determinants of pathogenicity. Furthermore, we investigated the determinants of Venus expression and stability in vitro and in vivo. These findings have advanced our understanding of influenza virus pathogenesis in mammals and are beneficial for the development of influenza virus-related vaccines and treatments.

[0119] Materials and Methods Cells. Human embryonic kidney 293 and 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, and Madin-Darby canine kidney (MDCK) cells were maintained in minimal essential medium (MEM) supplemented with 5% newborn bovine serum. All cells were incubated at 37°C in 5% CO.

[0120] Plasmid construction. Plasmids for virus rescue were constructed as described by Neumann et al. (1999). To measure viral polymerase activity, the open reading frames of influenza virus PB1, PB2, PA, and NP were amplified by PCR with gene-specific primers and cloned into the pCAGGS / MCS protein expression plasmid (Dias et al., 2009). The primer sequences are shown below. [Table 2]

[0121] All of the constructs were fully sequenced to ensure the absence of unwanted mutations.

[0122] Plasmid-based reverse genetics. Influenza A viruses were generated using plasmid-based reverse genetics as previously described (Murakami, 2008; Ozawa et al., 2007). Viral titers of rescued viruses were determined using plaque assays in MDCK cells. All rescued viruses were sequenced to confirm the absence of undesired mutations.

[0123] Mouse experiments. Six-week-old female C57 / BL6 (B6) mice (Japan SLC Co., Ltd., Shizuoka, Japan) were used in this study. To measure viral replication in mice, six mice in each group were anesthetized with isoflurane and then 10 5 PFU (50 μL) of the virus was inoculated intranasally. On days 1 and 3 post-infection (pi), three mice were euthanized and their organs, including lungs, kidneys, spleen, and brain, were collected and titrated in MDCK cells. The 50% mouse lethal dose (MLD) of the virus was 0.1 mg / mL. 50 To determine the serologic status, 4 mice from each group were injected with 10 0 ~10 5Ten-fold serial dilutions containing 50 μL of virus were inoculated intranasally. Body weight and survival rate were monitored daily for 14 days. The MLD was calculated using the method of Reed and Muench (1938). 50 All mouse experiments were performed in accordance with the regulations of the Animal Experimentation Committee of the University of Tokyo and were approved by the Animal Experimentation Committee of the Institute of Medical Science (University of Tokyo).

[0124] Virus passage in mice and MDCK cells. Mouse adaptation of the virus was performed as described in Example I. For virus passage in MDCK cells, confluent MDCK cells were infected with the virus at a multiplicity of infection (MOI) of 0.0001. At 48 hours post-infection (hpi), the supernatant was collected and titrated in MDCK cells. The collected fresh virus was used to infect MDCK cells for the next passage. This procedure was repeated five times.

[0125] Growth kinetics assay. Each virus was inoculated into triplicate wells of subconfluent MDCK cells at an MOI of 0.0001. The cells were supplemented with MEM containing 0.3% bovine serum albumin (BSA) and 1 μg / mL tosylsulfonylphenylalanyl chloromethyl ketone (TPCK) trypsin and incubated at 37°C in 5% CO2. Culture supernatants were harvested at the indicated times postinfection. The virus titers of the supernatants at different time points were determined by plaque assay in MDCK cells.

[0126] Minigenome luciferase assay. Polymerase activity was tested by minigenome assay using a dual-luciferase system, as previously described by Murakami (2008) and Ozawa et al. (2007). Briefly, 293 cells were transfected with viral protein expression plasmids for NP, PB1, PB2, and PA derived from WT-Venus-H5N1 or MA-Venus-H5N1 viruses (0.2 μg each), along with a plasmid expressing a reporter vRNA encoding the firefly luciferase gene under the control of the human RNA polymerase I promoter (pPolI / NP(0)Fluc(0), 0.2 μg) and pRL-null (Promega, 0.2 μg) encoding Renilla luciferase as an internal transfection control. Twenty-four hours after transfection, cell lysates were prepared using the Dual-Luciferase Reporter Assay System (Promega), and luciferase activity was measured using a GloMax96 microplate luminometer (Promega). The assay was normalized to Renilla luciferase activity. All experiments were performed in triplicate.

[0127] Laboratory facilities. All studies with H5N1 virus were conducted in an enhanced biosafety level 3 containment laboratory at the University of Tokyo (Tokyo, Japan), approved for such use by the Ministry of Agriculture, Forestry and Fisheries (Japan).

[0128] Statistical analysis. Data were analyzed using R software (www.r-project.org) (version 3.1). For comparisons of measurements from multiple groups collected at a single time point, we used one-way analysis of variance followed by Tukey's post hoc test. For comparisons of multiple groups with independently obtained measurements at different time points (i.e., mouse-derived virus growth curves obtained in MDCK cells), we used two-way analysis of variance followed by Tukey's post hoc test. For comparisons of multiple groups with dependent measurements (i.e., virus growth curves in cell cultures where aliquots were collected from the same culture at different time points), we used the R package NLME to fit a linear mixed-effects model to the data, taking into account time, virus strain, and the interaction between these two factors. Next, we used the R package PHIA to construct a contrast matrix to compare strains in a pairwise manner at the same time point (e.g., group_1 vs. group_2 at 24 hours postinfection, group_1 vs. group_3 at 24 hours postinfection, group_2 vs. group_3 at 24 hours postinfection). Because the comparisons were performed individually, the final p-values ​​were adjusted by using Holm's method to account for multiple comparisons. In all cases, results were considered statistically significant if we obtained a p-value (or adjusted p-value) < 0.05.

[0129] Sequence analysis. PB2 and PA sequences from the NCBI Influenza Virus Database were aligned using the MUSCLE program (Edgar, 2004) with default parameters and a maximum of 100 iterations. Alignments were visualized using Clustal X (Larkin et al., 2007), and frequencies of amino acid occurrence at specific positions were determined using custom-developed Perl scripts.

[0130] result Comparison between WT-Venus-H5N1 and MA-Venus-H5N1 viruses. As in Example I, the NS segment of A / Viet Nam / 1203 / 2004 (H5N1) (abbreviated as VN1203) was replaced with the Venus-fusion NS segment of Venus-PR8 virus by reverse genetics to obtain an H5N1 virus expressing the Venus fluorescent reporter gene (WT-Venus-H5N1 virus). Pathogenicity analysis in mice showed that this virus was resistant to 10 3 PFU's MLD 50 The results showed that the WT-Venus-H5N1 virus showed attenuated virulence in mice compared with the parental VN1203, which has a PFU value of 0.7 (compared to 0.7 PFU for VN1203) (Figures 6A and 6B and Hatta et al., 2007). Furthermore, the WT-Venus-H5N1 virus replicated primarily in the respiratory tract (Table 3), and its Venus expression was very weak in both MDCK cells (Figure 7) and mice after virus infection (Figure 8). [Table 3]

[0131] After six passages of the WT-Venus-H5N1 virus in mice, the MA-Venus-H5N1 virus was obtained. The MA-Venus-H5N1 virus had an MLD of 3.2 PFU. 50 The MA-Venus-H5N1 virus was lethal to mice at 1000 ng / mL (Example I). This virus replicated systemically in mice, with high viral titers detected in the lungs, spleen, and kidneys on day 1 postinfection, and virus could be detected in the brain on day 3 postinfection (Table 3). Furthermore, we detected high Venus expression in the MA-Venus-H5N1 virus in MDCK cells (Figure 7) and mice (Figure 8). Thus, compared with WT-Venus-H5N1, the MA-Venus-H5N1 virus exhibited significantly higher pathogenicity and higher replication capacity in mice. Furthermore, this virus exhibited high Venus expression during its replication in vitro and in vivo.

[0132] To identify genetic mutations that occurred during mouse adaptation, the genome of the MA-Venus-H5N1 virus was sequenced and compared with that of the WT-Venus-H5N1 virus. At the amino acid level, a total of seven differences were found between the two viruses in their PB1, PB2, PA, NA, M2, and NS1 genes (Table 4). Therefore, single or multiple amino acid changes among these seven different amino acids may contribute to the differences in mouse virulence and Venus expression between these two viruses. [Table 4]

[0133] V25A in PB2 and R443K in PA determine the virulence and Venus expression of Venus-H5N1 virus in mice. To investigate the genetic basis for the differences in virulence and Venus expression of the Venus-H5N1 virus after mouse adaptation, we established a reverse genetics system for the MA-Venus-H5N1 virus, designated the RG-MA virus. The RG-MA virus exhibited similar organ viral titers and MLD values ​​to those of its parent virus (MA-Venus-H5N1 virus). 50 The values ​​(Fig. 6C, Fig. 6D and Fig. 9) and Venus expression in mice (Fig. 9) are shown.

[0134] To identify the amino acids responsible for the differences in virulence and Venus expression between WT-Venus-H5N1 and MA-Venus-H5N1 viruses, six monogenic recombinant viruses were generated, each containing the PB2, PB1, PA, NA, M, or NS gene from MA-Venus-H5N1 virus and the other seven genes from WT-Venus-H5N1 virus. Recombinant viruses containing the PB1, NA, or NS gene of MA-Venus-H5N1 virus (referred to as WT+MA-PB1, WT+MA-NA, or WT+MA-NS) were able to express the virulence and Venus expression of WT-Venus-H5N1 (MLD).50 , 10 3 Although the reassortant strains harboring the PB2, PA, or M gene of MA-Venus-H5N1 (referred to as WT+MA-PB2, WT+MA-PA, or WT+MA-M) showed similar pathogenicity in mice as WT-Venus-H5N1 (Figure 9), the reassortant strains harboring the PB2, PA, or M gene of MA-Venus-H5N1 (referred to as WT+MA-PB2, WT+MA-PA, or WT+MA-M) showed higher pathogenicity in mice than WT-Venus-H5N1 (Figure 9). WT+MA-PB2 and WT+MA-PA replicated more efficiently in mouse lungs than WT-Venus-H5N1, and virus was also detected in the spleens of two of three mice infected with WT+MA-PB2.

[0135] The effects of the PB2 gene, PA gene, or M gene derived from WT-Venus-H5N1 on the virulence of MA-Venus-H5N1 were investigated by generating three monogenic recombinant viruses, each containing the PB2 gene, PA gene, or M gene from the WT-Venus-H5N1 virus and the remaining segments from the MA-Venus-H5N1 virus (referred to as MA+WT-PB2, MA+WT-PA, or MA+WT-M). 50 The value is MA-Venus-H5N1(MLD 50 , 3.2 PFU) was significantly higher than 10 2.3 Although the PFU was 100, the virulence of MA+WT-M in mice was similar to that of MA-Venus-H5N1 (Figure 9). These data suggest that the PB2 and PA genes played a role in the pathogenicity of MA-Venus-H5N1 virus in mice.

[0136] To evaluate the potential synergistic effect of the PB2 and PA genes on viral virulence in mice, reassortant carrying both the PB2 and PA genes of MA-Venus-H5N1 on the WT-Venus-H5N1 viral backbone [MA-(PB2+PA)] and reverse reassortant on the MA-Venus-H5N1 viral backbone [termed WT+MA-(PB2+PA) and MA+WT-(PB2+PA)] were rescued and evaluated for virulence in mice. Replacement of the PB2 and PA genes from MA-Venus-H5N1 virus with WT-Venus-H5N1 virus resulted in an MLD of 3.2 PFU. 50 PB2 significantly enhanced its virulence in mice at high levels (Figure 9), and also enhanced viral spread and replication in mice, similar to MA-Venus-H5N1 virus, and vice versa. Assuming the presence of single mutations in PB2 and PA after mouse adaptation, these data indicate that V25A in PB2 and PA R443K synergistically contribute to the virulence of MA-Venus-H5N1 virus in mice.

