Rotavirus is the most common cause of acute gastroenteritis in infants and children worldwide. The study is hospital-based surveillance of rotavirus diarrhea in children from Imphal, Manipur, India conducted from December 2015 to March 2019. The positivity rate was found to be high ∼69.25% (358/517) and proportion of diarrhea cases and rotavirus diarrhea was peak in winter months and mostly in children from 6 to 24 months. G3 (43%) was the most widely circulating genotype in Imphal followed by G1 (16%), G2 (8%), G9 (5%), G8 (3%), G10 (1%), and G4 (1%), while G12 (0.26%) was rarely detected. Among P-types, P[6] (22%) accounted for the highest prevalence followed by P[8] (11%) and P[4] (4%), P[11] (4%), P[10] (3%), P-type mixed infection 3%, while 53% were untypeable. In G/P combinations, we detected 22 different rotavirus strains at varying frequencies. Globally distributed G3P[8] and G1P[8] strains were observed in the study. G3P[6] emerged as the most predominant rotavirus strain followed by G3P[8], G1P[6], G1P[8], and G9P[6]. The common rotavirus strains distributed across the region namely G3P[8], G1P[8], G2P[4], G9P[4], G1P[4], G1P[6], and G9P[6] were also observed. Interestingly, our study has observed a high percentage of unusual strains namely G9P[4], G1P[11], G2P[11], G3P[10], G3P[11], G4P[11], G9P[10], G9P[11],G10P[6], and G10P[8]. Of note, the high frequency of non-typeable rotavirus P-types (56%) are suggestive of point mutations that might have accumulated in the primer-binding region of VP4 gene. The findings of the present study revealed the hospital-based prevalence of rotavirus disease and the circulating genotypes during the pre-vaccination period and highlights the need for continuous surveillance of rotavirus infection post-rotavac vaccine introduction in the state of Manipur, India.

The incidence and severity of begomovirus diseases have been increasing around the world recently, and the ridge gourd [Luffa acutangula (Roxb.) L.] is the latest example of a crop that has become highly susceptible to the outbreak of the tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) in India. Accurate diagnosis of causal agents is important in designing disease management strategies. In this study the coat protein (CP) gene from a ToLCNDV-Rg ridge gourd isolate was used to produce polyclonal antibodies (ToLCNDV-Rg-CP-PAb) in a rabbit. The antibodies successfully detected a 30.5 kDa ToLCNDV-Rg-CP in extracts of symptomatic ridge gourd leaf samples by several assays, such as Western Blotting (WB), Dot Immuno Binding Assay (DIBA), Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA), Immuno Capture Polymerase Chain Reaction (IC-PCR), and Immuno Capture Loop-Mediated Isothermal Amplification (IC-LAMP) assays. However, none of the negative samples tested positive in either of the detection methods. Among all the methods tested, the immunocapture assay, IC-LAMP, was the most sensitive in detecting ToLCNDV-Rg. Furthermore, antibodies generated in this study also detected other commonly occurring begomoviruses in South India, such as tomato leaf curl Palampur virus and squash leaf curl China virus in cucurbits. Together, ToLCNDV-Rg-CP-PAb can be used for detecting at least three species of begomoviruses infecting cucurbits. The obtained antibodies will contribute to monitoring disease outbreaks in multiple crops.

The COVID-19 pandemic highlights an urgent need for effective antivirals. Targeting host processes co-opted by viruses is an attractive antiviral strategy with a high resistance barrier. Picolinic acid (PA) is a tryptophan metabolite endogenously produced in mammals. Here, we report the broad-spectrum antiviral activity of PA against enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), flaviviruses, herpes simplex virus, and parainfluenza virus. Mechanistic studies reveal that PA inhibits enveloped virus entry by compromising viral membrane integrity, inhibiting virus-cellular membrane fusion, and interfering with cellular endocytosis. More importantly, in pre-clinical animal models, PA exhibits promising antiviral efficacy against SARS-CoV-2 and IAV. Overall, our data establish PA as a broad-spectrum antiviral with promising pre-clinical efficacy against pandemic viruses SARS-CoV-2 and IAV.

Gastrointestinal diseases including diarrhoea constitute a major cause of morbidity and mortality in infants and young children especially in developing countries. Worldwide deaths among all ages due to diarrhoea during 2015 were estimated to be about 1.31 million, diarrhoeal deaths in children below 5 years of age being 499 000. Rotavirus accounted for about 200 000 deaths. Although diarrhoeal deaths decreased significantly during the last two decades, they still represent the third largest cause of infantile deaths. Several bacterial, viral, parasitic, fungal and non-infectious diarrhoea causing agents have been identified, but 30% to 40% of diarrhoeal cases remain undiagnosed. Enteroviruses transmit by the faecal-oral route and replicate first in intestinal cells before spreading to the target organ. They have been associated with diarrhoea in a few studies, but their causative role in diarrhoea in humans has not been systematically demonstrated. In view of the recent demonstration that enteroviruses cause diarrhoea in newborn mice pups, thus validating Koch's postulates, the purpose of this review is to emphasise the importance of recognising enteroviruses as major gastrointestinal pathogens associated with acute and persistent diarrhoea and non-diarrhoeal increased frequency of bowel movements in infants, young children and adults. Our studies and several other subsequent studies reported from different countries should stimulate strategies to reduce the burden of infantile gastrointestinal disease, which has hitherto remained unaddressed.

Rotavirus is a major cause of severe acute gastroenteritis in the infants and young children. The past decade has evidenced the role of intrinsically disordered proteins/regions (IDPs)/(IDPRs) in viral and other diseases. In general, (IDPs)/(IDPRs) are considered as dynamic conformational ensembles that devoid of a specific 3D structure, being associated with various important biological phenomena. Viruses utilize IDPs/IDPRs to survive in harsh environments, to evade the host immune system, and to highjack and manipulate host cellular proteins. The role of IDPs/IDPRs in Rotavirus biology and pathogenicity are not assessed so far, therefore, we have designed this study to deeply look at the penetrance of intrinsic disorder in rotavirus proteome consisting 12 proteins encoded by 11 segments of viral genome. Also, for all human rotaviral proteins, we have deciphered molecular recognition features (MoRFs), which are disorder based binding sites in proteins. Our study shows the wide spread of intrinsic disorder in several rotavirus proteins, primarily the nonstructural proteins NSP3, NSP4, and NSP5 that are involved in viral replication, translation, viroplasm formation and/or maturation. This study may serve as a primer for understanding the role of IDPs/MoRFs in rotavirus biology, design of alternative therapeutic strategies, and development of disorder-based drugs.

