Plasmid vector for in-vitro transcription of mrna, construction method therefor and use thereof

Abstract

Provided is a nucleic acid molecule, comprising 1) a transcribable nucleic acid fragment and / or a nucleic acid used for introducing a transcribable nucleic acid fragment; 2) a polyA nucleic acid fragment; and 3) a gyrase binding site nucleic acid fragment. The nucleic acid molecule can improve the stability of polyA, thus solving the problem of polyA loss / shortening. Additionally, the present invention improves the stability of nucleic acid molecules, thus reducing the probability of recombination of plasmids during amplification, and facilitating industrial production of mRNA products.

Description

A plasmid vector for in vitro transcription of mRNA, construction method and application thereof Technical Field

[0001] The present invention belongs to the field of genetic engineering and specifically relates to an mRNA in vitro transcription vector capable of improving plasmid stability, a construction method and applications thereof. Background Art

[0002] mRNA technology has many advantages: first, mRNA does not integrate into the genome, thus avoiding concerns about insertional mutagenesis; more importantly, unlike relatively stable DNA, mRNA will subsequently degrade after performing its function, without causing other toxic or side effects to the human body; mRNA can be manufactured in a cell-free manner and can be rapidly and economically mass-produced through in vitro transcription technology. Its short R&D cycle allows for rapid, economical, and efficient production. Furthermore, a single mRNA can encode multiple antigens, proteins, or peptides, making the process highly adaptable and theoretically capable of expressing any protein.

[0003] The mRNA COVID-19 vaccine has proven the applicability of the mRNA technology platform in the vaccine field. Beyond COVID-19 vaccines, mRNA technology can also be widely used in infectious diseases, tumors, and protein replacement therapy.

[0004] For mRNA technology, a good production process begins with the design of a safe, stable, and efficient plasmid vector. Whether from a production process perspective, a therapeutic perspective, or a regulatory perspective, most non-essential sequences in plasmid DNA should be removed to minimize molecular weight for easier genetic manipulation. Large plasmids impose a significant metabolic burden on the host bacteria, reducing the cellular resources available for plasmid replication and leading to decreased plasmid DNA yield. Furthermore, in industrial production, plasmids must maintain a high degree of stability during continuous passage to ensure widespread application and stability of mRNA technology.

[0005] A challenge in the production of mRNA plasmid technology is that the plasmid's PolyA sequence shortens as the bacteria continue to multiply. This complicates the cloning and amplification of plasmids carrying the PolyA tail, making it impossible to achieve uniformity in the produced plasmid's PolyA sequence, thus failing to meet GMP-level production processes and pharmaceutical regulatory requirements. Furthermore, the PolyA sequence is a critical component for mRNA stability and translation efficiency. Therefore, addressing this issue of PolyA sequence shortening during plasmid fermentation production has been a constant focus for researchers.

[0006] To eliminate the instability of the PolyA tail on circular plasmids, Kyle Jacoby et al. developed the pEVL linear plasmid system, derived from the pJazz plasmid. Compared with traditional circular plasmids, the pEVL plasmid has a significant advantage in maintaining the length of the PolyA tail after continuous culture (Kyle Jacoby, 2016, Mol Ther Nucleic Acids). However, the copy number of the pEVL plasmid is very low, making it unsuitable for large-scale production. In 2019, Zeljka Trepotec et al. discovered that spacing the PolyA sequence can reduce recombination during plasmid DNA amplification and maintain the length of the PolyA tail. Representative examples of using segmented polyA tails include the technical solutions disclosed in BioNTech's patent US10717982B2 and Ethris' patent WO2020074642A1. Kristie Bloom et al. developed a plasmid that can increase the length of the PolyA tail through enzyme digestion and ligation, which serves as a template for in vitro co-transcription of mRNA to improve the shortening of the PolyA length during plasmid passage. In addition, other companies use shorter polyA sequences to achieve plasmid stability.

