Recombinant porcine rotaviruses and methods and systems for producing and using the same

Abstract

Disclosed are polynucleotides, and collections of polynucleotides, encoding segments of novel recombinant OSU strain rotaviruses and methods of making the same, systems for generating the same, as well as methods of using the same to elicit an immune response in a subject or to vaccinate a subject against rotavirus infection and infection.

Description

RECOMBINANT PORCINE ROTAVIRUSES AND METHODS AND SYSTEMS FORPRODUCING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 612.703, filed December 20, 2023, the content of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.SEQUENCE LISTING(0003] A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named “144578_00429.xml” which is 54,577 bytes in size and was created on December 17, 2024. The sequence listing is electronically submitted via Patent Center and is incorporated by reference herein in its entirety.BACKGROUND

[0004] Rotaviruses are a significant cause of severe potentially life-threatening gastroenteritis in the young of many economically important farm animals, including piglets. Although porcine rotavirus vaccines have been developed, both live oral and killed varieties, existing vaccines are generally ineffective in preventing rotavirus gastroenteritis in piglets. Therefore, there is a need in the art for novel rotavirus vaccines for porcine subjects, e.g., piglets.SUMMARY

[0005] In an aspect of the current disclosure, polynucleotides are provided. In some embodiments, the polynucleotides comprise a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments.the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises a sequence encoding an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.10006] In an aspect of the current disclosure, collections of polynucleotides are provided. In some embodiments, each of the polynucleotides in the collection comprises a sequence encoding at least one OSU strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4. and NSP5, wherein the polynucleotides of the collection encode each of the VP1, VP2. VP3, VP4, VP6, VP7, NSP1. NSP2, NSP3. NSP4, and NSP5 proteins, wherein each of the sequences encoding one rotavirus protein are operably linked to a promoter. In some embodiments, the sequences encoding at least one rotavirus protein selected from VP1, VP2, VP3, VP4, VP6,VP7, NSP1, NSP2, NSP3, NSP4. and NSP5 comprise SEQ ID NOs: 1-11, respectively. In some embodiments, the sequences encoding at least one rotavirus protein selected from VP1, VP2. VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, andNSP5 consist of SEQ ID NOs: 1-11, respectively. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.

[0007] In an aspect of the current disclosure, infectious particles are provided. In some embodiments, the infectious particles comprise a sequence encoding a recombinant OSU strainrotavirus protein, wherein the sequence encodes a positive-sense viral transcript. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.

[0008] In some embodiments, the infectious particles are made by transfecting cells with a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In someembodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.

[0009] In an aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one ofSEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea vims (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E vims (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E vims (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E vims (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.[001 Oj A method comprising: administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript to a subject. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide compnses any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises aheterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroententis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the subject is a pig. In some embodiments, the subject is a piglet, optionally, wherein the piglet is has not yet been weaned.[00111 In an aspect of the current disclosure, methods of eliciting an immune response to one or more microorganism in a subject are provided. In some embodiments, the methods comprise: administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript to a subject to elicit an immune response to the one or more microorganism. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1- 14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments,the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea vims (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E vims (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E vims (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E vims (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the one or more microorganisms comprises hepatitis E virus (HEV). In some embodiments, the one or more microorganisms comprises rotavirus and hepatitis E vims (HEV). In some embodiments, the subject is a pig. In some embodiments, the subject is a piglet, optionally, wherein the piglet is has not yet been weaned.

[0012] In some embodiments, methods are provided and comprise administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript to a subject. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positivesense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E vims (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E vims (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving pepti e sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the subject is a pig. In some embodiments, the subject is a piglet, optionally, wherein the piglet is has not yet been weaned.

[0013] In an aspect of the current disclosure, methods are provided and comprise: administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript to a subject to elicit an immune response in the subject to a pathogen or vaccinate the subject against one or more pathogens. In some embodiments, the recombinant OSU strainrotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea vims (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E vims (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E vims (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E vims (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the subject is a pig. In some embodiments, the subject is a piglet, optionally, wherein the piglet is has not yet been weaned.

[0014] In an aspect of the current disclosure, methods of vaccinating a subject against one or more pathogens are provided. In some embodiments, the methods comprise: administering a pharmaceutical composition comprising an infectious particle comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viraltranscript to a subject to vaccinate a subject against the one or more pathogens. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the one or more pathogens comprises hepatitis E virus (HEV). In some embodiments, the one or more pathogens comprises OSU strain rotavirus and hepatitis E virus (HEV). In some embodiments, the subject is a pig. In some embodiments, the subject is a piglet, optionally, wherein the piglet is has not yet been weaned.[00151 In an aspect of the current disclosure, cells are provided. In some embodiments, the cells comprise a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript or an infectious particle comprising a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.[00161 In some embodiments, the cells comprise a collection of polynucleotides, wherein each of the polynucleotides in the collection comprises a sequence encoding at least one OSU strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP 1, NSP2, NSP3, NSP4, and NSP5, wherein the polynucleotides of the collection encode each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins, wherein each of the sequences encoding one rotavirus protein are operably linked to a promoter. In some embodiments, the sequences encoding at least one rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 comprise SEQ ID NOs: 1-11, respectively. In some embodiments, the sequences encoding at least one rotavirus protein selected from VP 1 , VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, andNSP5 consist of SEQ ID NOs: 1-11, respectively. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavagesite is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the cell is an MA- 104 cell, a Vero cell or a BHK-1 cell. In some embodiments, the cell expresses a heterologous RNA polymerase. In some embodiments, the heterologous RNA polymerase is selected from T7 RNA polymerase and T3 RNA polymerase.

[0017] In an aspect of the current disclosure, methods of generating an OSU strain rotavirus in vitro are provided. In some embodiments, the methods comprise: introducing a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript into a cell; allowing the cell to express one or more rotavirus proteins selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5; incubating the cells for a sufficient time to produce rotavirus; and harvesting virus produced by the cells to generate the rotavirus in vitro. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7J genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide isoperably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the cell is an MA- 104 cell, a Vero cell or a BHK-1 cell. In some embodiments, the cell expresses a heterologous RNA polymerase. In some embodiments, the heterologous RNA polymerase is selected from T7 RNA polymerase and T3 RNA polymerase.

[0018] A method of generating an OSU strain rotavirus in vitro comprising: introducing a collection of polynucleotides, wherein each of the polynucleotides in the collection comprises a sequence encoding at least one OSU strain rotavirus protein selected from VP1, VP2. VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5, wherein the polynucleotides of the collection encode each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins, wherein each of the sequences encoding one rotavirus protein are operably linked to a promoter into a cell; incubating the cells for a sufficient time to produce rotavirus; and harvesting virus produced by the cells to generate the rotavirus in vitro. In some embodiments, the sequences encoding at least one rotavirus protein selected from VP1 , VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 comprise SEQ ID NOs: 1-11, respectively. In some embodiments, the sequences encoding at least one rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4. and NSP5 consist of SEQ ID NOs: 1-11, respectively. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, theheterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker. In some embodiments, the cell expresses a heterologous RNA polymerase. In some embodiments, the heterologous RNA polymerase is selected from T7 RNA polymerase and T3 RNA polymerase. In some embodiments, the cell comprises T7 RNA polymerase and, optionally, comprising African sw ine fever virus capping enzyme. In some embodiments, the cell is selected from an MA-104 cell, a Vero cell, and a BHK-1 cell. The method of claim 50, wherein the cell is a BHK-1 cell comprising T7 RNA polymerase and, optionally, comprising African swine fever virus capping enzyme.

[0019] In an aspect of the current disclosure, systems for generating recombinant rotavirus are provided. In some embodiments, the systems comprise: (a) a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript into a cell; and (b) cells capable of expressing the polynucleotide of (a). In some embodiments, the cells comprise a heterologous RNA polymerase and, optionally, comprising African swine fever virus capping enzy me. In some embodiments, the cells comprisea cells from a cell line selected from MA-104 cells, Vero cells, and BHK-1 cells. In some embodiments, the cells comprise BHK-1 cells comprising T7 RNA polymerase. In some embodiments, the cells comprise BHK-1 cells comprising T7 RNA polymerase, Vero cells, and MA-104 cells. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1- 14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7 promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.

[0020] In some embodiments, the systems for generating recombinant rotavirus comprise: (a) a collection of polynucleotides, wherein each of the polynucleotides in the collection comprises asequence encoding at least one OSU strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4. and NSP5, wherein the polynucleotides of the collection encode each of the VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins, wherein each of the sequences encoding one rotavirus protein are operably linked to a promoter; and (b) cells capable of expressing the collection of (a). In some embodiments, the cells comprise a heterologous RNA polymerase and. optionally, comprising African swine fever virus capping enzyme. In some embodiments, the cells comprise a cells from a cell line selected from MA-104 cells, Vero cells, and BHK-1 cells. In some embodiments, the cells comprise BHK-1 cells comprising T7 RNA polymerase. In some embodiments, the cells comprise BHK-1 cells comprising T7 RNA polymerase, Vero cells, and MA-104 cells. In some embodiments, the sequences encoding at least one rotavirus protein selected from VP1, VP2, VP3, VP4. VP6. VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 comprise SEQ ID NOs: 1-11, respectively. In some embodiments, the sequences encoding at least one rotavirus protein selected from VP 1 , VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, andNSP5 consist of SEQ ID NOs: 1-11, respectively. In some embodiments, the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein. In some embodiments, the polynucleotide encodes a positive sense viral transcript. In some embodiments, the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to one of SEQ ID NOs: 1-14. In some embodiments, the polynucleotide comprises an OSU strain NSP3 protein. In some embodiments, the polynucleotide further comprises a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF. In some embodiments, the heterologous polynucleotide encodes a peptide or protein. In some embodiments, the heterologous polynucleotide encodes a reporter. In some embodiments, the peptide or protein comprises a microorganismal peptide or protein. In some embodiments, the peptide or protein comprises a viral peptide or protein. In some embodiments, the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein. In some embodiments, the hepatitis E virus (HEV) protein comprises an HEV capsid protein. In some embodiments, the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19. In some embodiments, the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20. In some embodiments, the polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a T7promoter, optionally wherein the T7 promoter comprises SEQ ID NO: 17. In some embodiments, the promoter is a T3 promoter, optionally wherein the T3 promoter comprises SEQ ID NO: 18. In some embodiments, the polynucleotide comprises a sequence encoding a cleavage site. In some embodiments, the cleavage site is a protease cleavage site. In some embodiments, the cleavage site is a thrombin cleavage site (SEQ ID NO: 15). In some embodiments, the cleavage site is a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16). In some embodiments, the polynucleotide comprises a sequence encoding a linker. In some embodiments, the linker is a flexible linker selected from a GAG linker or a GSG linker.BRIEF DESCRIPTION OF THE FIGURES[00211 FIG. 1 shows a schematic of the vector pT7 / OSU NSP3. The full-length cDNA of rOSU gene segment 7 (encodes for NSP3) is positioned within a pT7 plasmid, which is ligated upstream with a promoter for T7 polymerase and downstream with a hepatitis delta virus (HDV) ribozyme. In the presence of the T7 RNA polymerase, the OSU pT7 plasmid produces a full-length gene segment 7 (+)RNA with authentic 5’ and 3’ termini.