Rotavirus; immune epitope; structural proteins; non-structural proteins. Rotavirus is the most common cause of acute gastroenteritis in infants and children worldwide. The functional correlation of B- and T-cells to long-lasting immunity against rotavirus infection in the literature is limited. In this work, a series of computational immuno-informatics approaches were applied and identified 28 linear B-cells, 26 conformational B-cell, 44 TC cell and 40 TH cell binding epitopes for structural and non-structural proteins of rotavirus. Further selection of putative B and T cell epitopes in the multi-epitope vaccine construct was carried out based on immunogenicity, conservancy, allergenicity and the helical content of predicted epitopes. An in-silico vaccine constructs was developed using an N-terminal adjuvant (RGD motif) followed by TC and TH cell epitopes and B-cell epitope with an appropriate linker. Multi-threading models of multi-epitope vaccine construct with B- and T-cell epitopes were generated and molecular dynamics simulation was performed to determine the stability of designed vaccine. Codon optimized multi-epitope vaccine antigens was expressed and affinity purified using the E. coli expression system. Further the T cell epitope presentation assay using the recombinant multi-epitope constructs and the T cell epitope predicted and identified in this study have not been investigated. Multi-epitope vaccine construct encompassing predicted B- and T-cell epitopes may help to generate long-term immune responses against rotavirus. The computational findings reported in this study may provide information in developing epitope-based vaccine and diagnostic assay for rotavirus-led diarrhea in children's.

Rotavirus nonstructural protein 4, the first viral enterotoxin to be identified, is a multidomain, multifunctional glycoprotein. Earlier, we reported a Ca2+-bound coiled-coil tetrameric structure of the diarrhea-inducing region of NSP4 from the rotavirus strains SA11 and I321 and a Ca2+-free pentameric structure from the rotavirus strain ST3, all with a parallel arrangement of α-helices. pH was found to determine the oligomeric state: a basic pH favoured a tetramer, whereas an acidic pH favoured a pentamer. Here, we report two novel forms of the coiled-coil region of NSP4 from the bovine rotavirus strains MF66 and NCDV. These crystallized at acidic pH, forming antiparallel coiled-coil tetrameric structures without any bound Ca2+ ion. Structural and mutational studies of the coiled-coil regions of NSP4 revealed that the nature of the residue at position 131 (Tyr/His) plays an important role in the observed structural diversity.

Rotavirus replicates in the cytoplasm of infected cells in unique virusinduced cytoplasmic inclusion bodies called viroplasms (VMs), which are nucleated by two essential viral nonstructural proteins, NSP2 and NSP5. However, the precise composition of the VM, the intracellular localization of host proteins during virus infection, and their association with VMs or role in rotavirus growth remained largely unexplored. Mass spectrometry analyses revealed the presence of several host heterogeneous nuclear ribonucleoproteins (hnRNPs), AU-rich element-binding proteins (ARE-BPs), and cytoplasmic proteins from uninfected MA104 cell extracts in the pulldown (PD) complexes of the purified viroplasmic proteins NSP2 and NSP5. Immunoblot analyses of PD complexes from RNase-treated and untreated cell extracts, analyses of coimmunoprecipitation complexes using RNase-treated infected cell lysates, and direct binding assays using purified recombinant proteins further demonstrated that the interactions of the majority of the hnRNPs and ARE-BPs with viroplasmic proteins are RNA independent. Time course immunoblot analysis of the nuclear and cytoplasmic fractions from rotavirus-infected and mock-infected cells and immunofluorescence confocal microscopy analyses of virus-infected cells revealed a surprising sequestration of the majority of the relocalized host proteins in viroplasms. Analyses of ectopic overexpression and small interfering RNA (siRNA)-mediated downregulation of expression revealed that host proteins either promote or inhibit viral protein expression and progeny virus production in virus-infected cells. This study demonstrates that rotavirus induces the cytoplasmic relocalization and sequestration of a large number of nuclear and cytoplasmic proteins in viroplasms, subverting essential cellular processes in both compartments to promote rapid virus growth, and reveals that the composition of rotavirus viroplasms is much more complex than is currently understood.

Enteroviruses consist of a large group of pathogenic viruses, poliovirus being the notorious member. For the last several decades, all the attention and resources were directed towards control and eradication of poliomyelitis in India. There is very little research on non-polio enteroviruses (NPEVs), the cousins of poliovirus, which are associated with a wide range of diseases, especially in infants and young children. We have established an active research group on NPEVs during the last six years and showed that about 35% of non-polio acute flaccid paralysis (NP-AFP) children were positive for NPEV infections, and detected 66 serotypes in NP-AFP children, EV71 being more frequently detected followed by Echovirus 13 and CVB5. Long-term comparative epidemiological studies on NPEVs and rotavirus in acute diarrhoea revealed for the first time, that, NPEV association is as significant as that of rotavirus. A surprising observation was the contrasting seasonal prevalence between enterovus- and rotavirus-associated diarrhoea, the former predominating in non-winter months and the latter occurring primarily in the winter season. NPEVs were associated with epidemics-like outbreaks during which they were detected in up to 50% children with acute diarrhoea. In recent years, enterovirus has been identified to be associated with acute encephalitis cases in Uttar Pradesh. HFMD outbreaks have been reported in recent years, including a major outbreak in Bangalore in 2013. Since, no studies exist in India on the biology of EVs, including HFMD, we have recently initiated work on this emerging disease. Our studies uncover an urgent need for detailed studies on these "so far" neglected and emerging viruses for effective child health management in the country.

