No description available.

Rotaviruses, members of the family Reoviridae, are the major etiologic agents of severe, acute dehydrating diarrhea in the young of many mammalian species, including humans, calves and foals. Recent estimates indicate an annual death toll of approximately 600,000 infants due to rotavirus, besides inflicting staggering economic burden worldwide. Most of these deaths occur in the developing countries and India is estimated to account for about a quarter of these deaths. Extensive molecular epidemiology studies carried out by our laboratory have revealed many interesting aspects about rotavirus diversity in this country. Molecular epidemiology of rotaviruses causing severe diarrhea in foals in two organized farms in northern India was carried out. These foal rotaviruses exhibited 5 different electropherotypes (E), E1-E5. Strains belonging to E1, E2 and E5 exhibited G10, P6[1]; G3 and G1 type specificities. Though the E1 strains possessed genes encoding G10 and P6[1] type outer capsid proteins, unlike the G10, P8[11] type strain I321, they exhibited high reactivity with the G6-specific MAb suggesting that the uncommon combination altered the specificity of the conformation-dependent antigenic epitopes on the surface proteins. Strains belonging to electropherotypes E3 and E4 were untypeable. Sequence analysis of the VP7 gene from E4 strains (Erv92 and Erv99), revealed that they represent a new VP7 genotype, G16. Nonstructural protein 4 (NSP4) of rotavirus is a multidomainal, multifunctional protein and is the first viral enterotoxin identified. We have recently reported that the diarrhea-inducing and double-layered particle (DLP)–binding properties of NSP4 are dependent on a structurally and functionally overlapping conformational domain that is conferred by cooperation between the N- and C-terminal regions of the cytoplasmic tail (Jagannath et al., J. Virol, pp 412-425, 2006). Further, a stretch of 40 amino acids (aa) from the C-terminus is predicted to be unstructured and highly susceptible to trypsin cleavage. We examined the role of this unstructured C-terminus of Hg18 NSP4 and SA11 NSP4 on the biological properties of NSP4 using a series of deletion and substitution mutants of the conserved proline and tyrosine residues in this region. Gel filtration, CD spectroscopy and Thioflavin T binding studies showed that these mutants have altered secondary structural contents and either failed to multimerize efficiently or multimerized with altered conformation. The C-terminal ten residues appear to play a regulatory role on multimerization. Proline 168, tyrosine 166 and methionine 175 appear to be critical determinants of DLP binding activity whereas, proline 165 and tyrosine 85 and 131 appears to determine the affinity of binding to DLP in the context of NSP4 ∆N72. Deletion and substitution mutants exhibited severely reduced diarrhea inducing ability and DLP binding property. Of great biological significance is the drastic decrease in the diarrhea inducing ability of the N- and C- terminal deletion mutant ∆N94 ∆C29 that exhibited about 11,000-fold increase in DD50 than the wild type (WT) ∆N72. These studies revealed that the predicted unstructured C-terminus is an important determinant of biological properties of NSP4. Extensive efforts to crystallize the complete cytoplasmic tail (CT) of NSP4 were unsuccessful and to date, the structure of only a synthetic peptide corresponding to aa 95-135 has been reported. Our recent studies indicate that the interspecies variable regions from aa 135-141 as well as the extreme C-terminus are critical determinants of virus virulence and diarrhea-inducing ability of the protein. Here, we examined the crystallization properties of several deletion mutants and report the structure of a mutant recombinant NSP4 from symptomatic (SA11) and asymptomatic (I321) strains that lacked the N-terminal 94 and C-terminal 29 aa (NSP4: 95-146) at 1.67 Å and 2.7Å, respectively. In spite of the high-resolution data, electron density for the stretch of 9 residues from the C-terminus could not be seen suggesting its highly flexible nature. The crystal packing showed a clear empty space for this region. Extension of the unstructured C-terminus beyond aa 146 hindered crystallization under the experimental conditions. The present structure revealed significant differences from that of the synthetic peptide in the conformation of amino acids at the end of the helix as well as crystal packing owing to the additional space required to accommodate the unstructured virulence-determining region. Conformational differences in this critical region effected by the presence or absence of proline or glycine at specific positions in the unstructured C-terminus, could form the basis for the wide range of variation seen in the diarrhea-inducing ability of NSP4 from different strains in newborn mouse pups. Although symptomatic and asymptomatic strains do not generally differ in the presence or absence of the conserved prolines or glycines, they contain a few additional changes that could alter the unique conformation required for optimal biological activity. In conclusion, we demonstrate that the predicted unstructured C-terminal region is indeed highly flexible and is an important determinant of biological functions of the NSP4, mutations in which probably correlates with the virulence properties of the virus.

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