[0007] However, most of the above methods are aimed at improving polyA itself. Even if a plasmid system is developed to improve the instability of PolyA, it has been proven to be unsuitable for large-scale production.

[0008] Summary of the Invention

[0009] The present invention conducts further research on plasmids suitable for RNA technology, breaking through most improvement methods that only target polyA. It considers solving the problem of PolyA loss from the perspective of the entire plasmid without affecting plasmid replication and yield, improving plasmid integrity and stability, and making it suitable for large-scale production.

[0010] The present invention provides a nucleic acid molecule for improving the stability of a PolyA tail during plasmid passage in bacterial fermentation and application thereof.

[0011] The term "vector" of the present invention is used herein in its general sense as known to those skilled in the art and includes any intermediate carrier of a nucleic acid, for example, which enables the nucleic acid to be introduced into a prokaryotic and / or eukaryotic host cell or, where appropriate, to be integrated into a genome, where the vector is preferably replicated and / or expressed.

[0012] The term "pharmaceutically acceptable carrier" in the present invention refers to an inactive substance formulated together with a pharmaceutically active substance to meet or promote the requirements of the active substance in terms of dosage, adsorption, solubility or pharmacokinetics.

[0013] The term "plasmid" according to the present invention generally relates to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of the chromosomal DNA.

[0014] The term "host cell" herein refers to any cell that can be transformed or transfected with exogenous nucleic acid, including prokaryotes (e.g., E. coli) or eukaryotic cells (e.g., yeast cells and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats, and primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines.

[0015] The terms "peptide" and "protein" of the present invention include substances containing not only amino acid components but also non-amino acid components such as sugar and phosphate structures, and also include substances containing bonds such as ester bonds, thioether bonds or disulfide bonds.

[0016] The term "nucleotide fragment encoding a peptide or protein" according to the present invention refers to a nucleotide fragment that, if present in an appropriate environment, preferably in a cell, can direct the translation of the assembly of amino acids to produce a peptide or protein.

[0017] The term "immune response" herein relates to the immune system's response, for example, to an immunogenic organism, such as a bacterium or virus, a cell, or a substance. The term "immune response" encompasses both innate and adaptive immune responses. Preferably, the immune response involves activation of immune cells, induction of cytokine biosynthesis, and / or antibody production. Preferably, the immune response includes the steps of activation of antigen-presenting cells, such as dendritic cells and / or macrophages, presentation of antigens or fragments thereof by the antigen-presenting cells, and activation of cytotoxic T cells as a result of this presentation.

[0018] In a first aspect, the present invention provides a nucleic acid molecule comprising 1) a transcribable nucleic acid segment or a nucleic acid for introducing a transcribable nucleic acid segment; 2) a nucleic acid segment comprising polydeoxyadenylic acid polyA; and 3) a nucleic acid segment comprising a gyrase binding site (SGS).

[0019] The transcribable nucleic acid fragment includes a nucleic acid fragment encoding a peptide or protein.

[0020] The nucleic acid for introducing a transcribable nucleic acid segment includes a multiple cloning site or a homologous recombination site.

[0021] The polyA comprises at least 50 consecutive deoxyadenylic acid nucleotides.

[0022] The polyA-containing nucleic acid fragment includes at least two elements with a length of at least 20 consecutive deoxyadenylic acid sequences, and at least one non-T deoxynucleotide is spaced between the two elements.

[0023] In some specific embodiments, the nucleic acid fragment containing the gyrase binding site is derived from a virus or a bacterium; preferably, the nucleic acid fragment containing the gyrase binding site is derived from a virus; more preferably, the nucleic acid fragment containing the gyrase binding site is derived from a bacteriophage.

[0024] In some specific embodiments, the nucleic acid fragment comprising the gyrase binding site is derived from a plasmid; preferably, the nucleic acid fragment comprising the gyrase binding site is derived from a ColE1 type plasmid.

