Strain SA11 Research Articles

Preparation of 4-arm star PELA and its encapsulation of rotavirus for drug delivery

A relatively high molecular weight of 4-arm star PELA was obtained by ring-opening polymerization of l-lactic acid O-carboxyanhydride with 4-arm-PEG in the presence of DMAP as an initiator. The results via1H NMR and 13C NMR show that the end of the star PELA chain is a hydroxyl group and the central core is a PEG group. Rotavirus (strain SA11) was incorporated into 4-arm star PELA microspheres formulated by the water in oil in water emulsification solvent extraction method. The microspheres produced were spherical, and the mean diameter was 1.34μm with a narrow size distribution. The drug release profile displayed a low burst release effect of 1.8% on the first day and a sustained release of antigen over 100 days. After the immunization of mice, the microsphere-entrapped RV elicited improved and long-lasting IgA and IgG antibody response in serum detected by ELISA in comparison to the free RV antigen. This study shows that 4-arm-PEG is an effective initiator for the ring-opening polymerization of Lac-OCA by DMAP as an initiator and that the resulting polymer is useful as a delivery system for the rotavirus vaccine.

Whole Genomic Analysis of Human G12P[6] and G12P[8] Rotavirus Strains that Have Emerged in Myanmar.

G12 rotaviruses are emerging rotavirus strains causing severe diarrhea in infants and young children worldwide. However, the whole genomes of only a few G12 strains have been fully sequenced and analyzed. In this study, we sequenced and characterized the complete genomes of six G12 strains (RVA/Human-tc/MMR/A14/2011/G12P[8], RVA/Human-tc/MMR/A23/2011/G12P[6], RVA/Human-tc/MMR/A25/2011/G12P[8], RVA/Human-tc/MMR/P02/2011/G12P[8], RVA/Human-tc/MMR/P39/2011/G12P[8], and RVA/Human-tc/MMR/P43/2011/G12P[8]) detected in six stool samples from children with acute gastroenteritis in Myanmar. On whole genomic analysis, all six Myanmarese G12 strains were found to have a Wa-like genetic backbone: G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strains A14, A25, P02, P39, and P43, and G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1 for strain A23. Phylogenetic analysis showed that most genes of the six strains examined in this study were genetically related to globally circulating human G1, G3, G9, and G12 strains. Of note is that the NSP4 gene of strain A23 exhibited the closest relationship with the cognate genes of human-like bovine strains as well as human strains, suggesting the occurrence of reassortment between human and bovine strains. Furthermore, strains A14, A25, P02, P39, and P43 were very closely related to one another in all the 11 gene segments, indicating derivation of the five strains from a common origin. On the other hand, strain A23 consistently formed distinct clusters as to all the 11 gene segments, indicating a distinct origin of strain A23 from that of strains A14, A25, P02, P39, and P43. To our knowledge, this is the first report on whole genome-based characterization of G12 strains that have emerged in Myanmar. Our observations will provide important insights into the evolutionary dynamics of spreading G12 rotaviruses in Asia.

Genetic Determinants of Japanese Encephalitis Virus Vaccine Strain SA14-14-2 That Govern Attenuation of Virulence in Mice.

The safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine are attributed to mutations that accumulated in the viral genome during its derivation. However, little is known about the contribution that is made by most of these mutations to virulence attenuation and vaccine immunogenicity. Here, we generated recombinant JEV (rJEV) strains containing JEV SA14-14-2 vaccine-specific mutations that are located in the untranslated regions (UTRs) and seven protein genes or are introduced from PCR-amplified regions of the JEV SA14-14-2 genome. The resulting mutant viruses were evaluated in tissue culture and in mice. The authentic JEV SA14-14-2 (E) protein, with amino acid substitutions L107F, E138K, I176V, T177A, E244G, Q264H, K279M, A315V, S366A, and K439R relative to the wild-type rJEV clone, was essential and sufficient for complete attenuation of neurovirulence. Individually, the nucleotide substitution T39A in the 5' UTR (5'-UTR-T39A), the capsid (C) protein amino acid substitution L66S (C-L66S), and the complete NS1/2A genome region containing 10 mutations each significantly reduced virus neuroinvasion but not neurovirulence. The levels of peripheral virulence attenuation imposed by the 5'-UTR-T39A and C-L66S mutations, individually, were somewhat mitigated in combination with other vaccine strain-specific mutations, which might be compensatory, and together did not affect immunogenicity. However, a marked reduction in immunogenicity was observed with the addition of the NS1/2A and NS5 vaccine virus genome regions. These results suggest that a second-generation recombinant vaccine can be rationally engineered to maximize levels of immunogenicity without compromising safety. The live-attenuated JEV SA14-14-2 vaccine has been vital for controlling the incidence of disease caused by JEV, particularly in rural areas of Asia where it is endemic. The vaccine was developed >25 years ago by passaging wild-type JEV strain SA14 in tissue cultures and rodents, with intermittent tissue culture plaque purifications, to produce a virus clone that had adequate levels of attenuation and immunogenicity. The vaccine and parent virus sequences were later compared, and mutations were identified throughout the vaccine virus genome, but their contributions to attenuation were never fully elucidated. Here, using reverse genetics, we comprehensively defined the impact of JEV SA14-14-2 mutations on attenuation of virulence and immunogenicity in mice. These results are relevant for quality control of new lots of the current live-attenuated vaccine and provide insight for the rational design of second-generation, live-attenuated, recombinant JEV vaccine candidates.

Biocatalytic desulfurization of thiophenic compounds and crude oil by newly isolated bacteria.

Microorganisms possess enormous highly specific metabolic activities, which enable them to utilize and transform nearly every known chemical class present in crude oil. In this context, one of the most studied biocatalytic processes is the biodesulfurization (BDS) of thiophenic sulfur-containing compounds such as benzothiophene (BT) and dibenzothiophene (DBT) in crude oils and refinery streams. Three newly isolated bacterial strains, which were affiliated as Rhodococcus sp. strain SA11, Stenotrophomonas sp. strain SA21, and Rhodococcus sp. strain SA31, were enriched from oil contaminated soil in the presence of DBT as the sole S source. GC-FID analysis of DBT-grown cultures showed consumption of DBT, transient formation of DBT sulfone (DBTO2) and accumulation of 2-hydroxybiphenyl (2-HBP). Molecular detection of the plasmid-borne dsz operon, which codes for the DBT desulfurization activity, revealed the presence of dszA, dszB, and dszC genes. These results point to the operation of the known 4S pathway in the BDS of DBT. The maximum consumption rate of DBT was 11 μmol/g dry cell weight (DCW)/h and the maximum formation rate of 2-HBP formation was 4 μmol/g DCW/h. Inhibition of both cell growth and DBT consumption by 2-HBP was observed for all isolates but SA11 isolate was the least affected. The isolated biocatalysts desulfurized other model DBT alkylated homologs. SA11 isolate was capable of desulfurizing BT as well. Resting cells of SA11 exhibited 10% reduction in total sulfur present in heavy crude oil and 18% reduction in total sulfur present in the hexane-soluble fraction of the heavy crude oil. The capabilities of the isolated bacteria to survive and desulfurize a wide range of S compounds present in crude oil are desirable traits for the development of a robust BDS biocatalyst to upgrade crude oils and refinery streams.