[0022] FIG. 2 shows a schematic of the vector pT7 / OSU NSP3-2A. The full-length cDNA of rOSU gene segment 7 (encodes for NSP3) is positioned within a pT7 plasmid, which is ligated upstream with a promoter for T7 polymerase and downstream with a hepatitis delta virus (HDV) ribozyme. In the presence of the T7 RNA polymerase, the OSU pT7 plasmid produces a full- length gene segment 7 (+)RNA with authentic 5’ and 3‘ termini. The porcine teschovirus 2A-like (2A) element is positioned at the 3‘ end of the NSP3 coding sequence. The rOSU pT7 gene segment 7 plasmid that expresses NSP3-2A was generated with in-fusion cloning[002 [ FIG. 3 shows a schematic of the vector pT7 / OSUNSP3-2A-fUnaG. The full-length cDNA of rOSU gene segment 7 (encodes for NSP3) is positioned within a pT7 plasmid, which is ligated upstream with a promoter for T7 polymerase and downstream with a hepatitis delta virus (HDV) ribozyme. In the presence of the T7 RNA polymerase, the OSU pT7 plasmid produces a full- length gene segment 7 (+)RNA with authentic 5’ and 3’ termini. The porcine teschovirus 2A-like (2A) element is positioned at the 3’ end of the NSP3 coding sequence followed by the in-frame coding sequence of the FLAG-tagged UnaG (fUnaG). Due to the activity7of the 2A element, translation of the RNA produces two proteins. NSP3 with remnants of the 2A element and thefUnaG. The rOSU pT7 gene segment 7 plasmid that expresses NSP3-2A-fUnaG was generated with in-fusion cloning

[0024] FIG. 4 shows a schematic of the vector pT7 / OSU NSP3-2A-HEV CP. The full-length cDNA of rOSU gene segment 7 (encodes for NSP3) is positioned within a pT7 plasmid, which is ligated upstream with a promoter for T7 polymerase and downstream with a hepatitis delta virus (HDV) riboz me. In the presence of the T7 RNA polymerase, the OSU pT7 plasmid produces a full-length gene segment 7 (+)RNA with authentic 5’ and 3’ termini. The porcine teschovirus 2A- like (2A) element is positioned at the 3’ end of the NSP3 coding sequence lol I ow ed by the inframe coding sequence of the Hepatitis E virus (HEV) capsid protein (CP). Due to the activity of the 2A element, translation of the RNA produces two proteins. NSP3 with remnants of the 2A element and the HEV CP. The rOSU pT7 gene segment 7 plasmid that expresses NSP3-2A-HEV CP w as generated with in-fusion cloning.

[0025] FIG. 5 shows a polyacrylamide gel loaded with RNA demonstrating the genome profiles of rOSU, rOSU-2A, rOSU-2A-fUnaG, and rOSU-2A-HEV CP compared to rSAl l and MAI 04 cell culture-adapted OSU. Viral dsRNA was recovered from MA104 cells infected with rSAl 1, MAI 04 cell culture-adapted OSU, rOSU, or modified rOSU isolates, resolved on a 10% polyacrylamide gel by electrophoresis, and detected by ethidium-bromide staining on a Bio-Rad ChemiDoc Imaging System. The migration of rSAl 1 RNA segments is indicated on the left side of the panel and the migration of rOSU RNA segments is indicated on the right side of the panel. The migration of modified rOSU gene segments 7 is indicated with red arrows.

[0026] FIG. 6 shows fluorescent images showing the expression of fUnaG. MAI 04 cells were infected with rOSU, rOSU-2A, or rOSU-2A-fUnaG at a multiplicity of infection of 5 plaque forming units per cell. At 9 hours post infection, the cells w ere imaged using a ZOE Fluorescent Cell Imager under the brightfield and green (excitation: 480, emission: 517) channels. The scale bars represent 100 pm.

[0027] FIG. 7 shows Western blots showing the expression of fUnaG and the activity of the 2A translational stop-start element. MA104 cells were infected with rOSU, rOSU-2A, or rOSU-2A- fUnaG at a multiplicity of infection of 5 plaque forming units per cell. At 9 hours post infection, the cells were lysed and resolved on a 10% polyacrylamide gel by electrophoresis. The resolved lysates were transferred to a nitrocellulose membrane and probed with FLAG M2 antibody (F 1804, Sigma, 1:2,000), 2A antibody (NBP2-59627, Novus, 1: 1,000), rotavirus NSP3 antibody (lot55068, 1 :2,000), rotavirus VP6 antibody (lot 53963, 1 :2,000), or P-actin antibody (8H10D10, Cell Signaling Technology, 1:2,000). The bound primary antibodies were detected using 1: 10.000 dilutions of horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-mouse IgG [CST], goat anti -guinea pig IgG [KPL], or goat anti-rabbit IgG [CST], HRP signals were developed using the Bio-Rad Clarity Western ECL substrate and developed using a Bio-Rad ChemiDoc imaging system. The migration of NSP3-2A-fUnaG, fUnaG, NSP3-2A, VP6, and - actin are indicated on the left side of the panels.[00281 FIGs. 8A and 8B show production of OSU G5P[7] porcine rotavirus. (A) Rotavirus reverse genetics system

[0029] , Recombinant rotavirus was prepared by transfecting BHK-T7 cells with 11 T7 plasmids, which contain full-length cDNAs of rotavirus genome segments, and the CMV-NP868R plasmid, which encodes the African Swine Fever Virus capping enzyme

[0028] , The BHK-T7 cells were overseeded 2 days post-transfection with MA104 cells to facilitate the spread and amplification of recombinant rotavirus. At 3 days post-overseeding, recombinant virus in the cells lysates was amplified on MAI 04 cells. The amplified virus was analyzed by RNA gel electrophoresis followed by plaque purification. Image was adapted from

[0029] (B) Recovery’ of recombinant SAI 1 / OSU monoreassortants by reverse genetics. Viral dsRNAs from rSAl 1, rOSU, and 11 rSAl 1 / OSU monoreassortants were resolved by electrophoresis on an 8% polyacrylamide gel and stained with ethidium bromide. The migrations of rOSU gene segments in rSAl 1 / OSU monoreassortants are indicated with red arrows. Genome segments 1-11 of rSAl l are indicated on the left side of the panel. Genome segments 1-11 of rOSU are indicated on the right side of the panel.

[0029] FIG. 9A, 9B, and 9C show production of recombinant OSU that encodes a foreign protein. (A) Modifications of rotavirus genome segment 7. The schematics indicate the nucleotide positions of the coding sequences for NSP3, the porcine teschovirus 2A element, 3X FLAG, and the fluorescent reporter UnaG (green). The red arrows indicate the positions of the 2A translational stop-restart elements, and the asterisks indicate the ends of the open reading frames. (B) Recovery of recombinant OSU-2A-UnaG by reverse genetics. Viral dsRNAs from OSU-tc_(MA104), rOSU, rOSU-2A, and rOSU-2A-UnaG were resolved by electrophoresis on an 8% polyacrylamide gel and stained with ethidium bromide. The migrations of modified genome segment 7 are indicated with red arrows. Genome segments 1-11 of rOSU are indicated on the left side of the panel. (C) Genetic stability. rOSU-2A and rOSU-2A-UnaG were serially passaged onMA104 cells. Viral dsRNAs from a total of 5 passages (P) were resolved by electrophoresis on an 8% polyacrylamide gel and stained with ethidium bromide. The migrations of modified genome segment 7 are indicated with red arrow. Genome segments 1-11 of rOSU are indicated on the left side of the panels.

[0030] FIGs. 10A, 10B, 10C, 10D, 10E, and 10F show characterization of recombinant OSU that expresses a foreign protein. (A and B) Production of infectious virus. MAI 04 cells were infected with the indicated viruses at an MOI of 5 PFU / cell. In panel A, titers were determined by plaque assay at the indicated times post infection. In panel B, titers were determined by plaque assay upon complete cytopathic effect (typically 3-5 days). Error bars indicate the standard deviations (A); horizontal bars indicate the means (B); in panel A, statistical analyses show the comparisons between OSU-tc (MA104) and rOSU, rOSU-2A, and rOSU-2A-UnaG; *, P < 0.05 (n = 3 biological replicate). (C and D) Plaque morphologies and sizes. In panel C, plaques on MA 104 cells were detected by crystal violet staining. In panel D, plaque diameters were measured using ImageJ software (30). 50 plaques were measured for each virus; *, P < 0.05 (n = 3 biological replicates). (E and F) Fluorescent reporter expression and activity of the 2A translational stoprestart element. In panel E, MAI 04 cells were infected with the indicated viruses at an MOI of 5 PFU / cell. At 9 hours post infection, infected cell lysates were prepared and analyzed by immunoblot. The migrations of molecular w eight markers are indicated on the left side of the panels. The migrations of NSP3-2A-FLAG-UnaG, FLAG-UnaG. and NSP3-2A are indicated on the right side of the panels (n = 3 biological replicates). In panel F, MAI 04 were infected with the indicated viruses at an MOI of 0.05 PFU / cell. At 28 hours post infection, the infected cells were imaged at 10X magnification under phase and green channels. Scale bars represent 400 pm (n = 3 biological replicates).DETAILED DESCRIPTION[00311 Disclosed herein are polynucleotides that encode a segment of the OSU strain of rotavirus and can be used in a reverse genetics system to generate recombinant OSU strain rotaviruses, and collections of polynucleotides. Also disclosed herein are methods of using the polynucleotides, or collections, to generate recombinant OSU strain rotaviruses, pharmaceutical compositions comprising the recombinant rotaviruses, and methods of using the same to elicit an immune response and / or to vaccinate a subject against porcine rotavirus strains, e.g.. OSU strain.[0032 [ The inventors discovered that the NSP3 segment of the OSU strain rotavirus may further comprise a heterologous polynucleotide sequence, e.g., encoding a peptide or protein from another source, e.g., a pathogen.|OO33] The disclosed polynucleotides, collections, methods, recombinant rotaviruses, and pharmaceutical compositions may be useful to vaccinate piglets, e.g., pre-weaning piglets, against the OSU strain of rotavirus, as well as one or more additional pathogens, e.g., HEV, porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus, African swine fever virus (ASFV), porcine enteric caliciviruses, pseudorabies, classical swine fever, bacterial pathogens, e.g., swine brucellosis, and / or a coronavirus.Polynucleotides

[0034] Accordingly, in a first aspect, polynucleotides comprising a sequence encoding a recombinant rotavirus protein are disclosed herein.[00351 As used herein, ‘'OSU” or “OSU strain” refers to a Rotavirus A strain with a G5P[7] genotype, the genotype associated with porcine rotaviruses that most frequently cause disease in piglets. In contrast, the instant disclosure provides “recombinant OSU” rotaviruses produced by the disclosed reverse genetics systems, which are distinguished from cell-culture adopted OSU strains isolated by traditional means from OSU-infected animals, i.e., by reproducing virus recovered from animals on suitable host cells.

