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With the changing hepatitis A epidemiology in India, focal viral outbreaks are being reported from different parts of the country. This study presents Hepatitis A Virus (HAV) strain characterization (period 2009-2020) from 18 states of India. For that, blood and stool samples (n ​= ​280) were screened for HAV RNA and sequences for 5'non-coding and VP3 regions were generated from positive samples (n ​= ​68). Presence of a single IIIA genotype in all samples indicated IIIA being the only HAV genotype currently circulating in India. Interestingly, it was evident that these strains form two distinct groups suggesting independent evolution of these two clusters.

Introduction: High prevalence of Hepatitis C virus (HCV) has been reported among the dialysis patients throughout the world. Aim of the study was to evaluate prevalence of HCV in haemodialysis patients at tertiary care hospitals of Pune, Maharashtra, India. Materials and Methods: A cross sectional study design was conducted among 250 patients from five dialysis centres in pune city including both government and corporate hospitals who reported to the nephrology department for haemodialysis in a period of one year. Cases were studied in detail about complete history, clinical and demographical profile as well as several biochemical parameters. A predesigned, pretested questionnaire was used for data collection. SPSS 21.0 version software was used to analyze the data. Result: Prevalence of HCV Infection in patients on hemodialysis was found to be (18.8%). Out of total anti-HCV antibody positive patients (59.6%) were found to be male and (40.4%) were female. Out of total anti-HCV antibody positive cases (72.3%) had been on dialysis for more than 5 years. About (68.1%) of total positive cases received blood transfusion once or more in life time. Conclusion: In developing countries with a high endemic background of HCV infection surveillance programs and efforts to increase awareness, improve diagnosis and facilitate treatment of acute HCV will have far reaching implications for the management of chronic HCV. Thus preventive measures and the adherence to universal precautions for HCV control remain a priority. Keywords: Seroprevalence, Hepatitis C, Haemodialysis, Tertiary care hospitals.

B.1.617 lineage is becoming a dominant SARS-CoV-2 lineage worldwide and was the dominant lineage reported in second COVID-19 wave in India, which necessitated studying the properties of the variant. We evaluated the pathogenicity and virus shedding of B.1.617.2 (Delta) and B.1.617.3 lineage of SARS-CoV-2 and compared with that of B.1, an early virus isolate with D614G mutation in Syrian hamster model. Viral load, antibody response and lung disease were studied. No significant difference in the virus shedding pattern was observed among these variants studied. A significantly high SARS-CoV-2 sub genomic RNA could be detected in the respiratory tract of hamsters infected with Delta variant for 14 days. Delta variant induced lung disease of moderate severity in 40% of infected animals. The neutralizing capability of the B.1, Delta and B.1.617.3 variant infected animals were found significantly lower with the B.1.351 (Beta variant). The findings of the study support the attributed disease severity and the increased transmission potential of the Delta variant.

An acute gastroenteritis outbreak at Devli Karad village, Maharashtra, India with an attack rate of 22.6% affected mainly adolescent and adult population. The viral investigations conducted on fecal specimens of patients hospitalized indicated the presence of rotavirus B (RVB) using RNA polyacrylamide gel electrophoresis and reverse transcription polymerase chain reaction. The samples collected from the source of drinking water also showed the presence of the only RVB. Absence of other viral agents and identification of RVB of genotype G2 as the etiological agent of the acute gastroenteritis outbreak highlights, the necessity of monitoring RVB, the viral agent known for its large outbreak potential., Highlight Acute gastroenteritis outbreak in western India with an attack rate of 22.6%.Presence of Rotavirus B (RVB) genotype G2 in all specimens as well as in source of drinking water.Absence of other viral causative agents indicated RVB as the main etiological agent.

