Rosenbergiella bacteria and use thereof in prevention and control of arboviruses

Abstract

Rosenbergiella bacteria and a use thereof in the prevention and control of arboviruses. A strain, the preservation number thereof being CGMCC No. 29922. Any of the following uses (i) to (iii) of Rosenbergiella sp. and / or a product thereof: (i) a use in the preparation of a product for controlling the propagation of arboviruses; (ii) a use in the preparation of a product for inhibiting the susceptibility of a propagation medium to the arboviruses; and (iii) a use of in the prevention and control of the arboviruses. Rosenbergiella sp. and glucose dehydrogenase produced thereby can efficiently and widely inhibit the replication and propagation of various mosquito-borne Flaviviruses. Therefore, Rosenbergiella sp. has good application potentials in terms of the prevention and control of Flaviviruses and the like.

Description

Rosenbergiella bacteria and their application in the prevention and control of arboviruses

[0001] Priority and related applications

[0002] The present invention claims priority to Chinese patent application No. 202410282184.9, entitled “Rosenbergiella bacteria and their application in insect-borne virus prevention and control”, filed on March 12, 2024. The entire contents of this application, including the appendix, are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of microbial technology, in particular to Rosenbergiella bacteria and applications thereof in the prevention and control of insect-borne viruses, and in particular to applications of a novel bacterium, Rosenbergiella sp., and its product, glucose dehydrogenase, in the prevention and control of flaviviruses. Background Art

[0004] Arboviruses, such as mosquito-borne flaviviruses, are causative agents of serious human diseases, including hemorrhagic fever, diphasic fever, arthritis, and neurotropic disorders, resulting in hundreds of millions of infections and numerous deaths annually. Dengue virus is the most prevalent mosquito-borne flavivirus, having emerged or re-emerged in over 100 countries worldwide and potentially causing 390 million infections annually. It primarily occurs in subtropical and tropical regions, including Yunnan and Guangdong provinces in my country. From 2015 to 2017, Zika virus epidemics in the Pacific Islands and the Americas included over 223,000 confirmed cases; these epidemics triggered a Public Health Emergency of International Concern in 2016. Severe neurological complications, such as Guillain-Barré syndrome in adults and microcephaly in newborns, have been associated with Zika virus infection. Accelerated outbreaks of these mosquito-borne flavivirus diseases have become a major public health concern worldwide. These flaviviruses maintain their life cycle between vertebrate hosts and mosquitoes. Mosquitoes acquire infectious virus particles from infected hosts through a blood meal. The acquired viral particles enter the mosquito gut epithelial cells, subsequently infect the hemocoel tissue, and migrate to the salivary glands, allowing the infected mosquito to transmit the virus in the next bite. Therefore, manipulating the interaction between the mosquito gut and viruses may be a viable strategy to intervene and protect insects from flavivirus infection.

[0005] The mosquito gut harbors a rich microbial community. Symbiotic microbiota maintain a balance with the mosquito and play a complex role in determining the mosquito's ability to transmit viruses. For example, a Talaromyces fungus in the gut of Aedes aegypti increases the mosquito's tolerance to dengue virus infection by regulating digestive enzyme and trypsin activity in the mosquito gut. Similarly, Serratia marcescens promotes dengue and Zika virus infection by secreting bacterial effectors called deglycosylated membrane-bound mucins onto the mosquito gut epithelium. Conversely, Wolbachia, an endosymbiotic microbe, can also interrupt viral replication by modulating the mosquito's immune or physiological responses. The interaction between mosquitoes, their gut microbiota, and viruses has garnered significant attention in recent years, as modulation of the gut microbiota modulates the mosquito's ability to transmit pathogenic viruses. Therefore, manipulation of the mosquito gut microbiota could be a potential tool for controlling the spread of flavivirus diseases.

[0006] Therefore, new strains or microbial agents that can control the spread of flavivirus diseases by controlling the mosquito gut microbiota remain to be developed.

[0007] Summary of the Invention

[0008] Problems to be solved by the invention

[0009] In view of the lack of new strains or microbial preparations in the prior art for controlling the spread of flavivirus diseases by regulating the intestinal flora of mosquitoes, the present invention has discovered a new species of Rosenbergiella sp., which is effective in preventing and controlling flaviviruses.

[0010] Solutions for solving problems

[0011] [1]. A strain, whose deposit number is CGMCC No.29922.

[0012] [2] A composition comprising the strain described in [1] and / or its product;

[0013] Optionally, the composition is a microbial preparation;

[0014] Optionally, the composition is used to control arboviruses;

[0015] Optionally, the strain and / or its product is used as an active ingredient in the composition.

[0016] [3] Any of the following uses (i) to (iii) of Rosenbergiella sp. and / or its products:

[0017] (i) Use in the preparation of products for controlling the spread of arboviruses;

[0018] (ii) use in the preparation of a product for inhibiting the susceptibility of a vector to an arbovirus;

[0019] (iii) Application in the prevention and control of arboviruses.

[0020] [4] The application according to [3], wherein:

[0021] The Rosenbergiella sp. includes strains whose 16S rRNA gene sequence has at least 97% homology to the sequence shown in SEQ ID NO: 1;

[0022] Preferably, the Rosenbergiella sp. includes a strain whose 16S rRNA gene sequence comprises the sequence shown in SEQ ID NO: 1;

[0023] More preferably, the Rosenbergiella sp. includes Rosenbergiella sp. YN46 strain, whose deposit number is CGMCC No.29922.

[0024] [5] The use according to [3] or [4], wherein the product of Rosenbergiella sp. comprises glucose dehydrogenase;

[0025] Optionally, the glucose dehydrogenase comprises one or more of the following sequences:

[0026] 1) the amino acid sequence shown in SEQ ID NO: 2;

[0027] 2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, and which retains the activity of the amino acid sequence of SEQ ID NO: 1;

[0028] 3) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted into the amino acid sequence shown in SEQ ID NO: 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 2;

[0029] 4) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 2 under stringent conditions, and the amino acid sequence retains the activity of the amino acid sequence as shown in SEQ ID NO: 2, wherein the stringent conditions are moderate stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0030] [6] The use according to any one of [3] to [5], wherein the arbovirus comprises one or more of the families Bunyaviridae, Flaviviridae, Reoviridae and Togaviridae;

[0031] Preferably, the arbovirus comprises one or more of the genus Flavivirus, genus Alphavirus and genus Orthobunyavirus.

[0032] [7] The use according to any one of [3] to [6], wherein the arbovirus is a flavivirus;

[0033] Optionally, the arbovirus comprises dengue virus and / or Zika virus.

[0034] [8] The use according to any one of [3] to [7], wherein the vectors of the arbovirus include one or more of mosquitoes, ticks or sand flies;

[0035] Preferably, the vector of the insect-borne virus is mosquito.

[0036] More preferably, the mosquito comprises one or more of Culex, Culiseta, Anopheles and Aedes;

[0037] Optionally, the mosquito is an Aedes mosquito, including one or more of Aedes aegypti, Aedes albopictus, Aedes polynesiensis, Aedes australis, Aedes cinereus, Aedes rusticus and Aedes vexans.