Cordycepin an Adenosine Analogue Executes Anti Rotaviral Effect by Stimulating Induction of Type I Interferon

Abstract Objectives: Rotavirus is the single most common etiological pathogen of severe diarrhea in infants causing death of over half a million infants a year. In spite of such a huge disease burden, still no effective antivirals are available against rotavirus. Cordycepin (3’-deoxyadenosine), an adenosine analogue, has been reported to modulate cell proliferation, platelet aggregation and provide protection against HIV infection. Herein, attempts were made to analyze efficacy of cordycepin for protection against rotavirus and underlying mechanism. Methods Cordycepin’s anti-rotaviral activity was elucidated against simian rotavirus strain SA11 (H96), rhesus strain RRV, human strain Wa (in vitro) and murine strain EW (in BALB/c). Plaque assay, qPCR for viral transcripts and immunoblotting of viral proteins were done to examine viral abundance. Effect of cordycepin on Interferon pathway was analyzed by immunoblotting, co-immunoprecipitation and molecular docking. Results Cordycepin was found to reduce propagation of different rotavirus strains such as SA11, RRV, Wa at 64μM with minimal cytotoxicity in vitro and EW in BALB/c mice. Protection was obtained upto 24 hours, after virus infection at both low and high multiplicity of infection. Sensitivity of rotavirus infection to Interferons has been documented. In cordycepin treated cells increased induction of Interferon and downstream antiviral proteins were observed. Cordycepin treatment leads to RIGI-MAVS interaction, IRF3 activation, and Interferon induction. By using different software probable docking sites and orientation of cordycepin and RIG-I were predicted. Cordycepin was found to have relatively higher binding probability for ATP binding domain than other domains. In Vero cells,deficient in Interferon signalling, cordycepin failed to protect against rotavirus. This suggests that though cordycepin may have other effects but anti-rotaviral effects were due to boosting of cellular innate immune responses mediated by Interferon. Conclusions Overall this study highlights that cordycepin exerts its anti-rotaviral activity by modulating immune signalling through enhanced Interferon production.

Structural Plasticity of the Coiled-Coil Domain of Rotavirus NSP4

Rotavirus (RV) nonstructural protein 4 (NSP4) is a virulence factor that disrupts cellular Ca(2+) homeostasis and plays multiple roles regulating RV replication and the pathophysiology of RV-induced diarrhea. Although its native oligomeric state is unclear, crystallographic studies of the coiled-coil domain (CCD) of NSP4 from two different strains suggest that it functions as a tetramer or a pentamer. While the CCD of simian strain SA11 NSP4 forms a tetramer that binds Ca(2+) at its core, the CCD of human strain ST3 forms a pentamer lacking the bound Ca(2+) despite the residues (E120 and Q123) that coordinate Ca(2+) binding being conserved. In these previous studies, while the tetramer crystallized at neutral pH, the pentamer crystallized at low pH, suggesting that preference for a particular oligomeric state is pH dependent and that pH could influence Ca(2+) binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca(2+) or pH. Biochemical, biophysical, and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high-affinity binding to Ca(2+) at neutral pH and forms a tetramer, it does not bind Ca(2+) at low pH and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca(2+) binding is necessary for the tetramer formation, as an E120A mutant forms a pentamer. We propose that the structural plasticity of NSP4 regulated by pH and Ca(2+) may form a basis for its pleiotropic functions during RV replication. The nonstructural protein NSP4 of rotavirus is a multifunctional protein that plays an important role in virus replication, morphogenesis, and pathogenesis. Previous crystallography studies of the coiled-coil domain (CCD) of NSP4 from two different rotavirus strains showed two distinct oligomeric states, a Ca(2+)-bound tetrameric state and a Ca(2+)-free pentameric state. Whether NSP4 CCD from the same strain can exist in different oligomeric states and what factors might regulate its oligomeric preferences are not known. This study used a combination of biochemical, biophysical, and crystallography techniques and found that the NSP4 CCD can undergo a reversible transition from a Ca(2+)-bound tetramer to a Ca(2+)-free pentamer in response to changes in pH. From these studies, we hypothesize that this remarkable structural adaptability of the CCD forms a basis for the pleiotropic functional properties of NSP4.

Involvement of aquaporins in a mouse model of rotavirus diarrhea.