Background & objectives: Avian influenza (AI) viruses have been a major cause of public health concern. Wild migratory birds and contaminated environmental sources such as waterbodies soiled with bird droppings play a significant role in the transmission of AI viruses. The objective of the present study was to develop a sensitive and user-friendly method for the concentration and detection of AI viruses from environmental water sources. Methods: Municipal potable water, surface water from reservoirs and sea were spiked with low pathogenic AI viruses. To concentrate the viruses by precipitation, a combination of potassium aluminium sulphate with milk powder was used. Real-time reverse transcription-polymerase chain reaction was performed for virus detection, and the results were compared with a virus concentration method using erythrocytes. Drinking water specimens from poultry markets were also tested for the presence of AI viruses. Results: A minimum of 101.0 EID50 (50% egg infectious dose)/ml spiked H5N1 and 101.7 EID50/ml spiked H9N2 viruses were detected from spiked potable water; 101.0 and 102.0 EID50/ml spiked H5N1 virus was detected from surface water and seawater samples, respectively. The present method was more sensitive than the erythrocyte-binding method as approximately 10-fold higher infectious virus titres were obtained. AI H9N2 viruses were detected and isolated from water from local poultry markets, using this method. Interpretation & conclusions: Viability and recovery of the spiked viruses were not affected by precipitation. The present method may be suitable for the detection of AI viruses from different environmental water sources and can also be applied during outbreak investigations.

Background Worldwide prevalence of Hepatitis C virus (HCV) infection hemodialysis (HD) ranges from 1 to 84.6% with serious complications. Assessment of prevalence, risk factors, and genotyping of HCV infection in patient on HD was carried out at Pune, India. Methods A total of 250 patients on HD from five HD centers were recruited and tested for anti-HCV antibody using third-generation enzyme-linked immunosorbent assay (ELISA). Qualitative HCV RNA detection was carried out by nested reverse transcriptase polymerase chain reaction (RT-PCR). Genotyping and sequencing were carried out using the BigDye Terminator cycle sequencing ready reaction kit. Results Mean age of patients was 47.3 years. Forty-seven cases out of a total of 250 were reactor for HCV antibody. Overall prevalence rate was 18.8% ranging from 6.7% to 35.6% in the five centers. Of total, 44.1% of females and 13.5% of males were HCV infected. The mean duration of HD in HCV-infected patients was 6.03 years. Prevalence was higher in patients aged > 5 years on HD with higher number of blood transfusions. Thirty-six cases were positive for HCV RNA. Only one HCV RNA was detected among the 203 anti-HCV negative samples. Discordance between antibody and HCV RNA positivity was noted. Seventeen infected cases had changed dialysis centers four times. Thirteen cases were HBsAg positive, of which six cases were coinfected with HCV. Thirty-seven samples were genotyped. Conclusion The predominant genotype was 1a (54.1%) followed by 1b (43.2%) and 3a (2.7%). Highest prevalence of HCV (35.6%) and intracenter PNI of 99.3% of genotype 1b (84.6%) in center 3 indicates a possible nosocomial transmission.

Seasonal outbreaks of acute encephalitis syndrome (AES) at Gorakhpur, India have been recognized since 2006. So far, the causative agent has not been identified. Use of next generation sequencing identified human parvovirus 4 (HPARV4) sequences in a CSF/plasma pool. These sequences showed highest identity with sequences earlier identified in similar patients from south India. Real-time PCR detected HPARV4 DNA in 20/78 (25.6%) CSF and 6/31 (19.3%) plasma of AES patients. Phylogenetic analysis classified three almost complete genomes and 24 partial NS1 sequences as genotype 2A. The observed association of HPARV4 with AES needs further evaluation. ELISAs for the detection of IgM and IgG antibodies against scrub typhus (Orientia tsutsugamushi, OT) showed ∼70% IgM/IgG positivity suggestive of etiologic association. Prospective, comprehensive studies are needed to confirm association of these agents, singly or in combination with AES in Gorakhpur region.

Many SARS-CoV-2 variants of concern has been reported recently which were linked to increased transmission. In our earlier study on virus shedding using VOC 202012/01(UK variant) and D614G variant in hamster model, we observed significantly higher viral RNA shedding through nasal wash in case of UK variant. Hence, we compared the transmission of both the UK and D614G variant by various routes in Syrian hamsters to understand whether the high viral RNA shedding could enhance the transmission efficiency of the variant. The current study demonstrated comparable transmission efficiency of both UK and D614G variants of SARS-CoV-2 in Syrian hamsters.