[0038] [9] The use according to any one of [3] to [8], wherein the product comprises one or more of a reagent or a kit; optionally, the reagent is a microbial preparation.

[0039]

[0010] Any one of the following methods (a) to (c):

[0040] (a) A method for controlling the transmission of arboviruses, said method comprising applying Rosenbergiella sp. and / or its products, or the composition as described in [2] to the transmission vector;

[0041] (b) a method for inhibiting the susceptibility of a vector to an arbovirus, said method comprising administering Rosenbergiella sp. and / or a product thereof, or a composition as described in [2] to the vector;

[0042] (c) A method for controlling insect-borne viruses, comprising spraying Rosenbergiella sp. and / or its products onto an area to be controlled, or spraying the composition as described in [2] onto an area to be controlled.

[0043] Effects of the Invention

[0044] This study reports for the first time a new species of the genus Rosenbergiella, which exhibits excellent control effects against arboviruses, such as mosquito-borne flaviviruses (Zika and dengue). Furthermore, the glucose dehydrogenase produced by this strain can also inhibit flavivirus infection in mosquitoes. The discovery of this strain enriches the resource of beneficial microorganisms. Its stable, highly effective, and environmentally friendly inhibition of flaviviruses suggests promising applications in the prevention and control of mosquito-borne flavivirus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a schematic diagram of the morphology of Rosenbergiella_YN46 under a scanning electron microscope.

[0046] Figure 2 is a schematic diagram of the 16S rRNA phylogenetic tree of Rosenbergiella_YN46.

[0047] FIG3 is a schematic diagram of the growth curve of Rosenbergiella_YN46 in brain heart infusion liquid culture medium.

[0048] Figure 4 is a schematic diagram of the inhibitory effect of Rosenbergiella_YN46 on dengue virus infection in Aedes albopictus.

[0049] Figure 5 is a schematic diagram of the inhibitory effect of Rosenbergiella_YN46 on Zika virus infection in Aedes albopictus.

[0050] FIG6 is a schematic diagram showing the inhibitory effect of Rosenbergiella_YN46 on dengue virus infection in Aedes aegypti.

[0051] Figure 7 is a schematic diagram of the inhibitory effect of Rosenbergiella_YN46 on Zika virus infection in Aedes aegypti.

[0052] FIG8 is a schematic diagram showing the inhibitory effect of glucose dehydrogenase on dengue virus infection in Aedes albopictus.

[0053] Figure 9 is a schematic diagram of the inhibitory effect of glucose dehydrogenase on Zika virus infection in Aedes albopictus.

[0054] FIG10 is a schematic diagram showing the inhibitory effect of glucose dehydrogenase on dengue virus infection in Aedes aegypti.

[0055] Figure 11 is a schematic diagram of the inhibitory effect of glucose dehydrogenase on Zika virus infection in Aedes aegypti. DETAILED DESCRIPTION

[0056] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0057] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0058] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0059] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0060] References throughout this specification to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the particular elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0061] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0062] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0063] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0064] In this application, "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0065] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0066] As used herein, "hybridizable," "complementary," or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) comprises a nucleotide sequence that enables the nucleic acid to non-covalently bind (i.e., form Watson-Crick base pairs and / or G / U base pairs), "anneal," or "hybridize" with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to the complementary nucleic acid) under appropriate in vitro and / or in vivo temperature and solution ionic strength conditions. Standard Watson-Crick base pairing includes: adenine (A) pairs with thymidine (T), adenine (A) pairs with uracil (U), and guanine (G) pairs with cytosine (C). In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization between DNA molecules and RNA molecules (e.g., when a DNA target nucleic acid base pairs with a guide RNA): guanine (G) can also pair with uracil (U). For example, in the case of tRNA anticodons base pairing with codons in mRNA, G / U base pairing is at least partially responsible for the degeneracy of the genetic code.

[0067] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 of this reference; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0068] As used herein, "moderately stringent conditions," "medium-high stringency conditions," "high stringency conditions," or "very high stringency conditions" describe conditions for nucleic acid hybridization and washing. Guidance for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Both aqueous and nonaqueous methods are described in this document, and either method can be used. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions are in 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2×SSC, 0.1% SDS at at least 50°C (the wash temperature can be increased to 55°C for low stringency conditions); (2) moderate stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C, and preferably; (4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2×SSC, 1% SDS at 65°C.

[0069] Hybridization requires two nucleic acids to contain complementary sequences, but the mispairing between the bases is possible. The conditions suitable for hybridization between two nucleic acids depend on the length and the degree of complementarity of nucleic acid, and the length and the degree of complementarity of nucleic acid are variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the melting temperature (Tm) value of the hybrid with the nucleic acid of these sequences. For hybridization between nucleic acids with short chain complementarity (for example, 35 or less, 30 or less, 25 or less, 22 or less, 20 or less or 18 or less complementarity of nucleotides), the mispairing position may become important (see Sambrook et al., supra, 11.7-11.8). Usually, the length of hybridizable nucleic acids is 8 nucleotides or more (for example, 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more or 30 nucleotides or more). Temperature, wash solution salt concentration, and other conditions can be adjusted as desired depending on factors such as the length of the region of complementarity and the degree of complementarity.

[0070] In the present invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0071] In the present invention, the term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in proteins. For example, a group of amino acids with aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains consists of serine and threonine; a group of amino acids with amide-containing side chains consists of asparagine and glutamine; a group of amino acids with aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains consists of lysine, arginine, and histidine; a group of amino acids with acidic side chains consists of glutamic acid and aspartic acid; and a group of amino acids with sulfur-containing side chains consists of cysteine ​​and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.

[0072] In the present invention, polynucleotide or polypeptide and another polynucleotide or polypeptide have certain " sequence identity " percentage, this means that when comparing, the percentage of base or amino acid is identical, and when comparing two sequences, the base or amino acid is in the same relative position. Sequence identity can be determined in many different ways. In order to determine sequence identity, various convenient methods and computer programs (for example, BLAST, T-COFFEE, MUSCLE, MAFF T etc.) can be used to compare sequences, and the methods and computer programs can be obtained by the world wide web at the website including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / . See, for example, Altschul et al. (1990), J.Mol.Bioi.215:403-10.

[0073] The technical solution of the present invention is described in detail below.

[0074] <Rosenbergiella sp.>

[0075] In some aspects of the present invention, a new species of the genus Rosenbergiella, Rosenbergiella sp., is provided. The morphological characteristics of the Rosenbergiella sp. are as follows: Gram staining is negative, and single colonies are round, white, smooth, and opaque.

[0076] The YN46 strain from Rosenbergiella sp. was deposited with the General Microbiology Center of the China Culture Collection Administration on March 1, 2024, under the classification name Rosenbergiella sp.; the deposit number is CGMCC No. 29922; and the depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. In the present invention, the YN46 strain from Rosenbergiella sp. is also referred to as the Rosenbergiella sp. YN46 strain, or simply as Rosenbergiella_YN46 or YN46 strain.

[0077] In some alternative embodiments, Rosenbergiella sp. is not limited to the above deposited strains, but may also be strains that are substantially identical in nature to the above deposited strains. Strains that are substantially identical in nature refer to strains classified as Rosenbergiella sp., and refer to strains that at least have the same degree of biological function as the strains deposited in the present invention.

