Human rotavirus (HRV) is a major causative agent of acute gastroenteritis (AGE), which causes severe dehydrating diarrhea in children under the age of five and results in up to 215,000 deaths worldwide each year. There are two live oral attenuated vaccines licensed for use in the United States that are highly effective in high-income countries but much less so in low-and middle-income countries (LMICs). Several factors contributing to decreased efficacy in these areas include chronic malnutrition, gut dysbiosis, and concurrent viral infection. Along with this, currently used vaccines require constant cold-chain storage to maintain vaccine stability, and those resources can be scarce in LMICs. These areas continue to maintain a high burden of HRV morbidity and mortality, and more efficacious vaccines are needed. The gnotobiotic (Gn) pig model of HRV infection and diarrhea has long been used in the evaluation of novel HRV vaccines due to Gn pigs' susceptibility to HRV infection, development of clinical signs, histopathological changes in the intestine, and the infection kinetics that mimic those seen in human infants. The first project in this dissertation used the Gn pig model to evaluate a thermostable live oral attenuated vaccine administered as a dissolvable film. Two doses of the tetravalent dissolvable film vaccine conferred significant protection from virus shedding by delaying its onset and reducing peak titers in feces. It also significantly delayed the onset of diarrhea and reduced the duration and area under the curve (AUC) of diarrhea. The dissolvable film was highly immunogenic, inducing high titers of serum virus neutralizing (VN) antibodies specific to each of the four G-types included in the vaccine formulation, HRV-specific serum IgA and IgG, and intestinal IgA. These data confirm the thermostable platform as a useful alternative to liquid vaccines that require cold-chain. The second project evaluated three mRNA-based nonreplicating vaccine candidates in the Gn pig model. All three mRNA candidates encoded a universal CD4+ T cell epitope, P2, derived from tetanus toxoid, fused with the encoded VP8* from P[4], P[6], and P[8] HRVs. Two candidates also encoded for a lumazine synthase (LS) domain fused with the P2-VP8*. A dose response study of the LS-P2-VP8* candidates was conducted simultaneously. Significant protection against virus shedding was induced by all three candidates, with LS-P2-VP8* candidates inducing significantly higher VP8*-specific serum IgG. All three candidates induced significantly higher numbers of P[8]-VP8*-specific IgG antibody-secreting cells (ASCs) and IFN-γ-producing T cells in the ileum, spleen and blood. These data provide guidance for further development of the relatively new mRNA-based technology for use in HRV vaccine development. In the final study of this dissertation, we used the Gn pig model of both P[8] and P[6] HRV infection to evaluate a cocktail nanoparticle-based HRV vaccine. This vaccine was made up of an S60 nanoparticle, self-assembled from the S domain of the human norovirus capsid protein. The exposed C-termini on the S60 nanoparticle were utilized as an antigen display platform, where VP8* from P[4], P[6] and P[8] HRVs was fused. This vaccine was tested as both a two-dose intramuscular (IM) regimen, or as an IM booster preceded by an oral priming immunization with commercial monovalent Rotarix®. Pigs were challenged with either P[6] or P[8] HRV to evaluate cross-protection of the nanoparticle vaccine. Both regimens were highly immunogenic, inducing high titers of serum VN, IgG and IgA antibodies. Furthermore, the prime-boost regimen conferred significant protection against virus shedding in P[8] HRV-challenged pigs as evidenced by the shortened duration of fecal virus shedding. There was also significant protection in P[6] HRV-challenged pigs vaccinated with the prime-boost regimen, as evidenced by the shortened duration, reduced mean peak titer and AUC of virus shedding. Prime-boost-vaccinated pigs challenged with P[8] HRV had significantly higher P[8]-specific IgG ASCs in the spleen post-challenge. Prime-boost-vaccinated pigs challenged with P[6] HRV had significantly higher numbers of P[6] and P[8]-specific IgG ASCs in the ileum, as well as significantly higher numbers of P[8]-specific IgA ASCs in the spleen post-challenge. Oral priming followed by parenteral boosting appears to be a promising vaccination strategy for HRV and these data warrant further investigation into this regimen. Through these studies, we improved our understanding of the effect of different vaccination routes and formulations in the effectiveness of conferring protection against an enteric virus. The knowledge will facilitate the development