Infections with Basal Core Promoter (BCP) (A1762T/G1764A) and Pre-Core (PC) (G1896A) hepatitis B virus HBeAg mutants are associated with severe liver injury. We analysed host cell responses in HepG2/C3A, hepatoma cells transfected with infectious clones developed from genotype D wild type (WT) and BCP/PC mutant (MT) viruses isolated from an acute resolved and an acute liver failure hepatitis B case respectively. Cells transfected with MT virus construct showed ~55 % apoptosis and with WT ~30 % apoptosis at 72 h. To determine possible roles of HBe and HBx proteins in apoptosis, we cloned these genes and co-transfected cells with WT+HBe/HBx or MT+HBe/HBx constructs. Co-expression of HBe protein improved cell viability significantly in both WT and MT virus constructs, indicating an important role of HBe in protecting cells. RNA sequencing analysis carried out at 12 and 72 h post-transfection with WT virus construct showed enrichment of innate/adaptive immune response-activating signal transduction, cell survival and amino acid/nucleic acid biosynthetic pathways at 12 and 72 h. By contrast, MT virus construct showed enrichment in host defence pathways and some biosynthetic pathways at the early time point (12 h), and inflammatory response, secretary granule, regulation of membrane potential and stress response regulatory pathways at the late time point (72 h). There was a significant down-regulation of genes involved in endoplasmic reticulum and mitochondrial functions and metabolism with MT construct and this possibly led to induction of apoptosis in cells. Considering rapid apoptotic changes in cells transfected with MT construct, it can be speculated that HBeAg plays a crucial role in cell survival. It enhances induction of metabolic and synthetic pathways and facilitates management of cellular stress that is induced due to hepatitis B virus infection/replication.

Background & objectives: Several phylogenetic classification systems have been devised to trace the viral lineages of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, inconsistency in the nomenclature limits uniformity in its epidemiological understanding. This study provides an integration of existing classifications and describes evolutionary trends of the SARS-CoV-2 strains circulating in India. Methods: The whole genomes of 330 SARS-CoV-2 samples were sequenced using next-generation sequencing (NGS). Phylogenetic and sequence analysis of a total of 3014 Indian SARS-CoV-2 sequences from 20 different States/Union Territories (January to September 2020) from the Global Initiative on Sharing All Influenza Data (GISAID) database was performed to observe the clustering of Nextstrain and Phylogenetic Assignment of Named Global Outbreak LINeages (Pangolin) lineages with the GISAID clades. The identification of mutational sites under selection pressure was performed using Mixed Effects Model of Evolution and Single-Likelihood Ancestor Counting methods available in the Datamonkey server. Results: Temporal data of the Indian SARS-CoV-2 genomes revealed that except for Uttarakhand, West Bengal and Haryana that showed the circulation of GISAID clade O even after July 2020, the rest of the States showed a complete switch to GR/GH clades. Pangolin lineages B.1.1.8 and B.1.113 identified within GR and GH clades, respectively, were noted to be indigenous evolutions. Sites identified to be under positive selection pressure within these clades were found to occur majorly in the non-structural proteins coded by ORF1a and ORF1b. Interpretation & conclusions: This study interpreted the geographical and temporal dominance of SARS-CoV-2 strains in India over a period of nine months based on the GISAID classification. An integration of the GISAID, Nextstrain and Pangolin classifications is also provided. The emergence of new lineages B.1.1.8 and B.1.113 was indicative of host-specific evolution of the SARS-CoV-2 strains in India. The hotspot mutations such as those driven by positive selection need to be further characterized.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis and a major public health problem in India. There are four mammalian HEV genotypes worldwide. In India, genotype 1 (HEV-1) is restricted to humans whereas genotype 4 (HEV-4) circulates in pigs. Studies from our laboratory have shown that HEV-4 (swine) virus can establish experimental infection in rhesus monkeys; however, HEV-1 (human) virus cannot infect pigs. Viral and/or cellular factors responsible for this host specificity are not yet known. We developed 12 different genotype 1-4 chimeric full genome clones with pSK-HEV2 as the backbone and by replacing structural (ORF2 and ORF3), non-structural (ORF1) and non-coding regions (NCR) with corresponding segments from the HEV-4 clone. S10-3 (human hepatoma) and PK-15 (pig kidney) cells were transfected with transcripts generated from the above clones to test their replication competence. Transfected cells were monitored for successful virus replication by detecting replicative intermediate RNA and capsid protein (immunofluorescence assay). All the chimeric constructs were able to replicate in S10-3 cells. However, only two chimeric clones, HEV-1 (HEV-4 5'NCR-ORF1) and HEV-1 (HEV-4 ORF1), containing 5'NCR-ORF1 and ORF1 regions from the HEV-4 clone, respectively, were able to replicate in PK-15 cells. We demonstrate for the first time the crucial role of ORF1 polyprotein in crossing the species barrier at the cellular level. These results indicate the importance of interactions between ORF1 protein domains and host cell specific factors during HEV replication and the critical role of cellular factors as post-entry barrier/s in virus establishment.