We have recently reported significant association of non-polio enteroviruses (NPEVs) with acute and persistent diarrhea (18-21% of total diarrheal cases), and non-diarrheal Increased Frequency of Bowel Movements (IFoBM-ND) (about 29% of the NPEV infections) in children and that the NPEV-associated diarrhea was as significant as rotavirus diarrhea. However, their diarrhea-causing potential is yet to be demonstrated in an animal model system. Since the determination of virus titers by the traditional plaque assay takes 4-7 days, there is a need for development of a rapid method for virus titer determination to facilitate active clinical research on enterovirus-associated diarrhea.The goal of this study is to develop a cell-based rapid detection and enumeration method and to demonstrate the diarrhea-inducing potential of purified and characterized non-polio enteroviruses, which were isolated from diarrheic children. Here we describe generation of monoclonal and polyclonal antibodies against purified strains belonging to different serotypes, and development of an enzyme-linked immuno focus assay (ELIFA) for detection and enumeration of live NPEV particles in clinical and purified virus samples, and a newborn mouse model for NPEV diarrhea. Plaque-purified NPVEs, belonging to different serotypes, isolated from children with diarrhea, were grown in cell culture and purified by isopycnic CsCl density gradient centrifugation. By ELIFA, NPEVs could be detected and enumerated within 12. h post-infection. Our results demonstrated that Coxsackievirus B1 (CVB1) and CVB5 strains, isolated from diarrheic children, induced severe diarrhea in orally-inoculated 9-12 day-old mouse pups, fulfilling Koch's postulates. The methods described here would facilitate studies on NPEV-associated gastrointestinal disease.

We recently reported significant association of non-polio enteroviruses (NPEVs) with acute diarrhea in children. Persistent diarrhea (PD) remains a major cause of morbidity and mortality in infants below two years of age in developing countries. Understanding age-dependent frequency and duration of NPEV infections is important to determine their association with persistent diarrhea and disease burden. Objectives: A cohort of 140 infants was followed for 6 months to 2 years of age to determine the frequency, duration, and association with PD of NPEV infections in comparison with rotavirus and other agents. Study design: Stool samples were collected every 14 days, and diarrheal episodes and their duration were recorded. Enteroviruses were characterized by RT-PCR and VP1 gene sequence analysis, rotavirus by electropherotyping, and other agents by PCR. Results: Of 4545 samples, negative for oral polio vaccine strains, 3907 (85.96%) and 638 (14.04%) were NPEV-negative and NPEV-positive, respectively, representing 403 (8.87%) infection episodes. About 68% of NPEV infections occurred during the first year with every child having at least one episode lasting between four days and four months. Approximately 38% and 22% of total diarrheal episodes were positive for NPEV and RV, respectively. While about 18% of NPEV infection episodes were associated with diarrhea, 6% being persistent, 13% of total diarrheal episodes were persistent involving infections by monotype NPEV strains or sequential infections by multiple strains and other agents. Conclusions: This is the first report revealing NPEVs as the single most frequently and persistently detected viral pathogen in every PD episode. © 2014 Elsevier B.V.

Objective: Infectious and non-infectious causes are associated with increased frequency of bowel movements (IFoBM). But, a viral aetiology to nondiarrhoeal IFoBM (IFoBM-ND) has not been described. Owing to an accidental infection by an echovirus 19 strain, persistent diarrhoea-Associated virus, isolated from a child with persistent diarrhoea, DCR experienced persistent IFoBM-ND with an urgency to pass apparently normal stools more than once each day for about 3 months. A follow-up study was undertaken to determine the prevalence of IFoBM-ND, and association of non-polio enteroviruses (NPEVs) with the symptom in infants from birth to 2 years. Design: A cohort of 140 newborns was followed for 6 months to 2 years from birth for IFoBM-ND. Stool samples collected every 14 days were examined for NPEVs, rotavirus and other viral/bacterial agents for their possible association with IFoBM-ND and diarrhoea. Results: Of 403 NPEV infection episodes among 4545 oral polio vaccine strains-negative stool samples, approximately 29% were associated with IFoBM-ND (15% acute and 14% persistent), including resolution of 74% of constipation episodes, and 18% with diarrhoea, suggesting that about 47% of NPEV infection episodes in children below 2 years of age are associated with gastrointestinal symptoms. About 83% of IFoBM-ND episodes are associated with the NPEV infection and 17% of the episodes are of unknown aetiology. Conclusions: NPEV is the single most frequently detected viral agent in children with IFoBM-ND and its association with the symptom is highly significant, warranting detailed investigations on the role of NPEVs in gastrointestinal diseases.

A 5-year comparative study on acute diarrhea due to NPEV and rotavirus was conducted.NPEVs and rotavirus were detected in 17.2% and 13.8% diarrheal stools, respectively.During the study, NPEVs were detected in 17% diarrheic and 6.16% healthy children.37 NPEV serotypes with E11 being more prevalent were detected in diarrheal children.NPEV-associated diarrhea is as significant as that of rotavirus but with contrasting seasonality. A causative agent in approximately 40% of diarrheal cases still remains unidentified. Though many enteroviruses (EVs) are transmitted through fecal-oral route and replicate in the intestinal cells, their association with acute diarrhea has not so far been recognized due to lack of detailed epidemiological investigations. This long-term, detailed molecular epidemiological study aims to conclusively determine the association of non-polio enteroviruses (NPEVs) with acute diarrhea in comaparison with rotavirus (RV) in children. Diarrheal stool specimens from 2161 children aged 0-2. years and 169 children between 2 and 9. years, and 1800 normal stool samples from age-matched healthy children between 0 and 9. years were examined during 2008-2012 for enterovirus (oral polio vaccine strains (OPVs) and NPEVs). Enterovirus serotypes were identified by complete VP1 gene sequence analysis. Enterovirus and rotavirus were detected in 19.01% (380/2330) and 13.82% (322/2330) diarrheal stools. During the study period, annual prevalence of EV- and RV-associated diarrhea ranged between 8% and 22%, but with contrasting seasonal prevalence with RV predominating during winter months and NPEV prevailing in other seasons. NPEVs are associated with epidemics-like outbreaks during which they are detected in up to 50% of diarrheic children, and in non-epidemic seasons in 0-10% of the patients. After subtraction of OPV-positive diarrheal cases (1.81%), while NPEVs are associated with about 17% of acute diarrhea, about 6% of healthy children showed asymptomatic NPEV excretion. Of 37 NPEV serotypes detected in diarrheal children, seven echovirus types 1, 7, 11, 13, 14, 30 and 33 are frequently observed, with E11 being more prevalent followed by E30. In conclusion, NPEVs are significantly associated with acute diarrhea, and NPEVs and rotavirus exhibit contrasting seasonal predominance. This study signifies the need for a new direction of research on enteroviruses involving systematic analysis of their contribution to diarrheal burden. © 2013 Elsevier B.V.