[0137] When we confirmed Venus expression of the above reassortant in MDCK cells, we found that the MA-PB2 gene significantly increased Venus expression (Fig. 7). In addition, Venus expression of WT+MA-PB2 virus in the lungs was also significantly enhanced (Fig. 8). While other single gene substitutions, including MA-PA, did not affect Venus expression, the double substitution of MA-PB2 and MA-PA on the WT-Venus-H5N1 virus backbone increased Venus expression in MDCK cells and mouse lungs compared to that achieved by WT-Venus-H5N1 virus and WT+MA-PB2 virus (Fig. 7 and Fig. 8). These data indicate that V25A of the PB2 protein plays an essential role in Venus expression of MA-Venus-H5N1 virus in vitro and in vivo, and that R443K of the PA protein enhances the effect of PB2 on Venus expression.

[0138] The amino acid at position 25 in the PB2 protein significantly enhances viral replication in mammalian cells. The replication abilities of these viruses were further examined in MDCK cells. We found that the MA-Venus-H5N1 virus had similar replication abilities to the RG-MA virus and grew more efficiently than the WT-Venus-H5N1 virus. The titers of the MA-Venus-H5N1 virus were significantly higher than those of the WT-Venus-H5N1 virus at 36 and 48 hpi (Fig. 10). We then examined the contributions of the PB2 and PA viral segments to the replication of the two viruses. Although significantly higher titers of WT+MA-PB2 and WT+MA-(PB2+PA) were observed compared with those of the WT-Venus-H5N1 virus at several time points postinfection, the replication efficiency of WT+MA-PA was comparable to that of the WT-Venus-H5N1 virus (Fig. 10). Although the titers of WT+MA-(PB2+PA) were higher than those of WT+MA-PB2 at 36 and 48 hpi, the difference was not statistically significant (Fig. 10). These results indicate that the MA-PB2 gene enhances Venus-H5N1 virus replication in MDCK cells, and this increase can be further enhanced in the presence of MA-PA, whereas MA-PA alone does not alter virus replication in MDCK cells.

[0139] Mutations in the polymerase gene after mouse adaptation reduce viral polymerase activity in mammalian cells. The polymerase activity of viral ribonucleoprotein (RNP) complexes has been correlated with viral replication and virulence (Gabriel et al., 2005; Leung et al., 2010; Li et al., 2008; Salomen et al., 2006). We determined the activity of eight RNP combinations, PB1, PB2, and PA, derived from WT-Venus-H5N1 or MA-Venus-H5N1 viruses by measuring luciferase activity. The polymerase activity of the mouse-adapted virus was nearly fourfold lower than that of the WT-Venus-H5N1 virus (Figure 11). Any MA gene substitution reduced the activity of the WT-Venus-H5N1 virus polymerase complex, but the polymerase activity of the complex containing the MA-PB2 and MA-PA double substitution was significantly reduced compared to that of the WT-Venus-H5N1 virus and similar to that of the MA-Venus-H5N1 virus. These results indicate that the polymerase activity of the RNP complex was specifically reduced after mouse adaptation, which is not consistent with the enhanced replication and virulence.

[0140] Molecular determinants of Venus stability in Venus-H5N1 viruses in vitro and in vivo. To evaluate the in vitro Venus stability of WT-Venus-H5N1 and RG-MA viruses, the two viruses were passaged five times in MDCK cells. During these passages, Venus-negative plaques were obtained from the WT-Venus-H5N1 virus but not from the RG-MA virus, suggesting that the Venus gene is more stable after mouse adaptation (Table 5). To identify the molecular determinants of this Venus stability, various reassortants were passaged five times in MDCK cells. Venus-negative plaques were obtained from reassortants harboring the MA-PB1, MA-NA, or MA-M genes, but we did not obtain any Venus-negative plaques from the fifth passage of Venus-H5N1 viruses harboring the MA-PB2, MA-PA, MA-(PB2+PA), or MA-NS genes (Table 5). These data suggest that the MA-PB2, MA-PA, and MA-NS genes play a role in Venus stability. [Table 5]

[0141] To further evaluate the role of these different genes on Venus stability, we amplified the NS segments of fifth-passage stocks derived from different reassortants using PCR and NS-specific primers. With the exception of the Venus NS segment (1.9 kb), the deleted NS segments were detectable at levels similar to those for the PR8 NS segment, which was less than 1 kb. The deleted NS segments of WT-Venus-H5N1 and reassortants containing the MA-NA and MA-M genes were significantly brighter than those of other reassortants (Fig. 12), further implying that the MA-NA and MA-M genes do not contribute to Venus stability in vitro. Although the RG-MA virus and reassortants containing MA-NS, MA-PA, or MA-PB2 were more stable, we still amplified the deleted Venus NS segments, albeit to a lesser extent, by PCR (Fig. 12). The deleted NS segments from various reassortants were extracted and sequenced, and the different deletion forms were identified from the different reassortants (Fig. 13).

[0142] In addition, to examine the in vivo stability of Venus, we transfected B6 mice with 10 5Mice were inoculated with PFU of WT-Venus-H5N1 virus, RG-MA virus, or WT+MA-(PB2+PA) virus. Before the mice were killed, lungs were collected on day 4 postinfection and homogenized in PBS. The supernatant was inoculated into MDCK cells, and Venus-negative plaques were harvested 48 hpi and amplified in MDCK cells. It should be noted that the Venus signal of plaques may correlate with the culture cell condition and detection time. Therefore, the Venus expression of amplified Venus-negative plaques was reconfirmed in MDCK cells to exclude false negatives. More than 95 plaques were detected from each lung, with only one plaque lacking Venus expression obtained from one of three mice infected with RG-MA virus, 12 Venus-negative plaques obtained from three mice infected with WT+MA-(PB2+PA), and more than 15 Venus-negative plaques obtained from each mouse infected with WT-Venus-H5N1 virus (Table 6). These results indicate that WT-Venus-H5N1 virus is the most unstable of these viruses in vivo, and that the PB2 and PA genes derived from MA-Venus-H5N1 virus enhance Venus stability, albeit to a lesser extent than occurs in MA-Venus-H5N1 virus. Therefore, mutations in PB1, PB2, PA, and NS may synergistically contribute to the Venus stability of MA-Venus-H5N1 virus in vivo. [Table 6]

[0143] Consideration Previously, we constructed a visualized H5N1 virus expressing a Venus reporter gene, which became more lethal to mice and more stable after mouse adaptation (Example I). In this study, we sequenced the entire genome of this virus (MA-Venus-H5N1) and identified seven amino acids that differed from the WT-Venus-H5N1 viral sequence. To explore the molecular determinants for the differences in virulence and Venus expression in mice between these two viruses, we generated a series of reassortants of both viruses using reverse genetics. We found that the double mutations in PB2 (V25A) and PA (R443K) significantly enhanced the pathogenicity of WT-Venus-H5N1 in mice. The V25A mutation in PB2 also significantly increased Venus expression and viral replication in MDCK cells and mice, and the R443K mutation in PA further enhanced these effects. The stability of different reassortants was examined in vitro, and reassortants containing MA-PB2, MA-PA, or MA-NS were found to be more stable. These results suggest that the PB2 and PA proteins play a role in the pathogenicity of Venus-expressing H5N1 viruses and Venus stability in mammalian hosts.

[0144] The virulence of highly pathogenic H5N1 avian influenza viruses in mammals is determined by multiple viral genes. For example, the HA protein plays an important role in the systemic replication and lethal infection of H5 subtype viruses in chickens (Kawaoka and Webster, 1988) and mammals (Hatta et al., 2001; Suguitan et al., 2012). The HA and NS genes of H5N1 viruses also contribute to their high virulence in ferrets (Imai et al., 2010). The NS1 protein helps to subvert the host's antiviral immune response and is essential for the pathogenicity of H5N1 viruses in mice (Jiao et al., 2005). Mutations in the M1 protein also affect the virulence of H5N1 viruses in mice (Fan et al., 2009). Amino acids at positions 627 and 701 of PB2 are major determinants of the high virulence of H5N1 influenza viruses in mammals (Hatta et al., 2001; Li et al., 2005). Finally, the PA protein has been reported to contribute to the virulence of H5N1 avian influenza virus in ducks (Song et al., 2011) and mice (Hu et al., 2013), where V25A of PB2 and R443K of PA were found to synergistically contribute to the pathogenicity of H5N1 virus in mice.

[0145] Based on all influenza virus sequences available in the public database (www.fludb.org) (23,514 PB2 proteins and 24,240 PA proteins), we found that 25V in PB2 and 443R in PA are highly conserved, whereas 25A in PB2 is present in only two viruses [A / Mallard / ON / 499 / 2005(H5N1), accession number EF392844; and A / Zhejiang / 92 / 2009(H1N1), accession number CY095997], and 443K in PA is present in only one strain isolated from a quail [A / Quail / Shantou / 1425 / 2001(H9N2), accession number EF154846]. Although the virulence of these viruses in mice is unknown, this study is the first to suggest that the combination of 25A in PB2 and 443K in PA contributes to increased virulence of the virus in mice and is an inherent property of the MA-Venus-H5N1 virus.

[0146] The influenza A virus RNA polymerase consists of the PB1, PB2, and PA subunits, which are involved in many essential processes in the viral life cycle (Naffakh et al., 2005). PB1 performs polymerase and endonuclease activities, PB2 is responsible for capped RNA recognition, and PA is involved in RNA replication and proteolytic activity (Obayasjo et al., 2005). The interface between these polymerase subunits is essential for transcription initiation (He et al., 2008; Sugiyama et al., 2009). Residues 1 to 37 at the N-terminus of the PB2 protein play an essential role in binding to the PB1 protein and affecting RNA polymerase activity, and these residues are highly conserved among all influenza virus subtypes (Sugiyama et al., 2009). The amino acid at position 25 of PB2 is located within the third α-helix (amino acids 25 to 32) of its PB1-binding domain (Sugiyama et al., 2009). In this study, the amino acid at position 25 in PB2 was found to be unstable, and V25A in PB2 was found to increase viral replication in mammalian cells and mice, thereby making H5N1 viruses more pathogenic in mice. The R443 residue in the PA protein also plays a role in replication activity (Obayashi et al., 2008; Regan et al., 2006), and the R443A mutation in PA blocks the production of infectious viruses (Regan et al., 2006). In this study, reassortants with R443K in their PA protein were rescued, demonstrating that R443K in PA enhanced viral replication in mouse lungs, highlighting the virulence of H5N1 viruses in mice. Therefore, this data further emphasizes the role of the amino acid at position 443 in the PA protein for influenza viruses.

[0147] Earlier reports have shown that the polymerase activity of viral RNP complexes is strictly correlated with viral replication and virulence (Gabriel et al., 2005; Leung et al., 2010; Li et al., 2008; Salomon et al., 2006). Viruses with higher polymerase activity in mammalian cells generally exhibit higher virulence in mice (Zhang et al., 2014) and ferrets (Salomon et al., 2006). However, viruses with high polymerase activity are not necessarily lethal to mice, suggesting that high pathogenicity of a virus in its host may require a certain level of polymerase activity (Gabriel et al., 2005). In this study, the MA-Venus-H5N1 virus was more lethal to mice than its wild-type counterpart, yet it had significantly lower polymerase activity, and any RNP combination with the polymerase gene derived from MA-Venus-H5N1 was also less active. These results may imply that the polymerase activity of the vRNP complex is strictly correlated with the viral genome and that a lower level of polymerase activity is more compatible with the reassembled genome of Venus-H5N1, which may benefit its high pathogenicity in mice.

[0148] The ability to visualize influenza viruses carrying fluorescent reporter genes through the development of live in vivo imaging would be a great benefit to influenza virus-related research (Heaton et al., 2013; Helft et al., 2012; Manicassamy et al., 2010; Pan et al., 2013; Example I). An effective virus for this purpose should have good replication ability and exhibit significant pathogenicity in its host. Moreover, it should express its fluorescent reporter protein highly and stably. Many attempts to construct influenza A viruses carrying a GFP reporter gene have been reported (Kittel et al., 2004; Manicassamy et al., 2010). However, some of these viruses showed poor replication or poor pathogenicity in mice (Kittel et al., 2004), while others generated relatively low fluorescent signals or did not stably express GFP during viral replication in vitro and in vivo (Manicassamy et al., 2010). The present data show that the MA-Venus-H5N1 virus, which is highly pathogenic to mice, also highly and stably expresses the Venus fluorescent protein in vitro and in vivo. In this analysis of the molecular determinants of Venus expression and stability, we found that V25A in PB2 played a role in determining Venus expression, which was further enhanced by the presence of R443K in PA. Analysis of Venus stability revealed that a single gene, MA-PB1, MA-PB2, MA-PA, or MA-NS, determines Venus stability in vitro, but in vivo the situation is more complex, and mutations in PB1, PB2, PA, and NS can synergistically co-determine Venus stability in MA-Venus-H5N1 virus.