[0078] In some alternative embodiments, Rosenbergiella sp. may include strains having a 16S rRNA gene sequence with at least 97% homology, preferably at least 98% homology, more preferably at least 98.5%, at least 98.7%, at least 99%, at least 99.5%, at least 99.9% homology to the 16S rRNA gene sequence of the strain deposited in the present invention. The 16S rRNA gene sequence of the strain deposited in the present invention is as follows (SEQ ID NO:1):

[0079] SEQ ID NO:1

[0080] In some preferred embodiments, Rosenbergiella sp. includes strain YN46. In some more preferred embodiments, Rosenbergiella sp. is strain YN46.

[0081] <Composition>

[0082] In some aspects of the present invention, there is provided a composition comprising Rosenbergiella sp. and / or its products as described above.

[0083] In some embodiments, the composition is a microbial preparation.

[0084] In some embodiments, the composition is used for preventing and controlling arboviruses.

[0085] In some embodiments, Rosenbergiella sp. and / or its products serve as the active ingredient of the composition.

[0086] <Applications and methods of Rosenbergiella sp. and / or its products>

[0087] In some aspects of the present invention, there is provided any one of the following applications (i)-(iii) of Rosenbergiella sp. or its products:

[0088] (i) Application in the preparation of a product for controlling the transmission of arboviruses;

[0089] (ii) use in the preparation of a product for inhibiting the susceptibility of a vector to an arbovirus;

[0090] (iii) Application in the prevention and control of arboviruses.

[0091] In some aspects of the present invention, any of the following methods (a) to (c) is also provided:

[0092] (a) a method for controlling the transmission of arboviruses, said method comprising applying Rosenbergiella sp. and / or a product thereof, or a composition as described above, to a transmission vector;

[0093] (b) a method of inhibiting the susceptibility of a vector to an arbovirus, said method comprising administering to the vector a Rosenbergiella sp. and / or a product thereof, or a composition as described above;

[0094] (c) A method for controlling insect-borne viruses, the method comprising spraying Rosenbergiella sp. and / or its products onto the area to be controlled, or spraying the composition as described above onto the area to be controlled, preferably, contacting the transmission vector in the control area with Rosenbergiella sp. and / or its products, or the composition as described above.

[0095] Rosenbergiella sp. and compositions as described above<Rosenbergiella sp.> and as described in the <Composition> section.

[0096] In some embodiments, as described above, the Rosenbergiella sp. includes strain YN46, with a deposit number of CGMCC No. 29922.

[0097] In some embodiments, the product of Rosenbergiella sp. comprises glucose dehydrogenase.

[0098] In some optional embodiments, the glucose dehydrogenase comprises one or more of the following sequences:

[0099] 1) the amino acid sequence shown in SEQ ID NO: 2;

[0100] 2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, and which retains the activity of the amino acid sequence of SEQ ID NO: 1;

[0101] 3) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted into the amino acid sequence shown in SEQ ID NO: 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 2;

[0102] 4) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 2 under stringent conditions, and the amino acid sequence retains the activity of the amino acid sequence as shown in SEQ ID NO: 2, wherein the stringent conditions are moderate stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0103] SEQ ID NO:2

[0104] In some exemplary embodiments, a protein tag can be fused to the carboxyl terminus and / or amino terminus of the amino acid sequence shown in SEQ ID NO: 2 to obtain a fusion protein. A protein tag refers to a polypeptide or protein that is fused and expressed with a target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein.

[0105] In some embodiments, nucleic acid molecules encoding the above-mentioned glucose dehydrogenase also fall within the scope of protection of the present invention.

[0106] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0107] The nucleic acid molecule may specifically be a gene encoding glucose dehydrogenase as shown in SEQ ID NO: 3 below:

[0108] SEQ ID NO:3

[0109] In the above aspects of the invention, a fundamental characteristic of arboviruses is that they replicate in mammalian and insect cells. Many arboviruses are transmitted to mammals by mosquitoes, ticks, sand flies, and the like. In some embodiments, the arboviruses include one or more of the families Bunyaviridae, Flaviviridae, Reoviridae, and Togaviridae. In some specific embodiments, the arboviruses include one or more of the genera Flavivirus, Alphavirus, and Orthobunyavirus.

[0110] In some more specific embodiments, examples of arboviruses include African Swine Fever virus, Tick-borne Encephalitis virus, Rift Valley Fever virus, Colorado Tick Fever virus, Equine Encephalosis virus, Chikungunya virus, Dengue virus (DV), Zika virus (ZV) and West Nile virus.

[0111] In some preferred embodiments, the arbovirus is from the genus Flaviviridae. In some more preferred embodiments, the arbovirus is dengue virus (e.g., dengue virus type-2, New Guinea C strain) and / or Zika virus (e.g., Zika virus GZ01 strain).

[0112] In the above aspects of the present invention, the vector of the arbovirus can be mosquitoes, ticks or sand flies. In some embodiments, the vector of the arbovirus is mosquitoes (insects of the family Culicidae), and accordingly, the arbovirus can be a mosquito-borne virus.

[0113] In some embodiments of the present invention, "mosquito" or "mosquito" refers to an insect of the family Culicidae. The mosquitoes of the present invention may include adult mosquitoes, mosquito larvae, pupae, or their eggs. Generally, the life cycle of a mosquito consists of four separate and distinct stages: egg, larva, pupa, and adult. Thus, the mosquito life cycle begins when eggs are placed on the surface of water (e.g., Culex, Culiseta, and Anopheles) or on moist soil that is flooded with water (e.g., Aedes). Most eggs hatch into larvae within 48 hours. The larvae live in the water, feeding on microorganisms and organic matter, and then surface to breathe. They will molt four times, growing larger after each molt, and on the fourth molt, the larvae become pupae. The pupal stage is a resting, non-feeding period of about two days. At this time, the mosquito becomes an adult. When development is complete, the pupal skin bursts open and the adult mosquito emerges.

[0114] In some embodiments of the present invention, the mosquito belongs to one or more of the subfamily Anophelinae and the subfamily Culicinae. In some specific embodiments of the present invention, the mosquito comprises one or more of the species Culex, Culiseta, Anopheles, and Aedes.

[0115] In some more specific embodiments of the present invention, the mosquito includes, but is not limited to, Aedes, such as one or more of Aedes aegypti, Aedes albopictus, Aedes polynesiensis, Aedes australis, Aedes cinereus, Aedes rusticus, and Aedes vexans. In some preferred embodiments of the present invention, the mosquito is Aedes aegypti and / or Aedes albopictus.

[0116] In the above aspects of the invention, the term "control" refers to reducing, inhibiting, regulating and / or blocking the progression and / or spread of pathogens / diseases, in particular the progression and / or spread of arboviruses / diseases caused by arbovirus infection.

[0117] In the above aspects of the invention, "host" refers to a unicellular or multicellular organism in which an arbovirus can replicate, including cell lines and animals. In some embodiments of the invention, the host can be any mammal that can serve as a host for an arbovirus, such as a primate, preferably a human. In other embodiments, the host can also be a rodent, such as a mouse or rat.