[0025] In some embodiments, the SGS nucleic acid fragment is constructed upstream of the T7 promoter in the plasmid vector; in some embodiments, the SGS nucleic acid fragment is constructed downstream of Poly A in the plasmid vector. Those skilled in the art can select an appropriate position to insert SGS according to their needs based on the disclosure of this application.

[0026] On the other hand, the present invention provides a DNA expression vector or recombinant plasmid, wherein the DNA expression vector or recombinant plasmid sequence comprises 1) a first nucleic acid fragment encoding an mRNA molecule; 2) a second nucleic acid fragment comprising polyadenosine deoxyribonucleic acid polyA; and 3) a third nucleic acid fragment comprising a gyrase binding site.

[0027] The present invention also provides a method for preparing an expression vector or a recombinant plasmid, which comprises transforming the expression vector or recombinant plasmid into competent Escherichia coli cells, applying an appropriate amount of the expression vector or recombinant plasmid onto a resistant LB medium plate for culture, picking a monoclonal colony, sequencing it, obtaining a positive bacterial solution with correct sequencing, and carrying out large-scale culture.

[0028] The present invention provides a method for amplifying a nucleic acid molecule, comprising replicating the nucleic acid molecule in a host cell and propagating the host cell.

[0029] In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell; preferably, the host cell is Escherichia coli.

[0030] The present invention provides a method for obtaining RNA, comprising amplifying a nucleic acid molecule according to the above method, and using the nucleic acid molecule as a template to transcribe into RNA in vitro.

[0031] The present invention provides a method for obtaining a peptide or protein, comprising obtaining mRNA encoding the peptide or protein according to the above method for obtaining RNA, and translating the mRNA into peptide or protein amino acid fragments.

[0032] The present invention provides a system for producing eukaryotic translatable mRNA, comprising 1) a vector or plasmid containing the above nucleic acid molecule; and 2) a host cell.

[0033] On the other hand, the present invention provides a use of a nucleic acid nucleotide fragment encoding a gyrase binding site in constructing a plasmid or vector, wherein the plasmid or vector is used for in vitro transcription into RNA.

[0034] In another aspect, the present invention provides a composition comprising the RNA, peptide or protein obtained according to the above method.

[0035] In some embodiments, the composition comprises one or more of pharmaceutically acceptable excipients, carriers, buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivators.

[0036] The buffer of the present invention includes one or more of HEPES, HIS, TRIS, PB, succinic acid, and citric acid; the protective agent includes one or more of gelatin, ethanol, diethylaminotetraacetic acid (EDTA), disodium diethylaminotetraacetic acid (EDTA-2Na), and magnesium chloride; the stabilizer includes one or more of sucrose, mannitol, fucose, and maltose; the surfactant includes one or more of Tween, Span, and glycerol; the osmotic pressure regulator includes sodium chloride or is omitted; and the auxiliary material components include one or more of mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, and glycerol.

[0037] In some specific embodiments, the composition of the present invention can be prepared as a mucosal immune preparation, a humoral immune preparation, a cellular immune preparation, or a skin immune preparation.

[0038] In some specific embodiments, the composition of the present invention can be prepared as a liquid dosage form, a solid dosage form, a semisolid dosage form, a gas dosage form, or an inhalation dosage form.

[0039] In some specific embodiments, the composition is administered by intravenous injection, intramuscular injection, subcutaneous injection, oral administration, buccal administration, sublingual administration, rectal administration, respiratory tract administration, or transdermal administration. Respiratory tract administration is by inhalation through the mouth, inhalation through the nose, or inhalation after atomization by a nebulizer.

[0040] The nucleic acid molecules of the present invention, and plasmids or vectors containing the nucleic acid molecules, can be used to express recombinant proteins in cells for transcription and expression. More specifically, when producing recombinant proteins, the expression vectors of the present invention can be used to transcribe recombinant nucleic acids and express recombinant proteins in cell-based systems. For example, recombinant antibodies, hormones, cytokines, enzymes, etc. can be prepared.