[0036] As used herein, an “OSU strain rotavirus protein” refers to a protein derived from the OSU strain of rotavirus.

[0037] In some embodiments, the polynucleotides are operably linked to a promoter to allow the expression of said polynucleotides in a cell, e.g., a mammalian cell.[0038| As used herein, “operably linked” refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, this refers to the functional relationship of transcriptional regulatory element (promoter) to a transcribed sequence. For example, a promoter is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate cell. Generally, promoter transcriptional regulatory elements that are operably linked to a sequence are physically contiguous to the transcribed sequence, i.e., they are cis acting. However, some transcriptional regulatory elements, such as enhancers, need not bephysically contiguous or located in close proximity to the coding sequences whose transcription they enhance. Exemplary promoters include a T7 bacteriophage promoter (SEQ ID NO: 17) and a T3 bacteriophage promoter (SEQ ID NO: 18). A suitable promoter may be chosen from promoters known in the art. In some embodiments, the cells are mammalian cells and are selected from MA-104 cells, Vero cells and BHK-1 cells.

[0039] In some embodiments, the polynucleotides comprise a sequence encoding a rotaviral protein, i.e., are selected from a sequence encoding rotavirus VPI, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5. The sequences encoding the OSU strain VPI, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5 proteins are provided herein as SEQ ID NOs: 1-11, respectively. Thus, the inventors contemplate that the disclosed polynucleotides may comprise a sequence comprising any one of SEQ ID NOs: 1-11, or functional variants thereof (e.g., nucleic acid sequence variants that encode the same amino acid due to the redundancy in the genetic code, for example, or variants that result in different amino acid sequence(s) but encode a protein or polypeptide having the same function), or variants having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%. at least about 86%, at least about 87, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to any one of the polypeptides encoded by SEQ ID NOs: 1-11.

[0040] The inventors discovered that a reverse genetics system can be used to generate OSU strain rotavirus expressing heterologous polynucleotide sequences by fusing the heterologous polynucleotide sequences to the sequence encoding the rotaviral NSP3 protein. Thus, the inventors disclose herein that, in some embodiments, the disclosed polynucleotides comprise a sequence encoding NSP3, e.g.. (SEQ ID NO: 9), and farther comprises a heterologous polynucleotide sequence.

[0041] The heterologous polynucleotide may encode a viral protein or peptide, e.g., hepatitis E virus (HEV) protein, whose amino acid sequence is SEQ ID NO: 19, or a sequence with 85% similarity, 86% similarity, 87% similarity, 88% similarity, 89% similarity, 90% similarity. 91% similarity, 92% similarity, 93% similarity, 94% similarity, 95% similarity, 96% similarity. 97% similarity, 98% similarity’, or 99% similarity to SEQ ID NO: 19, which may be encoded by SEQ ID NO: 20, respectively, or a sequence with 85% similarity’, 86% similarity, 87% similarity, 88%similarity, 89% similarity, 90% similarity, 91% similarity, 92% similarity, 93% similarity, 94% similarity, 95% similarity, 96% similarity, 97% similarity. 98% similarity, or 99% similarity to SEQ ID NO: 19 (see Table 1 for full sequences).Table 1. Exemplary heterologous amino acid sequences encoded by heterologous polynucleotides.

[0042] In some embodiments, the heterologous polynucleotide encodes a protein or peptide. In some embodiments, the sequence encoding NSP3, e.g., SEQ ID NO: 9, further comprises the heterologous polynucleotide fused to the 3 ’ end of the sequence such that the heterologous polynucleotide encodes a protein or peptide in frame with the NSP3 sequence, thereby allowing transcription of a single mRNA that encodes both NSP3 and the heterologous polynucleotide.10043 In some embodiments, the polynucleotide comprising a sequence encoding NSP3 and a heterologous polynucleotide comprise a sequence encoding a cleavage site. In some embodiments, the cleavage site is a self-cleaving peptide, e.g., porcine teschovirus P2A element (SEQ ID NO: 16). Thus, in some embodiments, the disclosed polynucleotides comprise, from 5‘ to 3’, apolynucleotide encoding NSP3, fused in-frame to a sequence encoding a self-cleaving peptide which is fused in-frame to a heterologous polynucleotide sequence encoding a peptide or protein. Accordingly, transcription and translation of such polynucleotides results in production of a fusion protein comprising, from N- to C-terminus, a rotavirus NSP3 protein fused to a self-cleaving peptide, e.g., SEQ ID NO: 16, which is fused to a peptide or protein encoded by the heterologous polynucleotide, in a cell; following translation, the fusion protein self-cleaves resulting in two separate proteins (1) a functional rotavirus NSP3 protein and (2) the protein or peptide encoded by the heterologous polynucleotide.

[0044] In some embodiments, the polynucleotides further comprise a sequence encoding a linker, e.g., a flexible linker located 3’ to, and in frame with, the sequence encoding NSP3 protein and 5’ to a cleavage site. Without being limited by any theory or mechanism, the addition of a flexible linker between the NSP3 protein and the cleavage site may improve cleavage. In some embodiments, the linker is a (GAG)nlinker (also referred to as a GAG linker), wherein n=l, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, or a (GSG)n linker (also referred to as a GSG linker), wherein n=l, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0045] In some embodiments, the sequence encoding a cleavage site encodes a protease cleavage site, e.g., a thrombin cleavage site, e.g., SEQ ID NO: 15.|0046] The heterologous polynucleotide sequence described above may comprise sequences encoding proteins or peptides derived from infectious organisms, e.g., HEV, porcine epidemic diarrhea virus (PEDV). porcine transmissible gastroenteritis virus (TGEV). porcine reproductive and respirator}' syndrome virus (PRRSV), swine influenza virus, African swine fever virus (ASFV), or a coronavirus. Therefore, in some embodiments, the disclosed compositions comprise sequences encoding rotavirus NSP3 fused, in-frame, to a heterologous polynucleotide encoding a porcine hepatitis E virus (HEV) peptide or protein, porcine epidemic diarrhea virus (PEDV) peptide or protein, porcine transmissible gastroenteritis virus (TGEV) peptide or protein, porcine reproductive and respiratory syndrome virus (PRRSV) peptide or protein, swine influenza virus peptide or protein, African swine fever virus (ASFV) peptide or protein, or coronavirus peptide or protein.

[0047] In some embodiments, the polynucleotides comprise a polynucleotide comprising a sequence encoding a recombinant rotavirus NSP3, e.g., SEQ ID NO: 9, wherein the polynucleotide further comprises a heterologous polynucleotide in frame with the sequenceencoding a recombinant rotavirus NSP3, wherein the heterologous polynucleotide encodes a peptide or protein comprising an HEV peptide or protein, or a fragment thereof. In some embodiments, the polynucleotides further comprise a sequence encoding a self-cleaving peptide, e.g., a sequence encoding SEQ ID NO: 16, fused, in-frame, between the polynucleotide and the heterologous polynucleotide. Therefore, transcription and translation of said composition results in, with reference to an HEV peptide being selected as the heterologous peptide encoded by the heterologous polynucleotide, from N- to C-terminus, a functional rotavirus NSP3 protein, selfcleaving peptide, e.g., SEQ ID NO: 16, and an HEV protein or peptide, e g., HEV capsid, e.g., SEQ ID NO: 19, or a fragment thereof.100481 SEQ ID NO: 12 encodes NSP3 fused in-frame to a P2A sequence (SEQ ID NO: 16). SEQ ID NO: 13 encodes NSP3 fused in-frame to a P2A sequence and the reporter fUnaG. SEQ ID NO: 14 encodes NSP3 fused in-frame to a P2A sequence and HEV capsid protein. The polynucleotides may comprise one of SEQ ID NOs: 12-14, or a sequence with at least 85% similarity, at least 86% similarity, at least 87% similarity, at least 88% similarity', at least 89% similarity, at least 90% similarity, at least 91% similarity, at least 92% similarity, at least 93% similarity, at least 94% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity, or at least 99% similarity’ to SEQ ID NOs: 12-14.|0049] Sequences of the instant disclosure include sequences with either a particular amount of “similarity'’ or “identity ” to the disclosed sequences.Infectious particles

[0050] In another aspect of the current disclosure, infectious particles are provided. In some embodiments, the infectious particles comprise a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, e.g., one of SEQ ID NOs 1-14, or a sequence with at least 85% similarity, at least 86% similarity, at least 87% similarity’, at least 88% similarity’, at least 89% similarity’, at least 90% similarity, at least 91% similarity, at least 92% similarity, at least 93% similarity, at least 94% similarity, at least 95% similarity, at least 96% similarity, at least 97% similarity, at least 98% similarity, or at least 99% similarity to one of SEQ ID NOs: 1- 14.

[0051] The disclosed polynucleotides differ from naturally occurring OSU strain disclosed in, e.g., Guo et al. “Amino Acid Substitutions in Positions 385 and 393 of the Hydrophobic Regionof VP4 May Be Associated with Rotavirus Attenuation and Cell Culture Adaptation.” Viruses 2020, 72(4), 408. which is incorporated herein by reference in its entirety. See Tables 2 and 3 below.Table 2. Sequence similarity7of disclosed sequences to those taught in Guo et al.Table 3. Portions of OSU sequences absent from those disclosed in Guo et al. but present in the disclosed sequences.[00521 As used herein, "infectious particles” refers to any particle capable of causing an infection of a cell or an organism. Exemplary infectious particles include, but are not limited to, viralparticles, or virions and the like. The terms “virus,” “viral particle,” and “virion” are used interchangeably herein.[00531 Without wishing to be limited, the instant disclosure provides polynucleotides comprising sequences encoding rotavirus proteins operably linked to a promoter, e.g., a T7 promoter (SEQ ID NO: 16). In some embodiments, the polynucleotides may be used in a reverse genetics approach to generate recombinant rotavirus, e.g., recombinant rotavirus strain OSU. Thus, in some embodiments, the recombinant rotavirus may comprise the disclosed polynucleotides.[0054| The disclosed infectious particles, e.g., recombinant rotaviruses, may comprise one or more heterologous proteins or peptides, e.g., a porcine hepatitis E virus (HEV) peptide or protein, porcine epidemic diarrhea virus (PEDV) peptide or protein, porcine transmissible gastroenteritis virus (TGEV) peptide or protein, porcine reproductive and respiratory syndrome virus (PRRSV) peptide or protein, swine influenza virus peptide or protein, African swine fever virus (ASFV) peptide or protein, or coronavirus peptide or protein, as described above. The heterologous proteins or peptides may be encoded in the infectious particle, e.g., viral genome, and subsequently produced during viral replication. Such infectious particles comprising a heterologous protein or peptide may be advantageous in eliciting an immune response in a subject or as a vaccine composition.Pharmaceutical compositions[0055| The inventors disclose herein compositions, methods, and systems useful in making recombinant rotavirus that may be suitable for administration to subjects. Therefore, in another aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise an infectious particle comprising a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein. In some embodiments, the pharmaceutical compositions comprise an infectious particle made by transfecting a cell with a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.| 056] The disclosed compositions and methods may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the disclosed compositions. Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administeredpharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed composition, which effective amount is related to the daily dose of the composition to be administered. Each dosage unit may contain the daily dose of a given composition or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each composition to be contained in each dosage unit can depend, in part, on the identity of the particular composition chosen for the therapy and other factors, such as the indication for which it is given. The disclosed pharmaceutical compositions may be formulated to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.|O057| The pharmaceutical compositions may be utilized in methods of eliciting an immune response or vaccinating against a pathogen, e.g., porcine hepatitis E virus (HEV), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus, African swine fever virus (ASFV), or a coronavirus.[0058| As used herein, the terms "treating" or 'to treat" each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow- the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration. By way of example, a subject may be at risk for infection by a pathogen, e.g., porcine hepatitis E virus (HEV). porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), swdne influenza virus, African swine fever virus (ASFV), or a coronavirus, and administration of the disclosed pharmaceutical compositions elicits a protective immune response or vaccinates against the pathogen.|0059] As used herein, a '‘subject” or a “subject in need thereof’ may refer to a porcine subject, e.g., an adult pig, an adolescent pig, a piglet, e.g., a piglet that has not yet been weaned.