[0118] In the above aspects of the present invention, the product can be a reagent or a kit, as well as any other suitable form of product. In some specific embodiments, the reagent is a microbial preparation.

[0119] Example

[0120] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0121] Example 1: Isolation and identification of Rosenbergiella_YN46

[0122] Adult female Aedes albopictus mosquitoes were collected using mosquito traps in Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province (104°49′E, 22°59′N). The mosquitoes were surface-disinfected with 75% ethanol, rinsed twice with sterile phosphate-buffered saline solution, and the midgut was dissected. The mosquitoes were then homogenized in 300 μl of sterile phosphate-buffered saline solution using a grinder. The homogenized solution was diluted 10 -1 to 10 -4 Afterwards, 100 μL was spread onto a blood plate and incubated at 30°C for 24 h. After 24 h, newly grown bacteria on the plate were transferred to brain heart infusion liquid medium (Beijing Solebeau Technology Co., Ltd., Catalog No. LA0360) and incubated in a constant temperature shaker at 30°C and 220 rpm until turbidity. The isolated bacteria were then classified by 16S rRNA gene sequencing and comparison, resulting in strain Rosenbergiella_YN46.

[0123] Rosenbergiella_YN46 was Gram-negative. Single colonies of the bacterium on blood agar plates (Guangdong Huankai Microbiology Technology Co., Ltd., Catalog No. 024070) or brain heart infusion agar plates appeared round, white, smooth, and opaque. The morphology of strain Rosenbergiella_YN46 was also identified by environmental scanning electron microscopy (Quanta 200), as shown in Figure 1.

[0124] Example 2: 16S rRNA gene sequence analysis of Rosenbergiella_YN46

[0125] The genomic DNA of strain Rosenbergiella_YN46 was extracted and purified using a bacterial DNA extraction kit (D3350-02, Omega). Using purified genomic DNA as a template, the 16S rRNA gene sequence of strain Rosenbergiella_YN46 was amplified using bacterial 16S rRNA gene amplification universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO: 4) and 1492R (5'-TACGACTTAACCCCAATCGC-3'; SEQ ID NO: 5). After amplification, an appropriate amount of PCR product was taken for agarose gel electrophoresis detection. When the target band was determined to be present, the remaining PCR product was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. The sequence was shown in SEQ ID NO.1, with a length of 1414 bp. The 16S rRNA gene sequence of Rosenbergiella_YN46 was uploaded to the CNGBdb database (https: / / db.cngb.org / ) and assigned the sequence number (N_001485395). This sequence was also uploaded to the GenBank database (https: / / www.ncbi.nlm.nih.gov / genbank / ) and assigned the sequence number (PP213046). The 16S rRNA gene sequence of strain Rosenbergiella_YN46 was submitted to the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) for sequence homology comparison. Phylogenetic analysis was performed with the 16S rRNA gene sequence of Rosenbergiella_YN46, along with other bacterial 16S rRNA gene sequences with high homology. Multiple sequence alignments were performed using ClustalW. A phylogenetic tree was constructed using MEGA XI software using a neighbor-joining algorithm with 1000 bootstrap iterations to visualize its evolutionary position and relationships with other species. The resulting phylogenetic tree is shown in Figure 2.

[0126] Example 3: Full gene sequence analysis of Rosenbergiella_YN46

[0127] A sequencing library for Rosenbergiella_YN46 was prepared using the MGIEasy Universal DNA Library Preparation Kit. Subsequently, the library was sequenced on the DNBSEQ T-series platform, obtaining 150-base-pair end-read data. To prepare the genome assembly data, read adapters and low-quality reads were removed using fastp software. Next, the genome was assembled using SPAdes (version 3.15.3) software with default settings. After assembly, proteins in the bacterial genome were predicted and annotated using Prokka (version 1.14.6) and the eggNOG database (version 5.0). Default settings were also used for the annotation process. Finally, the sequencing data were deposited in the CNSA (CNGB Sequence Archive) of CNGBdb (China National Gene Bank Database, https: / / db.cngb.org / cnsa / ); project number: CNP0005149) for subsequent data use. This sequence was also uploaded to the GenBank database (https: / / www.ncbi.nlm.nih.gov / genbank / ) and obtained the sequence number (JAZHEE000000000).

[0128] The results showed that the genome sequence of strain Rosenbergiella_YN46 contained 308 contigs, with a total genome length of 4,893,755 bp and a G+C content of 55.17%.

[0129] Example 4: Growth performance evaluation of Rosenbergiella_YN46

[0130] The preserved strain Rosenbergiella_YN46 was inoculated onto a blood plate using the streak method for activation. The blood plate was then incubated at 30°C for 24 hours. After 24 hours, a single colony that had grown on the plate was transferred to brain heart infusion liquid medium and incubated in a constant temperature shaker at 30°C and 220 rpm. 1 mL of the bacterial solution was collected at 0, 6, 12, 24, 48, 72, and 96 hours after the start of the culture. The bacterial solution was then diluted 10 -1 to 10 -20 100 μL of the solution was then spread onto brain heart infusion plates and incubated at 30°C for 24 hours. Colony forming units (CFUs) were then calculated by counting the colonies on the plates, and the growth performance of Rosenbergiella_YN46 was quantified using CFUs, as shown in Figure 3.

[0131] Example 5: Identification of the inhibitory effect of Rosenbergiella_YN46 on dengue virus infection in Aedes albopictus

[0132] Sterile cotton balls were soaked in 10% sucrose solution with or without bacteria Rosenbergiella_YN46 (bacterial concentration was 1×10 8CFUs / mL). Then, use this as food for Aedes albopictus, and continue to feed them for 7 days to colonize the bacteria Rosenbergiella_YN46 in the midgut of Aedes albopictus. Next, extracorporeal membrane feeding of blood is performed. One day in advance, the Aedes albopictus is placed in a paper cup without adding food, and the surface is covered with a moist paper towel to keep it moist. The seed liquid of dengue virus type 2 (DENV2) New Guinea C strain (abbreviated as DENV2 in the embodiment, the same in subsequent embodiments) is inoculated into Vero cells, and the cell supernatant, namely DENV2, is collected after 5 days. DENV2 and human blood are mixed in equal volumes, mixed evenly, and added to the reservoir of the extracorporeal membrane feeding system, and placed on a paper cup. The Aedes albopictus in the paper cup begins to suck blood. After 30 minutes, the reservoir is removed and soaked in 10% 84 disinfectant. Aedes albopictus mosquitoes were anesthetized in a 4°C refrigerator for 15 minutes. Blood-feeding mosquitoes were then picked and placed in new mosquito cups for another 8 days. Mosquitoes were then sacrificed and the DENV2 infection rate was assessed by real-time quantitative PCR. The primer sequences for quantitative PCR were as follows: DENV2-F: 5-CATTCCAAGTGAGAATCTCTTTGTCA-3' (SEQ ID NO: 6); DENV2-R: 5-CAGATCTCTGATGAATAACCAACG-3' (SEQ ID NO: 7). Thermal cycling consisted of an initial denaturation step at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, and annealing / extension at 60°C for 30 seconds. The amount of viral genomic RNA in each mosquito was determined by constructing a target gene standard curve and converting the cycle threshold (Ct) value to the gene concentration for each sample. These standard curves were generated by amplifying viral genomes containing the qPCR target using primers labeled with a T7 promoter. Reverse transcription was performed using the MegaScript T7 transcription kit (Ambion, AM1333) and purification was performed using MegaClear (Ambion, AM1908). Total RNA concentration was quantified using Nanodrop 2000 (Thermo Scientific, E112352) to determine the copy number. An absolute standard curve was constructed for gene quantification. The detection limit of viral genome copies was set to 1000 copies. In the case where uninfected mosquitoes showed a cycle threshold (Ct) of "N / A", these negative test data were replaced with the maximum possible Ct value (Ct=40). When the viral genome copy number was greater than 1000 copies, the mosquito was considered to be infected with the virus. Infection rate = (number of mosquitoes infected with the virus / total number of mosquitoes) × 100%.