Hepatitis E Virus (HEV) is the major causative agent of acute hepatitis in developing countries. Its genome has three open reading frames (ORFs)—called as ORF1, ORF2, and ORF3. ORF1 encodes nonstructural polyprotein having multiple domains, namely: Methyltransferase, Y domain, Protease, Macro domain, Helicase, and RNA-dependent RNA polymerase. In the present study, we show that HEV-macro domain specifically interacts with light chain subunit of human ferritin (FTL). In cultured hepatoma cells, HEV-macro domain reduces secretion of ferritin without causing any change in the expression levels of FTL. This inhibitory effect was further enhanced upon Brefeldin-A treatment. The levels of transferrin Receptor 1 or ferroportin, two important proteins in iron metabolism, remained unchanged in HEV-macro domain expressing cells. Similarly, there were no alterations in the levels of cellular labile iron pool and reactive oxygen species, indicating that HEV-macro domain does not influence cellular iron homeostasis/metabolism. As ferritin is an acute-phase protein, secreted in higher level in infected persons and HEV-macro domain has the property of reducing synthesis of inflammatory cytokines, we propose that by directly binding to FTL, macro domain prevents ferritin from entering into circulation and helps in further attenuation of the host immune response. Electronic supplementary material The online version of this article (doi:10.1007/s11010-016-2715-0) contains supplementary material, which is available to authorized users.

The molecular mechanisms of liver pathology and clinical disease in hepatitis E virus (HEV) infection remain unclear. MicroRNAs (miRNAs) are known to modulate viral pathogenesis either by directly altering viral gene expression or by enhancing cellular antiviral responses. Given the importance of microRNA-122 (miR-122) in liver pathobiology, we investigated possible role of miR-122 in HEV infection. In silico predictions using HEV genotype 1 (HEV-1), HEV-2, HEV-3, and HEV-4 sequences showed that the majority of genomes (203/222) harbor at least one miR-122/microRNA-122-3p (miR-122*) target site. Interestingly, HEV-1 genomes showed a highly (97%) conserved miR-122 target site in the RNA-dependent RNA polymerase (RdRp) region (RdRp c ). We analyzed the significance of miR-122 target sites in HEV-1/HEV-3 (HEV-1/3) genomes by using a replicon-based cell culture system. HEV infection did not change the basal levels of miR-122 in hepatoma cells. However, transfection of these cells with miR-122 mimics enhanced HEV-1/3 replication and depletion of miR-122 with inhibitors led to suppression of HEV-1/3 replication. Mutant HEV-1 replicons with an altered target RdRp c sequence (CACTCC) showed a drastic decrease in virus replication, whereas introduction of alternative miR-122 target sites in mutant replicons rescued viral replication. There was enrichment of HEV-1 RNA and miR-122 molecules in RNA-induced silencing complexes in HEV-infected cells. Furthermore, pulldown of miR-122 molecules from HEV-infected cells resulted in pulldown of HEV genomic RNA along with miR-122 molecules. These observations indicate that miR-122 facilitates HEV-1 replication, probably via direct interaction with a target site in the viral genome. The positive role of miR-122 in viral replication presents novel opportunities for antiviral therapy and management of hepatitis E. IMPORTANCE Hepatitis E is a problem in both developing and developed countries. HEV infection in most patients follows a self-limited course; however, 20% to 30% mortality is seen in infected pregnant women. HEV superinfections in patients with chronic hepatitis B or hepatitis C virus infections are associated with adverse clinical outcomes, and both conditions warrant therapy. Chronic HEV infections in immunocompromised transplant recipients are known to rapidly progress into cirrhosis. Currently, off-label use of ribavirin (RBV) and polyethylene glycol-interferon (PEG-IFN) as antiviral therapy has shown promising results in both acute and chronic hepatitis E patients; however, the teratogenicity of RBV limits its use during pregnancy, while alpha IFN (IFN-α) increases the risk of transplant rejections. Experimental data determined with genotype 1 virus in the current study show that miR-122 facilitates HEV replication. These observations present novel opportunities for antiviral therapy and management of hepatitis E.