[0060] As used herein the term “effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) for eliciting an immune response to a pathogen, e.g., porcine hepatitis E virus (HEV), porcineepidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respirator}’ syndrome vims (PRRSV), sw ine influenza virus, African swine fever vims (ASFV), or a coronavims, or vaccinating against the pathogen.10061] An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of composition administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular composition administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0062] Oral administration is an illustrative route of administering the compositions and methods disclosed herein. Other illustrative routes of administration include transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited by the physical properties of the compounds being employed and the convenience of the subject and the caregiver.10063] As one skilled in the art will appreciate, suitable formulations include those that are suitable for more than one route of administration. For example, the formulation can be one that is suitable for both intrathecal and intracerebral administration. Alternatively, suitable formulations include those that are suitable for only one route of administration as w ell as those that are suitable for one or more routes of administration, but not suitable for one or more other routes of administration. For example, the formulation can be one that is suitable for oral, transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, and / or intrathecal administration but not suitable for intracerebral administration. f 0064] The inert ingredients and manner of formulation of the pharmaceutical compositions are conventional. The usual methods of formulation used in pharmaceutical science may be used here. All of the usual types of compositions may be used, including tablets, chewable tablets, capsules, solutions, parenteral solutions, intranasal sprays or powders, troches, suppositories, transdennal patches, and suspensions. In general, compositions contain from about 0.5% to about 50% of the compound in total, depending on the desired doses and the type of composition to be used. Theamount of the compound, however, is best defined as the “effective amount’", that is, the amount of the compound which provides the desired dose to the patient in need of such treatment. The activity of the compounds employed in the compositions and methods disclosed herein are not believed to depend greatly on the nature of the composition, and, therefore, the compositions can be chosen and formulated primarily or solely for convenience and economy.[0065} Capsules are prepared by mixing the compound with a suitable diluent and filling the proper amount of the mixture in capsules. The usual diluents include inert powdered substances (such as starches), powdered cellulose (especially crystalline and microcrystalline cellulose), sugars (such as fructose, mannitol and sucrose), grain flours, and similar edible powders.[0066| Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants, and disintegrators (in addition to the compounds). Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such as sodium chloride), and powdered sugar. Powdered cellulose derivatives can also be used. Typical tablet binders include substances such as starch, gelatin, and sugars (e.g., lactose, fructose, glucose, and the like). Natural and synthetic gums can also be used, including acacia, alginates, methylcellulose, polyvinylpyrrolidine, and the like. Polyethylene glycol, ethylcellulose, and waxes can also serve as binders.[0067} Tablets can be coated with sugar, e g., as a flavor enhancer and sealant. The compounds also may be formulated as chewable tablets, by using large amounts of pleasant-tasting substances, such as mannitol, in the formulation. Instantly dissolving tablet-like formulations can also be employed, for example, to assure that the patient consumes the dosage form and to avoid the difficulty that some patients experience in swallowing solid objects.[00681 A lubricant can be used in the tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid, and hydrogenated vegetable oils.

[0069] Tablets can also contain disintegrators. Disintegrators are substances that swell when wetted to break up the tablet and release the compound. The}’ include starches, clays, celluloses, algins, and gums. As further illustration, com and potato starches, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, and carboxymethylcellulose can be used.[00701 Compositions can be formulated as enteric formulations, for example, to protect the active ingredient from the strongly acid contents of the stomach. Such formulations can be created by coating a solid dosage form with a film of a polymer which is insoluble in acid environments and soluble in basic environments. Illustrative films include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate.[0071 [ Transdermal patches can also be used to deliver the compounds. Transdemial patches can include a resinous composition in which the compound will dissolve or partially dissolve; and a film which protects the composition, and which holds the resinous composition in contact with the skin. Other, more complicated patch compositions can also be used, such as those having a membrane pierced with a plurality’ of pores through which the drugs are pumped by osmotic action. [0072[ As one skilled in the art will also appreciate, the formulation can be prepared with materials (<?.g., actives excipients, carriers (such as cyclodextrins), diluents, etc.) having properties (e.g. , purity) that render the formulation suitable for administration to humans. Alternatively, the formulation can be prepared with materials having purity and / or other properties that render the formulation suitable for administration to non-human subjects, but not suitable for administration to humans.Methods of generating recombinant rotavirus[0073[ In another aspect of the current disclosure, methods of generating OSU strain rotavirus in vitro are provided. In some embodiments, the methods comprise introducing a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein; allowing the cell to express one or more rotavirus proteins selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5; incubating the cells for a sufficient time to produce rotavirus; and harvesting virus produced by the cells to generate OSU strain rotavirus in vitro.Cells[00741 The inventors disclose herein cells comprising the disclosed polynucleotides, which may also be used in the disclosed methods and systems. Accordingly, in another aspect of the current disclosure, cells are provided. In some embodiments, the cells comprise a polynucleotidecomprising a sequence encoding a recombinant OSU strain rotavirus protein. In some embodiments, the cells are selected from MA-104 cells, Vero cells, and BHK-1 cells.

[0075] Rotavirus vaccine strains have traditionally been grown using Vero cells. This method of producing rotavirus has been found to be suitable for generation of rotavirus for administration to subjects. Therefore, in some embodiments, the cells are Vero cells.[0076} In some embodiments, the cells disclosed herein further comprise a heterologous RNA polymerase, wherein the heterologous RNA polymerase binds to the promoter in the disclosed polynucleotides and catalyzes sequence-dependent RNA polymerization based on the composition when the composition is introduced into the cell. As used herein, “heterologous RNA polymerase” refers to an RNA polymerase not present in the cell without introduction through molecular biological techniques, e.g., transduction, transfection, lipofection. etc. In some embodiments, the heterologous RNA polymerase comprises T7 bacteriophage RNA polymerase or T3 bacteriophage RNA polymerase, more commonly known as simply T7 polymerase and T3 polymerase, respectively.[0077 } Accordingly, in some embodiments, the cells further comprise T7 RNA polymerase or T3 RNA polymerase. In some embodiments, such cells are referred to as, e.g., BHK-T7 cells, because they are derived from BHK-1 cells, but express the heterologous RNA polymerase T7 bacteriophage RNA polymerase. Thus, as used herein, “BHK-T7 cells” are BHK-1 cells that express the heterologous RNA polymerase T7 bacteriophage RNA polymerase.Methods of eliciting an immune response

[0078] The instant disclosure provides polynucleotides, methods of making the same, and infectious particles. Therefore, in another aspect of the current disclosure, methods of eliciting an immune response are provided. In some embodiments, the methods comprise administering a pharmaceutical composition comprising an infectious particle comprising a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.|00791 In some embodiments, the methods comprise administering a pharmaceutical composition comprising an infectious particle made by transfecting cells with a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.

[0089] In some embodiments, methods of eliciting an immune response are provided. In some embodiments, the methods of eliciting an immune response comprise administering apharmaceutical composition comprising an infectious particle comprising a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein. In some embodiments, methods of eliciting an immune response comprise administering a pharmaceutical composition comprising an infectious particle made by transfecting cells with a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.Methods of vaccinating a subject

[0081] In another aspect of the current disclosure, methods of vaccinating a subject against one or more pathogens are provided. In some embodiments, the methods comprise administering a pharmaceutical composition comprising an infectious particle comprising a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein. In some embodiments, the methods comprise administering a pharmaceutical composition comprising an infectious particle made by transfecting cells with a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein.Systems for generating recombinant rotavirus|0082] In another aspect of the current disclosure, systems for generating recombinant rotavirus are provided. In some embodiments, the systems comprise: (a) a polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein; and (b) cells capable of expressing the polynucleotides of (a).Definitions

[0083] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.1 084] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0085] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary7skill in the art and will vary' to some extent on the context inwhich they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about’ ' and "‘approximately” will mean up to plus or minus 10% of the particular term and ‘‘substantially” and ‘‘significantly” will mean more than plus or minus 10% of the particular term.

[0086] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0087] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0088] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g.. “a system having at least one of A, B and C” would include but not be limited to systems that have A alone. B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0089] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.[0090 | The modal verb "may" refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.” [0091 [ A “subject in need thereof’ as utilized herein may refer to a subject at risk for rotavirus infection. In some embodiments, the disclosed compositions, methods, and infectious particles comprise heterologous polynucleotides that encode for additional, non-rotaviral, proteins or peptides. Therefore, in some embodiments, a subject in need thereof may refer to a subject at risk of rotaviral infection and / or infection by another pathogen, wherein the heterologous polynucleotide encodes an antigen, e.g., protein or peptide, from the pathogen which is not a rotavirus.[00921 The term “subject” may be used interchangeably with the tenns “individual” and “patient” and includes human and non-human mammalian subjects.[0093| The phrases “% sequence identity,” “percent identity,” or “% identity” refer to the percentage of amino acid residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail below, generally presen e the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664. which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastp,” that is used to align a known ammo acid sequence with other amino acids sequences from a variety of databases.[00941 Nucleic acids, proteins, and / or other compositions described herein may be purified. As used herein, “purified” means separate from the majority of other compounds or entities, and encompasses partially purified or substantially purified. Purity may be denoted by a weight by weight measure and may be determined using a variety of analytical techniques such as but not limited to mass spectrometry, HPLC, etc.[0095} Polypeptide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.[0096} As used herein, the term "polypeptide," protein" and "peptide" are used interchangeably and refer to a polymer of 3 or more amino acids. Thus, for example, a protein may include two proteins that are joined (fused) together. Moreover, a protein may refer to a portion or fragment of a protein, e.g., SARS-CoV-2 SI protein, which is a fragment of the SARS-CoV-2 surface glycoprotein or “S” protein.[0097} The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Nucleic acids generally refer to polymers comprising nucleotides or nucleotide analogs joined together through backbone linkages such as but not limited to phosphodi ester bonds. Nucleic acids include deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) such as messenger RNA (mRNA), transfer RNA (tRNA), etc. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and / ordouble-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA. rRNA, siRNA, snRNA. a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or include non-naturally occurring nucleotides or nucleosides. Furthermore, the terms "nucleic acid,” "DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides, (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadeno sine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g.. methylated bases); intercalated bases; modified sugars (e g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).[00981 The term “hybridization”, as used herein, refers to the fonnation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between fully complementary nucleic acid strands or between “substantially complementary” nucleic acid strands that contain minor regions of mismatch. Conditions under which hybridization of fully complementary nucleic acid strands is strongly preferred are referred to as "stringent hybridization conditions” or "sequence-specific hybridization conditions”. Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch tolerated can be controlled by suitableadjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length and base pair composition of the oligonucleotides, ionic strength, and incidence of mismatched base pairs, following the guidance provided by the art (see, e.g., Sambrook et al., 1989, Molecular Cloning-A Laboratory7Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3 / 4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference).EXAMPLES[0099| The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.Example 1- Development of a reverse genetics system for OSU porcine rotavirus and its potential application for the production of combination vaccines against other porcine viruses10100] Background