[0149] In summary, we identified molecular determinants in mouse-adapted Venus-H5N1 virus that play important roles in the virus's pathogenicity in mice and its Venus expression and stability in vitro and in vivo. These molecular markers will benefit future research in the development of anti-influenza virus drugs and vaccines.

[0150] Example III Materials and Methods Cells and viruses. Madin-Darby canine kidney (MDCK) cells were maintained in minimum essential medium (MEM) containing 5% newborn calf serum (neurocutaneous NCS). Human embryonic kidney 293T (HEK293T) and HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS). A / Puerto Rico / 8 / 34 (H1N1; PR8) (Horimoto et al., 2007) and NS1-Venus PR8 viruses were generated using reverse genetics and propagated in MDCK cells in MEM containing L-(tosylamido-2-phenyl)ethyl chloromethyl ketone (TPCK)-treated trypsin (0.8 μg / mL) and 0.3% bovine serum albumin (BSA) (Sigma-Aldrich) for 48 h at 37°C.

[0151] Adaptation of NS1-Venus PR8 virus to mice. Six to eight-week-old female C57BL / 6 mice (Japan SLC) were infected with 50 μL of 2.3 × 10 6 Mice were infected intranasally with plaque-forming units (PFU) of NS1-Venus PR8 virus. Lungs were harvested 3–6 days postinfection (dpi) and homogenized in 1 mL of phosphate-buffered saline (PBS). To obtain clones with high growth potential and Venus expression, lung homogenates were plaque-purified using MDCK cells. Large, highly Venus-expressing plaques were harvested, and the cloned viruses were propagated in MDCK cells at 37°C for 48 hours. 50 μL of the supernatant was then used as an inoculum for the next passage. These procedures were repeated six times.

[0152] Sequence analysis. Viral RNA sequence analysis was performed as previously described (Sakabe et al., 2011). Briefly, viral RNA was extracted using the QIAamp Viral RNA mini kit (QIAGEN), and viral RNA was reverse transcribed using Superscript III™ reverse transcriptase (Invitrogen) and an oligonucleotide complementary to a 12-nucleotide sequence at the 3' end of the viral RNA (Katz et al., 1990). Each segment was amplified by PCR using Phusion High Fidelity DNA polymerase (Finnzymes) and primers specific to each segment of the PR8 virus. PCR products were purified and sequenced using an ABI 3130xl (Applied Biosystems).

[0153] Plasmid construction and reverse genetics. For reverse genetics and as a template for mutagenesis, a plasmid containing the cloned cDNA of the PR8 gene between the human RNA polymerase I promoter and the mouse RNA polymerase I terminator (referred to as the PolI plasmid) was used. Using site-directed mutagenesis, mutations found in the NS1-Venus PR8 virus after passage were introduced into the PR8 plasmid constructs (referred to as pPolIR-PR8-PB2-E712D and pPolIR-PR8-HA-T380A, respectively). Reverse genetics was performed as previously described (Neumann et al., 1999). The eight PolI plasmids were cotransfected into HEK293T cells with eukaryotic protein expression plasmids for PB2, PB1, PA, and NP derived from PR8 using TransIT-293 transfection reagent (Mirus). Forty-eight hours after transfection, the supernatant was collected and propagated once in MDCK cells in MEM containing TPCK-treated trypsin (0.8 μg / mL) and 0.3% BSA at 37°C for 48 hours. Cell debris was removed by centrifugation at 2,100 × g for 20 minutes at 4°C, and the supernatant was stored at -80°C until use. Virus titers were determined by plaque assay using MDCK cells.

[0154] Multinucleate formation assay. Multinucleate formation assays were performed as previously described (Imai et al., 2012) with modifications. HEK293 cells grown in 24-well plates were infected with wild-type PR8 or PR8 carrying the hemagglutinin (HA) mutation found in the NS1-Venus PR8 MA virus in DMEM containing 10% FCS at a multiplicity of infection (MOI) of 10. 18 hours after infection, cells were washed with MEM containing 0.3% BSA and treated with TPCK-treated trypsin (1 μg / mL) in MEM containing 0.3% BSA for 15 minutes at 37°C to cleave HA on the cell surface into HA1 and HA2. Trypsin was inactivated by washing the cells with DMEM containing 10% FCS. To initiate multinucleated cell formation, cells were exposed to a low pH buffer (145 mM NaCl, 20 mM sodium citrate (pH 6.0-5.4)) for 2 min at 37 °C. The low pH buffer was then replaced with DMEM containing 10% FCS, and the cells were incubated for 2 h at 37 °C. Cells were then fixed with methanol and stained with Giemsa's solution. Photographs were obtained using a microscope equipped with a digital camera (Nikon).

[0155] Western blotting. MDCK cells were infected with each virus at an MOI of 1 without trypsin. 12 hours postinfection, cells were lysed with Novex® Tris-Glycine SDS sample buffer (Invitrogen) and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were then transferred to a PVDF membrane in transfer buffer (100 mM Tris, 190 mM glycine). After membrane blocking, the membrane was incubated with a rabbit anti-GFP polyclonal antibody (MBL) or rabbit antiserum against A / WSN / 33 (H1N1) (R309), which was available in our laboratory. This antiserum reacts with influenza virus proteins, including HA, NP, and matrix protein (M1). After incubation with the primary antibody, the membrane was washed with PBS containing 0.05% Tween-20 (PBS-T), and then incubated with ECL™ anti-rabbit IgG HRP-conjugated whole antibody (GE Healthcare). Finally, specific proteins were detected using the ECL Plus Western Blotting Detection System (GE Healthcare).Photographic images were obtained using a VersaDoc Imaging System (Bio-Rad).

[0156] Virus virulence and replication in mice. Six-week-old female C57BL / 6 mice were infected with 50 μL of 10 3 , 10 4 or 10 5 Four mice per group were intranasally infected with PFU of each virus. Survival and weight changes were monitored for 14 days after infection. Three mice per group were infected with 10 3 Mice were infected with PFU of each virus and euthanized on the indicated days, their lungs were harvested, and viral titers were determined by plaque assay on MDCK cells.

[0157] Immunofluorescence assay. Six-week-old female C57BL / 6 mice were injected with 50 μL of 10 4Mice were infected intranasally with PFU of each virus. Three mice per group were euthanized on the indicated days. To fix the lungs, 800 μL of 4% paraformaldehyde (PFA) in phosphate buffer solution was injected intratracheally into the lungs, followed by removal of the lungs. After incubation with 10 mL of 4% PFA for 4 hours at 4°C, the buffer was gradually exchanged with 10%, 20%, and 30% sucrose in PBS. The lungs were then embedded in Optimum Cutting Temperature (OCT) Compound (Tissue-Tek) and frozen in liquid nitrogen. Frozen sections (6 μm thick) were permeabilized with 0.2% Triton X-100 in PBS and incubated with primary antibodies for 12 hours at 4°C. The primary antibodies were goat anti-Clara cell 10 kDa protein (CC10) (Santa Cruz, sc-9772), rabbit anti-surfactant protein C (SP-C) (Santa Cruz, sc-13979), golden Syrian hamster anti-podoplanin (eBioscience, eBio8.1.1), and rabbit anti-calcitonin gene-related peptide (CGRP) (Sigma-Aldrich, C8198). After washing with PBS, sections were incubated with species-specific fluorochrome-conjugated secondary antibodies for 30 min at room temperature. Nuclei were stained with Hoechst 33342 (Invitrogen). Sections were observed using a Nikon A1 confocal microscope (Nikon).

[0158] Preparation of transparent samples. Transparent samples were prepared using a SCALEVIEW A2 (Olympus) according to a previous report (Hama et al., 2012). Six-week-old female C57BL / 6 mice were inoculated with 50 μL of 10 5Mice were infected intranasally with PFU of each virus. On the indicated days, intracardiac perfusion was performed, and lungs were fixed with 4% PFA in PBS for 4 hours at 4°C. Lungs were incubated with 10%, 20%, and 30% sucrose in PBS as described above, embedded in OCT compound, and frozen in liquid nitrogen. After thawing and rinsing with PBS, the lungs were fixed again with 4% PFA in PBS for 30 minutes at room temperature. Lungs were then transferred to a SCALEVIEW A2 and incubated at 4°C for at least two weeks. The SCALEVIEW A2 was replaced every two to three days. Cleared samples were observed using a stereofluorescence microscope (Leica M205FA) equipped with a digital camera (DFC365FX) and a GFP3 filter (480 / 40LP510).

[0159] Flow cytometry. To prepare single-cell suspensions, lungs were minced with scissors and digested with 20 mg of collagenase D (Roche) and 200 units of DNase (Worthington) for 30 minutes at 37°C. Samples were then passed through a 100 μm cell strainer, and red blood cells were lysed with red blood cell lysis buffer (Sigma-Aldrich). Single-cell suspensions were stained with a combination of the following antibodies: allophycocyanin-conjugated anti-F4 / 80 (eBioscience, BM8), allophycocyanin-cyanin 7-conjugated anti-CD11b (BioLegend, M1 / 70), phycoerythrin-cyanin 7-conjugated anti-CD11c (BD PharMingen, HL3), and eFluor 450-conjugated CD45 (eBioscince, 30-F11). Dead cells were stained with ViaProbe (Becton Dickinson). Stained samples were analyzed using a FACSAria II (Becton Dickinson and Company) and FlowJo software (TreeStar).

[0160] RNA isolation and integrity. Venus-positive and Venus-negative cells from three pooled lungs were collected in TRIzol Reagent (Invitrogen). Total RNA was extracted by isopropanol precipitation with glycogen as a carrier. The integrity of the isolated total RNA was assessed by determining the UV260 / 280 absorbance ratio and examining the 28S / 18S ribosomal RNA bands using an Agilent 2100 Bioanalyzer (Agilent Technologies) according to the manufacturer's instructions.

[0161] Microarray analysis. Forty nanograms of total RNA was amplified using the Arcturus® Riboamp® Plus RNA Amplification Kit (Life Technologies). Cy3-labeled complementary RNA probe synthesis was initiated with 100 ng of total RNA using the Agilent Low Input Quick Amp Labeling kit (one color) (Agilent Technologies) according to the manufacturer's instructions. Agilent SurePrint G3 Gene Mouse GE 8 × 60K microarrays were also used. Slides were scanned using an Agilent High-Resolution Microarray Scanner, and image data was processed using Agilent Feature Extraction software version 10.7.3.1. All data were then uploaded to GeneSpring GX version 12.5 for data analysis. For data analysis, each gene expression array dataset was normalized in silico to the pool of samples from PBS-inoculated mice. Statistically significant differences in gene expression between Venus-positive and Venus-negative cells were determined using one-way analysis of variance (ANOVA) followed by a Tukey HSD post-hoc test (P < 0.05) and Benjamin-Hochberg false discovery rate correction. Differentially expressed genes were further selected to include genes whose expression changed 2.0-fold compared to levels in the PBS group. Genes that passed statistical analysis were further assigned to a Gene Ontology (GO) group.