The region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 has been purified and crystallized and diffraction data have been collected to a resolution of 2.6 Å. Several attempts to solve the structure by the molecular-replacement method using the available tetrameric structures of this domain were unsuccessful despite a sequence identity of 73% to the already known structures. A more systematic approach with a dimer as the search model led to an unexpected pentameric structure using the program Phaser. The various steps involved in arriving at this molecular-replacement solution, which un-ravelled a case of subtle variation between different oligo-meric states unknown at the time of solving the structure, are presented in this paper. © 2012 International Union of Crystallography Printed in Singapore - all rights reserved.

The crystal structure of the region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 was determined at a resolution of 2.5 Å. Severe diffraction anisotropy, rotational pseudo-symmetry and twinning complicated the refinement of this structure. A systematic explanation confirming the crystal pathologies and describing how the structure was successfully refined is given in this report. © 2012 International Union of Crystallography Printed in Singapore-all rights reserved.

A novel pentameric structure which differs from the previously reported tetrameric form of the diarrheainducing region of the rotavirus enterotoxin NSP4 is reported here. A significant feature of this pentameric form is the absence of the calcium ion located in the core region of the tetrameric structures. The lysis of cells, the crystallization of the region spanning residues 95 to 146 of NSP4 (NSP4 95-146) of strain ST3 (ST3:NSP4 95-146) at acidic pH, and comparative studies of the recombinant purified peptide under different conditions by size-exclusion chromatography (SEC) and of the crystal structures suggested pH-, Ca 2+-, and protein concentration-dependent oligomeric transitions in the peptide. Since the NSP4 95-146 mutant lacks the N-terminal amphipathic domain (AD) and most of the C-terminal flexible region (FR), to demonstrate that the pentameric transition is not a consequence of the lack of the N- and C-terminal regions, glutaraldehyde cross-linking of the ΔN72 and ΔN94 mutant proteins, which contain or lack the AD, respectively, but possess the complete C-terminal FR, was carried out. The results indicate the presence of pentamers in preparations of these longer mutants. Detailed SEC analyses of ΔN94 prepared under different conditions, however, revealed protein concentrationdependent but metal ion- and pH-independent pentamer accumulation at high concentrations which dissociated into tetramers and lower oligomers at low protein concentrations. While calcium appeared to stabilize the tetramer, magnesium in particular stabilized the dimer. ΔN72 existed primarily in the multimeric form under all conditions. These findings of a calcium-free NSP4 pentamer and its concentration-dependent and largely calcium-independent oligomeric transitions open up a new dimension in an understanding of the structural basis of its multitude of functions. © 2011, American Society for Microbiology.

Rotavirus is a major cause of acute infantile diarrhoea worldwide. The virus genome consists of 11 segments of double-stranded RNA that codes for six structural proteins (VP1-6) and six non-structural proteins (NSP1-6). NSPs are proteins expressed from the virus genome in the infected cell, but are not incorporated into the mature virus particle. NSPs play an essential role in virus replication, morphogenesis and pathogenesis, and most of them exhibit multifunctional properties. Structure-function analysis of the NSPs is essential for understanding the molecular mechanisms by which the virus circumvents host innate immune responses, inhibits cellular protein synthesis, hijacks the protein synthetic machinery for its own propagation and manifests the disease process. Because of their essential roles in virus biology, NSPs represent potential targets for the development of antiviral agents. Determination of the three-dimensional structure of NSPs has been hindered due to low-level expression and aggregation. To date, the complete three-dimensional structure of only NSP2 has been determined. The structures of the N- and C-terminal domains of NSP3 and the diarrhoea-inducing domain of NSP4 have also been determined. This review primarily covers the structural and biological functions of the NSPs whose three-dimensional structural aspects have been fully or partially understood, but provides a brief account of other NSPs and the structural features of the mature virion as determined by electron cryomicroscopy.

The rotavirus non-structural protein NSP4 functions as the viral enterotoxin and intracellular receptor for the double-layered particles (DLP). The full-length protein cannot be expressed and/or purified to homogeneity from bacterial or insect cells. However, a bacterially expressed and purified mutant lacking the N-terminal 72 aa (ΔN72) was recently obtained from strains Hg18 and SA11 exhibiting approximately 17-20-, 150-200- and 13166-15800-fold lower DD50 (50% diarrhoea-inducing dose) values in suckling mice compared with that reported for the partially pure, full-length protein, a C-terminal M1751 mutant and a synthetic peptide comprising aa 114-135, respectively, suggesting the requirement for a unique conformation for optimal functions of the purified protein. The stretch of approximately 40 aa from the C terminus of the cytoplasmic tail of the endoplasmic reticulum-anchored NSP4 is highly flexible and exhibits high sequence variation compared with the other regions, the significance of which in diarrhoea induction remain unresolved. Here, it was shown that every amino acid substitution or deletion in the flexible C terminus resulted in altered conformation, multimerization, trypsin resistance and thioflavin T (ThT) binding, and affected DLP binding and the diarrhoea-inducing ability of the highly diarrhoeagenic SA11 and Hg18 ΔN72 in suckling mice. These studies further revealed that high ThT fluorescence correlated with efficient diarrhoea induction, suggesting the importance of an optimal ThT-recognizable conformation in diarrhoea induction by purified NSP4. These results based on biological properties provide a possible conformational basis for understanding the influence of primary sequence variations on diarrhoea induction in newborn mice by purified NSP4s that cannot be explained by extensive sequence analyses. © 2008 SGM.