[0101] Rotaviruses are a significant cause of severe potentially -life threatening gastroenteritis in the young of many economically important farm animals, including piglets. Although porcine rotavirus vaccines have been developed, both live oral and killed varieties, existing vaccines are generally ineffective in preventing rotavirus gastroenteritis in piglets. The establishment of more effective porcine rotavirus vaccines could be advanced through the application of reverse genetics technologies to create recombinant porcine rotavirus vaccine candidates that are superior in generating protective immunological responses. Through the use of such technology to develop porcine rotavirus as a vaccine vector, it may also be possible to generate combination vaccines capable of protecting pigs against diseases caused by other major pathogenic porcine viruses, including porcine hepatitis E virus (HEV), porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus, African swine fever virus (ASFV) and others.[0.1021 We report the invention of a robust reverse genetics system for the OSU strain of porcine rotavirus. The is the first description of a porcine rotavirus reverse genetics system. OSU rotavirus represents a. Rotavirus A species with a G5P[7] genotype, the genotype associated with porcine rotaviruses that most frequently cause disease in piglets. The OSU reverse genetics system is similar to rotavirus reverse genetics systems that have been previously established for other Rotavirus A species, including those infecting non-human primates (SAI 1 and RRV), humans (KU, CDC-9, Odelia, and RIX4414-like), cattle (UK and RF), mice (rD6 / 2-2g), and birds (PO- 13). The OSU reverse genetics system was developed based on sequence information that we determined through Nanopore sequencing of an OSU lab strain.|O IO3| Recombinant OSU rotaviruses generated by the OSU reverse genetics system can serve as candidates for the production of next-generation live oral vaccines. In addition, we have determined that the OSU reverse genetics system can be used to generate recombinant OSU rotaviruses that function as expression vectors of foreign proteins, including the capsid protein of porcine HEV. This is the first report describing the use of a recombinant rotavirus as a vector encoding the capsid protein of Hepatitis E virus (HEV). Our findings provide proof-of-concept data that it may be possible to generate combination vaccines using recombinant OSU rotaviruses that protect pigs against diseases caused by other pathogenic viruses.|0104] The approach that we used to develop OSU rotavirus as an expression vector of foreign capsid proteins mirrors approaches that we have used previously. We have shown before that it is possible to develop rotaviruses as an expression vector of the norovirus capsid proteins and the SARS-CoV-2 spike SI protein. As part of the work reported in this invention, we generated recombinant OSU rotavirus expressing foreign proteins by replacing the single NSP3 open reading frame (ORF) in the viral segment 7 RNA with a modified ORF that encodes NSP3 fused to a foreign protein. The introduction of a 2A translational stop-restart element between NSP3 ORF and the foreign protein ORF, allowed the modified OSU segment 7 RNA to express NSP3 and the foreign protein (e.g., UnaG green fluorescent protein) as separate products.

[0105] Methodology

[0106] The OSU reverse genetics system was developed as follows. Sequences for the eleven genome segments w ere determined by Nanopore sequencing of the MAI 04 cell culture-adaptedOSU strain (see Supplement 1). The sequences were used by Azenta, Inc. to construct eleven pUC19- based T7 transcription plasmids, each expressing one of the eleven OSU plus-sense RNAs. For the expression of foreign proteins, the pUC 19-based T7 transcription plasmid corresponding to NSP3 was modified by in-fusion cloning. The porcine teschovirus 2A-like (2A) element was positioned at the 3' end of the NSP3 coding sequence followed by the in-frame coding sequence of the foreign protein; we modified viral segment 7 to express fUnaG (FLAG- tagged green fluorophore) and HEV CP (viral capsid protein) (FIGs. 1-4). The OSU pT7 plasmids were then used to produce recombinant OSU following the reverse genetics protocols that we previously described (Philip AA, Patton JT. Expression of Separate Heterologous Proteins from the Rotavirus NSP3 Genome Segment Using a Translational 2A Stop-Restart Element. J Virol. 2020 Aug 31;94(18):e00959-20.). The OSU pT7 transcription plasmids and the pCMV- NP868R capping enzyme plasmid were transfected into BHK-T7 cells. Two days later, the transfected BHK-T7 cells were overseeded with MAI 04 cells. Three days later, the BHK- T7 / MA104 cell culture was lysed and fresh MAI 04 cells were infected with the lysates. When the cells culture reached complete infection, lysates were prepared and virus was recovered by plaque isolation. The RNA genomes of rOSU, rOSU-2A. rOSU-2A-fUnaG, and rOSU-2A-HEV CP are shown below (FIG. 5). The expression of fUnaG and the activity of the 2A translational stop-start element during infection are shown below (FIGs. 6 and 7).

[0107] Future Plans

[0108] We will generate and test whether recombinant OSU rotaviruses, including those expressing foreign capsid proteins (HEV, PRRSV, TGEV, porcine enteric caliciviruses, classical swine fever (CSF) virus, and pseudorabies virus, etc.) or immunogenic antigens or toxins of bacteria, capable of inducing protective immune responses in piglets), can induce protective immune responses in piglets. In addition, we will generate addition recombinant OSU rotaviruses that express fluorescent reporter proteins, such as mRuby, mKate, GFP, or BFP. The potential useful of these as diagnostic tools will be evaluated.Advantages of the technology disclosed herein

[0109] This is the first development of a reverse genetics system for porcine rotavirus.[01101 The reverse genetics system allows genetic modification of the OSU strain of porcine rotavirus.

[0111] OSU rotavirus genotype (G5P[7]) is representative of most porcine rotaviruses causing disease in piglets.

[0112] The disclosed OSU reverse genetics system allows development of OSU rotavirus as an expression vector of foreign proteins.

[0113] Recombinant OSU rotaviruses were made that encode the capsid protein of other pathogenic porcine viruses.

[0114] Recombinant OSU rotaviruses were made that encode the capsid protein of HEV, a major cause of porcine disease.

[0115] Recombinant OSU rotaviruses may be used in the development of live-oral combination vaccines, such as an OSU-based rotavirus-HEV vaccine.

[0116] Recombinant OSU rotaviruses were made that expressed the fluorescent reporter protein, UnaG.[01.17} Recombinant OSU rotaviruses that express fluorescent reporter proteins may be used as diagnostic tools, for detection of infection and measurement of immunological responses.Example 2 - Production of OSU G5P[7] Porcine Rotavirus Expressing a Fluorescent Reporter by Reverse Genetics

[0118] Reference is made to Snyder et al. ‘’Production of OSU G5P[7] Porcine Rotavirus Expressing a Fluorescent Reporter via Reverse Genetics” Viruses, 2024, 16, 411, which is a publication authored by the inventors and is incorporated by reference herein in its entirety.

[0119] Rotaviruses are a significant cause of severe, potentially life-threatening gastroenteritis in the young of many economically important animals. Although vaccines against porcine rotavirus exist, both live oral and inactivated, their effectiveness in preventing gastroenteritis is less than ideal. Thus, a need remains for the development of new generations of porcine rotavirus vaccines. The Ohio State University (OSU) rotavirus strain represents & Rotavirus A species with a G5P[7] genotype, the genotype most frequently associated with rotavirus disease in piglets. Using complete genome sequences that were determined by Nanopore sequencing, we developed a robust reverse genetics system enabling recovery of recombinant (r)OSU rotavirus. Although rOSU grew to high titers (~107plaque-forming units / ml), its growth kinetics were modestlydecreased in comparison to laboratory-adapted OSU vims. The reverse genetics system was used to generate rOSU rotavirus that served as an expression vector for foreign protein. Specifically, by engineering a fused NSP3-2A-UnaG open reading frame into the segment 7 RNA, we produced genetically stable rOSU that expressed the fluorescent UnaG protein as a functional separate product. Together, these findings raise the possibility of producing improved live oral porcine rotavirus vaccines through reverse genetics-based modification or combination porcine rotavirus vaccines that can express neutralizing antigens of other porcine enteric diseases.

[0120] 1. Introduction

[0121] Reverse genetics systems have been developed for several Rotavirus A strains, including those that infect non-human primates (SA11 and RRV), humans (KU, CDC-9, Odelia, and RIX4414-like). cattle (UK and RF), mice (rD6 / 2-2g), and birds (PO-13) [1-9], These systems can be used for producing next-generation live oral vaccines, dual vaccine platforms that express foreign proteins, and diagnostic tools. Rotavirus (RV) accounts for a significant disease burden within important livestock, highlighting the need for effective animal rotavirus vaccines. Nonetheless, no reverse genetics systems currently exist for any porcine rotaviruses, including those with the G5P[7] genotype, which represents the most frequent cause of disease in porcine populations [10,11],|(H 22] RV is a major cause of acute gastroenteritis in piglets [10,11], The vims is transmitted by the fecal-oral route and damages small intestinal enterocytes; as such, milk consumed by nursing piglets is not digested or absorbed into the intestines

[0012] , Moreover, RV is resistant to environmental factors, such as temperature, pH, and common disinfectants, which creates a persistent risk of infection [13,14], Although RV -induced diarrhea is associated with low mortality and high morbidity, productivity losses create a significant economic burden on the global pork industry’. Cunent vaccines only control diarrhea among infected populations [10,11], Thus, aneed exists for vaccines that are more effective.