[0133] The results showed that compared with Aedes albopictus not colonized with Rosenbergiella_YN46 (control group in Figure 4), the viral load and infection rate of DENV2 in Aedes albopictus colonized with Rosenbergiella_YN46 (Rosenbergiella_YN46 group in Figure 4) were significantly reduced, as shown in Figure 4. Therefore, Rosenbergiella_YN46 can inhibit dengue virus infection in Aedes albopictus.

[0134] Example 6: Identification of the inhibitory effect of Rosenbergiella_YN46 on Zika virus infection in Aedes albopictus

[0135] Sterile cotton balls were soaked in 10% sucrose solution with or without bacteria Rosenbergiella_YN46 (bacterial concentration was 1×10 8CFUs / mL). Then, use this as food for Aedes albopictus, and continue to feed them for 7 days to colonize the bacteria Rosenbergiella_YN46 in the midgut of Aedes albopictus. Next, extracorporeal membrane feeding of blood is performed. Place the Aedes albopictus in a paper cup one day in advance without adding food, and cover it with a moist paper towel to keep it moist. The Zika virus (Zikv virus, ZIKV) GZ01 strain (abbreviated as ZIKV in the examples, the same as in subsequent examples) seed liquid is inoculated into Vero cells, and the cell supernatant, namely ZIKV, is collected after 4 days. ZIKV and human blood are mixed in equal volumes, mixed evenly, and added to the reservoir of the extracorporeal membrane feeding system, and placed on a paper cup. The Aedes albopictus in the paper cup begins to suck blood. After 30 minutes, remove the reservoir and soak it in 10% 84 disinfectant. Aedes albopictus mosquitoes were anesthetized in a 4°C refrigerator for 15 minutes. Blood-feeding mosquitoes were then picked and placed in new mosquito cups for another 8 days. Mosquitoes were then sacrificed and ZIKV infection rates were determined using real-time quantitative PCR. The primer sequences for the quantitative PCR were as follows: ZIKV-F: 5-CCGCTGCCCAACACAAG-3' (SEQ ID NO: 8); ZIKV-R: 5-CCACTAACGTTCTTTTGCAGACAT-3' (SEQ ID NO: 9). Thermal cycling consisted of an initial denaturation step at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, and annealing / extension at 60°C for 30 seconds. The amount of viral genomic RNA in each mosquito was determined by constructing a target gene standard curve and converting the cycle threshold (Ct) value to the gene concentration for each sample. These standard curves were generated by amplifying viral genomes containing the qPCR target using primers labeled with a T7 promoter. Reverse transcription was performed using the MegaScript T7 transcription kit (Ambion, AM1333) and purification was performed using MegaClear (Ambion, AM1908). Total RNA concentration was quantified using Nanodrop 2000 (Thermo Scientific, E112352) to determine the copy number. An absolute standard curve was constructed for gene quantification. The detection limit of viral genome copies was set to 1000 copies. In the case where uninfected mosquitoes showed a cycle threshold (Ct) of "N / A", these negative test data were replaced with the maximum possible Ct value (Ct=40). When the viral genome copy number was greater than 1000 copies, the mosquito was considered to be infected with the virus. Infection rate = (number of mosquitoes infected with the virus / total number of mosquitoes) × 100%.

[0136] The results showed that compared with Aedes albopictus not colonized with Rosenbergiella_YN46 (the control group in Figure 5), the viral load and infection rate of ZIKV in Aedes albopictus colonized with Rosenbergiella_YN46 (the Rosenbergiella_YN46 group in Figure 5) were significantly reduced, as shown in Figure 5. Therefore, Rosenbergiella_YN46 can inhibit Zika virus infection in Aedes albopictus.

[0137] Example 7: Identification of the inhibitory effect of Rosenbergiella_YN46 on dengue virus infection in Aedes aegypti

[0138] Sterile cotton balls were soaked in 10% sucrose solution with or without bacteria Rosenbergiella_YN46 (bacterial concentration was 1×10 8CFUs / mL). Then, this is used as food for Aedes aegypti mosquitoes, and they are fed for 7 days to colonize the bacteria Rosenbergiella_YN46 in the midgut of Aedes aegypti mosquitoes. Next, extracorporeal membrane feeding of blood is performed. One day in advance, the Aedes aegypti mosquitoes are placed in a paper cup without food, and the surface is covered with a moist paper towel to keep it moist. The DENV2 seed liquid is inoculated into Vero cells, and the cell supernatant, namely DENV2, is collected after 5 days. DENV2 and human blood are mixed in equal volumes, mixed evenly, and added to the reservoir of the extracorporeal membrane feeding system, and placed on the paper cup. The Aedes aegypti mosquitoes in the paper cup begin to suck blood. After 30 minutes, the reservoir is removed and soaked in 10% 84 disinfectant. The Aedes aegypti mosquitoes are placed in a 4°C refrigerator for anesthesia for 15 minutes, and then the mosquitoes that have sucked blood are picked and placed in a new mosquito cup for continued breeding for 8 days. Finally, the mosquitoes are killed and the DENV2 infection rate is detected by real-time fluorescence quantitative PCR. Primer sequences for quantitative PCR are as follows: DENV2-F: 5-CATTCCAAGTGAGAATCTCTTTGTCA-3' (SEQ ID NO: 6); DENV2-R: 5-CAGATCTCTGATGAATAACCAACG-3' (SEQ ID NO: 7). Thermal cycling consisted of an initial denaturation step at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60°C for 30 seconds. The amount of viral genomic RNA per mosquito was determined by constructing a target gene standard curve and converting the cycle threshold (Ct) value to the gene concentration for each sample. These standard curves were generated by amplifying viral genomes containing the qPCR target using primers labeled with a T7 promoter. Reverse transcription was performed using the MegaScript T7 Transcription Kit (Ambion, AM1333) and purified using MegaClear (Ambion, AM1908). Total RNA concentration was quantified using Nanodrop 2000 (Thermo Scientific, E112352) to determine the copy number. An absolute standard curve was constructed for gene quantification. The detection limit of viral genome copies was set to 1000 copies. In the case where uninfected mosquitoes showed a cycle threshold (Ct) of "N / A", these negative test data were replaced with the maximum possible Ct value (Ct = 40). When the viral genome copy number was greater than 1000 copies, the mosquito was considered to be infected with the virus. Infection rate = (number of mosquitoes infected with the virus / total number of mosquitoes) × 100%.