[0041] The nucleic acid molecules, plasmids or vectors containing the nucleic acid molecules of the present invention can be used, for example, for transient expression of genes. Possible application areas are RNA-based vaccines, which are transfected into cells in vitro or administered directly to transiently express functional proteins in vivo or in vitro.

[0042] The present invention provides uses of the above-mentioned composition, including for use in medicine, for delivering transgenes into host cells, for eliciting a primary immune response in an animal to treat or prevent at least one disease, for boosting an immune response in an animal, and for inducing an immune response in an animal to break tolerance to self-antigens. Such immune responses include vaccines for cancer prevention, therapeutic tumor vaccines, and vaccines for preventing viral, bacterial, and fungal infections.

[0043] The present invention provides use of any of the above nucleic acid molecules, expression vectors or compositions in the preparation of drugs or vaccines for treating or preventing diseases.

[0044] In some embodiments, the disease is caused by one or more of the following viruses, bacteria, or protozoa: novel coronavirus, dengue virus, malarial parasite, herpes zoster virus, feline parvovirus, feline calicivirus, feline herpesvirus, feline leukemia virus, feline coronavirus, porcine reproductive and respiratory syndrome virus, herpes simplex virus type 1, herpes simplex virus type 2, encephalitis virus, papillomavirus, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, norwalk virus, coxsackievirus, hepatitis A virus, rhinovirus, severe acute respiratory syndrome virus, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2, SARS-CoV-2 Severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, hepatitis E virus, human immunodeficiency virus (HIV), influenza virus type A or type B, Guanarito virus, Junin virus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipah virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, Escherichia coli virus, human enterovirus, Japanese encephalitis virus, herpes simplex virus, Eastern equine encephalitis virus, hantavirus, Middle East respiratory coronavirus, chikungunya virus, or Banna virus.

[0045] The beneficial effects of the present invention include:

[0046] First, the stability of Poly A is improved through the SGS sequence to solve the problem of Poly A loss / shortening.

[0047] Secondly, the stability of nucleic acid molecules is improved through the SGS sequence.

[0048] Thirdly, the SGS sequence reduces the probability of plasmid recombination during amplification, further improving the safety of plasmid application.

[0049] Fourthly, through the SGS sequence, RNA molecules, peptides or proteins with higher purity that meet GMP-level production processes and drug regulatory requirements can be obtained, making them suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1. Effects of SGS sequences from different sources on the stability of polyA sequences under non-resistant conditions.

[0051] Figure 2. Effect of SGS sequences from different sources on the stability of polyA sequences under Kana resistance conditions.

[0052] Figure 3. Effect of SGS Mu398 sequence on the stability of PolyA of different length types under non-resistant conditions.

[0053] Figure 4. Effect of SGS Mu398 sequence on the stability of PolyA of different length types under Kana resistance conditions.

[0054] Figure 5. Applicability of the SGS Mu398 sequence to different target antigen sequences under resistance-free conditions.

[0055] Figure 6. Applicability of the Mu398 SGS sequence to different target antigen sequences under Kana resistance conditions.

[0056] Figure 7. Results of mRNA integrity test of the 10th generation of F001 recombinant plasmid transcription.

[0057] Figure 8. Results of mRNA integrity test of the 10th generation of F002 recombinant plasmid transcription.

[0058] Figure 9. Results of mRNA integrity test of the 10th generation of F003 recombinant plasmid transcription.

[0059] Figure 10. Results of mRNA integrity test of the 10th generation of F004 recombinant plasmid transcription.