Rotavirus diarrhea is a major worldwide cause of infantile gastroenteritis; however, the mechanism responsible for intestinal fluid loss remains unclear. Water transfer across the intestinal epithelial membrane seems to occur because of aquaporins (AQPs). Accumulating evidence indicates that alterations in AQPs may play an important role in pathogenesis. Here, we focus on changes in AQPs in a mouse model of rotavirus diarrhea. In the present study, 32 of 35 mice developed diarrhea and mild dehydration within 24 hours after infection with rotavirus strain SA11. Intestinal epithelial cells demonstrated cytoplasmic vacuolation, malaligned villi, and atrophy. AQP1 expression was significantly attenuated in the ileum and colon in comparison with controls; likewise, AQP4 and -8 protein expression were significantly decreased in the colon of rotavirus diarrhea-infected mice. In contrast, AQP3 protein expression was significantly increased in the colon of rotavirus-infected mice in comparison with controls. These results indicate that rotavirus diarrhea is associated with the downregulation of AQP1, -4, and -8 expression. Therefore, AQPs play an important role in rotavirus diarrhea.

Characterization of live-attenuated Japanese encephalitis vaccine virus SA14-14-2

The live attenuated Japanese encephalitis (JE) vaccine SA14-14-2 was licensed decades ago and now approved for clinical use in most JE endemic countries. Large-scale clinical trials demonstrate ideal safety and efficacy profile of this Chinese vaccine. The SA14-14-2 vaccine was derived from a virulent strain SA14 after hundreds of serial passaging in cells and animals, concern about virulence reversion remains exist. In the present study, to study the in vitro and in vivo genetic and attenuation stability of the vaccine virus, SA14-14-2 was serially passaged in Vero cells and mouse brain followed by sequence comparison and attenuation phenotype analysis. The results showed that no significant mutation was acquired after serial passaging in Vero cells except a single Ser66Leu mutation within capsid protein, which had no effect on viral virulence in mice. Importantly, serial passaging of SA14-14-2 in suckling mouse brain resulted in emergence of adaptive mutations and increased virulence in mice. Population and plaque-purified clone consensus sequence analysis showed four adaptive mutations in envelope (E) protein, F107L, K138E, T226R and I270T, sequentially occurred and become predominant during serial passaging in suckling mouse brain. Especially, these adaptive mutations were close related with the enhanced neurovirulence and neuroinvasiveness in mice. Our results provide experimental evidence of highly genetic and attenuation stability of SA14-14-2 following passaging in Vero cells, and reveal the potential virulence reversion during passaging in mouse brain in association with critical adaptive mutations in E protein. These findings are important for quality control and evaluation of live JE vaccines and will help understand the attenuation mechanism of flavivirus vaccine.

Sequence and structural analyses of NSP4 proteins from human group A rotavirus strains detected in Tunisia

BackgroundThe NSP4 protein of group A rotavirus (RVA) has been recognized as a viral enterotoxin and plays important roles in viral pathogenesis and morphogenesis. Domains involved in structural and functional interactions have been proposed mainly based on the simian SA11 strain. MethodsNSP4 has been classified into 15 different genotypes (E1-E15), and the aim of this study was to analyze the sequences of 46 RVA strains in order to determine the aminoacid (aa) differences between E1 and E2 genotypes. Another aspect was to characterize the structural and physicochemical properties of these strains. ResultsComparison of deduced aa sequences of the NSP4 protein showed that divergences between NSP4 genotypes E1 and E2 were mostly observed in the VP4-binding, the interspecies variable domain (ISVD) and the double-layered particle (DLP) binding domains. Interestingly, uncommon variations in residues 131 and 138, which are known to be important aa in pathogenesis, were found in one unusual animal derived strain belonging to the E2 genotype. Concerning the structural aspect, no significant differences were noted. ConclusionThe presence of punctual aa variations in the NSP4 genotypes may indicate that NSP4 mutates mainly via accumulation of point mutations.