[0162] result Establishment of mouse-adapted NS1-Venus PR8 virus. We successfully rescued the NS1-Venus PR8 WT virus by reverse genetics, and this virus was avirulent in mice (MLD). 50 :>10 5PFU), Venus expression was very weak in MDCK cells and lung sections of mice infected with this virus. To increase the virulence and Venus expression of the NS1-Venus PR8 WT virus, the virus was serially passaged in mice by intranasal infection with a plaque-purified, high-Venus-expressing clone (see Examples I and II). After six serial passages, the virulence of the virus appeared to increase; therefore, this mouse-adapted NS1-Venus PR8 WT virus was sequenced to explore mutations.

[0163] Sequence analysis showed that two amino acid substitutions occurred after passaging (Table 7). [Table 7]

[0164] One of the mutations was in PB2 (glutamic acid to aspartic acid substitution at position 712), and the other was in HA (threonine to alanine substitution at position 380). To confirm their contribution to virulence in mice, these mutations were introduced into the corresponding polI plasmid, and reverse genetics was used to generate NS1-Venus PR8 carrying the two mutations (referred to as NS1-Venus PR8 MA virus). The pathogenicity of the NS1-Venus PR8 MA virus was higher than that of the NS1-Venus PR8 WT virus (MLD 0.01). 50 :2.1×10 4 Furthermore, the Venus signal was strong in the lungs from mice infected with NS1-Venus PR8 MA virus, whereas no Venus signal was detected in lungs infected with NS1-Venus PR8 WT or PR8 (data not shown). Thus, NS1-Venus PR8 MA showed promise as a useful reporter virus.

[0165] Comparison of mutant virus replication in MDCK cells. To compare the growth of these viruses in cell lines, two single-gene reassortants were generated, each carrying the PB2 or HA gene of the NS1-Venus PR8 MA virus and the remaining genes from the NS1-Venus PR8 WT virus for use in experiments with the NS1-Venus PR8 MA virus. MDCK cells were infected with these viruses at an MOI of 0.001, and virus titers in the supernatants were determined by plaque assay every 12 hours (FIG. 14). The NS1-Venus PR8 WT virus was infected at an MOI of 10 6.5 The NS1-Venus PR8 MA virus grew to 10 PFU / mL, which is comparable to the wild-type PR8 virus. 8 The virus titers of the NS1-Venus PR8 PB2 virus and the NS1-Venus PR8 HA virus were approximately 10 7.5 Although the viral titers reached PFU / mL, these were lower than those of the NS1-Venus PR8 MA virus. Therefore, the growth ability of the NS1-Venus PR8 MA virus was significantly improved in MDCK cells, and the mutations in the PB2 and HA genes had additive effects.

[0166] Comparison of pathogenicity and replication of mutant viruses in mice. 5 PFU, 10 4 PFU or 10 3 PFU of these viruses were infected into C57BL / 6 mice, and their weight and survival rate were monitored (Figure 15). 5 Mice infected with PFU of these viruses lost significant body weight, and one of the four mice infected with the NS1-Venus PR8 WT virus and all mice infected with the NS1-Venus PR8 PB2 and NS1-Venus PR8 MA viruses had to be euthanized during the observation period. 4Mice infected with PFU of NS1-Venus PR8 PB2 virus and NS1-Venus PR8 MA virus showed significant weight loss, and one of four mice infected with NS1-Venus MA virus and two of four mice infected with NS1-Venus PR8 PB2 virus succumbed to the infection. 4 Although the weight of mice infected with PFU of NS1-Venus PR8 HA virus and NS1-Venus PR8 WT virus was slightly reduced, all of the mice survived. 3 In the case of infection with PFU, the weight of mice infected with NS1-Venus PR8 PB2 and NS1-Venus PR8 MA was slightly reduced, but all of these mice also survived. 3 Mice infected with PFU of NS1-Venus PR8 WT and NS1-Venus PR8 HA showed little weight loss, and all of the mice survived. The viral titers of these viruses were determined in the lungs of mice (Figure 16). 3 PFU of virus was infected, and lungs were collected on days 3, 5, and 7 after infection. The maximum viral lung titers in mice infected with the NS1-Venus PR8 PB2 virus were similar to those in mice infected with the NS1-Venus PR8 MA virus. 6 The viral titers in lungs from mice infected with NS1-Venus PR8 WT and NS1-Venus PR8 HA viruses were significantly lower than those in lungs from mice infected with NS1-Venus PR8 PB2 and NS1-Venus PR8 MA viruses at all time points. Finally, at 7 days postinfection, no virus was detected in lungs from mice infected with NS1-Venus PR8 WT. Collectively, these results indicate that only the PB2 mutation affected the virulence and replication of NS1-Venus PR8 MA virus in mice.

[0167] Stability of Venus expression by NS1-Venus PR8 MA virus during replication in vitro and in vivo. In Manicassamy's study (Manicassamy et al., 2010), the proportion of GFP-negative viruses increased over time. This is one of the obstacles to using this virus for live imaging studies. We evaluated the stability of Venus expression by NS1-Venus PR8 MA virus during replication in MDCK cells (Figure 17A). Even 72 hours after infection, more than 90% of plaques were Venus-positive. We monitored the positive rate of Venus expression during repeated passage of the virus in cell culture (Figure 17B). Approximately 90% of plaques expressed Venus even after five passages, suggesting that Venus expression by NS1-Venus PR8 MA virus was stable in cell culture. Finally, we confirmed that Venus expression was stable during viral replication in vivo (Figure 17C). Plaque assays were performed using lung homogenates, and the percentage of Venus-positive plaques was estimated essentially as described above. At 3 days postinfection, the percentage of Venus-positive plaques was over 85%, whereas at 7 days postinfection, the percentage of Venus-positive plaques was approximately 75%. Collectively, these results indicate that Venus expression by the NS1-Venus PR8 MA virus was stable during replication in vitro, and the percentage of Venus-positive plaques in mouse lungs was similar to that previously reported (Manicassamy et al., 2010).

[0168] The PB2-E712D substitution contributes to high Venus expression. The Venus expression level of the NS1-Venus PR8 MA virus was substantially higher than that of the NS1-Venus PR8 WT virus. Because PB2 is one of the subunits of influenza virus polymerase, it was hypothesized that the PB2-E712D substitution is important for the increased Venus expression. To compare Venus protein expression, Western blot analysis of viral proteins and Venus in infected cells was performed (Fig. 18A). At 12 hours postinfection, the amount of M1 protein was similar for all viruses, but the amount of Venus protein was higher in cells infected with the NS1-Venus PR8 PB2 virus and the NS1-Venus PR8 MA virus compared with the other two viruses that had the parental PB2 gene. Venus expression in infected cells was also observed using confocal laser scanning microscopy (Fig. 18B). As expected, the Venus signal in cells infected with the NS1-Venus PR8 PB2 and NS1-Venus PR8 MA viruses was stronger than that in cells infected with the two viruses carrying the parental PB2 gene. Collectively, these results indicate that the PB2-E712D substitution was responsible for the high Venus expression.

[0169] To demonstrate that the PB2-E712D mutation increased Venus expression levels, MDCK cells were infected with the indicated viruses at an MOI of 1 and subjected to confocal microscopy 12 hours later (Fig. 18C). As expected, the levels of NS1-Venus fusion protein were higher in cells infected with MA-Venus-PR8 or PB2-Venus-PR8 than in cells infected with WT-Venus-PR8 or HA-Venus-PR8 (Fig. 18C).

[0170] Collectively, the data indicate that the PB2-E712D substitution is primarily responsible for the increased replication capacity, Venus expression, and virulence of the MA-Venus-PR8 virus in mice. To assess whether the PB2-E712D mutation directly affects viral polymerase activity in minireplicon assays, HEK293 cells were transfected with viral protein expression plasmids for NP, PA, PB1, and PB2 or PB2-E712D, along with a plasmid expressing vRNA encoding the firefly luciferase gene, while a pRL-null luciferase protein expression plasmid (Promega) served as a transfection control. Luciferase activity was measured 48 hours after transfection using the Dual-Glo Luciferase Assay System (Promega) (Ozawa et al., 2007). Unexpectedly, the polymerase activity of PB2-E712D was lower than that of the parental PB2 (Figure 18D). Similar results were obtained with canine MDCK cells (data not shown). In the context of a minireplicon measuring viral replication and transcription, the PB2-E712D mutation is thus attenuated; in contrast, this mutation enhances viral growth in the context of replicating viruses. These findings indicate that the PB2 protein not only functions in viral replication / transcription but also plays additional roles in the viral life cycle.

[0171] The HA-T380A substitution raises the threshold for membrane fusion. MA-Venus-PR8 HA vRNA did not significantly increase the toxicity of WT-Venus-PR8 in mice. However, HA-Venus-PR8 virus grew more efficiently in MDCK cells than WT-Venus-PR8 (Figure 14), suggesting the contribution of the HA-T380A mutation to viral replication, at least in cultured cells. Because the HA-T380A substitution is located on the a-helix in the HA2 subunit (Gamblin et al., 2004), its effect on HA membrane fusion activity was assessed using a multinuclear cell formation assay (Imai et al., 2012). Briefly, HEK293 cells were infected with WT-PR8 or mutant PR8 viruses encoding HA-T380A at an MOI of 10. After 18 h, cells were treated with TPCK-treated trypsin (1 μg / mL) for 15 min at 37°C, exposed to low-pH buffer (145 mM NaCl, 20 mM sodium citrate (pH 6.0–5.4)) for 2 min, incubated in maintenance medium at 37°C for 2 h, fixed with methanol, and stained with Giemsa's solution. Wild-type HA had a threshold for membrane fusion at pH 5.5, whereas the threshold for HA-T380A was pH 5.8 (Figure 19), which led to a conformational change in HA at an earlier stage of endosomal maturation during influenza virus entry (Lozach et al., 2011). The shift in the pH threshold for membrane fusion may affect HA thermostability (Ruigrok et al., 1986), an effect we did not observe at 50°C (data not shown).

[0172] Time course observation of virus growth in whole mouse lungs. The NS1-Venus PR8 MA virus allows observation of virus-infected cells without immunostaining because the Venus expression by this virus is high enough to allow visualization of infected cells by microscopy. To observe how influenza virus grows in the lungs, the cleared lungs are treated with SCALEVIEW A2, a reagent that makes samples optically transparent without reducing fluorescence intensity (Figure 20). Mice are infected with 10 5Mice were intranasally infected with PFU of PR8 virus, NS1-Venus PR8 WT virus, or NS1-Venus PR8 MA virus, and lungs were harvested on days 1, 3, and 5 postinfection. After treatment with SCALEVIEW A2, the samples were observed using a stereofluorescence microscope. Directly observed Venus signals were unclear due to insufficient transparency. Therefore, the transparent samples were longitudinally cut open to expose the bronchi (Figure 20, lower panel, "Cut"). Venus expression was not observed in the transparent samples from mice infected with the NS1-Venus PR8 WT virus at any time point (Figures 20G and 20H). Samples harvested 3 days postinfection are shown. In lungs infected with the NS1-Venus PR8 MA virus, no Venus signal was observed 1 day after infection (Figures 20A and 20B). However, Venus expression was clearly observed in a large portion of bronchial epithelial cells 3 days after infection (Figures 20C and 20D). Occasionally, Venus expression was also observed in alveolar epithelial cells surrounding the bronchi. Five days after infection, most Venus-positive cells found in the bronchial epithelium had disappeared, while the number of Venus-positive cells in the bronchioles and alveoli had increased (Figures 20E and 20F). Based on these observations, Venus-positive cells found in the bronchi 3 days after infection may have died, and influenza virus may have spread from the bronchi to the bronchioles and alveoli over time. No clear Venus signal was observed in the cleared lungs from mice inoculated with PR8 or PBS (Figures 20I–L). These results demonstrate that the NS1-Venus PR8 MA virus and the transparency reagent SCALEVIEW A2 enable visualization of the dynamics of influenza virus infection in all lung lobes.