Rotaviruses causing severe diarrhea in foals in two organized farms in northern India, during the period from 2003 to 2005, were characterized by electropherotyping, serotyping, and sequence analysis of the genes encoding the outer capsid proteins. Of 137 specimens, 47 (34.31%) were positive for rotavirus and exhibited at least five different electropherotypes (E), E1 to E5. Strains belonging to different electropherotypes exhibited either a different serotype/genotype specificity or a lack of reactivity to typing monoclonal antibodies (MAbs) used in this study. Strains belonging to E1, E2, and E5 exhibited genotype G10,P6[1], G3, and G1 specificities and accounted for 19.0, 42.9, and 9.5% of the isolates, respectively. Though they possessed G10-type VP7, the E1 strains exhibited high reactivity with the G6-specific MAb, suggesting that the uncommon combination of the outer capsid proteins altered the specificity of the conformation-dependent antigenic epitopes on VP7. E3 and E4 strains accounted for 28.6% of the isolates and were untypeable. Sequence analysis of VP7 from E4 strains (Erv92 and Erv99) revealed that they represent a new VP7 genotype, G16. The detection of unexpected bovine rotavirus-derived G10,P6[1] reassortants, G1 serotype strains, and a new genotype (G16) strain in two distant farms reveals an interesting epidemiological situation and diversity of equine rotaviruses in India. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Rotavirus nonstructural protein 4 (NSP4) is a multidomainal and multifunctional protein and is recognized as the first virus-encoded enterotoxin. Extensive efforts to crystallize the complete cytoplasmic tail (CT), which exhibits all the known biological functions, have been unsuccessful, and to date, the structure of only a synthetic peptide corresponding to amino acids (aa) 95-137 has been reported. Recent studies indicate that the interspecies-variable domain (ISVD) from aa 135 to 141 as well as the extreme C-terminus are critical determinants of virus virulence and the diarrhea-inducing ability of the protein. Among the five NSP4 genotypes identified, those belonging to genotypes A1, B and C possess either a proline at position 138 or a glycine at 140, while those of A2, D and E lack these residues in the ISVD, suggesting conformational differences in this region among different NSP4s. Here, we examined the crystallization properties of several deletion mutants and report the structure of a recombinant mutant, NSP4:95-146, lacking the N-terminal 94 and C-terminal 29 aa, from SA11 (A1) and I321 (A2) at 1.67 and 2.7 Å, respectively. In spite of the high resolution of one of the structures, electron density for the C-terminal 9 residues could not be seen for either of the mutants, and the crystal packing resulted in the creation of 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 the crystal packing owing to the additional space required to accommodate the un structured virulence-determining region. The crystal structure and secondary structure prediction of the NSP4:95-146 mutants from different genotypes suggest that the region C-terminal to aa 137 in all the NSP4 proteins is likely to be unstructured, and this might be of structural and biological functional significance. © 2007 Springer-Verlag.

Rotavirus NSP4 is a multifunctional endoplasmic reticulum (ER)-resident nonstructural protein with the N terminus anchored in the ER and about 131 amino acids (aa) of the C-terminal tail (CT) oriented in the cytoplasm. Previous studies showed a peptide spanning aa 114 to 135 to induce diarrhea in newborn mouse pups with the 50% diarrheal dose approximately 100-fold higher than that for the full-length protein, suggesting a role for other regions in the protein in potentiating its diarrhea-inducing ability. In this report, employing a large number of methods and deletion and amino acid substitution mutants, we provide evidence for the cooperation between the extreme C terminus and a putative amphipathic α-helix located between aa 73 and 85 (AAH73-85) at the N terminus of ΔN72, a mutant that lacked the N-terminal 72 aa of nonstructural protein 4 (NSP4) from Hg18 and SA11. Cooperation between the two termini appears to generate a unique conformational state, specifically recognized by thioflavin T, that promoted efficient multimerization of the oligomer into high-molecular-mass soluble complexes and dramatically enhanced resistance against trypsin digestion, enterotoxin activity of the diarrhea-inducing region (DIR), and double-layered particle-binding activity of the protein. Mutations in either the C terminus, AAH73-85, or the DIR resulted in severely compromised biological functions, suggesting that the properties of NSP4 are subject to modulation by a single and/or overlapping highly sensitive conformational domain that appears to encompass the entire CT. Our results provide for the first time, in the absence of a three-dimensional structure, a unique conformation-dependent mechanism for understanding the NSP4-mediated pleiotropic properties including virus virulence and morphogenesis. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

We evaluated safety and immunogenicity of two orally administered human rotavirus vaccine candidates 116E and I321. Ninety healthy infants aged 8 weeks received a single dose of 116E (105 FFu (florescence focus units)), I321 (105 FFu) or placebo. There were no significant differences in the number of adverse events. Fever was reported by 6/30, 1/30 and 5/30 in the 116E, I321 and placebo groups; the corresponding figures for diarrhoea were 5/30, 8/29 and 3/30. Serum IgA seroconversion rates were 73%, 39% and 20% in the 116E, I321 and placebo groups, respectively. Vaccine virus was shed on days 3, 7 or 28 in 11/30 infants of the 116E and none in the other two groups. The 116E strain is attenuated, clinically safe and highly immunogenic with a single dose. © 2006 Elsevier Ltd. All rights reserved.

The need for a rotavirus vaccine in India is based on the enormous burden associated with the >100,000 deaths due to rotavirus diarrhea that occur annually among Indian children. Two rotavirus strains identified during nosocomial outbreaks of rotavirus infection in New Delhi and Bangalore, India, more than a decade ago are being developed as live oral vaccines. Infected newborns had no symptoms, shed virus for up to 2 weeks after infection, mounted a robust immune response, and demonstrated protection against severe rotavirus diarrhea after reinfection. The 2 strains are naturally occurring bovine-human reassortants. The New Delhi strain, 116E, is characterized as having a P[11],G9 genotype, and the Bangalore strain, 1321, is characterized as having a P[11],G10 genotype. The strains have been prepared as pilot lots for clinical trials to be conducted in New Delhi. This unique project, which is developing a new rotavirus vaccine in India with the use of Indian strains, an Indian manufacturer, and an Indian clinical development program, aims to expedite introduction of rotavirus vaccines in India. © 2005 by the Infectious Diseases Society of America. All rights reserved.