Inhibitory Effect of a Hot-Water Extract of Leaves of Japanese Big-Leaf Magnolia (Magnolia obovata) on Rotavirus-Induced Diarrhea in Mouse Pups.

The leaf of Japanese big-leaf magnolia (Magnolia obovata Thunb.) has long been used as a natural packaging material for traditional foods in Japan. However, many of the physiological functions of the leaves against oral infection and resultant illness remain unclear. The aim of the present study was to investigate the effects of a hot-water extract of the leaves of Magnolia obovata on diarrhea induced by rotavirus (RV), a major cause of acute diarrhea. RV strain SA11 was mixed with the M. obovata leaf extract and inoculated orally to neonatal BALB/c mouse pups. Simultaneous inoculation of SA11 with the extract significantly decreased the incidence of diarrhea. In addition, the extract significantly inhibited cytopathic effects and mRNA expression of viral proteins in SA11-infected MA104 cells. Two flavonoid glycosides, quercitrin and rutin, were strongly suggested to be major anti-RV agents in the extract by serial solvent extraction and reversed-phase HPLC-ESI-MS analysis. Our results suggest that the hot-water extract of M. obovata leaves can be used as a medicine or food additive to prevent and ameliorate RV-induced diarrhea in individuals that may have difficulty in benefitting from the RV vaccines.

Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A

A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102M<NS4A-R79K<NS2B-T102M+NS4A-R79K. An in vivo virulence assay in mice showed that the recombinant virus containing double mutations showed similar virulence to the WT SA14 (GenBank accession no. M55506). This study reports the first chemically synthesized JEV. A reverse genetics assay demonstrated that substitutions of NS2B-T102M and NS4A-R79K altered JEV replication.

Analysis of Seroreactivity against Cell Culture–DerivedBartonellaspp. Antigens in Dogs

BackgroundLittle is known about the specificity of Bartonella spp. immunofluorescent antibody (IFA) assays in dogs. Bacteremia in sick dogs most often has been associated with Bartonella henselae (Bh), Bartonella vinsonii subspecies berkhoffii (Bvb), and Bartonella koehlerae (Bk). Clarification of the diagnostic utility of IFA serology when testing against these organisms is needed.ObjectiveTo evaluate the specificity of Bartonella IFA assays utilizing 6 cell culture–grown antigen preparations.AnimalsArchived sera from SPF dogs (n = 29) and from dogs experimentally infected with Bvb (n = 10) and Bh (n = 3).MethodsAntibodies (Abs) to Bvb genotypes I, II, and III, Bh serotype I, strains H‐1 and SA2, and to Bk were determined by IFA testing.ResultsSerum from naïve SPF dogs shown to be negative for Bartonella bacteremia did not react with any of the 6 Bartonella antigens by IFA testing. Dogs experimentally infected with Bvb genotype I developed Abs against homologous antigens, with no cross‐reactivity to heterologous Bvb genotypes, Bh H‐1, SA2 strains, or to Bk. Dogs experimentally infected with Bh serotype I developed Abs against Bh H‐1, but not to Bh SA2 strain with no cross‐reactive Abs to Bvb genotypes I–III or to Bk.Conclusions and Clinical Importance Bartonella spp. Ab responses during acute experimental infections are species and type specific.

Flavonoid-associated direct loss of rotavirus antigen/antigen activity in cell-free suspension