[0060] Figure 11. Results of mRNA integrity test of the 10th generation of F005 recombinant plasmid transcription. DETAILED DESCRIPTION

[0061] Example 1 Effect of SGS sequences from different sources on the stability of plasmid polyA

[0062] To investigate the effect of the SGS sequence on plasmid stability, SGS sequences from different sources were inserted downstream of the vector polyA or upstream of the T7 promoter, where the polyA consisted of a continuous 100A sequence. The target gene sequence was also inserted into the plasmid vector to observe the effect of the SGS sequence on plasmid stability. The design methods are shown in Table 1. The results showed that the insertion of the SGS sequence significantly improved the passage stability of the polyA under both antibiotic-free and Kana-resistant conditions (Figures 1 and 2).

[0063] The transcription vector of the present invention comprises a 5'UTR sequence, a Kozak sequence, an antigen coding sequence, a 3'UTR sequence, a polyA tail sequence and an SGS sequence, wherein the sequences are synthesized into a pUC57-kana vector by Nanjing KingSher Biotechnology Co., Ltd., and then connected to a universal transcription vector in sequence by homologous recombination. The constructed plasmid is transferred into Trans1-Blue Escherichia coli competent cells, and an appropriate amount is applied to a Kana-resistant LB culture medium plate and cultured at 37°C for a certain period of time. Monoclonal colonies are picked and sequenced, and the positive bacterial solution with correct sequencing is preserved. Subsequently, the bacterial solution containing the positive plasmid is shaken at 37°C, and the plasmid is extracted to obtain an in vitro transcription template, and mRNA is prepared by co-transcription capping.

[0064] Table 1. Design methods of the effects of SGS sequence on plasmid stability

[0065] Example 2 Effect of SGS sequence on the stability of PolyA of different length types

[0066] To further verify whether the SGS sequence can improve the stability of PolyA sequences of different lengths, the Mu398 SGS sequence and different types of polyA sequences were inserted into the above-mentioned plasmid vectors to observe the stability of the polyA sequences. The specific design is shown in Table 2.

[0067] Table 2. Design methods for the effects of SGS sequences on the stability of PolyA of different length types

[0068] The results showed that the addition of the Mu398 SGS sequence could increase the stability of various types of polyA sequences and maintain their integrity for up to 10 generations (Figures 3 and 4).

[0069] Example 3 Determination of a universal high-stability plasmid vector

[0070] In order to further verify the versatility of the above-mentioned plasmid vector, the present invention inserted the target gene sequences from five different pathogens, including novel coronavirus (COVID-19), dengue virus (DENV), malaria, varicella zoster virus (VZV) and feline parvovirus (FPV), into the above-mentioned vector containing the SGS sequence, and constructed them into expression vectors F001, F002, F003, F004 and F005, respectively. The passage stability of 100A in a row during the passage of the plasmid was observed. The results showed that the SGS sequence can be applied to different target gene sequences and can ensure the passage stability of each recombinant plasmid polyA within 10 generations (as shown in Figures 5 and 6).

[0071] In order to further observe the transcription effect of the transcription vector, the 10th generation recombinant vector plasmid constructed above was subjected to in vitro transcription after large-scale plasmid extraction. The results showed that each plasmid could transcribe high-quality mRNA (as shown in Figures 7-11, and the data can be seen in Table 3).

[0072] Table 3. Test results data of Figures 7-11

[0073] In summary, the present invention provides a universal and highly stable plasmid vector, which has universal applicability and can solve the problem of plasmid instability caused by polyA sequences in the mRNA field.

Claims

1. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises 1) a transcribable nucleic acid segment and / or a nucleic acid for introducing a transcribable nucleic acid segment; 2) a nucleic acid segment comprising polydeoxyadenylic acid polyA; and 3) a nucleic acid segment comprising a gyrase binding site.

2. The nucleic acid molecule according to claim 1, wherein The transcribable nucleic acid segment encodes a peptide or protein.

3. The nucleic acid molecule according to claim 1 or 2, characterized in that The nucleic acid fragment for introducing a transcribable nucleic acid fragment comprises a multiple cloning site or a homologous recombination site.

4. The nucleic acid molecule according to any one of claims 1 to 3, characterized in that The polyA comprises at least 50 consecutive deoxyadenylic acid nucleotides.