of more effective vaccination strategies against HRV, the leading cause of infantile diarrhea in LMICs, as well as other enteric viruses. / Doctor of Philosophy / Human rotavirus (HRV) is a major causative agent of acute gastroenteritis (AGE) in children under the age of five. Acute gastroenteritis is characterized by nausea, vomiting, and potentially deadly dehydrating diarrhea. There are two highly effective vaccines licensed for use in the United States; however, these vaccines are much less effective in low- and middle-income countries (LMICs), where HRV disease burden is the highest. There are several reasons thought to be responsible for the decrease in effectiveness seen in these areas, including chronic malnutrition and gut dysbiosis. Non-biological reasons for decreased efficacy may include the breakdown of cold-chain storage for these vaccines, which require constant low temperature storage that is often unavailable in LMICs. Thermostable vaccines are necessary for increasing vaccine distribution and efficacy in these areas. Because many of the biologic factors thought to interfere with the effectiveness of these vaccines appear to be confined to the gastrointestinal tract, development of next generation HRV vaccines has focused on the parenteral route of administration. The gnotobiotic (Gn) pig model is a highly relevant animal model that has been used for decades to evaluate novel HRV vaccine efficacy. Our first study evaluated a thermostable, dissolvable live oral vaccine administered as a dissolvable film in our Gn pig model. Two doses of this vaccine significantly reduced the severity of diarrhea and virus shedding in the stool. Our second study evaluated three mRNA-based intramuscular (IM) vaccines in the Gn pig model. Three doses of all mRNA candidates provided significant protection from virus shedding in the stool, as well as inducing the production of strong HRV-specific antibodies in the serum and high numbers of virus-specific T cells in the tissues. In our final study, we evaluated a nanoparticle-based vaccine as a two-dose IM regimen or as an IM booster preceded by an oral immunization using the commercially available Rotarix® vaccine. The prime-boost regimen significantly shortened the duration and severity of virus shedding in the stool. We also detected more cross-strain HRV-specific antibody-secreting cells in the tissues. All three vaccines evaluated in this dissertation offer differing novelty in the field of HRV vaccine development, and the Gn pig model has been instrumental in the evaluation of these vaccines.

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Human rotavirus (HRV) and human norovirus (HuNoV) are the most common causative agents of acute gastroenteritis- (AGE) related morbidity and mortality around the world. Gnotobiotic (Gn) pigs are the ideal large-animal model that allows for accurate, and precise, preclinical evaluation of vaccine efficacy. Similarities in gastrointestinal anatomy, physiology, and immune system allows for direct translation of results from Gn pigs to humans. Commercially available HRV vaccines perform significantly poorer in low- and middle- income countries as compared with developed countries. Non-replicating rotavirus vaccines (NRRVs) have been proposed as a viable solution to the problems facing currently available live-, attenuated oral vaccines and evaluation of a NRRV was the first research project in this dissertation. Three doses of a novel parenterally administered nanoparticle-based RV vaccine, P24-VP8*, adjuvanted with Al(OH)3 adjuvant, was able to prime VP8*-specific mucosal and systemic T cell responses (IFN-γ producing CD4+ and CD8+ T cells), and to induce strong systemic B cell responses (IgA, IgG and serum neutralizing antibodies). A significant reduction in the mean diarrhea duration, fecal virus shedding titers, and significantly lower fecal cumulative consistency scores was observed among vaccinated pigs demonstrating the efficacy of the vaccine against RV infection and diarrhea. Next, we determined the median infectious dose (ID50) and median diarrhea dose (DD50) of the GII.4/2003 Cin-1 variant of HuNoV in Gn pigs to better standardize the pig model for HuNoV vaccine evaluation. Gn pigs were inoculated with 7 different doses of Cin-1 at 33-34 days of age. Pigs were monitored daily from post-inoculation day (PID) 1 to 7, for fecal virus shedding and fecal consistency to evaluate the virus infectiousness and associated diarrhea. The Log10 ID50 and DD50 were determined based on various mathematical models to be between 3.11 to 3.76, and 3.37 to 4.87 RNA copies, respectively. The Beta-Poisson was identified to be the best-fitting statistical model for estimating both the ID50 and DD50 