[0173] Identification of target cells of NS1-Venus PR8 MA virus in mouse lungs. Transparent lungs infected with NS1-Venus PR8 MA virus revealed that influenza virus first infected the bronchial epithelium and then invaded the alveoli over time. Next, to identify target cells of NS1-Venus PR8 MA virus, we performed immunofluorescence assays of frozen sections using several antibodies specific to lung cells (Figure 21). Epithelial cells of bronchi and bronchioles include Clara cells, ciliated cells, goblet cells, and a small number of neuroendocrine cells, whereas alveoli contain type I and type II pneumocytes. Because Clara cells constitute the bulk of the lumen of bronchi and bronchioles (Rawlins et al., 2006) and type II pneumocytes have previously been reported to be targets of influenza viruses (Baskin et al., 2009), we determined that type I and type II pneumocytes were concentrated in Clara cells and alveolar epithelial cells, respectively. At 3 days after infection, a significant proportion of bronchiolar cells were Venus-positive, and almost all of these cells were CC10-positive (Fig. 21A). In addition, cuboidal Venus signals in the alveolar region were associated with SP-C-positive cells (Fig. 21B, white arrows). Although rare, Venus-positive type I alveolar epithelial cells were observed 5 days after infection (Fig. 21B, white arrows). However, no Venus expression was detected in neuroendocrine cells (data not shown).

[0174] Flow cytometry was performed to determine whether alveolar macrophages and monocytes were infected with the NS1-Venus PR8 MA virus, because these immune cells reside in the lung and serve as the first line of defense against inhaled microorganisms and particulate matter. Alveolar macrophages express F4 / 80 + Based on the CD11b expression levels in the population, the cells were predominantly monocytes (Figure 22A). 5Mice were infected with PFU of PR8 virus or NS1-Venus PR8 MA virus, and the total numbers of these cells were compared. After influenza virus infection, the number of alveolar macrophages was only slightly different from that of the control group. However, the number of monocytes significantly increased due to infiltration of monocytes from the blood vessels into the infection site (Figures 22B and 22C). Regarding the proportion of Venus-positive cells, 3 days after infection, 3.16% ± 0.59% of alveolar macrophages were Venus-positive cells, and 1.55% ± 0.07% of monocytes were Venus-positive (Figures 22D and 22E). Furthermore, the number of Venus-positive cells slightly decreased 3 to 5 days after NS1-Venus PR8 MA virus infection. After PR8 infection, the number of Venus-positive cells was comparable to that in mock-treated mice. Taken together, these results indicate that Clara cells in the bronchi and bronchioles, type II alveolar epithelial cells, monocytes, and alveolar macrophages in the alveolar region of the lung are target cells for influenza virus.

[0175] F4 / 80 + Differential gene expression between Venus-positive and Venus-negative cells in cell populations. Because alveolar macrophages and monocytes act as the first line of defense against inhaled microorganisms, it is possible that infection of these cells with influenza virus may affect their ability to prevent the spread of infection. To assess this, we compared the gene expression profiles between Venus-positive and Venus-negative cells in the alveolar macrophage and monocyte populations by microarray analysis. Since the number of Venus-positive alveolar macrophages and monocytes that could be recovered from one mouse by flow cytometry was too small to perform microarray analysis, we subcultured these cells as F4 / 80 + Cells were analyzed together and pooled from three mice. Live mononuclear cells were CD45 + and Via-Probe - As shown in Figure 22A, the cells were gated as F4 / 80 +Alveolar macrophages and monocytes were identified based on the CD11b expression level in the population. Venus-positive F4 / 80 cells and Venus-negative F4 / 80 cells were sorted from the live mononuclear cell fraction using a FACSAria II. 高 Because alveolar macrophages have high autofluorescence, there was a possibility of overlap with the Venus signal. Therefore, CD11c with moderate expression of Venus was not detected. 高 Alveolar macrophages were excluded from the Venus-positive fraction (Fig. 23A). Confocal microscopic observation of sorted cells demonstrated that these cells could be appropriately recovered based on Venus expression (Fig. 23B). In addition, given that Venus expression was observable throughout the cells, these cells were infected with the virus but did not phagocytose infected cells. Microarray analysis demonstrated that expression was consistent with that of F4 / 80 cells derived from PBS-inoculated mice. + We identified thousands of genes whose expression was statistically altered by at least 2.0-fold compared to the cellular level (data not shown). Among these genes, the expression of the Venus-positive F4 / 80 + Cell and Venus negative F4 / 80 +633 genes were identified that were statistically different by at least 4.0-fold between the cells (Figure 24A). Gene ontology analysis revealed that these genes were involved in extracellular activity (Figure 24B). For genes annotated with "cytokine activity," a total of 24 genes had altered expression levels, including several cytokines and chemokines such as type I interferon (IFN) (Figure 24C). All of these genes, except for those for interleukin (IL)-4 and Cxcl13 [chemokine (C-X-C motif) ligand 13], were upregulated in Venus-positive cells relative to Venus-negative cells. Furthermore, when type I clustered on genes annotated in "response to wounding," most genes, including collagen type 1 alpha 1 (Col1a1), collagen type 3 alpha 1 (Col3a1), collagen type 5 alpha 1 (Col5a1), hyaluronoglucosamidase 1 (Hyal1), and fibrinogen gamma chain (Fgg), were identified as Venus-positive F4 / 80. + In summary, these results suggest that F4 / 80 + This indicates that a small number of cells were infected with influenza virus compared to the total number of cells, and the gene expression levels of several cytokines and chemokines were enhanced in virus-infected cells at the site of infection. Furthermore, F4 / 80 cells infected with NS1-Venus PR8 MA virus were significantly increased in the NS1-Venus PR8 MA virus-infected cells. + The cells had enhanced expression of genes involved in responding to wounds caused by infection and inflammation.

[0176] Example IV A vector capable of expressing a heterologous gene product from a fusion construct with the viral NS1 protein has been described above (Figure 26). In particular, the PB2-E712D mutation stabilized the expression of the heterologous gene product. Serial passages of the test virus (WT-Venus-PR8) were performed to identify other mutations in the polymerase complex that contribute to stabilization (Figure 27).

[0177] Example V An E-to-D mutation at position 712 of the polymerase subunit PB2 (PB2-E712D) stabilized the inserted Venus gene (Fukuyama et al., 2015; Katsura et al., 2016). We also prepared an H5N1 virus (Venus-H5N1) carrying the Venus gene inserted into the NS segment from PR8 (Fukuyama et al., 2015). However, like WT-Venus-PR8, WT-Venus-H5N1 exhibited moderate virulence and low Venus expression. However, we obtained a mutant that became more lethal to mice and stably expressed Venus after mouse adaptation. A V-to-A mutation at position 25 of the polymerase subunit PB2 and an R-to-K mutation at position 443 of the polymerase subunit PA contributed to the stable maintenance of the Venus gene (Zhao et al., 2015). These results indicated that the composition of the viral polymerase plays a role in stabilizing the inserted foreign gene, but the mechanism by which the Venus gene can be deleted and how polymerase mutations stabilize the Venus gene remained unclear.

[0178] As described below, we investigated the mechanism of Venus gene stabilization by comparing events during infection with WT-Venus-PR8 and Venus-PR8 carrying the PB2-E712D mutation (Venus-PR8-PB2-E712D). We examined polymerase fidelity and RNA and protein expression in infected cells and performed sequencing analysis in conjunction with coinfection experiments to determine how the Venus gene is deleted. Furthermore, to further our understanding of the stabilization mechanism, we identified additional mutations that contribute to Venus gene stabilization.

[0179] Materials and Methods Cells and viruses. Madin-Darby canine kidney (MDCK) cells were cultured in minimum essential medium (Gibco) with 5% newborn calf serum at 37°C in 5% CO2. Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Using reverse genetics (Neumann et al., 1999), WT-Venus-PR8 and a Venus-PR8 mutant carrying the NS segment encoding the Venus fluorescent protein (Fukuyama et al., 2015) were generated and grown in MDCK cells at 37°C.

[0180] Venus Stability. MDCK cells were infected with WT-Venus-PR8 or each Venus-PR8 mutant at an MOI of 0.001. Supernatants were collected 48 hours postinfection and titrated by plaque assay in MDCK cells. The resulting viruses were passaged four times in the same manner. The percentage of Venus-expressing plaques in virus stocks from different passages was determined in MDCK cells by observing more than 65 plaques in each virus stock using fluorescence microscopy. To exclude false-positive plaques, Venus-negative plaques were picked, amplified in MDCK cells, and re-evaluated for Venus expression.

[0181] Large-scale sequencing analysis. WT-PR8, PR8-PB2-E712D, PR8-PB1-V43I (Cheung et al., 2014; Naito et al., 2015), and PR8-PB1-T123A (Pauly et al., 2017) were generated by reverse genetics (Neumann et al., 1999) and infected MDCK cells at an MOI of 0.001. Supernatants were collected 48 h postinfection and titrated using plaque assays in MDCK cells. The resulting viruses were passaged five times in the same manner. Viral RNA was extracted from the virus before passage and from the virus after five passages using the QIAamp Viral RNA Mini Kit (Qiagen). Reverse transcription-PCR (RT-PCR) was performed using the Superscript III High-Fidelity RT-PCR Kit (Invitrogen). DNA amplicons were purified using 0.45x Agencourt AMpure XP magnetic beads (Beckman Coulter), and 1 ng was used for barcoded library preparation with the Nextera XT DNA kit (Illumina). After bead-based normalization (Illumina), the library was sequenced on the MiSeq platform using the MiSeq v2 300-cycle reagent kit (Illumina) with paired-end operation. Raw sequence reads were analyzed using the ViVan pipeline (Isakov et al., 2015). Here, a 1% cutoff was used as the minimum frequency. Furthermore, we defined an empirical cutoff for minimum read coverage as follows: for a variant with a frequency of 1%, at least 1,000 reads should cover the region. Similarly, for a variant with a frequency of 0.1%, 10,000 reads should cover the region. Variants were removed if coverage was less than 1,000 / (frequency). The sequencing data for the virus that had been passaged five times was compared to the sequencing data for the virus before passage, and the number of nucleotide mutations that were not observed before passage but were observed only after passage were counted.The number of mutations per nucleotide was calculated for each segment and the average values ​​for all eight segments in each virus were compared.

[0182] Quantitative real-time PCR. MDCK cells were infected with WT-Venus-PR8 or Venus-PR8-PB2-E712D at an MOI of 1, or mock infected with medium. Total RNA was extracted from cells 9 hours postinfection using an RNeasy mini kit (Qiagen). RNA quantification was performed as previously described (Kawakami et al., 2011). Primers for IFN-β, NS vRNA, NP vRNA, and β-actin were previously described (Kawakami et al., 2011; Kupke et al., 2018; Park et al., 2015). Data were analyzed using a 2-well platelet count (PBC). -ΔΔCT The results were analyzed by the method ( Livak et al., 2001 ) and normalized to the expression of β-actin mRNA.

[0183] Western blotting. MDCK cells were infected with each virus at an MOI of 1 or mock infected with medium. At the indicated time points, cells were lysed with Tris-glycine SDS sample buffer (Invitrogen). Cell lysates were sonicated, heated at 95°C for 10 min, and then subjected to SDS-PAGE. SDS-PAGE was performed on Any kD Mini-PROTEAN TGX precast protein gels (Bio-Rad). Proteins on the SDS-PAGE gel were transferred to polyvinylidene difluoride membranes (Millipore) and detected with the indicated primary antibodies (rabbit anti-NS1 [GeneTex], mouse anti-Aichi NP [2S 347 / 4], mouse anti-β-actin [Sigma-Aldrich]) followed by secondary antibodies (sheep horseradish peroxidase [HRP]-conjugated anti-mouse IgG [GE Healthcare] or donkey HRP-conjugated anti-rabbit IgG [GE Healthcare]). Specific protein signals were detected using ECL Prime Western blotting detection reagent (GE Healthcare). Images were captured using a ChemiDoc Touch imaging system (Bio-Rad) and quantified using Image Lab software (Bio-Rad).