[0123] Through modification of the RV genome by reverse genetics, the virus can be used as an expression vector of foreign proteins [2,3,15-20], The RV genome is composed of 11 segments of double-stranded (ds)RNA. Each segment contains the coding sequence for a single protein except for segment 11, which expresses two proteins

[0021] , As one approach for using RV as an expression vector, the NSP3 open reading frame (ORF) in the segment 7 RNA is replaced with a modified ORF that encodes NSP3 fused to a foreign protein. Insertion of the 2A translational stop-restart element allows for the modified segment 7 ORF to separately express NSP3 and the foreign protein [16-18.22-24], This approach has been used to generate recombinant RV that express fluorescent reporters, such as UnaG (green), mRuby (red), and TagBFP (blue), and other viral proteins, such as the norovirus VP1 capsid protein and respirator}' syndrome coronavirus 2 SI spike domain [3,15-18], Such recombinant viruses may be used to generate combination vaccines capable of inducing protective immune responses against RV and a second pathogenic virus.|0124] We report a robust reverse genetics system for The Ohio State University (OSU) G5P[7] porcine RV. The complete sequences of its 11 dsRNA genome segments were determined by Nanopore sequencing of a laboratory-adapted OSU strain [25-27], Using T7 expression plasmids that contain coding sequences for each genome segment, we generated recombinant OSU (rOSU) and 11 rSAl l / OSU monoreassortants. Single step growth curves, end point titers, and plaque morphologies revealed a modest reduction in growth kinetics for rOSU compared to the laboratory-adapted strain. To determine if the reverse genetics system allowed for the development of the OSU virus as an expression vector, we generated rOSU with a modified segment 7 RNA that encoded the fluorescent reporter UnaG (rOSU-2A-UnaG). Immunoblot analysis and fluorescence microscopy revealed the expression of a self-cleaved and functional UnaG protein following rOSU-2A-UnaG infection. Together, this w ork reveals (i) the first reverse genetics system for a porcine RV, (ii) a platform for making targeted genetic modifications of OSU. and (iii) a system that allows for the expression of foreign proteins during OSU infection.

[0125] 2. Materials and Methods

[0126] 21. Cells and virus

[0127] Embryonic monkey kidney (MA104) cells were grown in Dulbecco’s modified eagle medium (DMEM) containing 5% fetal bovine serum (FBS, Gibco) and 1% penicillinstreptomycin at 37°C in a 5% CO2 incubator. Baby hamster kidney cells that constitutively express the T7 RNA polymerase (BHK-T7) were provided by Dr. Ulla Buchholz (Laboratory of Infectious Diseases, NIAID, NIH) and were grown in Glasgow minimum essential medium (GMEM) containing 5% heat-inactivated FBS, 1% penicillin-streptomycin, 2% nonessential amino acids (Gibco), and 1% glutamine at 37°C in a 5% CO2 incubator. BHK-T7 cells were grown in medium supplemented with 2% Geneticin (Invitrogen) in every other passage.

[0128] RVA / Pig-tc / USA / 1975 / OSU / G5P[7] was provided by Dr. Taka Hoshino (Laboratory of Infectious Diseases, NIAID, NIH). This virus w as activated by adjusting to a final concentrationof 10 g / mL porcine pancreatic trypsin, type IX (Millipore Sigma) and incubating at 37°C for 60 min. The activated virus was then propagated in MA 104 cells maintained in serum-free DMEM with 0.5 pg / rnL trypsin. The infected cells lysates were clarified by low-speed centrifugation at 1500xg for 15 min at 4°C. Virus was isolated from the clarified lysates by extraction with an equal volume of Vertrel-XF (TMC Industries) followed by ultracentrifugation at 100.000 xg for 2 h at 4°C. The pelleted virus [OSU-tc(MA104)] virus was resuspended in 500 pL of Tris-buffered saline and stored at -80°C.

[0129] 2.2. Nanopore sequencing

[0130] OSU-tc_(MA104) dsRNA was extracted from 250 pL of clarified infected cell lysate using a Zymo Research Direct-zol RNA Miniprep Kit following the manufacturer’s instructions. Prior to library preparation, the dsRNA was denatured with dimethylsulfoxide and poly(A)-tailed using New England Biolabs Escherichia coli Poly (A) polymerase following the manufacturer's instructions. The poly(A)-tailed RNA was subjected to library preparation using an Oxford Nanopore Technologies direct cDNA sequencing kit (SQK-DCS109) following the manufacturer’s instructions. The library’ w as sequenced using an Oxford Nanopore Technologies Mini ON sequencer. Sequence assemblies of the 11 genome segments of OSU-tc_(MA104) were prepared using Geneious Primer software version 2023.2.1.

[0011] 2.3. OSU sequences used in the generation ofT7 expression plasmids

[0132] The sequences of the OSU-tc_(MA104) genome segments w ere deposited in GenBank under the accession numbers OP978238-OP978248. The sequences of modified segment 7 RNAs of OSU NSP3-2A (PPI 12343) and OSU NSP3-2A-UnaG (PPI 12344) were also deposited in GenBank.

[0013] 2.4. Plasmids used in this study

[0134] Recombinant SA11 (rSAl l) viruses were prepared using the plasmids pT7 / SAHVPl, pT7 / SAl 1VP2. pT7 / SAl 1VP3, pT7 / SAl 1VP4, pT7 / SAl 1VP6, pT7 / SAl 1VP7, pT7 / SAl 1NSP1. pT7 / SAl 1NSP2. pT7 / SAl 1NSP3, pT7 / SAl 1NSP4, and pT7 / SAl 1NSP5 and pCMV / NP868R (1. 28). Recombinant OSU (rOSU) viruses were prepared using the plasmids pT7 / OSUVPl, pT7 / OSUVP2, pT7 / OSUVP3, pT7 / OSUVP4, pT7 / OSUVP6, pT7 / OSUVP7, pT7 / OSUNSPl, pT7 / OSUNSP2, pT7 / OSUNSP3, pT7 / OSUNSP4, and pT7 / OSUNSP5 and pCMV / NP868R

[0028] , The pT7 / OSU plasmids were made by Genewiz, Azenta Life Sciences, based on OSU sequences determined by Nanopore sequencing. The plasmid pT7 / SAl 1 NSP3-2A-UnaG was previouslydescribed

[0017] , The plasmids pT7 / OSU NSP3-2A and pT7 / OSU NSP3-2A-UnaG were produced by fusing DNA frag-ments for 2 A or 2A-3xFLAG-UnaG, respectively, to the 3 '-end of the OSU NSP3 open reading frame of pT7 / OSU NSP3 using the TaKaRa Bio In-Fusion cloning system. Primer synthesis and plasmid sequencing were performed by EuroFins Scientific (Table 2).

[0135] Table 2. Primers used in constructing OSU segment 7 expression platforms

[0136] 2.5. Isolation, amplification, and analysis of recombinant viruses[0137} Rotavirus reverse genetics was performed as previously described [1,29]. Briefly, BHK- T7 cells in 12-well plates were transfected with the 11 SAI 1 or OSU T7 plasmids, or combinations thereof, and with the capping enzy me plasmid, pCMV-NP868R, using Minis TransIT-LTl transfection reagent. Transfection mixtures contained 0.8 pg each of the pT7 plasmids except for pT7 / NSP2 and pT7 / NSP5 which were used at 3-fold higher concentrations. Two days post transfection, the BHK-T7 cells were overseeded with MAI 04 cells, and trypsin was added to the medium to a final concentration of 0.5 pg / mL. Three days later, the BHK-T7 / MA104 cell mixtures were freeze-thawed thrice, and the lysates were clarified by low-speed centrifugation at800xg for 5 min at 4°C. To amplify the recovered viruses, the lysates were adjusted to 10 pg / mL trypsin and incubated for 1 h at 37°C. MAI 04 cells in 6-well plates were then infected with 300 pL of the trypsin-treated lysates and incubated at 37°C in a 5% CO2 incubator until all cells were lysed (typically 3-5 days). Recombinant viruses were recovered from the lysates by plaque isolation on MAI 04 cells. Plaque-isolated viruses were initially grown on MAI 04 cells in 6-well plates and then, to generate larger pools, grown on MAI 04 cells in T175 tissue culture flasks at low multiplicity of infection (<1 PFU / cell). Briefly, 100 pL of the plaque-amplified lysates were activated by incubation with 10 ptymL trypsin (final concentration). The activated lysates were diluted into 10 mL of serum-free DMEM and then used as inoculum to infect MAI 04 cells in T175 flasks. The flasks were placed at 37°C in a 5% CO2 incubator for 1 h with rocking to ensure equal coverage of the inoculum over the monolayers. Following adsorption, the inoculum was removed and 25 mL of serum-free DMEM containing 0.5 pg / mL trypsin was added to each flask. The flasks were returned to the incubator until all cells were lysed (typically 3-5 days). The infected cell lysates were collected, clarified by low-speed centrifugation at 800xg for 5 min at 4°C, and stored at -80°C. Viral dsRNAs were recovered from the infected cell lysates by TRIzol extraction, resolved by electrophoresis on 8% polyacrylamide gels in Tris-glycine buffer, detected by staining with ethidium bromide, and visualized using a Bio-Rad ChemiDoc MP imaging system. Peak titers were determined from the infected cell lysates by plaque assay.

[0138] 2.6. Plaque assay

[0139] Rotavirus plaque assays were perfonned as previously described

[0029] , At 5 days post infection, MAI 04 monolayers with agarose overlays were incubated overnight with phosphate- buffered saline (PBS) containing 3.7% formaldehyde. The agarose overlays were then removed, and the monolayers were stained with 1% crystal violet in 5% ethanol for 3 h. The fixed and stained monolayers were rinsed with water and air dried. The plaque diameters were measured using ImageJ software

[0030] , Statistically significant differences in titer and plaque size were determined using ANOVA (GraphPad Prism).

[0140] 2. 7. Immunoblot analysis

[0141] Rotavirus infections were performed as previously described

[0029] , Briefly, MA104 cells in 6-well plates were infected with 5 PFU / cell of the indicated viruses. At 9 h post infection, the infected cells were scraped into cold PBS. The infected cells were then washed with cold PBS, pelleted by centrifugation at 5,000 X g for 5 min at 40C, and lysed by incubation withnondenaturing lysis buffer (300 mM NaCl, 100 mM Tris-HCl [pH 7.4], 2% Triton X-100, and IxEDTA-free protease inhibitor cocktail [Roche Complete]) for 30 min on ice. For immunoblot analysis, lysates were resolved by electrophoresis on 10% polyacrylamide gels in Tris-glycine buffer and transferred to nitrocellulose membranes. After blocking with PBS containing 0.1% Tween-20 and 5% nonfat dry milk, the blots were probed with FLAG M2 antibody (F 1804, Sigma Aldrich, 1:2,000), 2A antibody (NBP2-59627, Novus, 1: 1,000), rotavirus NSP3 antibody (lot 55068, 1:2,000), rotavirus VP6 antibody (lot 53963, 1:2,000), or [Lactin antibody (D6A8, Cell Signaling Technology, 1:2000). The bound primary antibodies were detected using 1: 10,000 dilutions of horseradish peroxidase (HRP)-conjugated secondary7antibodies (goat anti-mouse IgG [CST], goat anti-guinea pig IgG [KPL], or goat anti-rabbit IgG [CST]) in 5% nonfat dry milk. HRP signals were developed using the Bio-Rad Clarity Western ECL substrate and developed using a Bio-Rad ChemiDoc imaging system.