Hepatitis E virus (HEV) is a positive-sense RNA virus and member of the genus Orthohepevirus in the family Hepeviridae. Although HEV RNA-dependent RNA polymerase (HEV-RdRp) plays an important role in the HEV life cycle, its template specificities are not completely understood. We expressed HEV-RdRp protein with His-tag in a bacterial system and analysed template specificities using different putative cis-regulatory elements in the HEV genome. The enzyme showed highest affinity for the 3′ non-coding region (NCR), then for the 5′NCR and least for the putative subgenomic promoter (SgP). The enzyme could co-bind to 3′NCR and putative SgP templates together, as evident from the supershift in binding assay, indicating presence of different binding sites for these elements. Proteomic analysis revealed that the RNA elements share two common peptides for binding, while a third peptide, which is highly conserved across different HEV genotypes, is specific for 3′NCR. We propose that, during the early phases of replication, as negative sense antigenome copies accumulate at the replication site, they probably initiate promoter swapping from 3′NCR to SgP, to favour synthesis of subgenomic RNA and to prevent synthesis of genomic RNA. The conserved site for 3′NCR binding could be potential antiviral target and needs further evaluation.

Hepatitis E virus (HEV) is a significant health problem in developing countries causing sporadic and epidemic forms of acute viral hepatitis. Hepatitis E is a self-limiting disease; however, chronic HEV infections are being reported in immunocompromised individuals. The disease severity is more during pregnancy with high mortality (20–25%), especially in third trimester. Early cellular responses after HEV infection are not completely understood. We analyzed innate immune responses associated with genotype-I HEV replication in human hepatoma cell lines (Huh7, Huh7.5 and HepG2/C3A) using HEV replicon system. These cells supported HEV replication with different efficiencies due to the cell type specific innate immune responses. HepG2/C3A cells were less supportive to HEV replication as compared to Huh7.5 and S10-3 cells. Reconstitution of the defective RIG-I and TLR3 signaling in Huh7.5 cells enabled them to induce higher level antiviral responses and restrict HEV replication, suggesting the involvement of both RIG-I and TLR3 in sensing HEV RNA and downstream activation of interferon regulatory factor 3 (IRF3) to generate antiviral responses. Inhibition of IRF3 mediated downstream responses in HepG2/C3A cells by pharmacological inhibitor BX795 significantly improved HEV replication efficiency implying the importance of this study in establishing a better cell culture system for future HEV studies.

Hepatitis E virus (HEV) ORF1 protein (pORF1) contains methyltransferase (MetT), papain-like cysteine protease (PCP), RNA helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains. ORF1 sequence analysis showed two consensus LXGG cleavage sites at 664 and 1205. LXGG sequence is recognized by viral and cellular deubiquitinating enzymes. The protein encompassing the predicted MetT-PCP domains of HEV ORF1 was tested for deubiquitinating activity using fluorogenic substrates – ubiquitin-7-amino-4-methylcoumarin (AMC), IFN-stimulated gene 15 (ISG15)-AMC, Nedd8-AMC and SUMO-AMC. MetT-PCP cleaved all four substrates but processing of ISG15-AMC was more robust. There was no processing of the Hel and RdRp domains having the conserved (1205) LXGG site by the protein. MetT-PCP carried out deISGylation of the ISG15-conjugated cellular proteins, suggesting a possible role in combating cellular antiviral pathways.

Hepatitis E virus (HEV) has a positive-sense RNA genome with a 5′-m7G cap. HEV open reading frame 1 (ORF1) encodes a polyprotein with multiple enzyme domains required for replication. HEV helicase is a nucleoside triphosphatase (NTPase) with the ability to unwind RNA duplexes in the 5′-to-3′ direction. When incubated with 5′-[γ- 32 P]RNA and 5′-[α- 32 P]RNA, HEV helicase released 32 P only from 5′-[γ- 32 P]RNA, showing specificity for the γ-β-triphosphate bond. Removal of γ-phosphate from the 5′ end of the primary transcripts (pppRNA to ppRNA) by RNA triphosphatase is an essential step during cap formation. It is suggested that HEV employs the helicase to mediate the first step of 5′ cap synthesis.