of Cin-1. Determining the ID50 of the challenge virus strain is crucial for identifying the true infectiousness of HuNoVs and for accurate evaluation of protective efficacies in pre-clinical studies of therapeutics, vaccines and other prophylactics using this reliable animal model. The lack of an easily reproducible cell culture model for HuNoV has significantly delayed the development of effective vaccines. There is still no HuNoV vaccine available. Currently, the vaccine development efforts are mostly based on genetically engineered virus-like particles (VLPs) comprised of the major HuNoV capsid protein VP1. We tested the immunogenicity of a novel tetravalent VLP vaccine containing 4 major HuNoV genotypes (GI.1, GII.3, GII.4 and GII.17) using Gn pigs and evaluated its protective efficacy when challenged with GII.4 Cin-1 HuNoV. Three doses of the VLP vaccine with Al(OH)3 adjuvant administered to Gn pigs intramuscularly (IM), induced high levels of VLP-specific serum IgA and IgG antibody and hemagglutination inhibition antibody responses in the vaccinated pigs. VLP-specific IFN-γ producing CD4+ and CD8+ T cells were also elevated among vaccinated pigs at post-challenge day (PCD) 7 in the spleen and blood, but not in the ileum. However, the vaccinated pigs were not protected from infection and diarrhea when challenged with any one of the three different doses (2 x 105, 8 x 104, and 2 x 104 genome RNA copies) of Cin-1 HuNoV. These results indicated that the IM tetravalent VLP vaccine was highly immunogenic, but the presence of high levels of immune effectors induced by the vaccine were not sufficient for protecting the Gn pigs from Cin-1 challenge. Amino acid (aa) sequence analysis showed that the GII.4 Sydney 2012 strain which was included in the VLP vaccine, had 23 aa substitutions in the major receptor binding domain (P2) compared to the Cin-1, a GII.4 Farmington Hills 2002 strain. Our findings, for the first time, provided in vivo experimental evidence for the total lack of cross-genogroup, cross-genotype and cross-variant protection among HuNoV. This finding has importance implications for HuNoV vaccine development. HuNoV vaccines have to include multiple variants and have to be routinely updated in order to ensure sustained protection among the population. Together these three studies in this dissertation demonstrate the versatility of Gn pigs as a reliable large animal model for studying the pathogenesis and immunity of enteric viruses and the evaluation of immunogenicity and protective efficacy of novel enteric viral vaccines. / Doctor of Philosophy / People of all age groups are susceptible to acute gastroenteritis (AGE), a condition characterized by sudden onset of diarrhea, nausea and abdominal cramps. The two most important viral pathogens responsible for causing AGE are rotavirus (RV) and norovirus (NoV). Gnotobiotic (Gn) pigs have been valuable in helping us understand the mechanism of infection, pathogenesis, immunity and have played a key role in the expediting development of novel vaccines and therapeutics against both of these viruses. Live oral RV vaccines are available but they are not very effective in low income countries where the vaccines are needed the most. Next generation parenteral vaccines are proposed to improve the RV vaccine efficacy. Our first study showed that a nanoparticle-based intramuscular (IM) RV vaccine effectively reduced the duration and severity of human RV infection and diarrhea in Gn pigs. Secondly, we examined in detail the infectivity of HuNoV and identified accurately using different mathematical models on how much virus would be required to infect and cause diarrhea in naïve Gn pigs. This knowledge would greatly help in the accurate assessment of the efficacy of NoV vaccines. Third, we evaluated the immunogenicity and protective efficacy of a tetravalent IM NoV vaccine in Gn pigs. Although the vaccine was highly immunogenic, it did not confer any protection against infection and diarrhea upon challenge with the NoV at different doses. NoVs are so diverse that one year we might be infected with one strain and a few years later, we might be infected again with another strain, even though they belong to the same genotype, and experience the same symptoms. This is because, changes brought about due to mutation in the virus capsid protein allow the viruses to hide from neutralizing antibodies induced by previous infection or vaccination as we have revealed in this study. NoV diversity and lack of cross protection need to be taken into consideration during vaccine development. This thesis shows how Gn pigs can be used to study these components in order to further maximize our ability to understand and combat enteric viral diseases.