[0184] Coinfection analysis. Three synonymous nucleotide substitutions were made in the 3' or 5' region of the NS segment of WT-Venus-PR8 that do not overlap the packaging signal sequence (Fujii et al., 2005). These modified viruses were used to coinfect MDCK cells at an MOI of 0.001 or 5, respectively. Supernatants were collected 2 days or 8 hours postinfection, respectively, and used to infect MDCK cells. Venus-negative plaques were picked and amplified in MDCK cells, and the sequences of the NS segments in the resulting viruses were then analyzed.

[0185] Identification of additional mutations that stabilize the Venus gene. WT-Venus-PR8 was infected into MDCK cells at an MOI of 0.001. The supernatant was collected 2 days postinfection and used to infect MDCK cells. Venus-positive plaques were then picked and repeatedly amplified in MDCK cells until mutants stably expressing Venus fluorescence were obtained. Mutant sequences were analyzed to identify amino acid mutations in PB2, PB1, and PA. To determine whether these mutations contributed to the stability of the Venus gene, mutants containing each of the identified amino acid mutations were generated by reverse genetics (Neumann et al., 1999), and the Venus stability of each mutant was examined as described above. The identified amino acid positions were plotted on the crystal structure of the influenza virus polymerase complex (PDB ID 4WSB) using the PyMOL molecular graphics system. In Figure 32B, the polymerase internal tunnel was visualized using the MOLEonline web interface (Pravda et al., 2018), and the information was deposited in ChannelsDB (Pravda et al., 2018). The percentage of strains containing the identified amino acids was determined using the "Sequencing Feature Variant Type" tool in the Influenza Research Database (Zhang et al., 2017; Noronha et al., 2012).

[0186] Statistical analysis. Statistical differences between WT-Venus-PR8 and Venus-PR8-PB2-E712D were assessed using a two-tailed unpaired Student's t-test. A P value of less than 0.05 was considered significant.

[0187] result Loss of Venus expression in WT-Venus-PR8 restores replication efficiency. WT-Venus-PR8 and Venus-PR8-PB2-E712D were prepared using reverse genetics as previously described (Neumann et al., 1999). The gene encoding the Venus fluorescent protein was inserted into the NS segment (Fukuyama et al., 2015), as shown in Figure 28A. We first examined how rapidly Venus expression was lost in WT-Venus-PR8 and the relationship between Venus deletion and virus titer. The virus was passaged in MDCK cells at a multiplicity of infection (MOI) of 0.001, and the percentage of Venus-positive plaques was measured (Figure 28B). While Venus expression was immediately lost in WT-Venus-PR8, all plaques of Venus-PR8-PB2-E712D showed Venus expression after four passages. Although WT-Venus-PR8 showed lower titers than Venus-PR8-PB2-E712D in MDCK cells, as previously described (Katsura et al., 2016), the viral titer increased during virus passage as the percentage of Venus-positive plaques decreased (Figure 28C). This result suggests that loss of the Venus gene in mutant WT-Venus-PR8 restored viral replication efficiency.

[0188] The PB2-E712D mutation does not cause appreciable changes in polymerase fidelity. We hypothesized that the PB2-E712D mutation increases viral polymerase fidelity by maintaining the inserted Venus gene during passage. To test this hypothesis, we generated WT-PR8 and PR8-PB2-E712D, which differs from WT-PR8 only by having an aspartic acid at position 712 of PB2, by reverse genetics and compared their mutation rates. Here, we used a virus that did not contain the Venus gene to make it easier to measure mutation rates. PR8-PB1-V43I, a reported high-fidelity mutant virus (Cheung et al., 2014; Naito et al., 2015), and PR8-PB1-T123A, a reported low-fidelity mutant virus (Pauly et al., 2017), were also generated by reverse genetics and used as controls. To estimate the mutation rate, these viruses were passaged in MDCK cells at an MOI of 0.001 and subjected to large-scale genome-wide sequencing. The sequencing data for the five-passage virus was compared with that for the virus before passage. The number of nucleotide changes in the five-passage virus that were not present before passage was counted. The number of mutations introduced during the five passages is shown by segment in Figure 29A. The number of mutations per nucleotide was also calculated for normalization, and the average value for all eight segments was compared (Figure 29B). The high-fidelity control, PR8-PB1-V43I, showed fewer mutations than WT-PR8, while the low-fidelity control, PR8-PB1-T123A, showed more mutations than WT-PR8. PR8-PB1-V43I showed fewer mutations than WT-PR8, but the difference between PR8-PB1-V43I and WT-PR8 was small. Because a previous report suggested that PB1-V43I did not alter the mutation rate (Pauly et al., 2017), the effect of PB1-V43I on the mutation rate may have depended on the virus strain or experimental conditions. Moreover, there was no clear difference in the number of mutations between WT-PR8 and PR8-PB2-E712D.Although we cannot exclude the possibility that the PB2-E712D mutation affects viral polymerase fidelity, its effect is unlikely to be large enough to cause a significant difference in Venus stability. Therefore, this result suggests that the stability of the Venus gene is not affected by the fidelity of the viral polymerase.

[0189] Transcription / replication of the modified RNA segment is impaired in WT-Venus-PR8. Several reports suggest that recombinant viruses containing foreign gene insertions in their NS segments may grow more efficiently in interferon (IFN)-deficient Vero cells than in IFN-competent cells such as MDCK cells (Kittel et al., 2004; Ferko et al., 2001; Kuznetsova et al., 2014). Therefore, we quantified the expression level of IFN-β in virus-infected cells using quantitative real-time PCR. MDCK cells were infected with WT-Venus-PR8 or Venus-PR8-PB2-E712D at an MOI of 1 or mock-infected with medium alone, and the relative expression level of IFN-β in infected cells was quantified 9 hours after infection. WT-Venus-PR8 induced higher IFN-β expression levels than Venus-PR8-PB2-E712D (Figure 30A). This result suggests that WT-Venus-PR8 does not efficiently inhibit IFN-β expression. Given that NS1 plays a major role in suppressing IFN expression and IFN-mediated antiviral responses in the host (Garcia-Sastre et al., 1998; Opitz et al., 2007), we quantified NS vRNA in infected cells using influenza virus strand-specific real-time PCR (Kawakami et al., 2011; Kupke et al., 2018). Although the amount of NS vRNA in WT-Venus-PR8-infected cells was 90% lower than that in Venus-PR8-PB2-E712D-infected cells (Figure 30B), there was no significant difference in their NS vRNA expression levels (Figure 30C). The NS / NP vRNA ratio in WT-Venus-PR8-infected cells was 80% lower than that in Venus-PR8-PB2-E712D-infected cells (Figure 30D), suggesting that transcription / replication of the NS segment is specifically impaired in WT-Venus-PR8.All sequences of the plasmids used to generate viruses by reverse genetics were confirmed before use, and the NS segments of WT-Venus-PR8 and Venus-PR8-PB2-E712D were derived from the same NS-Venus plasmid. Therefore, it is unlikely that WT-Venus-PR8 or Venus-PR8-PB2-E712D have a mutated promoter sequence in their NS segments. Therefore, the difference in transcription / replication efficiency of the NS segments may be caused by PB2-E712D. Furthermore, the expression level of NS1 protein was confirmed by Western blotting (Figure 30E). Due to the reduced transcription / replication efficiency of the NS segments, the expression level of NS1 protein in WT-Venus-PR8-infected cells was much lower than that in Venus-PR8-PB2-E712D-infected cells. In contrast, the expression levels of NP were almost the same. The NS1 / NP ratio, quantified based on band intensity, was significantly reduced in WT-Venus-PR8-infected cells (Figure 30F). Therefore, low levels of NS1 expression likely lead to high expression of IFN-β in WT-Venus-PR8-infected cells. Furthermore, high expression of IFN-β leads to attenuation of WT-Venus-PR8, although other factors may be involved.

[0190] The inserted Venus gene is deleted by an internal deletion. To explore how the Venus gene deletion occurs, we sequenced the NS segment of WT-Venus-PR8, which had lost Venus expression after serial passage. Plaque assays were performed using three independently passaged WT-Venus-PR8 virus stocks, and the majority of plaques were found to be Venus-negative. More than five plaques from each stock were sequenced, revealing one or two deletion patterns in each virus stock. Large deletions occurred in the NS segment, resulting in the loss of most of the Venus sequence (Figure 31A). However, no specific patterns of deletions, such as the number of nucleotide deletions, the site(s) of the deletion(s), or the specific sequence(s) where the deletions occurred, were identified. The large deletion was hypothesized to result from an internal deletion caused by polymerase jumping, a known mechanism for defective interfering viral RNA production (Davis et al., 1980; Jennings et al., 1983), or genetic recombination, which plays a role in RNA virus adaptation through viral genome rearrangement (Xiao et al., 2016; Simon et al., 2011; Mitanul et al., 2000; Khatchikian et al., 1989). Synonymous mutations were introduced into the 3' or 5' region of the NS segment of WT-Venus-PR8 (Figure 31B). MDCK cells were then infected with each of these mutant viruses at an MOI of 0.001 or 5, and the supernatants were collected 2 days or 8 hours postinfection, respectively. The supernatants were then incubated with MDCK cells. Venus-negative plaques were picked and amplified in MDCK cells. We then determined the NS segment sequence in viruses that had lost Venus expression. We found that the truncated NS segments had synonymous mutations on only one side (Fig. 31C). No viruses were found with NS segments that had synonymous mutations on both sides or no synonymous mutations in both low- and high-MOI coinfections. This result indicates that the large deletions in the NS segments resulted from internal deletions in each NS segment and not from genetic recombination between NS segments.

[0191] Additional mutations stabilize the Venus gene. To further understand the mechanism of Venus deletion and stabilization, we attempted additional mutations in the polymerase complex that stabilize the Venus gene. MDCK cells were infected with WT-Venus-PR8 at an MOI of 0.001, and then Venus-positive plaques were picked and amplified in MDCK cells. After serial passage and plaque purification of the Venus-positive virus, mutants that stably expressed enhanced Venus fluorescence were obtained. Sequence analysis showed that mutations were introduced into the polymerase genes PB2, PB1, and PA of each mutant (Figure 32A). PA-180 and PA-200, like PB2-712, are located on the surface of the polymerase complex, whereas PB2-540, PB1-149, and PB1-684 are located inside the complex. PA-180 and PA-200 are located within the endonuclease domain, PB1-149 and PB1-684 are located near the exit site of the RNA template, and PB2-540 is located near the exit site of the newly synthesized RNA product (Figure 32B). However, the function of the region around PB2-712 remains unclear (Reich et al., 2014; Pflug et al., 2017; Gerlach et al., 2015). To determine whether these mutations contribute to Venus gene stabilization, we generated mutant viruses containing each mutation using reverse genetics and measured the Venus retention ratio after four passages in MDCK cells (Figure 32C). The mutant viruses showed enhanced Venus stability compared to WT-Venus-PR8, indicating that these amino acids play a role in Venus gene stabilization. Considering that PB2-540, PB1-149, and PB1-684 are located near the polymerase internal tunnel through which the template and product pass during the transcription / replication reaction (Reich et al., 2014; Pflug et al., 2017; Gerlach et al., 2015), these amino acids may affect the binding stability of the RNA template, product, and polymerase complex, although further analysis is needed to clarify how these amino acids contribute to the stability of the Venus gene.When we examined whether these mutations were found in previously isolated influenza A viruses in the Influenza Research Database (Figure 32D), we found that these amino acids were extremely rare, suggesting that they are not evolutionarily advantageous.