The region spanning the tetrameric coiled-coil domain and the interspecies-variable virulence-determining region of the cytoplasmic tail of rotaviral nonstructural protein NSP4 has been crystallized. The crystals belong to space group I222, with unit-cell parameters a = 30.70, b = 38.07, c = 181.62 Å, and contain two molecules in the asymmetric unit. Diffraction data have been collected utilizing a MAR imaging plate to a resolution of 2.2 Å. The tetramer is generated by the crystallographic dyad along the c axis. © 2004 International Union of Crystallography.

We sought to determine the proportion of rotavirus (RV) infections among children with severe diarrhea in Bangalore, India, and to determine the role of neonatal infection with the asymptomatic RV strain I321 in protection against subsequent RV diarrhea. At 2 major hospitals, there was a >42% decrease in diarrhea-specific admissions during the study period. At 6 hospitals, asymptomatic infections were found in 25%-50% of neonates, when screening was performed randomly, and in >58% of neonates, when screening was performed daily, with the majority of infections occurring within the first 7 days of life. All the RVs found in asymptomatic neonates were strain I321. A 24-month follow-up of a cohort of 44 children who had been neonatally infected with strain I321 and 28 children who had not (control group) revealed comparable rates of RV detection but a marked decrease in the number of RV diarrhea episodes in the strain I321-infected group (2.3%), compared with the control group (39.3%) (P<.0001). This preliminary study suggests a possible association between neonatal infection with strain I321 and protection against subsequent RV illness.

Previous studies have shown predominant association of G10P11 type bovine rotavirus-derived reassortant strains with asymptomatic infections in newborn children in India. To understand the epidemiological and genetic basis for the origin of these strains in humans, the relative frequencies of different serotypes among bovine rotaviruses (BRVs) isolated from southern, western and central regions of the country were determined by subgroup and serotype analysis as well as nucleotide (nt) sequence analysis of the genes encoding the outer capsid proteins VP4 and VP7. Since the human G10P11 asymptomatic neonatal strain I321 possessed NSP1 from a human rotavirus, to determine its genetic origin in the bovine strains, comparative analysis of partial gene sequences from representative G10P11 strains was also carried out. The following observations were of great epidemiological significance, (i) G10P11 strains predominated in all the three regions with frequencies ranging between 55.6% and 85.2%. In contrast to the high prevalence of G6 strains in other countries, only one G6 strain was detected in this study and G8 strains represented 5.8% of the isolates, (ii) among the G10 strains, in serotyping ELISA, four patterns of reactivity were observed that appeared to correlate with the differences in electropherotypic patterns and amino acid (aa) sequence of theVP7, (iii) surprisingly, strains belonging to serotype G3 were detected more frequently (10.7%) than those of serotypes G6 and G8 combined, while strains representing the new serotype (G15) were observed in a single farm in Bangalore, and (iv) about 3.9% of the isolates were nontypeable as they exhibited high cross-reactivity to the serotyping MAbs used in the study. Comparative analysis of the VP7 gene sequence from the prototype G3 MAb-reactive bovine strain J63 revealed greatest sequence relatedness (87.6% nt and 96.0% aa) with that of serotype G3 rhesus-monkey strain RRV. It also exhibited high sequence homology with the VP7 from several animal and animal rotavirus-related human G3 strains (Simian SA11; equine ERV316 and FI-14; canine CU-1 and K9; porcine 4F; Feline Cat2 and human HCR3, YO and AU1). Partial nucleotide sequence analysis of the NSP1 gene of J63 showed greatest nt sequence homology (95.9%) to the NSP1 gene allele of the Indian G8 strain, isolated from a diarrheic child, which is likely to have been transmitted directly from cattle and 92.6% homology to that of the bovine G8 strain A5-10 suggesting the likely origin of J63 by gene reassortment between a bovine G8 strain and a G3 animal strain. Prevalence of G10P11 strains in cattle and G10P11 or P11 type reassortant strains in asymptomatic neonates as well as detection of G8P[1] strains in diarrheic children support our hypothesis for bidirectional transmission of rotaviruses between humans and cattle and origin of novel strains catalyzed by the age-old traditions and socio-economic conditions in India.

In an epidemiological study of symptomatic human rotaviruses in Mysore, India during 1993 and 1994, isolates MP409 and MP480 were isolated from two children suffering from severe, acute dehydrating diarrhea. Both isolates exhibited 'long' RNA pattern and subgroup I specificity suggesting the likelihood of their animal origin. Both isolates did not react with monoclonal antibodies (MAbs) specific for serotypes G1 to G6 as well as G10. To determine the genetic origin of these isolates, complete nucleotide sequences of genes encoding the outer capsid proteins VP4 and VP7, nonstructural proteins NSP1 and NSP3 and viral enterotoxin protein NSP4 from MP409 and partial sequences of genes from MP480 were determined. Comparison of the 5′ and 3′ terminal sequences of 250 nucleotides revealed complete identity of the gene sequences in both strains suggesting that MP409 and MP480 are two different isolates of a single strain. Comparison of the nucleotide and deduced amino acid sequences of VP4, VP7, NSP1 and NSP3 of MP409 with published sequences of strains belonging to different serotypes revealed that both outer capsid proteins VP4 and VP7 and NSP1 are highly related to the respective proteins from the P6[1], G8 type bovine rotavirus A5 isolated from a calf with diarrhoea in Thailand and that the NSP3 is highly homologous to that of bovine rotaviruses. The NSP4 protein showed greatest sequence identity with NSP4s belonging to the KUN genetic group to which NSP4s from human G2 type strains and bovine rotaviruses belong. MP409 and MP480 likely signify interspecies transmission of P6[1], G8 type strains from cattle to humans and represent the first P6[1] type rotaviruses isolated in humans. These and our previous studies on the asymptomatic neonatal strain I321 are of evolutionary and epidemiological significance in the context of close association of majority of the Indian population with cattle.