With the absence of gut microbiota, gnotobiotic (Gn) pigs are a unique animal model for studying infection and immunity, and evaluating vaccine and therapeutics for human enteric pathogens. Here, we demonstrate Gn pigs as effective large animal models for human enteric viruses, through evaluating human enterovirus 71 (EV71) infection and immunity, and vaccine and therapeutics for human rotavirus (HRV). Gn pigs could be infected via oral or oronasal route, the natural route of infection. Infected pigs developed clinical signs including fever, neurological and respiratory signs, similar to those seen in human patients. Fecal shedding up to 18 days post infection and virus distribution in intestinal, respiratory and central nervous system tissues were observed. Strong mucosal and systemic T cell responses (IFN-γ producing CD4+ and CD8+ T cells) and systemic B cell responses (serum neutralizing antibodies) were also detected. The study demonstrates a novel large animal model for EV71 to investigate viral pathogenesis, immunity, and to evaluate vaccine and antiviral drugs. Using the well-established Gn pig model for HRV, the adjuvant and therapeutic effects of prebiotics rice bran (RB) and probiotics were evaluated. RB alone or RB plus probiotic Lactobacillus rhamnosus GG (LGG) and probiotic E. coli Nissle 1917 (EcN), were shown to protect against rotavirus diarrhea (80%-100% reduction in the incidence rate) significantly and display strong immune - stimulatory effects on the immunogenicity of an oral attenuated HRV (AttHRV) vaccine. Mechanisms for the adjuvant effect include stimulating the production of intestinal and systemic IFN-γ] producing T cells and promoting mucosal IgA antibody responses. The mechanisms for reducing rotavirus diarrhea include promoting LGG and EcN growth and colonization and host gut health, and maintaining gut integrity and permeability during rotavirus infection. We showed that RB plus LGG and EcN is a highly effective therapeutic regimen against HRV diarrhea. Together, these results indicated that Gn pigs may serve as an excellent animal model for the study of infection, immunity, vaccine and therapeutics for human enteric viruses. / Ph. D.

Rotaviruses (RVs) are medically significant gastrointestinal pathogens and are a leading cause of childhood mortality in many countries. The RV RNA-dependent RNA polymerase, VP1, synthesizes RNA during viral replication only in the presence of another RV protein, VP2, which comprises the innermost core shell layer of the virion. Though these VP1-VP2 interactions are essential for RV replication, the mechanism by which the core shell regulates polymerase activity remains incompletely understood. Here, we sought to identify and characterize specific regions of both VP1 and VP2 that are required for core shell dependent polymerase activity. First, we used bioinformatics approaches to analyze VP1 and VP2 sequence diversity across many RV strains and identify positional locations of critical amino acid changes within the context of known structural domains and motifs. We next tested how the identified sequence differences influenced VP2-dependent VP1 activity in vitro. These data revealed that VP1 and VP2 protein diversity correlates with functional differences between avian and mammalian RV strains. Then, we used these sequential and functional incompatibilities to map key regions of VP1 important for mediating RNA synthesis. To pinpoint critical interacting regions of VP1 and VP2, we used site directed mutagenesis to engineer several modified VP1 and VP2 proteins. Then, we employed an in vitro RNA synthesis assay to test how the introduced mutations influenced VP2-dependent VP1 activity. Altogether, our results revealed several functionally important VP1 residues critical for in vitro VP2-dependent VP1 activity, either individually or in combination with neighboring residues, including E265/L267, R614, and D971/S978/I980. Structural analyses show VP2 interactions at these surface-exposed VP1 sites, which altogether supports a direct contact model of core shell dependent RV polymerase activity. Moreover, recombinant VP1 proteins containing multiple mutations at buried residues were incapable of facilitating RNA synthesis in vitro under the assay conditions, indicating that an extensive intramolecular signaling network exists to mediate VP1 activity. Taken together, these results suggest that VP2 binding at the VP1 surface may induce intramolecular interactions critical for VP1 activity. Overall, results from these studies provide important insight into VP1-VP2 binding interface(s) that are necessary for RV replication. / Ph. D. / Rotaviruses (RVs) are clinically-significant gastrointestinal pathogens that cause severe diarrhea and dehydration in children. RVs encode a specialized polymerase enzyme, called VP1, which functions to synthesize RNA during viral replication. RNA synthesis activities of VP1 are tightly regulated by another RV protein, VP2, which comprises the innermost core shell layer of the virion. Though these VP1-VP2 interactions are essential for viral replication, the mechanism by which the core shell supports polymerase activity remains poorly understood. Here, we sought to identify and characterize specific regions of both VP1 and VP2 that are essential for polymerase activity in a test tube (i.e., in vitro). First, we analyzed VP1 and VP2 sequence diversity across many RV strains. Then, we tested how the identified sequence differences influenced VP2-dependent VP1 activation in vitro. To pinpoint critical regions of VP1 and VP2, we next engineered and assayed several mutant proteins. Altogether, our results revealed several functionally important residues of VP1 and VP2, which raises new ideas about VP1-VP2 binding interface(s) that are important for viral replication. Moreover, results from these studies may provide a scientific platform for the rational design of next-generation RV vaccines or antiviral therapeutics.