[0192] Consideration Recombinant influenza viruses expressing foreign genes are useful tools, but long insertions in the viral genome are often unstable, resulting in attenuation of the recombinant virus. We previously found that amino acids in the influenza virus polymerase complex play an important role in stabilizing foreign gene insertions. The Venus gene inserted into the NS segment was stabilized by PB2-E712D in H1N1 viruses (Fukuyama et al., 2015; Katsura et al., 2016) and PB2-V25A and PA-R443K in H5N1 viruses (Zhao et al., 2016). However, the mechanism by which these amino acids contribute to this stabilization remained unclear. In this study, we explored the mechanism of PB2-E712D-induced stabilization of the Venus gene inserted into the NS segment of H1N1 viruses. We found that the transcription / replication efficiency of the modified segment was significantly reduced in WT-Venus-PR8 compared to Venus-PR8-PB2-E712D. This finding suggests that the PB2-E712D mutation stabilizes the inserted foreign gene due to enhanced transcription / replication efficiency of the modified RNA segment. In contrast, the transcription / replication efficiency of segments without additional sequences remains unchanged regardless of the presence or absence of the PB2-E712D mutation. Furthermore, polymerase activity is reduced, not enhanced, by the PB2-E712D mutation in minireplicon assays (Katsura et al., 2016). These results indicate that the altered transcription / replication efficiency caused by PB2-E712D is specific to the modified RNA segment. Insertion of the foreign gene appears to impair transcription / replication of the modified segment, and the polymerase overcomes this obstacle in the presence of the PB2-E712D mutation.

[0193] In WT-Venus-PR8, where the Venus gene is inserted into the NS segment, the transcription / replication efficiency of this segment is significantly reduced. As a result, expression of the NS1 protein is also reduced. Because NS1 plays a role in inhibiting IFN-mediated antiviral responses (Garcia-Sastre et al., 2008; Optiz et al., 2007), WT-Venus-PR8 is unable to efficiently inhibit IFN-β expression, which may result in viral attenuation. The viral titer of WT-Venus-PR8 increases during serial passage in MDCK cells as the virus loses Venus expression (Figures 28B and 28C), suggesting that mutant WT-Venus-PR8 lacking the Venus gene grows more efficiently than the original WT-Venus-PR8. Therefore, the rapid loss of Venus expression in WT-Venus-PR8 may result from the selection of mutants lacking the Venus gene during serial passage. Venus-PR8-PB2-E712D restores the transcription / replication efficiency of the NS segment, resulting in efficient viral replication. Therefore, Venus-expressing viruses are not purged by selection pressure in the presence of the PB2-E712D mutation, allowing Venus-PR8-PB2-E712D to stably maintain the inserted Venus gene.

[0194] How does transcription / replication efficiency decrease specifically on modified RNA segments? And how is it enhanced by the PB2-E712D mutation? The answer to these questions may lie in the RNA secondary structure and the binding affinity between the polymerase complex and the RNA template. The insertion of a foreign gene must alter the RNA secondary structure, and transcription / replication by the viral polymerase complex may be negatively affected by this unusual RNA secondary structure. Although we do not know conclusively how the PB2-E712D mutation overcomes the transcription / replication obstacle, one possible explanation is that it increases the binding affinity between the polymerase complex and the RNA template.

[0195] Sequence analysis of WT-Venus-PR8, which lacks Venus expression, combined with co-infection experiments (Figures 29B and 29C) suggested that the inserted sequence was deleted due to an internal deletion. Internal deletions often occur during the influenza virus replication cycle, regardless of the presence of foreign gene insertions, and have been reported to play a role in viral adaptation (Lui et al., 1993; Lui et al., 1985; Yang et al., 1987) and the generation of defective interfering viral RNA (Davis et al., 1980; Jennings et al., 1983). Internal deletions are thought to be caused by dissociation of the polymerase complex from the RNA template during transcription / replication (Jennings et al., 1983; Lazarini et al., 2001; Dimmock et al., 2014; Lopez et al., 2014). Amino acid mutations in the polymerase complex affect the frequency of internal deletions (Fodor et al., 2003; Vasilijevic et al., 2017; Slaine et al., 2018; Te Velthuis et al., 2018). PB2-E712D may also be involved in the appearance of internal deletions. Therefore, the stabilization of the Venus gene in Venus-PR8-PB2-E712D may be caused not only by enhanced transcription / replication in the modified segment but also by a reduced frequency of internal deletions.

[0196] We identified additional mutations in the influenza virus polymerase complex that stabilize the inserted Venus gene, which may help us further understand the stabilization mechanism based on the location of these mutations in the viral polymerase complex. Some of the identified amino acids are located near the polymerase internal tunnel, which is close to the RNA template or newly synthesized RNA product during the transcription / replication reaction (Reich et al., 2014; Pflug et al., 2017; Gerlach et al., 2015). These amino acids may directly affect the binding affinity between the polymerase complex, template, and product. A previous report showing that PB2 amino acids located in the template exit channel are involved in the formation of short aberrant RNAs (Te Velthuis et al., 2018) supports the possibility that amino acids near the polymerase internal tunnel affect the binding affinity between the polymerase complex, template, and product. However, PA-180 and PA-200, located in the endonuclease domain, are not near the polymerase internal tunnel, which is also true for PB2-712. Therefore, these amino acids may indirectly affect binding affinity, or there may be other mechanisms involved in stabilizing the Venus gene. These mutations could be used to establish recombinant influenza viruses expressing foreign genes. However, because PB2-V25A, which stabilizes the Venus gene in Venus-H5N1, had a negative effect on viral replication in Venus-PR8 and did not cause Venus stabilization (our unpublished data), these amino acids may not necessarily cause the stabilization of foreign genes in all influenza virus strains.

[0197] Although the identified amino acids appear to enhance the genetic stability of the viral genome, they were rarely found in virus isolates (Figure 32D). Mutations that support the maintenance of inserted sequences may not be evolutionarily advantageous for the virus. The insertion of additional sequences into the viral genome is often deleterious to viral replication. These mutations are likely rare in virus populations, avoiding the accumulation of deleterious insertions. Viruses can purge deleterious insertions by reducing the transcription / replication efficiency of RNA segments containing insertions that form abnormal secondary structures. In conclusion, although the amino acid mutations we identified in this study are useful for generating recombinant viruses, they are unlikely to be advantageous to the virus in the long run.

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[0199] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described with reference to certain preferred embodiments thereof, but it will be apparent to those skilled in the art that many details have been set forth for purposes of illustration and that the invention is susceptible to further embodiments and that the specific details herein may be varied significantly without departing from the basic principles of the invention. Some aspects of the invention are described below. 1. An isolated recombinant influenza virus having a PA viral segment, a PB1 viral segment, a PB2 viral segment, an NP viral segment, an NS viral segment, an M viral segment, an NA viral segment, and an HA viral segment, wherein at least one of said viral segments is a PB2 viral segment encoding a PB2 having a residue at position 540 that is not asparagine, a PA viral segment encoding a PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or an asparagine. an isolated recombinant influenza virus, wherein the PB1 viral segment encodes a PB1 having a residue at position 685 that is not glutamic acid, or any combination thereof, wherein the recombinant influenza virus has enhanced stability and / or enhanced replication compared to a corresponding recombinant influenza virus having a residue at position 540 in PB2 that is asparagine, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid, or a residue at position 685 in PB1 that is aspartic acid. 2. An isolated recombinant influenza virus having a PA viral segment, a PB1 viral segment, a PB2 viral segment, an NP viral segment, an NS viral segment, an M viral segment, an NA viral segment, and an HA viral segment, wherein at least one of the viral segments is a PB2 viral segment encoding PB2 having a residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, and at least one of the other viral segments is a PA viral segment encoding PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a residue at position 149 that is not valine or a residue at position 684 that is not glutamic acid. 1. An isolated recombinant influenza virus comprising a PB1 viral segment encoding PB1 having a residue at position 540 in PB2 that is asparagine, a residue at position 712 in PB2 that is glutamic acid, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid, or a residue at position 685 in PB1 that is aspartic acid, or any combination thereof, wherein the recombinant influenza virus has enhanced stability and / or enhanced replication compared to a corresponding recombinant influenza virus having a residue at position 540 in PB2 that is asparagine, a residue at position 712 in PB2 that is glutamic acid, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid, or a residue at position 685 in PB1 that is aspartic acid. 3. An isolated recombinant influenza virus having a PA viral segment, a PB1 viral segment, a PB2 viral segment, an NP viral segment, an NS viral segment, an M viral segment, an NA viral segment, and an HA viral segment, wherein the recombinant virus does not comprise a PB2 viral segment encoding a PB2 having a residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, a PA viral segment encoding a PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid. 1. An isolated recombinant influenza virus having two or more viral segments, including a PB1 viral segment encoding a PB1 having a nucleotide sequence similar to that of position 540 of PB2, a PB1 viral segment encoding a PB1 having a nucleotide sequence similar to that of position 712 ... 4. The virus according to any one of items 1 to 3, wherein the residue at position 540 of PB2 is K, R, D, E, Q or H, the residue at position 712 of PB2 is D, N, Q, S, H, T, Y or C, the residue at position 180 in PA is R, K, D, E, N or H, the residue at position 200 in PA is A, I, L, C, S, M, F, P, G or V, the residue at position 149 in PB1 is A, T, I, L, C, S, M, F, P or G, the residue at position 684 is D, Q, S, H, T, Y, C, K, R or N, or the residue at position 685 in PB1 is E, N, R, H, K, S, T, Y, C or Q. 5. The virus according to any one of items 1 to 3, wherein the residue at position 540 of PB2 is K, R, H, D, S, H, T, Y or C, the residue at position 712 of PB2 is D, K, H, R, Q or N, the residue at position 180 in PA is R, K, D, N, S, H, T, Y or H, the residue at position 200 in PA is A, I, L, G, S, M or V, the residue at position 149 in PB1 is A, T, I, L, S, M or G, the residue at position 684 is D, Q, H, L, R or N, or the residue at position 685 in PB1 is E, N, R, H, K or Q. 6. The virus according to any one of items 1 to 3, wherein the residue at position 540 of PB2 is K, R or H, the residue at position 712 of PB2 is D or N, the residue at position 180 in PA is R, K or H, the residue at position 200 in PA is A, I, L, G or V, the residue at position 149 in PB1 is A, T, I, L or G, the residue at position 684 is D or N, or the residue at position 685 in PB1 is E or Q. 7. The virus of item 1 or 2, wherein PB2 has a residue at position 540 that is not asparagine, PA has a residue at position 180 that is not glutamine and a residue at position 200 that is not threonine, and PB1 has a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid. 8. The virus of item 1 or 2, wherein PB2 has a residue at position 540 that is not asparagine, PA has a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, and PB1 has a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid. 9. The virus of item 2 or 3, wherein PB2 has a residue at position 540 that is not asparagine or a residue at position 712 that is not aspartic acid, PA has a residue at position 180 that is not glutamine and a residue at position 200 that is not threonine, and PB1 has a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid. 10. The virus according to item 2 or 3, wherein PB2 has a residue at position 540 that is not asparagine and a residue at position 712 that is not aspartic acid, PA has a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, and PB1 has a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid. 11. The virus according to any one of items 1 to 10, wherein the PA further comprises a residue at position 443 that is not arginine, the PB1 further comprises a residue at position 737 that is not lysine, the PB2 further comprises a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid, the NS viral segment encodes an NS1 having a residue at position 167 that is not proline, and the HA viral segment encodes an HA having a residue at position 380 that is not threonine, or any combination thereof. 12. The virus of item 11, wherein the residue at position 443 of PA is K or H, the residue at position 737 of PB1 is H or R, the residue at position 25 of PB2 is A, L, T, I or G, the residue at position 712 of PB2 is D, and the residue at position 167 of NS1 is S, C, M, A, L, I, G or T, or any combination thereof. 13. The recombinant virus according to any one of items 1 to 12, wherein at least one of the viral segments comprises a heterologous gene sequence encoding a gene product. 14. The recombinant virus of item 13, wherein the heterologous sequence is within the NS viral segment, the M viral segment, the NP viral segment, the PA viral segment, the PB1 viral segment, or the PB2 viral segment. 15. The recombinant virus of item 13, wherein the heterologous sequence is 5' or 3' to the PA coding sequence in the PA viral segment and 5' or 3' to the PB1 coding sequence in the PB1 viral segment. 16. The recombinant virus of item 13, wherein the heterologous sequence is 5' or 3' to the PB2 coding sequence in the PB2 viral segment. 17. The recombinant virus of item 13, wherein the heterologous sequence is 5' or 3' to the NS1 coding sequence in the NS viral segment. 18. The recombinant virus according to any one of items 1 to 17, comprising an additional viral segment comprising a heterologous gene sequence encoding a gene product. 19. The recombinant virus according to item 18, wherein the additional viral segment is a NS viral segment, an M viral segment, an NP viral segment, a PA viral segment, a PB1 viral segment, or a PB2 viral segment. 20. The virus according to any one of items 1 to 19, having an HA that is H1, H2, H3, H5, H7, H9 or H10. 21. The virus according to any one of items 1 to 20, which is an influenza A virus. 22. A vaccine comprising the isolated recombinant virus according to any one of items 1 to 21. 23. The vaccine of item 22, wherein the virus encodes a non-influenza microbial protein. 24. The vaccine of item 22, wherein the virus encodes a heterologous influenza protein. 25. The vaccine of item 22, wherein the virus encodes a cancer-associated antigen. 26.a) A vector for vRNA production comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to influenza virus NS cDNA linked to a transcription termination sequence, wherein the PB1 DNA, the PB2 DNA, or the PA DNA in the vector for vRNA production the vector for producing vRNA, wherein the DNA encodes at least one of a PB2 viral segment encoding PB2 having a residue at position 540 that is not asparagine, a PA viral segment encoding PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or a PB1 viral segment encoding PB1 having a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid, or a combination thereof; and optionally b) a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NS2 A plurality of influenza virus vectors for preparing a reassortant, comprising: 27. The vector according to Item 26, wherein the PB1 DNA, the PB2 DNA, the PA DNA, the NP DNA, the NS DNA, and the M DNA in the vector for vRNA production have sequences corresponding to those encoding polypeptides having at least 95% amino acid sequence identity to the corresponding polypeptides encoded by SEQ ID NOs: 1 to 6 or 10 to 15. 28. The vector according to item 26 or 27, wherein the residue at position 540 in PB2 is K, R or H, the residue at position 180 in PA is R, K or H, the residue at position 200 in PA is A, I, L, G or V, the residue at position 149 in PB1 is A, T, I, L or G, the residue at position 684 is D or N, or the residue at position 685 in PB1 is E or Q. 29. The vector according to any one of items 26 to 28, wherein at least one of the viral segments comprises a heterologous gene sequence encoding a gene product. 30. The vector according to any one of items 26 to 29, comprising a further vector having a viral segment comprising a heterologous gene sequence encoding a gene product. 31. To the cells, an amount effective to produce infectious influenza virus, A vector for producing vRNA comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector for producing vRNA comprising a promoter operably linked to influenza virus NS DNA linked to a transcription termination sequence, wherein the PB1 DNA, the PB2 DNA, or the PA DNA in the vector for vRNA production is i) PB2 having a residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, and PA having a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or PB1 having a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid, or a residue at position 685 that is not aspartic acid, or at least one of any combination thereof; or ii) PB2 having a residue at position 540 that is not asparagine, a PA having a residue at position 180 that is not glutamine, or a residue at position 200 that is not threonine; or iii) PB2 having a residue at position 540 that is not asparagine or a residue at position 712 that is not glutamic acid, a residue at position 180 that is not glutamine or a residue at position 200 that is not threonine, or PB1 having a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid, or any combination thereof. and optionally, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NP, and optionally a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus HA, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NA, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M1, a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus M2, or a vector for producing mRNA comprising a promoter operably linked to a DNA segment encoding influenza virus NS2 10. A method for preparing an influenza virus, comprising contacting 32. The method according to item 31, wherein the cells are avian or mammalian cells. 33. The method according to item 32, wherein the cells are Vero cells, human cells or MDCK cells. 34. The method according to any one of items 31 to 33, wherein the PB1 DNA, the PB2 DNA, the PA DNA, the NP DNA, the NS DNA, and the M DNA in the vector for vRNA production have sequences corresponding to those encoding polypeptides having at least 95% amino acid sequence identity to the corresponding polypeptides encoded by SEQ ID NOs: 1 to 6 or 10 to 15. 35. The method of any one of items 31 to 34, wherein the residue at position 540 of PB2 is K, R or H, the residue at position 712 of PB2 is D or N, the residue at position 180 in PA is R, K or H, the residue at position 200 in PA is A, I, L, G or V, the residue at position 149 in PB1 is A, T, I, L or G, the residue at position 684 is D or N, or the residue at position 685 in PB1 is E or Q. 36. The method according to any one of items 31 to 35, wherein the influenza virus comprises a heterologous gene sequence encoding a gene product. 37. The method of claim 36, wherein the heterologous sequence is within the NS viral segment, the M viral segment, the NP viral segment, the PA viral segment, the PB1 viral segment, or the PB2 viral segment. 38. The method of item 36, wherein the heterologous sequence is 5' or 3' to the PA coding sequence in the PA viral segment and 5' or 3' to the PB1 coding sequence in the PB1 viral segment. 39. The method of item 36, wherein the heterologous sequence is 5' or 3' to the PB2 coding sequence in the PB2 viral segment. 40. The method of item 37, wherein the heterologous sequence is 5' or 3' to the NS1 coding sequence in the NS viral segment. 41. The method according to any one of items 31 to 40, comprising an additional viral segment comprising a heterologous gene sequence encoding a gene product. 42. The method of item 41, wherein the additional viral segment is a NS viral segment, an M viral segment, an NP viral segment, a PA viral segment, a PB1 viral segment, or a PB2 viral segment.