[0139] The results showed that compared with Aedes aegypti mosquitoes not colonized with Rosenbergiella_YN46 (control group in Figure 6), the viral load and infection rate of DENV2 in Aedes aegypti mosquitoes colonized with Rosenbergiella_YN46 (Rosenbergiella_YN46 group in Figure 6) were significantly reduced, as shown in Figure 6. Therefore, Rosenbergiella_YN46 can inhibit dengue virus infection in Aedes aegypti.

[0140] Example 8: Identification of the inhibitory effect of Rosenbergiella_YN46 on Zika virus infection in Aedes aegypti

[0141] Sterile cotton balls were soaked in 10% sucrose solution with or without bacteria Rosenbergiella_YN46 (bacterial concentration was 1×10 8CFUs / mL). Then, this is used as food for Aedes aegypti mosquitoes, and they are fed for 7 days to colonize the bacteria Rosenbergiella_YN46 in the midgut of Aedes aegypti mosquitoes. Next, extracorporeal membrane feeding of blood is performed. One day in advance, the Aedes aegypti mosquitoes are placed in a paper cup without food, and the surface is covered with a moist paper towel to keep it moist. The ZIKV seed liquid is inoculated into Vero cells, and the cell supernatant, namely ZIKV, is collected after 4 days. ZIKV and human blood are mixed in equal volumes, mixed evenly, and added to the reservoir of the extracorporeal membrane feeding system, and placed on the paper cup. The Aedes aegypti mosquitoes in the paper cup begin to suck blood. After 30 minutes, the reservoir is removed and soaked in 10% 84 disinfectant. The Aedes aegypti mosquitoes are placed in a 4°C refrigerator for anesthesia for 15 minutes, and then the mosquitoes that have sucked blood are picked and placed in a new mosquito cup for continued breeding for 8 days. Finally, the mosquitoes are killed and the infection rate of ZIKV is detected by real-time fluorescence quantitative PCR. Primer sequences for quantitative PCR were as follows: ZIKV-F: 5-CCGCTGCCCAACACAAG-3' (SEQ ID NO: 8); ZIKV-R: 5-CCACTAACGTTCTTTTGCAGACAT-3' (SEQ ID NO: 9). Thermal cycling consisted of an initial denaturation step at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60°C for 30 seconds. The amount of viral genomic RNA per mosquito was determined by constructing a target gene standard curve and converting the cycle threshold (Ct) value to the gene concentration for each sample. These standard curves were generated by amplifying viral genomes containing the qPCR target using primers labeled with a T7 promoter. Reverse transcription was performed using the MegaScript T7 Transcription Kit (Ambion, AM1333) and purified using MegaClear (Ambion, AM1908). Total RNA concentration was quantified using Nanodrop 2000 (Thermo Scientific, E112352) to determine the copy number. An absolute standard curve was constructed for gene quantification. The detection limit of viral genome copies was set to 1000 copies. In the case where uninfected mosquitoes showed a cycle threshold (Ct) of "N / A", these negative test data were replaced with the maximum possible Ct value (Ct = 40). When the viral genome copy number was greater than 1000 copies, the mosquito was considered to be infected with the virus. Infection rate = (number of mosquitoes infected with the virus / total number of mosquitoes) × 100%.

[0142] The results showed that the viral load and infection rate of ZIKV in Aedes aegypti colonized with Rosenbergiella_YN46 (the Rosenbergiella_YN46 group in Figure 7) were significantly decreased compared to Aedes aegypti not colonized with Rosenbergiella_YN46 (the control group in Figure 7), as shown in Figure 7. Therefore, Rosenbergiella_YN46 can inhibit the infection of Zika virus in Aedes aegypti.

[0143] Example 9: Purification and identification of glucose dehydrogenase

[0144] Genomic DNA from strain Rosenbergiella_YN46 was extracted using a bacterial DNA extraction kit (D3350-02, Omega) and assembled for genome sequencing. The Rosenbergiella_YN46 genome was assembled as follows: a sequencing library was prepared using the MGIEasy Universal DNA Library Preparation Kit and subsequently sequenced on the DNBSEQ T-Series platform, generating 150-base-pair end-reads. To prepare the assembly data, read adapters and low-quality reads were removed using fastp. The genome was assembled using SPAdes (version 3.15.3) with default settings. After assembly, proteins in the bacterial genome were predicted and annotated using Prokka (version 1.14.6) and the eggNOG database (version 5.0), using default settings. The sequencing data were deposited in the CNSA (CNGB Sequence Archive) of CNGBdb (China National Gene Bank Database, https: / / db.cngb.org / cnsa / ); project number: CNP0005149. The glucose dehydrogenase sequence of Rosenbergiella_YN46 was obtained through the annotation results. The glucose dehydrogenase sequence of Rosenbergiella_YN46 was also uploaded to the GenBank database (https: / / www.ncbi.nlm.nih.gov / genbank / ) and obtained the sequence number (PP296640).

[0145] For the purification of glucose dehydrogenase. The Escherichia coli expression system was used to express and purify the glucose dehydrogenase of Rosenbergiella_YN46. The amino acid sequence of glucose dehydrogenase is shown in SEQ ID No. 2. The pET-28a(+) plasmid vector (Merck, 69864) was selected to express glucose dehydrogenase. The plasmid vector contains a strong phage T7 promoter and a histidine tag (His tag) that can be bound to a Ni2+ column or a Co2+ column. The glucose dehydrogenase gene of Rosenbergiella_YN46 was inserted into the pET-28a(+) plasmid vector by PCR amplification, and then the plasmid was transformed into BL21 DE3 competent cells (Beijing Quanshijin Biotechnology Co., Ltd., CD601-02). The BL21 DE3 monoclonal bacteria transformed with the glucose dehydrogenase gene were picked and cultured at 37°C and 220rpm until the OD 600 When the pH value was between 0.6 and 0.8, IPTG was added to a final concentration of 0.1 mM to induce protein expression, and the culture was incubated at 16°C and 90 rpm for 16 h to induce the production of soluble glucose dehydrogenase. The purified glucose dehydrogenase was then added with 5 μM pyrroloquinoline quinone, 1 mM CaCl2, and 1 mM MgCl2 to obtain its active form.

[0146] For the identification of glucose dehydrogenase, the purified protein was added to a glucose dehydrogenase activity assay solution (5 mM pyrroloquinoline quinone, 5 mM p-benzoquinone, 150 mM NaCl2) at a final concentration of 10 μg / ml. After incubation at 30°C for 30 minutes, the glucose and gluconic acid in the reaction products were detected by liquid chromatography-mass spectrometry. The results showed that glucose dehydrogenase was able to catalyze the production of gluconic acid from glucose.