5. The nucleic acid molecule according to any one of claims 1 to 4, characterized in that The polyA-containing nucleic acid fragment includes at least two elements with a length of at least 20 consecutive deoxyadenylic acid sequences, and at least one non-T deoxynucleotide is spaced between the two elements.

6. The nucleic acid molecule according to any one of claims 1 to 5, characterized in that The nucleic acid fragment containing the gyrase binding site is derived from a virus or a bacterium; preferably, the nucleic acid fragment containing the gyrase binding site is derived from a virus; more preferably, the nucleic acid fragment containing the gyrase binding site is derived from a bacteriophage.

7. The nucleic acid molecule according to any one of claims 1 to 5, characterized in that The nucleic acid fragment containing the gyrase binding site is derived from a plasmid; preferably, the nucleic acid fragment containing the gyrase binding site is derived from a ColE1 type plasmid.

8. A plasmid vector, characterized in that The plasmid vector comprises 1) a first nucleic acid segment encoding an mRNA molecule; 2) a second nucleic acid segment comprising polyA; and 3) a third nucleic acid segment comprising a gyrase binding site.

9. A method for preparing a plasmid vector, characterized in that: The plasmid vector as claimed in claim 8 is transformed into competent E. coli cells, and an appropriate amount is applied to a resistant LB medium plate for culture, and a monoclonal colony is picked and sequenced to obtain a positive bacterial solution with correct sequencing, and then large-scale culture is performed.

10. A method for amplifying a nucleic acid molecule, characterized in that: The method comprises propagating the nucleic acid molecule of claims 1 to 7 in a host cell and propagating the host cell, wherein the host cell comprises bacteria or fungi; the host cell is preferably Escherichia coli.

11. A method for obtaining RNA, characterized in that: The method comprises amplifying a nucleic acid molecule according to the method of claim 10, and using the nucleic acid molecule as a template to transcribe into RNA in vitro.

12. A method for obtaining a peptide or protein, characterized in that: The method comprises obtaining mRNA encoding a peptide or protein according to the method of claim 11, and translating the mRNA into peptide or protein amino acid fragments.

13. A system for producing eukaryotic translatable mRNA, characterized in that: The invention comprises 1) a plasmid vector containing the nucleic acid molecule according to claims 1 to 7; and 2) a host cell.

14. Use of a nucleic acid fragment encoding a gyrase binding site in constructing a plasmid vector, characterized in that: The plasmid vector is used for in vitro transcription into RNA.

15. A composition, characterized in that Comprising RNA, peptide or protein obtained according to the method of claim 11 or 12.

16. The composition according to claim 15, characterized in that The composition comprises one or more of pharmaceutically acceptable auxiliary materials, carriers, buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivators.

17. Use of the nucleic acid molecule according to any one of claims 1 to 7, the plasmid vector according to claim 8 or the composition according to any one of claims 15 to 16 in the preparation of a drug or vaccine for treating or preventing a disease.

18. The use according to claim 17, characterized in that The disease is caused by one or more of the following viruses, bacteria or protozoa: novel coronavirus, dengue virus, malarial parasite, herpes zoster virus, feline parvovirus, feline calicivirus, feline herpesvirus, feline leukemia virus, feline coronavirus, porcine reproductive and respiratory syndrome virus, herpes simplex virus type 1, herpes simplex virus type 2, encephalitis virus, papillomavirus, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, West Nile virus, rubella virus, hepatitis E virus, human immunodeficiency virus (HIV), influenza virus type A or type B, Guanarito virus, Hu Nin virus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipah virus, rabies virus, hepatitis D virus, rotavirus, orbivirus, Escherichia coli virus, human enterovirus, Japanese encephalitis virus, herpes simplex virus, Eastern equine encephalitis virus, Hantavirus, Middle East respiratory coronavirus, Chikungunya virus, or Banna virus.

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