Norovirus and rotavirus are the most common viral causes of acute gastroenteritis among all age groups and in children under 5 years of age, respectively. Understanding the pathogenesis of the virus and correlates of protective immunity is fundamental to developing effective prevention and treatment strategies. Gnotobiotic (Gn) pigs are an attractive animal model for studying enteric viruses due to their similarities to humans, particularly in regards to the immune system and gastrointestinal anatomy and physiology. Here, to establish a reliable Gn pig model of human norovirus (HuNoV) infection and disease, we determined the median infectious dose (ID50) of a GII.4 2006b variant in pigs. We also evaluated the effects of age and administration of the cholesterol-lowering drug simvastatin on susceptibility to NoV infection. In neonatal pigs (4-5 days of age, the ID50 was determined to be 2.74 x 103 viral RNA copies. The ID50 was increased in 33-34 day old pigs (6.43 x 104), but decreased to <2.74 x 103 following simvastatin treatment in the same age group. Overall, the development of diarrhea, fecal virus shedding and small intestinal cytopathological changes confirmed the usefulness of the Gn pig as an appropriate animal model for studying HuNoVs. We also utilized the well-established Gn pig model of human rotavirus (HRV) infection and disease to evaluate adjunctive treatment options for HRV-induced diarrhea. We demonstrated that the anti-secretory drug racecadotril was capable of diminishing clinical signs of HRV infection and shortening duration of illness. Reduced dehydration in the racecadotril-treated pigs was evident by the significant gain in body weight compared to controls during the course of the study. We also determined that a high dose of the probiotic Lactobacillus acidophilus NCFM (LA) was able to reduce RV diarrhea severity and duration compared to a low dose. The difference in therapeutic potential was attributed to divergent effects in innate immunity pre- and post-challenge. High dose of LA (HiLA) induced an anti-inflammatory dendritic cell (DC) profile, characterized primarily by upregulation of TLR2 expression and production of cytokine IL-10. Conversely, low dose of LA (LoLA) upregulated TLR3 and TLR9 and increased secretion of cytokine IL-6. Additionally, HiLA induced both IFN-alpha and TNF-alpha responses in DCs, but LoLA was only able to increase the frequency of TNF-alpha-producing DCs. These results provide further support of Gn pigs as a highly applicable animal model for studying pathogenesis, innate immunity and therapeutics of human enteric viruses. / Ph. D.

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

Reverse genetics is an innovative molecular biology tool that enables the manipulation of viral genomes at the cDNA level in order to generate particular mutants or artificial viruses. The reverse genetics system for the influenza virus is arguably one of the best illustrations of the potential power of this technology. This reverse genetics system is the basis for the ability to regularly adapt influenza vaccines strains. Today, reverse genetic systems have been developed for many animal RNA viruses. Selection-free reverse genetics systems have been developed for the members of the Reoviridae family including, African horsesickness virus, bluetongue virus and orthoreovirus. This ground-breaking technology has led to the generation of valuable evidence regarding the replication and pathogenesis of these viruses. Unfortunately, extrapolating either the plasmid-based or transcript-based reverse genetics systems to rotavirus has not yet been successful. The development of a selection-free rotavirus reverse genetics system will enable the systematic investigation of poorly understood aspects of the rotavirus replication cycle and aid the development of more effective vaccines, amongst other research avenues. This study investigated the importance of co-expressed rotavirus proteins in the development of a selection-free rotavirus reverse genetics system. The consensus sequences of the rotavirus strains Wa (RVA/Human-tc/USA/WaCS/1974/G1P[8]) and SA11 (RVA/Simian-tc/ZAF/SA11/1958/G3P[2]) where used to design rotavirus expression plasmids. The consensus nucleotide sequence of a human rotavirus Wa strain was determined by sequence-independent cDNA synthesis and amplification combined with next-generation 454® pyrosequencing. A total of 4 novel nucleotide changes, which also resulted in amino acid changes, were detected in genome segment 7 (NSP3), genome segment 9 (VP7) and genome segment 10 (NSP4). In silico analysis indicated that none of the