[0147] Example 10: Identification of the inhibitory effect of glucose dehydrogenase on dengue virus infection in Aedes albopictus

[0148] Aedes albopictus mosquitoes were placed in a paper cup one day in advance, without food, and covered with a moistened paper towel for moisture. A DENV2 seed solution was inoculated into Vero cells, and after 5 days, the cell supernatant (DENV2) was harvested. Equal volumes of DENV2 and human blood were mixed, supplemented or not with 10 μg / ml purified glucose dehydrogenase. After mixing thoroughly, the mixture was added to the reservoir of an extracorporeal membrane feeding system and placed on a paper cup. The mosquitoes in the cup began to feed blood. After 30 minutes, the reservoir was removed and soaked in 10% 84 disinfectant. The mosquitoes were anesthetized in a 4°C refrigerator for 15 minutes. The mosquitoes that had fed blood were then picked and placed in a new mosquito cup for another 8 days. The mosquitoes were then sacrificed and the DENV2 infection rate was determined by real-time quantitative PCR. Primer sequences for quantitative PCR are as follows: DENV2-F: 5-CATTCCAAGTGAGAATCTCTTTGTCA-3' (SEQ ID NO: 6); DENV2-R: 5-CAGATCTCTGATGAATAACCAACG-3' (SEQ ID NO: 7). Thermal cycling consisted of an initial denaturation step at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60°C for 30 seconds. The amount of viral genomic RNA per mosquito was determined by constructing a target gene standard curve and converting the cycle threshold (Ct) value to the gene concentration for each sample. These standard curves were generated by amplifying viral genomes containing the qPCR target using primers labeled with a T7 promoter. Reverse transcription was performed using the MegaScript T7 Transcription Kit (Ambion, AM1333) and purified using MegaClear (Ambion, AM1908). Total RNA concentration was quantified using Nanodrop 2000 (Thermo Scientific, E112352) to determine the copy number. An absolute standard curve was constructed for gene quantification. The detection limit of viral genome copies was set to 1000 copies. In the case where uninfected mosquitoes showed a cycle threshold (Ct) of "N / A", these negative test data were replaced with the maximum possible Ct value (Ct = 40). When the viral genome copy number was greater than 1000 copies, the mosquito was considered to be infected with the virus. Infection rate = (number of mosquitoes infected with the virus / total number of mosquitoes) × 100%.

[0149] The results showed that compared with Aedes albopictus without glucose dehydrogenase supplementation (control group in Figure 8), the viral load and infection rate of DENV2 in Aedes albopictus supplemented with glucose dehydrogenase (glucose dehydrogenase group in Figure 8) were significantly reduced, as shown in Figure 8. Therefore, glucose dehydrogenase can inhibit dengue virus infection in Aedes albopictus.

[0150] Example 11: Identification of the inhibitory effect of glucose dehydrogenase on Zika virus infection in Aedes albopictus

[0151] Aedes albopictus mosquitoes were placed in a paper cup one day in advance, without food, and covered with a moistened paper towel for moisture. ZIKV seed stock was inoculated into Vero cells, and after four days, the cell supernatant (ZIKV) was harvested. Equal volumes of ZIKV and human blood were mixed, supplemented or not with 10 μg / ml purified glucose dehydrogenase. After thorough mixing, the mixture was added to the reservoir of an extracorporeal membrane feeding system and placed on a paper cup. The mosquitoes in the cup began feeding blood. After 30 minutes, the reservoir was removed and soaked in 10% 84 disinfectant. The mosquitoes were anesthetized in a 4°C refrigerator for 15 minutes. The mosquitoes that had fed blood were then picked and transferred to a new mosquito cup for further feeding for another eight days. The mosquitoes were then sacrificed and ZIKV infection rates were determined using real-time quantitative PCR. Primer sequences for quantitative PCR are as follows: ZIKV-F: 5-CCGCTGCCCAACACAAG-3' (SEQ ID NO: 8); ZIKV-R: 5-CCACTAACGTTCTTTTGCAGACAT-3' (SEQ ID NO: 9). Thermal cycling consisted of an initial denaturation step at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60°C for 30 seconds. The amount of viral genomic RNA in each mosquito was determined by constructing a target gene standard curve and converting the cycle threshold (Ct) value to gene concentration for each sample. These standard curves were generated by amplifying viral genomes containing the qPCR target using primers labeled with a T7 promoter. Reverse transcription was performed using the MegaScript T7 Transcription Kit (Ambion, AM1333) and purified using MegaClear (Ambion, AM1908). Total RNA concentration was quantified using a Nanodrop 2000 (Thermo Scientific, E112352) to determine copy number. An absolute standard curve was constructed for gene quantification. The detection limit for viral genome copies was set at 1000 copies. When the viral genome copy number exceeded 1000 copies, the mosquito was considered infected with the virus. All negative test data were replaced with one genome copy. Infection rate = (number of virus-infected mosquitoes / total number of mosquitoes) × 100%.

[0152] The results showed that compared with Aedes albopictus without glucose dehydrogenase supplementation (control group in Figure 9), the viral load and infection rate of ZIKV in Aedes albopictus supplemented with glucose dehydrogenase (glucose dehydrogenase group in Figure 9) were significantly reduced, as shown in Figure 9. Therefore, glucose dehydrogenase can inhibit Zika virus infection in Aedes albopictus.

[0153] Example 12: Identification of the inhibitory effect of glucose dehydrogenase on dengue virus infection in Aedes aegypti

[0154] Aedes aegypti mosquitoes were placed in a paper cup one day in advance, without food, and covered with a moistened paper towel for moisture. A DENV2 seed stock was inoculated into Vero cells, and after 5 days, the cell supernatant (DENV2) was harvested. Equal volumes of DENV2 and human blood were mixed, supplemented or not with 10 μg / ml purified glucose dehydrogenase. After thorough mixing, the mixture was added to the reservoir of an extracorporeal membrane feeding system and placed on a paper cup. The mosquitoes in the cup began feeding blood. After 30 minutes, the reservoir was removed and soaked in 10% 84 disinfectant. The mosquitoes were anesthetized in a 4°C refrigerator for 15 minutes. The mosquitoes that had fed blood were then picked and placed in a new mosquito cup for another 8 days. The mosquitoes were then sacrificed and the DENV2 infection rate was determined by real-time quantitative PCR. Primer sequences for quantitative PCR are as follows: DENV2-F: 5-CATTCCAAGTGAGAATCTCTTTGTCA-3' (SEQ ID NO: 6); DENV2-R: 5-CAGATCTCTGATGAATAACCAACG-3' (SEQ ID NO: 7). Thermal cycling consisted of an initial denaturation step at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, and annealing / extension at 60°C for 30 seconds. The amount of viral genomic RNA in each mosquito was determined by constructing a target gene standard curve and converting the cycle threshold (Ct) value to the gene concentration for each sample. These standard curves were generated by amplifying viral genomes containing the qPCR target using primers labeled with a T7 promoter. Reverse transcription was performed using the MegaScript T7 Transcription Kit (Ambion, AM1333) and purified using MegaClear (Ambion, AM1908). Total RNA concentration was quantified using Nanodrop 2000 (Thermo Scientific, E112352) to determine the copy number. An absolute standard curve was constructed for gene quantification. The detection limit of viral genome copies was set to 1000 copies. In the case where uninfected mosquitoes showed a cycle threshold (Ct) of "N / A", these negative test data were replaced with the maximum possible Ct value (Ct = 40). When the viral genome copy number was greater than 1000 copies, the mosquito was considered to be infected with the virus. Infection rate = (number of mosquitoes infected with the virus / total number of mosquitoes) × 100%.