During a limited epidemiological study, the serotype specificities of several isolates of bovine rotavirus, exhibiting identical electropherotypes, from a single cattle farm near Bangalore, India, could not be determined using a panel of serotyping monoclonal antibodies (MAbs) specific for G serotypes 1-6 and 10. To determine the genotypes of these isolates, the nucleotide sequences of the genes encoding the outer capsid proteins VP4 and VP7 of two representative isolates, Hg18 and Hg23, were determined. The corresponding gene sequences from the two isolates were identical, indicating that these isolates represented a single strain of bovine rotavirus. Comparison of the VP4 nucleotide (nt) and the deduced amino acid (aa) sequences with those of several human and animal rotavirus strains representing all of the currently recognized 20 different VP4 (P) genotypes revealed low nt and aa sequence identities of 61.0 to 74.2% and 57.9 to 78.2% for VP4. The percentages of amino acid homology for the VP8* and VP5* regions of VP4 were 37.7 to 67.9 and 68.1 to 84.2%, respectively. The nt and aa sequences of the VP7 gene were also distinct from those of human and animal strains belonging to the previously established 14 VP7(G) serotypes (65.9 to 755% nt and 59.5 to 77.6% aa identities). These findings suggest the classification of the VP4 and VP7 genes of the bovine isolates represented by Hg18 as new P and G genotypes and provide further evidence for the vast genetic/antigenic diversity of group A rotaviruses. (C) 2000 Academic Press.

Epidemiology of symptomatic rotaviruses from Bangalore and Mysore in Southern India was investigated. While serotype G3 predominated throughout the 7-year study period from 1988 to 1994 in Bangalore, serotype G1 was more predominant than serotype G3 in Mysore during 1993 and 1994. Serotype G2 strains were either not detected or infrequently observed in both the cities. However, several strains with subgroup I and 'short' RNA pattern that exhibited high reactivity with typing MAbs specific for serotype 2 as well as other serotypes were detected throughout the period. Among the nonserotypeable strains from both cities, several exhibited dual subgroup (SGI + II) or subgroup I specificity and 'long' RNA pattern indicating their probable animal origin. Notably, a gradual, yet highly significant reduction in rotavirus gastroenteritis, from 45.3% in 1988 to 1.8% during 1994, was observed in Bangalore in stark contrast to the consistently high (about 34%) incidence of asymptomatic infections among neonates by I321-like G10P11 type strains during the same period. Moreover, I321-like asymptomatic strains were not detected in children with diarrhea.

The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined. Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic. Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types. To date, expression of complete VP4 in E. coli has not been achieved. In this study we present successful expression in E. coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography. The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it. Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses. © Springer-Verlag 1996.

Thiobacillus ferrooxidans MAL4-1, an isolate from Malanjkhand copper mines, India, was adapted to grow in the presence of high concentration (30 gL-1) of Cu2+, resulting in a 15-fold increase in its tolerance to Cu2+. While wild-type T. ferrooxidans MAL4-1 contained multiple plasmids, cultures adapted to Cu2+ concentrations of 20 gL-1 or more showed a drastic reduction in the copy number of the plasmids. The reduction for three of the plasmids was estimated to be over 50-fold. Examination of the plasmid profiles of the strains adapted to high concentration of SO4/2anion (as Na2SO4 or ZnSO4) indicated that the reduction in plasmid copy number is not owing to SO4/2- anion, but is specific for Cu2+. The effect of mercury on the plasmids was similar to that of copper. Deadaptation of the Cu2+- or Hg2+-adapted T. ferrooxidans resulted in restoration of the plasmids to the original level within the first passage. The fact that the plasmid copy number, in general, is drastically reduced in Cu2+-adapted T. ferrooxidans suggests that resistance to copper is chromosome mediated. This is the first report of a selective negative influence of copper ions on the copy number of plasmids in T. ferrooxidans.

VP6, the intermediate capsid protein of the virion, specifies subgroup specificity of rotavirus. It is also the most conserved, both at nucleotide and amino acid levels, among group A rotaviruses and is the target of choice for rotavirus detection. In this study we report the sequence of the subgroup I (SGI)-specific VP6 from the serotype G2 strain IS2 isolated from a child suffering from acute diarrhoea in Bangalore and its comparison with the published VP6 sequences. Interestingly, IS2 gene 6 shared highest homology with that from bovine UK strain and the protein contained substitutions by lysine at amino acid positions 97 and 134. In contrast, the amino acids Met and Glu/Asp at these respective positions are highly conserved in all the other group A rotaviruses sequenced so far. These observations have obvious implications for the evolution of serotype G2 and G2-like strains circulating in India. The SGI VP6, of a human rotavirus, possessing epitopes that are conformationally similar to those found in the native protein in the virion, was successfully expressed in E. coli and purified for the first time by single-step affinity chromatography.

NSP3, an acidic nonstructural protein, encoded by gene 7 has been implicated as the key player in the assembly of the 11 viral plus-strand RNAs into the early replication intermediates during rotavirus morphogenesis. To date, the sequence of NSP3 from only three animal rotaviruses (SA11, SA114F, and bovine UK) has been determined and that from a human strain has not been reported. To determine the genetic diversity among gene 7 alleles from group A rotaviruses, the nucleotide sequence of the NSP3 gene from 13 strains belonging to nine different G serotypes, from both humans and animals, has been determined. Based on the amino acid sequence identity as well as phylogenetic analysis, NSP3 from group A rotaviruses falls into three evolutionarily related groups, i.e., the SA11 group, the Wa group, and the S2 group. The SA11/SA114F gene appears to have a distant ancestral origin from that of the others and codes for a polypeptide of 315 amino acids (aa) in length. NSP3 from all other group A rotaviruses is only 313 aa in length because of a 2-amino-acid deletion near the carboxy-terminus. While the SA114F gene has the longest 3′ untranslated region (UTR) of 132 nucleotides, that from other strains suffered deletions of varying lengths at two positions downstream of the translational termination codon. In spite of the divergence of the nucleotide (nt) sequence in the protein coding region, a stretch of about 80 nt in the 3′ UTR is highly conserved in the NSP3 gene from all the strains. This conserved sequence in the 3′ UTR might play an important role in the regulation of expression of the NSP3 gene. © 1995 Academic Press. All rights reserved.