detected nucleotide changes, and consequent amino acid variations, had any significant effect on viral structure. Evolutionary analysis indicated that the sequenced rotavirus WaCS was closely related to the ParWa and VirWa variants, which were derived from the original 1974 Wa isolate. Despite serial passaging in animals, as well as cell cultures, the Wa genome seems to be stable. Considering that the current reference sequence for the Wa strain is a composite sequence of various Wa variants, the rotavirus WaCS may be a more appropriate reference sequence. The rotavirus Wa and SA11 strains were selected for plasmid-based expression of rotavirus proteins, under control of a T7 promoter sequence, due to the fact that they propagate well in MA104 cells and the availability of their consensus sequences. The T7 RNA polymerase was provided by a recombinant fowlpox virus. After extensive transfection optimisation on a variety of mammalian cell lines, MA104 cells proved to be the best suited for the expression rotavirus proteins from plasmids. The expression of rotavirus Wa and SA11 VP1, VP6, NSP2 and NSP5 could be confirmed with immunostaining in MA104 and HEK 293H cells. Another approach involved the codon-optimised expression of the rotavirus replication complex scaffold in MA104 cells under the control of a CMV promoter sequence. This system was independent from the recombinant fowlpox virus. All three plasmid expression sets were designed to be used in combination with the transcript-based reverse genetics system in order to improve the odds of developing a successful rotavirus reverse genetics system. Rotavirus transcripts were generated using transcriptively active rotavirus SA11 double layered particles (DLPs). MA104 and HEK293H cells proved to be the best suited for the expression of rotavirus transcripts although expression of rotavirus VP6 could be demonstrated in all cell cultures examined (MA104, HEK 293H, BSR and COS-7) using immunostaining. In addition, the expression of transcript derived rotavirus VP1, NSP2 and NSP5 could be confirmed with immunofluorescence in MA104 and HEK 293H cells. This is the first report of rotavirus transcripts being translated in cultured cells. A peculiar cell death pattern was observed within 24 hours in response to transfection of rotavirus transcripts. This observed cell death, however does not seem to be related to normal viral cytopathic effect as no viable rotavirus could be recovered. In an effort to combine the transcript- and plasmid systems, a dual transfection strategy was followed where plasmids encoding rotavirus proteins were transfected first followed, 12 hours later, by the transfection of rotavirus SA11 transcripts. The codon- optimised plasmid system was designed as it was postulated that expression of the DLP-complex (VP1, VP2, VP3 and VP6), the rotavirus replication complex would form and assist with replication and/or packaging. Transfecting codon- optimized plasmids first noticeably delayed the mass cell death observed when transfecting rotavirus transcripts on their own. None of the examined coexpression systems were able to produce a viable rotavirus. Finally, the innate immune responses elicited by rotavirus transcripts and plasmid-derived rotavirus Wa and SA11 proteins were investigated. Quantitative RT-PCR (qRT-PCR) experiments indicated that rotavirus transcripts induced high levels of the expression of the cytokines IFN- α1, IFN-1β, IFN-λ1 and CXCL10. The expression of certain viral proteins from plasmids (VP3, VP7 and NSP5/6) was more likely to stimulate specific interferon responses, while other viral proteins (VP1, VP2, VP4 and NSP1) seem to be able to actively suppress the expression of certain cytokines. In the light of these suppression results, specific rotavirus proteins were expressed from transfected plasmids to investigate their potential in supressing the interferon responses provoked by rotavirus transcripts. qRT-PCR results indicated that cells transfected with the plasmids encoding NSP1, NSP2 or a combination of NSP2 and NSP5 significantly reduced the expression of specific cytokines induced by rotavirus transcripts. These findings point to other possible viral innate suppression mechanisms in addition to the degradation of interferon regulatory factors by NSP1. The suppression of the strong innate immune response elicited by rotavirus transcripts might well prove to be vital in the quest to better understand the replication cycle of this virus and eventually lead to the development of a selection-free reverse genetics system for rotavirus. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014