[0142] 2.8. Genetic stability) analysis

[0143] Genetic stability7experiments were performed as previously described [3,16-18], Briefly, the indicated viruses were serially passaged five times using 1 : 100 dilutions of infected cell lysates that were prepared in serum-free DMEM. When cytopathic effect reached completion (typically 3-5 days), the cells were freeze-thawed thrice. Viral dsRNAs were recovered from the infected cell lysates by TRIzol extraction

[0029] , resolved by electrophoresis on 8% polyacrylamide gels in Tris-glycine buffer, detected by staining with ethidium bromide, and visualized using a Bio-Rad ChemiDoc MP imaging system.

[0144] 2.9. Assessment of infectivity by infectious particle production

[0145] Rotavirus infections were performed as previously described

[0029] , Briefly, MAI 04 cells in 6-well plates were infected with 5 PFU / cell of the indicated viruses. Following adsorption, the infected cells were washed thrice with PBS and incubated in serum-free DMEM containing 0.5 pg / rnL trypsin at 37°C in a 5% CO2 incubator. At the indicated times post infection, the infected cells were freeze-thawed thrice, clarified by low-speed centrifugation at 800xg for 5 min at 4°C, and analyzed by plaque assay. Statistically significant differences in titer were determined using ANOVA.

[0146] 2.10. Assessment of fluorescent reporter expression

[0147] Rotavirus infections were performed as previously described

[0029] , Briefly, MAI 04 cells in 6-well plates were infected with 0.05 PFU / cell of rOSU and rOSU-2A-UnaG. Followingadsorption, the infected cells were washed thrice with PBS and incubated in serum-free DMEM containing 0.5 pg / mL trypsin at 37°C in a Sartorius IncuCyte S3 Live-Cell Analysis System. At 28 hours post infection, images were acquired at 10X magnification under phase and green (excitation [440-480 nm], emission [504-544 nm]) channels.

[0148] 2.11. Statistical analyses|0149] The results from all experiments represent three biological replicates. Horizontal bars indicate the means. Error bars indicate the standard deviations. P values were calculated using one-way analysis of variance with Bonferroni Correction (Graph Pad Prism).

[0150] 3. Results and Discussion

[0151] 3.1. Recovery of OSU G5P[7] porcine rotavirus by reverse genetics

[0152] Genotype G5P[7] is representative of most rotaviruses (RV) that cause acute gastroenteritis in suckling and weaned pigs, which leads to economic losses that plague the global pork industry' [10,11], Current treatments are generally ineffective at preventing disease [31-33]; thus, aneed exists for robust molecular tools to develop next-generation porcine rotavirus vaccines. Reverse genetics systems exist for several Rotavirus A strains [1-9]; however, no such system is available for a porcine RV. To address this knowledge gap, we utilized the well-studied Ohio State University (OSU) G5P[7] prototy pe strain [25-27], Laboratory-adapted OSU (OSU-tc_(MA104)) was grown in MA104 cells, and viral dsRNA was extracted from infected cell lysates. The isolated RNA was then processed for Nanopore sequencing to obtain the complete sequences of all 11 genome segments (deposited in GenBank). Notably, the Nanopore sequences for 7 OSU- tc_(MAl 04) genome segments (VP2, VP3, VP6, VP7, NSP2, NSP3, and NSP5) were identical to the virulent (RVA / Pig-tc / USA / 1975 / OSU / G5P7 / virulent) and attenuated (RVA / Pig- tc / USA / 1975 / OSU / G5P7 / attenuated) strains. In contrast, VP1, VP4. and NSP1 were >99% identical, whereas NSP4 was >95% identical (nucleotide and amino acid sequence comparisons)

[0027] , The OSU-tc_(MA104) sequencing information was used to construct plasmids for reverse genetics experiments. Full-length cDNAs of each genome segment were positioned within T7 plasmids in between of an upstream T7 polymerase promoter and a downstream hepatitis delta virus ribozyme. In the presence of the T7 RNA polymerase, the OSU T7 plasmids produce full- length, positive sense RNA with authentic 5’ and 3’ termini.

[0153] To confirm that each OSU T7 expression plasmid was functional, we generated 11 recombinant SAI 1 / OSU (rSAl 1 / OSU) monoreassortants. SAI 1 represents the prototype strain ofsimian RV [34-36], Reverse genetics experiments were perfonned as previously described (FIG 1A) [1,29], Briefly. 1 OSU T7 expression plasmid, 10 SAI 1 T7 expression plasmids, and pCMV- NP868R were transfected into BHK-T7 cells, which were subsequently overseeded with MA 104 cells. Recombinant viruses generated in the transfected cells were amplified and their dsRNA profiles analyzed by gel electrophoresis. All transfection mixtures designed to produce monoreassortants resulted in the recovery of infectious virus (i.e., viral dsRNA) and induced cytopathic effect within 5 days of infection. Thus, the genetic information obtained by Nanopore sequencing was functional for reverse genetics. By comparing the banding patterns to rSAl 1, we also determined the migration distance for each rOSU genome segment (FIG. 8B, see red arrows). Functional differences between the SAI 1 and OSU genome segments and protein products may be investigated using the monoreassortants discussed here. Strikingly, we recovered recombinant virus that was comprised of all 11 OSU genome segments (FIG. 8B, see rOSU lane). Thus, we have developed a reverse genetics system for G5P[7] porcine RV that may be used for the production of vaccines targeting the most common cause of porcine RV infections. In contrast to current vaccines against porcine RV. which have been developed by serially passaging virulent strains in tissue culture - a costly and time consuming process, the OSU reverse genetics system allows for the rapid generation of vaccine candidates and for the introduction of directed attenuating genetic mutations [10,37-40],

[0154] 3.2. Recovery of OSU G5P[7] porcine rotavirus encoding a foreign protein

[0155] RV can be modified to express fluorescent reporters, such as UnaG (green), mRuby (red), and TagBFP (blue), and other viral proteins, such as norovirus VP1 and SARS-COV-2 S 1 [2,3,15- 20]). These recombinant viruses are valuable for analyzing RV biology' by fluorescence-based imaging and may be used to induce protective immunity responses against multiple pathogenic viruses. As a proof-of-concept, we explored the possibility of expressing UnaG from OSU genome segment 7. The porcine teschovirus 2A-like (2A) element was positioned at the 3’ end of the NSP3 coding sequence followed by the in-frame coding sequence of FLAG-tagged UnaG. Due to the activity' of the 2A element [22-24], translation of the RNA produces two proteins: NSP3 with remnants of the 2A element and FLAG-tagged UnaG. We generated an additional expression plasmid with only the 2A element following NSP3 (FIG 9A). Using RV reverse genetics (FIG. 8A), we recovered the rOSU-2A and rOSU-2A-UnaG viruses. For each, we observed a shift in the migration pattern of genome segment 7 to a higher molecular form (FIG 9B, see red arrows).These shifts are presumably due to the extra genetic material introduced into the segment 7 RNA by addition of the 2A and 2A-FLAG-UnaG coding sequences. We also noted similar dsRNA banding patterns for OSU-tc_(MA104) and rOSU, providing additional evidence for the utility of the OSU reverse genetics system.

[0156] To develop OSU G5P[7] porcine rotavirus as a vector for expressing foreign protein, the recombinant virus must be genetically stable. This property is essential for scaling large quantities for vaccine production and for other applications. To examine genetic stability, we serially passaged rOSU-2A and rOSU-2A-UnaG at low multiplicity of infection. We observed no differences in the pattern of viral dsRNA recovered from the infected lysates during passage (FIG 9C, see red arrows). The 2A-FLAG-UnaG coding sequence is stable within the OSU background for at least 5 rounds of infection; however, larger inserts may be unstable. As such, future studies will evaluate the limits of OSU genome flexibility.

[0157] 3.3. Growth characteristics of rOSU G5P[7] rotaviruses

[0158] The recombinant viruses generated in this work were derived from sequencing information of the laboratory-adapted strain. To detennine if these viruses exhibit similar growth characteristics, we compared their growth kinetics and plaque morphologies. The analysis showed that rOSU was a well growing virus, reaching peak titers of ~107in MA104 cells. However, the peak titer produced reached by rOSU w as ~0.5 log less than that reached by the OSU-tc_(MAl 04) virus. Moreover, single-step grow th experiments indicated that rOSU grew slow er than the OSU- tc (MAI 04) virus. Plaque analysis also showed that the rOSU virus formed smaller plaques on MAI 04 cells than the OSU-tc_(MAl 04) virus (FIG 10A-D). These results suggest sequence differences exist between rOSU and OSU-tc_(MA104) that impact virus growth. We conclude from this that the consensus sequence information generated for OSU-tc_(MA104) genome by Nanopore sequencing may not fully reflect the distribution and combinations of sequence variations in the OSU-tc_(MA104) population associated with the fittest, best growing viruses. Indeed, the OSU-tc_(MA104) population likely represents a quasispecies, of which rOSU may, or may not, be a single variant.

[0159] We next examined the grow th characteristics of recombinant viruses that encode foreign protein. Compared to the rOSU, rOSU-2A-UnaG produced ~1 log-unit less infectious virus but generated similar plaque sizes (FIG. 10A-D). This result is consistent with previous studies thatshow addition of genetic material to the segment 7 RNA is correlated w ith reduced virus growth [3,16-18],

[0160] 3.4. Expression of foreign protein by rOSU G5P[7] porcine rotavirus|0161] To develop OSU as a dual vaccine platform, the vector must express a foreign protein. As such, the protein products made by rOSU-2A-UnaG following infection were probed by immunoblot with an anti-FLAG antibody (FIG. 10E). This assay revealed high levels of cleaved FLAG-UnaG (~18 kDa); rOSU-2A-UnaG directed expression of the foreign protein and a functional 2A element. We also detected minor amounts of uncleaved NSP3-2A-UnaG (~56 kDa), which likely represents a readthrough of the 2A element. Probing with an anti-2A antibody revealed a protein product that migrated at the expected molecular weight for NSP3 linked to the remnant residues of the 2A peptide (—38 kDa) (FIG. 10E). Viral protein VP6 and host protein 0- actin were detected under all infection conditions; however, NSP3-2A-UnaG, FLAG-UnaG, and NSP3-2A were not present in rOSU infected cell lysates. Finally, we used fluorescence microscopy to determine if the expressed and cleaved FLAG-UnaG was functional. In contrast to rOSU, rOSU-2A-UnaG produced high levels of fluorescent signal within live cells (FIG. 10F).

[0162] 4. Conclusions

[0013] The establishment of more effective porcine RV vaccines can be advanced through the application of reverse genetics technologies to create candidates that are superior in generating protective immunological responses. Such reverse genetics technologies allow for targeted genetic modifications and avoid the costly and time-consuming process of attenuating virulent strains through serially passage. Moreover, it may be possible to design combination vaccines that are capable of protecting individuals against RV and other pathogenic viruses. In this work, we developed a robust reverse genetics system for the G5P[7] OSU strain of porcine rotavirus. Using information obtained by Nanopore sequencing the laboratory-adapted strain, we constructed 11 T7 expression plasmids that were sufficient for generating recombinant OSU (FIG. 8). We leveraged the reverse genetics system to produce a recombinant virus that expressed UnaG within infected cells (FIGs. 9 and 10). Recombinant viruses that express fluorescent reporters are valuable tools for monitoring virus spread in infected animals and assessing immunological responses in pigs.