Human rotaviruses were isolated from asymptomatic neonates at various hospitals and clinics in the city of Bangalore, India, and were found to be subgroup I specific and possess long RNA patterns (M. Sukumaran, K. Gowda, P. P. Maiya, T. P. Srinivas, M. S. Kumar, S. Aijaz, R. R. Reddy, L. Padilla, H. B. Greenberg, and C. D. Rao, Arch. Virol. 126:239-251, 1992). Three of these strains were adapted to tissue culture and found by serotype analysis and neutralization assays to be of serotype 10, a serotype commonly found in cattle but infrequently found in humans and not previously identified in neonates. By RNA-RNA hybridization, a high level of relatedness to a serotype 10 bovine rotavirus strain and a low-to-medium level of relatedness to a human rotavirus strain were observed. Since this human isolate shares a genogroup with bovine rotavirus, it is likely that it originated by interspecies transmission. A human rotavirus strain isolated from asymptomatic neonates and similar to bovine rotavirus might represent a good vaccine candidate.

The nucleotide sequence of genes 4 and 9, encoding the outer capsid proteins VP4 and VP7 of a serotype 10 tissue culture-adapted strain, 1321, representative of asymptomatic neonatal rotaviruses isolated from neonates in Bangalore, India, were determined. Comparison of nucleotide and deduced amino acid sequences of 1321 VP4 and VP7 with previously published sequences of various serotypes revealed that both genes were highly homologous to the respective genes of serotype 10 bovine rotavirus, B223. The VP4 of 1321 represents a new human P serotype and the 1321 and related strains represent the first description of neonatal rotaviruses that appear to derive both surface proteins from an animal rotavirus. © 1993 Academic Press. All rights reserved.

A large number of stool specimes, of healthy new born infants, collected from various hospitals and clinics in Bangalore City, India, have been examined for the presence of asymptomatic rotaviral excretion. Out of 370 samples analysed during a three year period from 1988 to 1991, 133 specimens (36%) were positive for rotavirus RNA. All these asymptomatic neonatal strains, without exception, showed "long" RNA pattern, but subgroup I specificity. Serotype analysis by ELISA or by hybridization with serotype-specific probes indicated that these strains probably represent a new serotype in newborn children. We find an exclusive association of human rotaviruses having "long" RNA pattern and subgroup I specificity with asymptomatic neonatal infections in contrast to the earlier observations of association of such unusual strains with acute gastroenteritis in young children. © 1992 Springer-Verlag.

c-sis/platelet-derived growth factor 2 (PDGF-2) is a prototype growth factor with transforming potential. The c-sis/PDGF-2 transcript contains a long 5' untranslated sequence (UTS) that is highly G · C rich. To examine the influence of this sequence on sis/PDGF-2 expression, we localized the c-sis/PDGF-2 promoter and used this promoter or the simian virus 40 early promoter to drive expression of the bacterial chloramphenicol acutyltransferase or sis/PDGF-2 gene. The 5' UTS of c-sis/PDGF-2 mRNA had no effect on RNA expression but was shown to exert a potent inhibitory effect on translation. By deletion analysis, we demonstrated that the 5' UTS inhibited protein expression by as much as 40-fold. The inhibitory effect was independent of reporter gene, cell type, or promoter used. A highly G · C-rich 140-base-pair sequence immediately preceding the c-sis/PDGF-2 initiation codon was shown to be nearly as effective as the entire 5' UTS in translational inhibition. Transfection analysis demonstrated that the 5' UTS significantly reduced the transforming efficiency of the sis/PDGF-2 gene as well. Thus, our findings raise the possibility that changes in regulation at the level of sis/PDGF-2 translation may play a role in development of the neoplastic phenotype.

The DBL transforming gene was originally identified by transfection of NIH 3T3 cells with DNA from a human B-cell lymphoma. This gene was found to have arisen as a result of recombination of the 3' portion of the DBL protooncogene coding sequences with an unrelated segment of human DNA. It encodes a cytoplasmic protein that is equally distributed between cytosol and crude membrane fractions. To further characterize this transforming gene, a biologically active cDNA clone of the DBL transforming gene mRNA was isolated. Analysis of the sequence of the DBL oncogene cDNA revealed a long open reading frame that encodes a hybrid protein whose first 50 amino acids (at least) derive from a complete exon of a different locus. No significant homology with known oncogenes or any known protein sequences was demonstrated. The computer analysis of the predicted DBL protein indicated it is highly hydrophilic with no hydrophobic domains characteristic of a membrane-spanning region or signal peptide. Thus, the DBL oncoprotein is distinct among known transforming gene products.

The 3'-terminal sequences of the 10 double-stranded RNA genome segments of bluetongue virus (serotypes 10 and 11) were determined. The double-stranded RNAs were 3' labeled with [5'-32P]pCp and resolved into 10 segments by electrophoresis. After denaturation, the two complementary strands of segments 4 through 10 were resolved into fast- and slow-migrating species by polyacrylamide gel electrophoresis, and their 3' end sequences were determined. Complete RNase T1 digestion of the individual 3'-labeled double-stranded RNA segments yielded two labeled oligonucleotides, one of which migrated faster than the other on 20% polyacrylamide-7 M urea gels. Sequence analyses of the two oligonucleotides of segments 4 through 10 confirmed the corresponding RNA sequence data results. The 3'-terminal sequences of the fast-migrating RNA species were (HO)CAAUUU...; those of the slow-migrating RNA species were (HO)CAUUCACA.... Similar results were obtained for double-stranded RNA from bluetongue-virus serotypes 10 and 11. Beyond the common termini, the sequences for each segment varied considerably.

Four serotypes of bluetongue virus (BTV-10, 11, 13 and 17) have been identified in the United States. Analyses of the genome RNA segments and viral induced polypeptides of U.S. prototype BTV-17 virus by comparison with the corresponding macromolecules of earlier isolates of BTV-11 serotype support the hypothesis that BTV-17 originated by genotypic and antigenic drift from a BTV-11 serotype virus.