[0155] The results showed that compared with the Aedes aegypti without glucose dehydrogenase supplementation (control group in Figure 10), the viral load and infection rate of DENV2 in the Aedes albopictus supplemented with glucose dehydrogenase (glucose dehydrogenase group in Figure 10) were significantly reduced, as shown in Figure 10. Therefore, glucose dehydrogenase can inhibit dengue virus infection in Aedes aegypti.

[0156] Example 13: Identification of the inhibitory effect of glucose dehydrogenase on Zika virus infection in Aedes aegypti

[0157] Aedes aegypti mosquitoes were placed in a paper cup one day in advance, without food, and covered with a moistened paper towel for moisture. ZIKV seed stock was inoculated into Vero cells, and after four days, the cell supernatant (ZIKV) was harvested. Equal volumes of ZIKV and human blood were mixed, supplemented or not with 10 μg / ml purified glucose dehydrogenase. After thorough mixing, the mixture was added to the reservoir of an extracorporeal membrane feeding system and placed on a paper cup. The mosquitoes in the cup began feeding blood. After 30 minutes, the reservoir was removed and soaked in 10% 84 disinfectant. The mosquitoes were anesthetized in a 4°C refrigerator for 15 minutes. The mosquitoes that had fed blood were then picked and transferred to a new mosquito cup for another eight days. The mosquitoes were then sacrificed and ZIKV infection rates were determined by real-time quantitative PCR. Primer sequences for quantitative PCR are as follows: ZIKV-F: 5-CCGCTGCCCAACACAAG-3' (SEQ ID NO: 8); ZIKV-R: 5-CCACTAACGTTCTTTTGCAGACAT-3' (SEQ ID NO: 9). Thermal cycling consisted of an initial denaturation step at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60°C for 30 seconds. The amount of viral genomic RNA in each mosquito was determined by constructing a target gene standard curve and converting the cycle threshold (Ct) value to gene concentration for each sample. These standard curves were generated by amplifying viral genomes containing the qPCR target using primers labeled with a T7 promoter. Reverse transcription was performed using the MegaScript T7 Transcription Kit (Ambion, AM1333) and purified using MegaClear (Ambion, AM1908). Total RNA concentration was quantified using a Nanodrop 2000 (Thermo Scientific, E112352) to determine copy number. An absolute standard curve was constructed for gene quantification. The detection limit of viral genome copies was set at 1000 copies. In the case of uninfected mosquitoes showing a cycle threshold (Ct) value of "N / A", these negative test data were replaced with the maximum possible Ct value (Ct = 40). When the viral genome copy number is greater than 1000 copies, the mosquito is considered infected with the virus. Infection rate = (number of virus-infected mosquitoes / total number of mosquitoes) × 100%.

[0158] The results showed that compared with Aedes aegypti without glucose dehydrogenase supplementation (control group in Figure 11), the viral load and infection rate of ZIKV in Aedes aegypti supplemented with glucose dehydrogenase (glucose dehydrogenase group in Figure 11) were significantly reduced, as shown in Figure 11. Therefore, glucose dehydrogenase can inhibit Zika virus infection in Aedes aegypti.

[0159] These results demonstrate that the novel Rosenbergiella sp., a new species of the genus Rosenbergiella, exhibits significant inhibition of mosquito infection by the mosquito-borne flaviviruses Zika and dengue. This strain is a broad-spectrum, highly effective biocontrol strain that can be used for the prevention and control of these viruses by direct spraying in mosquito breeding areas, or for the production of microbial agents for the prevention and control of these viruses. The novel Rosenbergiella sp. produces glucose dehydrogenase, which has great potential for catalyzing gluconic acid production and for the prevention and control of mosquito-borne flaviviruses.

[0160] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0161] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A strain, whose deposit number is CGMCC No.29922.

2. A composition comprising the strain according to claim 1 and / or its product; Optionally, the composition is a microbial preparation; Optionally, the composition is used to control arboviruses; Optionally, the strain and / or its product is used as an active ingredient in the composition.

3. Any of the following uses (i) to (iii) of Rosenbergiella sp. and / or its products: (i) Use in the preparation of products for controlling the spread of arboviruses; (ii) use in the preparation of a product for inhibiting the susceptibility of a vector to an arbovirus; (iii) Application in the prevention and control of arboviruses.

4. The use according to claim 3, wherein: The Rosenbergiella sp. includes strains whose 16S rRNA gene sequence has at least 97% homology to the sequence shown in SEQ ID NO: 1; Preferably, the Rosenbergiella sp. includes a strain whose 16S rRNA gene sequence comprises the sequence shown in SEQ ID NO: 1; More preferably, the Rosenbergiella sp. includes Rosenbergiella sp. YN46 strain, whose deposit number is CGMCC No.29922.

5. The use according to claim 3 or 4, wherein: The product of the Rosenbergiella sp. comprises glucose dehydrogenase; Optionally, the glucose dehydrogenase comprises one or more of the following sequences: 1) the amino acid sequence shown in SEQ ID NO: 2; 2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, and which retains the activity of the amino acid sequence of SEQ ID NO: 1; 3) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted into the amino acid sequence shown in SEQ ID NO: 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 2; 4) an amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence being identical to the amino acid sequence encoded by SEQ ID The polynucleotide sequence of the amino acid sequence shown in NO:2 hybridizes under stringent conditions, and the amino acid sequence retains the activity of the amino acid sequence shown in SEQ ID NO:2, wherein the stringent conditions are moderately stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

6. The use according to any one of claims 3 to 5, wherein The arbovirus includes one or more of the families Bunyaviridae, Flaviviridae, Reoviridae and Togaviridae; Preferably, the arbovirus comprises one or more of the genus Flavivirus, genus Alphavirus and genus Orthobunyavirus.

7. The use according to any one of claims 3 to 6, wherein The arbovirus is of the genus Flavivirus; Optionally, the arbovirus comprises dengue virus and / or Zika virus.

8. The use according to any one of claims 3 to 7, wherein The vectors of the arbovirus include one or more of mosquitoes, ticks or sand flies; Preferably, the vector of the arbovirus is mosquito; More preferably, the mosquito comprises one or more of Culex, Culiseta, Anopheles and Aedes; Optionally, the mosquito is an Aedes mosquito, including one or more of Aedes aegypti, Aedes albopictus, Aedes polynesiensis, Aedes australis, Aedes cinereus, Aedes rusticus and Aedes vexans.

9. The use according to any one of claims 3 to 8, wherein The product comprises one or more of a reagent or a kit; optionally, the reagent is a microbial preparation.

10. Any of the following methods (a) to (c): (a) a method for controlling the transmission of arboviruses, said method comprising applying Rosenbergiella sp. and / or its products, or the composition according to claim 2, to the transmission vector; (b) a method for inhibiting the susceptibility of a vector to an arbovirus, said method comprising administering to the vector a Rosenbergiella sp. and / or a product thereof, or a composition as claimed in claim 2; (c) A method for controlling insect-borne viruses, comprising spraying Rosenbergiella sp. and / or its products onto an area to be controlled, or spraying the composition according to claim 2 onto an area to be controlled.

Images