Secondary plant metabolites, such as the flavonoids epigallocatechin gallate (EGCG) and proanthocyanidins (PAC) have been linked to numerous health promoting activities. Studies addressing flavonoid antiviral activity, however, have been directed towards testing infectivity and investigating inhibition of metabolic processes associated with viral growth in the host cell. Few studies have addressed the direct loss of viral activity by plant metabolites in a cell-free system. In the current study, the extent of antiviral activity by a structurally diverse group of flavonoids present in cranberry juice and grape juice (proanthocyanidins), citrus fruits (hesperidin, naringin, and diosmin), and green tea (catechins) was investigated using a quantitative antigen capture enzyme linked immunoassay (ELISA) in a cell-free system of simian rotavirus strain SA-11 (RTV). The RTV, representative of an enteric virus, was placed in MA104 cell cultures and tested by endpoint dilution. PACs at 100 and 200 g/mL caused a direct loss of virus activity at 56% and 92%, respectively, as compared with the control. Use of EGCG at 80 and 160 g/mL resulted in a loss of RTV infectivity of 45 and >98%. Individual tests with the PAC components catechin and epicatechin (EC) required increased concentrations (ca. 5000 to 10,000 g/mL) to cause an RTV antigen loss approaching 50%. The combined use of catechin and EC had no significant synergistic effect on the loss of RTV capsid antigen. Tests with the flavanones (hesperidin and naringin) and the semisynthetic isoflavone (diosmin) displayed antiviral activity at a concentration of 1200 g/mL. PAC extract at 200 g/L inhibited RTV-induced hemagglutination, suggesting an inhibitory effect on the binding of RTV antigenic determinants to RBC receptors. Gold-labeled immunoelectron microscopy showed RTV particles entrapped or sequestered with PAC aggregates. These findings suggest two things: first, recognition that the potential antiviral efficacy of flavonoids is related to structural differences between groups and within subgroups, and second, antiviral (anti-RTV) activity occurs at the virusflavonoid interface in the absence of the viral host

Experimental infection of dogs with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii

The lack of a suitable infection model remains an important obstacle for the pathophysiological understanding of Bartonella spp. The following pilot study was designed to determine whether cell culture-grown Bartonella henselae SA2 and Bartonella vinsonii subsp. berkhoffii genotype III would cause persistent bacteremia in dogs. Pre-inoculation screening established that two laboratory-raised Golden retrievers were naturally-infected with Bartonella koehlerae. Despite prior infection, one dog each was inoculated subcutaneously with 5×104B. henselae (SA2 strain) or 3×104B. vinsonii subsp. berkhoffii genotype III. Dogs were bled weekly for serological testing and culture using Bartonella alpha proteobacteria growth medium (BAPGM) diagnostic platform. Dog 1 seroconverted to B. henselae and Dog 2 seroconverted to B. vinsonii subsp. berkhoffii genotype III. Throughout the study period, Bartonella spp. DNA was neither amplified nor isolated in ante-mortem BAPGM enrichment blood cultures. B. henselae SA2 was isolated from a postmortem bone marrow from Dog 1 and B. koehlerae DNA was amplified from postmortem lung from Dog 2 following BAPGM enrichment culture. Limitations include lack of uninfected controls, a potentially suboptimal B. vinsonii subsp. berkhoffii inoculum and a relatively short duration of study. We conclude that following intradermal infection, sequestration of Bartonella spp. in tissues may limit diagnostic detection of these bacteria in dog blood samples.

Rotavirus core shell subdomains involved in polymerase encapsidation into virus-like particles

The triple-layered rotavirus virion encases an 11-segmented, dsRNA genome and 11-12 copies of the viral polymerase (VP1). VP1 transcribes and replicates the genome while tethered beneath the VP2 core shell. Genome replication (i.e. minus-strand RNA synthesis) by VP1 occurs in association with core assembly. During this process, VP2 directly engages VP1, thereby (i) packaging the polymerase into a nascent core and (ii) triggering the enzyme to initiate minus-strand RNA synthesis on bound plus-strand RNA templates. Recent work has shed light on VP2 regions important for VP1 enzymic activity. In the current study, we sought to investigate VP2 subdomains involved in the encapsidation of VP1 into recombinant virus-like particles (VLPs), which are formed of VP2 and the middle layer virion protein (VP6). We showed that strain SA11 VLPs efficiently encapsidated SA11 VP1, but not the genetically divergent Bristol VP1. VLPs made with an SA11 VP2 mutant lacking residues 1-10 of the amino-terminal domain (NTD) were still able to encapsidate VP1; however, removal of the entire NTD (residues 1-102) completely abolished polymerase packaging. We also showed that a chimeric VP2 protein containing the NTD and dimer-forming subdomain of strain Bristol VP2 can efficiently encapsidate SA11 VP1. These results suggest that the VP2 NTD and dimer-forming subdomain play important, albeit non-specific, roles in both VP1 packaging and activation. When combined with previous work, the results of this study support the notion that the same VP2 regions that engage VP1 during activation are also involved in packaging the enzyme into the core.