[0164] The development of the porcine OSU reverse genetics system also has possible application for understanding the biology7of human RVs, given the close similarity of the genotypeconstellations of the OSU virus and the human Wa-like genogroup RVs (e.g., G1P[8], G3P[8], and G12P[8] RVs)

[0041] , Notably, the genotype of the genome segments for the nonstructural proteins and the core structural proteins of OSU are generally the same as found for the Wa-like viruses: R1-C1-M1-A1-N1-T1-E1-H1. Indeed, phylogenetic analyses have indicated that the human Wa-like viruses evolved from OSU-like porcine RV s rotaviruses

[0042] . Unlike many animal viruses, the NSP1 protein of the OSU virus reportedly relies on a mechanism similar to the Wa- like viruses to antagonize the interferon signaling system

[0043] , As a result of the ease of growing porcine rotaviruses and their genetic similarity to Wa-like viruses, the possibility of generating human RV vaccines from porcine virus strains has been explored

[0044] ,[01651 In the foregoing description, it will be readily apparent to one skilled in the art that vary ing substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0166] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

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Development of a live attenuated trivalent porcine rotavirus A vaccine against disease caused bv recent strains most prevalent in South Korea. Vet Res 50:2. Bull JJ. 2015. Evolutionary reversion of live viral vaccines: Can genetic engineering subdue it? Virus Evol 1.40. Hanley KA. 2011. The double-edged sword: How evolution can make or break a live- atenuated virus vaccine. Evolution (NY) 4:635-643.41. Mathijnssens J, Ciarlet M, McDonald SM, Atoui H, Banyai K, Brister JR, Buesa J, Esona MD, Estes MK. Gentsch JR, Iturriza-Gomara M, Johne R. Kirkwood CD, Martella V, Mertens PP, Nakagomi O, Parreno V, Rahman M, Ruggeri FM, Saif LJ, Santos N, Steyer A, Taniguchi K, Paton JT, Desselberger U, Van Ranst M. Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group (RCWG). Arch Virol. 2011 Aug;156(8):1397-413.42. Mathijnssens J, Ciarlet M, Heiman E, Arijs I, Delbeke T, McDonald SM, Palombo EA, Iturriza-Gomara M, Maes P, Patton JT, Rahman M, Van Ranst M. Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS- 1 -like and bovine rotavirus strains. J Virol. 2008 Apr;82(7):3204-19.43. Morelli M, Dennis AF, Paton JT. Putative E3 ubiquitin ligase of human rotavirus inhibits NF-KB activation by using molecular mimicry to target P-TrCP. mBio. 2015 Jan 27;6(l):e02490-14.44. Hoshino Y, Jones RW, Ross J, Kapikian AZ. Porcine rotavirus strain Gottfried-based human rotavirus candidate vaccines: construction and characterization. Vaccine. 2005 May 31;23(29):3791-9.|OI68| Sequences

Claims

CLAIMSWhat is claimed is:

1. A polynucleotide comprising a sequence encoding a recombinant OSU strain rotavirus protein, wherein the sequence encodes a positive-sense viral transcript.

2. The polynucleotide of claim 1, wherein the recombinant OSU strain rotavirus protein is a G5P[7] genotype rotavirus protein.

3. The polynucleotide of claim 1, wherein the polynucleotide encodes a positive sense viral transcript.

4. The polynucleotide of claim 1 , wherein the polynucleotide comprises any one of SEQ ID NOs 1-14, or a sequence with at least 85% identity to any one of SEQ ID NOs: 1-14.

5. The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence encoding an OSU strain NSP3 protein.

6. The polynucleotide of claim 1, wherein the polynucleotide further comprises a heterologous polynucleotide.

7. The polynucleotide of claim 6, wherein the heterologous polynucleotide encodes a protein in-frame with the OSU strain NSP3 ORF.

8. The polynucleotide of claim 7, wherein the heterologous polynucleotide encodes a peptide or protein.

9. The polynucleotide of claim 7, wherein the heterologous polynucleotide encodes a reporter.

10. The polynucleotide of claim 8, wherein the peptide or protein comprises a microorganismal peptide or protein.

11. The polynucleotide of claim 10, wherein the peptide or protein comprises a viral peptide or protein.

12. The polynucleotide of claim 11 , wherein the peptide or protein comprises a hepatitis E virus (HEV) protein, an epidemic diarrhea virus (PEDV) protein, a transmissible gastroenteritis virus (TGEV) protein, or a coronavirus protein.

13. The polynucleotide of claim 12, wherein the hepatitis E virus (HEV) protein comprises an HEV capsid protein.

14. The polynucleotide of claim 13, wherein the hepatitis E virus (HEV) capsid protein comprises SEQ ID NO: 19.

15. The polynucleotide of claim 13, wherein the hepatitis E virus (HEV) capsid protein is encoded by SEQ ID NO: 20.

16. The polynucleotide of claim 1, wherein the polynucleotide is operably linked to a promoter.

17. The polynucleotide of claim 16, wherein the promoter is a T7 promoter, optionally, wherein the T7 promoter comprises SEQ ID NO: 17.

18. The polynucleotide of claim 16, wherein the promoter is a T3 promoter, optionally, wherein the T3 promoter comprises SEQ ID NO: 18.

19. The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence encoding a cleavage site.

20. The polynucleotide of claim 19, wherein the cleavage site is a protease cleavage site.

21. The polynucleotide of claim 20, wherein the cleavage site is a thrombin cleavage site (SEQ ID NO: 15).

22. The polynucleotide of claim 19, wherein the cleavage site is a self-cleaving peptide sequence.

23. The polynucleotide of claim 22, wherein the self-cleaving peptide sequence is porcine teschovirus 2A element (SEQ ID NO: 16).

24. The polynucleotide of any one of claims 1 -23, the polynucleotide comprises a sequence encoding a linker.

25. The polynucleotide of claim 24, the linker is a flexible linker selected from a GAG linker or a GSG linker.

26. A collection of polynucleotides, wherein each of the polynucleotides in the collection comprises a sequence encoding at least one OSU strain rotavirus protein selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5, wherein the polynucleotides of the collection encode each of the VP1, VP2, VP3, VP4. VP6. VP7. NSP1, NSP2, NSP3, NSP4. and NSP5 proteins, wherein each of the sequences encoding one rotavirus protein are operably linked to a promoter.

27. The collection of claim 26, wherein the sequences encoding at least one OSU strain rotavirus protein selected from VP1, VP2. VP3. VP4. VP6. VP7. NSP1, NSP2, NSP3. NSP4, and NSP5 comprise SEQ ID NOs: 1-11, respectively.

28. The collection of claim 27, wherein the sequences encoding at least one OSU strain rotavirus protein selected from VP1, VP2. VP3. VP4. VP6. VP7. NSP1, NSP2, NSP3. NSP4, and NSP5 consist of SEQ ID NOs: 1 -11 , respectively.

29. An infectious particle comprising the polynucleotide of claims claim 1.

30. An infectious particle comprising the polynucleotide of claim 11.

31. An infectious particle made by transfecting cells with the polynucleotide of any one of claim 1 or claim 11.

32. A pharmaceutical composition comprising the infectious particle of claim 29 or 30.

33. A method comprising: administering the pharmaceutical composition of claim 32 to a subject.

34. A method of eliciting an immune response to one or more microorganism in a subject, the method comprising: administering the pharmaceutical composition of claim 32 to a subject to elicit an immune response to the one or more microorganism.

35. The method of claim 33, wherein the one or more microorganisms comprises hepatitis E virus (HEV).

36. The method of claim 33, wherein the one or more microorganisms comprises rotavirus and hepatitis E virus (HEV).

37. A method comprising: administering the pharmaceutical composition of claim 32 to a subject.

38. A method comprising: administering the pharmaceutical composition of claim 32 to a subj ect to elicit an immune response in the subj ect to a pathogen or vaccinate the subj ect against one or more pathogens.

39. A method of vaccinating a subject against one or more pathogens, the method comprising: administering the pharmaceutical composition of claim 32 to a subject to vaccinate a subject against the one or more pathogens.

40. The method of claim 37 or 38, wherein the one or more pathogens comprises hepatitis E virus (HEV).

41. The method of claim 37, wherein the one or more pathogens comprises OSU strain rotavirus and hepatitis E virus (HEV).

42. The method of any one of claims 33-41, wherein the subject is a pig.

43. The method of claim 42, wherein the subject is a piglet, optionally, wherein the piglet is has not yet been weaned.

44. A cell comprising the polynucleotide of claim 1.

45. A cell comprising the collection of claim 26.

46. The cell of claim 44 or 45, wherein the cell is an MA-104 cell, a Vero cell or a BHK-1 cell.

47. The cell of claim 46, wherein the cell expresses a heterologous RNA polymerase.

48. The cell of claim 47. wherein the heterologous RNA polymerase is selected from T7 RNA polymerase and T3 RNA polymerase.

49. A method of generating an OSU strain rotavirus in vitro comprising: introducing the polynucleotide of any one of claims 1-25 into a cell; allowing the cell to express one or more rotavirus proteins selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4, and NSP5; incubating the cells for a sufficient time to produce rotavirus; and harvesting virus produced by the cells to generate the rotavirus in vitro.

50. A method of generating an OSU strain rotavirus in vitro comprising: introducing the collection of claim 26 into a cell; incubating the cells for a sufficient time to produce rotavirus; and harvesting virus produced by the cells to generate the rotavirus in vitro.

51. The method of claim 49 or 50, wherein the cell comprises T7 RNA polymerase and. optionally, comprising African swine fever virus capping enzyme.

52. The method of claim 49, wherein the cell is selected from an MA-104 cell, a Vero cell, and a BHK-1 cell.

53. The method of claim 49, wherein the cell expresses a heterologous RNA polymerase.

54. The method of claim 53, wherein the heterologous RNA polymerase is selected from T7 RNA polymerase and T3 RNA polymerase.

55. The method of claim 50, wherein the cell is a BHK-1 cell comprising T7 RNA polymerase and, optionally, comprising African swine fever virus capping enzyme.

56. A system for generating recombinant rotavirus comprising:(a) the polynucleotide of claim 1; and(b) cells capable of expressing the polynucleotides of (a).

57. A system for generating recombinant rotavirus comprising:(a) the collection of claim 26; and(b) cells capable of expressing the collection of (a).

58. The system of claim 56 or 57, wherein the cells comprise a heterologous RNA polymerase and, optionally, comprising African swine fever virus capping enzyme.

59. The system of claim 58, wherein the cells comprise a cells from a cell line selected from MA-104 cells, Vero cells, and BHK-1 cells.

60. The system of claim 59, wherein the cells comprise BHK-1 cells comprising T7 RNA polymerase.61 . The system of claim 60, wherein the cells comprise BHK-1 cells comprising T7 RNA polymerase, Vero cells, and MA-104 cells.

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