Claims

1. 1. An isolated reassortant influenza virus having a PA viral segment, a PB1 viral segment, a PB2 viral segment, an NP viral segment, an NS viral segment, an M viral segment, an NA viral segment, and an HA viral segment; i) at least one of the viral segments is a PB2 viral segment encoding PB2 having a residue at position 540 that is K, a PA viral segment encoding PA having a residue at position 180 that is R or a residue at position 200 that is A, or a PB1 viral segment encoding PB1 having a residue at position 149 that is A, a residue at position 684 that is D, or a residue at position 685 that is E, or any combination thereof, and the reassortant influenza virus has enhanced genetic stability and / or enhanced replication compared to a corresponding influenza virus having a residue at position 540 in PB2 that is asparagine, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid, or a residue at position 685 in PB1 that is aspartic acid; ii) at least one of the viral segments is a PB2 viral segment encoding PB2 having a residue at position 540 that is K, and at least one of the other viral segments is a PA viral segment encoding PA having a residue at position 180 that is R or a residue at position 200 that is A, or a PB1 viral segment encoding PB1 having a residue at position 149 that is A, a residue at position 684 that is D, or a residue at position 685 that is E, or any combination thereof, and the reassortant influenza virus has enhanced genetic stability and / or enhanced replication compared to a corresponding influenza virus having a residue at position 540 in PB2 that is asparagine, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid, or a residue at position 685 in PB1 that is aspartic acid; or iii) the reassortant influenza virus has two or more viral segments, including a PB2 viral segment encoding PB2 having a residue at position 540 that is K or a residue at position 712 that is D, a PA viral segment encoding PA having a residue at position 180 that is R or a residue at position 200 that is A, or a PB1 viral segment encoding PB1 having a residue at position 149 that is A, a residue at position 684 that is D, or a residue at position 685 that is E, or any combination thereof, 1. An isolated reassortant influenza virus having enhanced genetic stability and / or enhanced replication compared to a corresponding influenza virus having a residue at position 540 in PB2 that is asparagine, a residue at position 712 in PB2 that is glutamic acid, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glutamic acid, or a residue at position 685 in PB1 that is aspartic acid.

2. The virus of claim 1, wherein the PA further comprises a residue at position 443 that is not arginine, the PB1 further comprises a residue at position 737 that is not lysine, the PB2 further comprises a residue at position 25 that is not valine, the NS viral segment encodes an NS1 having a residue at position 167 that is not proline, and the HA viral segment encodes an HA having a residue at position 380 that is not threonine, or any combination thereof.

3. The virus of claim 2, wherein the residue at position 443 of PA is K or H, the residue at position 737 of PB1 is H or R, the residue at position 25 of PB2 is A, L, T, I or G, the residue at position 712 of PB2 is D, and the residue at position 167 of NS1 is S, C, M, A, L, I, G or T, or any combination thereof.

4. 4. The virus of claim 1, wherein at least one of the viral segments comprises a heterologous gene sequence encoding a gene product.

5. 5. The virus of claim 4, wherein the heterologous gene sequence is present within the NS viral segment, the M viral segment, the NP viral segment, the PA viral segment, the PB1 viral segment, or the PB2 viral segment.

6. The virus of claim 5, wherein the heterologous gene sequence is 5' or 3' to the PA coding sequence in the PA viral segment, 5' or 3' to the PB1 coding sequence in the PB1 viral segment, 5' or 3' to the PB2 coding sequence in the PB2 viral segment, or 5' or 3' to the NS1 coding sequence in the NS viral segment.

7. 7. The virus of claim 1, further comprising a viral segment comprising a heterologous gene sequence encoding a gene product.

8. 8. The virus of claim 7, wherein the additional viral segment is an NS viral segment, an M viral segment, an NP viral segment, a PA viral segment, a PB1 viral segment, or a PB2 viral segment.

9. 9. A vaccine comprising the isolated virus of any one of claims 1 to 8, wherein the virus optionally encodes a non-influenza microbial protein, a heterologous influenza protein, or a cancer-associated antigen.

10. A vector for producing vRNA comprising a promoter operably linked to influenza virus PA DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus PB1 DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus PB2 DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus HA DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus NP DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus NA DNA linked to a transcription termination sequence, a vector for producing vRNA comprising a promoter operably linked to influenza virus M DNA linked to a transcription termination sequence, and a vector for producing vRNA comprising a promoter operably linked to influenza virus NS cDNA linked to a transcription termination sequence, wherein the PB1 DNA, the PB2 DNA, or the PA DNA in the vector for vRNA production the vector for vRNA production, wherein the DNA encodes at least one of a PB2 viral segment encoding PB2 having a residue at position 540 that is K or a residue at position 712 that is D, a PA viral segment encoding PA having a residue at position 180 that is R or a residue at position 200 that is A, or a PB1 viral segment encoding PB1 having a residue at position 200 that is A, a residue at position 684 that is D, or a residue at position 685 that is E, or a combination thereof.

9. A plurality of influenza virus vectors for preparing the virus of any one of claims 1 to 8, comprising:

11. 11. The vector of claim 10, wherein the PB1 DNA, the PB2 DNA, the PA DNA, the NP DNA, the NS DNA, and the M DNA in the vector for vRNA production have sequences corresponding to those encoding polypeptides having at least 95% amino acid sequence identity to the corresponding PB1, PB2, PA, NP, NS, and M polypeptides encoded by one of SEQ ID NOs: 1-6 or 10-15.

12. 12. The vector of claim 10 or 11, wherein at least one of the viral segments comprises a heterologous gene sequence encoding a gene product.

13. 13. The vector of claim 12, wherein the heterologous gene sequence is present within an NS viral segment, an M viral segment, an NP viral segment, a PA viral segment, a PB1 viral segment, or a PB2 viral segment.

14. The vector of claim 13, wherein the heterologous gene sequence is 5' or 3' to the PA coding sequence in the PA viral segment, 5' or 3' to the PB1 coding sequence in the PB1 viral segment, 5' or 3' to the PB2 coding sequence in the PB2 viral segment, or 5' or 3' to the NS1 coding sequence in the NS viral segment.

15. 15. The vector of any one of claims 10 to 14, comprising a further vector comprising a viral segment comprising a heterologous gene sequence encoding a gene product.

16. 16. The vector of claim 15, wherein the additional viral segment is an NS viral segment, an M viral segment, an NP viral segment, a PA viral segment, a PB1 viral segment, or a PB2 viral segment.

17. 20. An in vitro method for preparing influenza virus, comprising contacting a cell with a vector of any one of claims 10 to 16 in an amount effective to produce infectious influenza virus.

18. 18. The method of claim 17, wherein the cell is an avian cell or a mammalian cell that is a Vero cell, a human cell, or an MDCK cell.

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