Identification of Cellular Calcium Binding Protein Calmodulin as a Regulator of Rotavirus A Infection during Comparative Proteomic Study

Rotavirus (RV) being the major diarrhoegenic virus causes around 527000 children death (<5years age) worldwide. In cellular environment, viruses constantly adapt and modulate to survive and replicate while the host cell also responds to combat the situation and this results in the differential regulation of cellular proteins. To identify the virus induced differential expression of proteins, 2D-DIGE (Two-dimensional Difference Gel Electrophoresis) based proteomics was used. For this, HT-29 cells were infected with RV strain SA11 for 0 hours, 3 hours and 9 hours post infection (hpi), differentially expressed spots were excised from the gel and identified using MALDI-TOF/TOF mass spectrometry. 2D-DIGE based proteomics study identified 32 differentially modulated proteins, of which 22 were unique. Some of these were validated in HT-29 cell line and in BALB/c mice model. One of the modulated cellular proteins, calmodulin (CaM) was found to directly interact with RV protein VP6 in the presence of Ca2+. Ca2+-CaM/VP6 interaction positively regulates RV propagation since both CaM inhibitor (W-7) and Ca2+ chelator (BAPTA-AM) resulted in decreased viral titers. This study not only identifies differentially modulated cellular proteins upon infection with rotavirus in 2D-DIGE but also confirmed positive engagement of cellular Ca2+/CaM during viral pathogenesis.

Whole-genome consensus sequence analysis of a South African rotavirus SA11 sample reveals a mixed infection with two close derivatives of the SA11-H96 strain

Whole-genome, sequence-independent amplification and 454(®) pyrosequencing of a rotavirus SA11 cell culture sample with an unknown passage history yielded consensus sequences of twelve complete genome segments. Two distinct sequences for genome segment 8 (encoding NSP2) were present, indicating a mixed infection with two rotavirus SA11 strains. The genotypes of the viruses were G3-P[2]-I2-R2-C5-M5-A5-Nx-T5-E2-H5, where x was either 5 or 2. The strains were named RVA/Simian-tc/ZAF/SA11-N5/1958/G3P[2] and RVA/Simian-tc/ZAF/SA11-N2/1958/G3P[2]. The genotype (N2) and sequence of genome segment 8 of RVA/Simian-tc/ZAF/SA11-N2/1958/G3P[2] were identical to that of the bovine rotavirus O agent. Five novel amino acids were detected in minor population variants of three genome segments. Genome segment 1 (VP1) has a high nucleotide substitution rate, but the substitutions are synonymous. Distance matrices and Bayesian molecular clock phylogenetics showed that SA11-N2 is a reassortant containing genome segment 8 from the O agent, whereas SA11-N5 is a very close derivative of the prototype SA11-H96.

A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1’ formation and contributes to attenuation

Japanese encephalitis (JE) remains the leading cause of viral encephalitis in the Asia-Pacific region, and the live vaccine SA14-14-2 is currently recommended by WHO and widely used in Asian countries with a good safety and efficacy profile. In this study, we demonstrated that SA14-14-2 failed to produce NS1', the larger NS1-related protein, compared with its parental strain SA14 in various cells. Sequence analysis and secondary structure prediction identified a single silent mutation G66A in the NS2A-coding region of SA14-14-2 destabilized the conserved pseudoknot structure, which was associated with a -1 ribosomal frame shift event. Using reverse genetic technology and animal study, we provided solid evidence that this single silent mutation G66A in the NS2A gene abolished the production of NS1' in vitro and reduced neurovirulence and neuroinvasiveness in mice. These findings provide critical information in understanding the molecular mechanism of JE vaccine attenuation and is critical for JE vaccine quality control.

A rapid, automated approach for quantitation of rotavirus and reovirus infectivity

Current microscopy-based approaches for immunofluorescence detection of viral infectivity are time consuming and labor intensive and can yield variable results subject to observer bias. To circumvent these problems, we developed a rapid and automated infrared immunofluorescence imager-based infectivity assay for both rotavirus and reovirus that can be used to quantify viral infectivity and infectivity inhibition. For rotavirus, monolayers of MA104 cells were infected with simian strain SA-11 or SA-11 preincubated with rotavirus-specific human IgA. For reovirus, monolayers of either HeLa S3 cells or L929 cells were infected with strains type 1 Lang (T1L), type 3 Dearing (T3D), or either virus preincubated with a serotype-specific neutralizing monoclonal antibody (mAb). Infected cells were fixed and incubated with virus-specific polyclonal antiserum, followed by an infrared fluorescence-conjugated secondary antibody. Well-to-well variation in cell number was normalized using fluorescent reagents that stain fixed cells. Virus-infected cells were detected by scanning plates using an infrared imager, and results were obtained as a percent response of fluorescence intensity relative to a virus-specific standard. An expected dose-dependent inhibition of both SA-11 infectivity with rotavirus-specific human IgA and reovirus infectivity with T1L-specific mAb 5C6 and T3D-specific mAb 9BG5 was observed, confirming the utility of this assay for quantification of viral infectivity and infectivity blockade. The imager-based viral infectivity assay fully automates data collection and provides an important advance in technology for applications such as screening for novel modulators of viral infectivity. This basic platform can be adapted for use with multiple viruses and cell types.

Effects of the viability ofLactobacillus rhamnosusGG on rotavirus infection in neonatal rats

To study the effects of live and dead Lactobacillus rhamnosus GG (GG) on rotavirus infection in a neonatal rat model. At the age of 2 d, suckling Lewis rat pups were supplemented with either live or dead GG and the treatment was continued daily throughout the experiment. At the age of 5 and 6 d the pups received oral rotavirus (RV) SA-11 strain. The pups were sacrificed at the age of 7 or 8 d by decapitation. The gastrointestinal tract was removed and macroscopic observations were done. The consistency of feces in the colon was classified using a four-tier system. RV was detected from the plasma, small intestine, colon and feces by real-time quantitative polymerase chain reaction (PCR). In this neonatal rat model, RV induced a mild-to-moderate diarrhea in all except one pup of the RV-inoculated rats. RV moderately reduced body weight development from day 6 onwards. On day 7, after 2 d of RV infection, live and dead GG groups gained significantly more weight than the RV group without probiotics [36% (P = 0.001) and 28% (P = 0.031), respectively]. In addition, when compared with the RV control group, both live and dead GG reduced the weight ratio of colon/animal body weight to the same level as in the healthy control group, with reductions of 22% (P = 0.002) and 28% (P < 0.001), respectively. Diarrhea increased moderately in both GG groups. However, the diarrhea incidence and severity in the GG groups were not statistically significantly different as compared with the RV control group. Moreover, observed diarrhea did not provoke weight loss or death. The RV control group had the largest amount of RV PCR-positive samples among the RV-infected groups, and the live GG group had the smallest amount. Rats receiving live GG had significantly less RV in the colon (P = 0.027) when compared with the RV control group. Live GG was also more effective over dead GG in reducing the quantity of RV from plasma (P = 0.047). Both live and dead GG have beneficial effects in RV infection. GG may increase RV clearance from the body and reduce colon swelling.