Background: Increased incidence of HCV infection is noted universally throughout the world in patients undergoing haemodialysis. Genotyping and gene sequencing of Hepatitis C virus in individuals with positive HCV-RNA helps in determining the optimal type, duration and response to therapy. Aim of the study was to determine gene sequencing which is considered as gold standard for determination of genotypes.Methods: A cross sectional study design was conducted among 250 patients from five dialysis centres in Pune city in a period of one year. Qualitative HCV RNA detection was carried out by nested RT-PCR. Genotyping and sequencing was carried out using the Big-Dye Terminator cycle sequencing ready reaction kit. SPSS 21.0 version software was used to analyze the data.Results: Out of total 250 patients 47(18.8%) were anti HCV antibody positive and 37 (14.8%) were HCV RNA positive. Out of 47 patients who tested positive for anti-HCV antibody 36 showed presence of HCV RNA (76.6% positive correlation). Out of 203 ELISA negative patients only 1 was found to be positive. Out of total 37 patients predominant genotype was found to be 1a (54.1%) followed by 1b (43.2%) and 3a (2.7%).Conclusions: Genotyping and gene sequencing in patients with HCV RNA positivity revealed predominant genotype 1a and 1b. Detailed phylogenetic tree analysis revealed clustering of same genotypes in centre likely suggesting common source of infection prevalent in dialysis units and nosocomial transmission of virus.

Hepatitis E virus (HEV) is a major cause of enterically transmitted acute hepatitis in developing nations and occurs in sporadic and epidemic forms. The disease may become severe with high mortality (20%) among pregnant women. Due to lack of efficient cell culture system and small animal model, early molecular events of HEV infection are not yet known. In the present study, human lung epithelial cells, A549, were infected with HEV to monitor expression levels of genes/proteins in antiviral pathways. Both live and UV inactivated virus elicited robust induction of inflammatory cytokines/chemokines such as IL-6, IL-8, TNF-α, and RANTES within 12 h of infection. Cells exposed to soluble capsid protein showed no induction suggesting the capsid structure and not the protein being detected as the pathogen pattern by cells. A delayed up-regulation of type I interferon genes only by the live virus at 48 h post HEV infection indicated the need of virus replication. However, absence of secreted interferons till 96 h suggested possible involvement of post-transcriptional regulation of type I IFN expression. HEV infected cells showed activation of both NF-κB and IRF3 transcription factors when seen at protein levels; however, reporter gene assays showed predominant expression via NF-κB promoter as compared to IRF3 promoter. Knockdown experiments done using siRNAs showed involvement of MyD88 and TRIF adaptors in generating antiviral response thus indicating role of TLR2, TLR4 and TLR3 in sensing viral molecules. MAVS knockdown surprisingly enhanced only proinflammatory cytokines and not type I IFNs. This suggested that HEV not only down-regulates RIG-I helicase like receptor mediated IFN induction but also employs MAVS in curtailing host inflammatory response. Our findings uncover an early cellular response in HEV infection and associated molecular mechanisms suggesting the potential role of inflammatory response triggered by HEV infection in host immune response and pathogenesis.

Current hepatitis E virus (HEV) negative-sense RNA detection assays have the drawback of false positivity. cDNA synthesis using tag-based primer and Superscript RT-III followed by exonuclease I treatment increased the specificity. Assays could detect as few as 10 copies of negative-sense RNA and could be used in detecting low levels of HEV replication in cells. Virus particles in stool samples of hepatitis E patients showed encapsidation of negative-sense RNA along with HEV genomic RNA.

Chikungunya virus (CHIKV) is an insect borne virus (genus: Alphavirus) which causes acute febrile illness in humans followed by a prolonged arthralgic disease that affects the joints of the extremities. Re-emergence of the virus in the form of outbreaks in last 6-7 years has posed a serious public health problem. CHIKV has a positive sense single stranded RNA genome of about 12,000 nt. Open reading frame 1 of the viral genome encodes a polyprotein precursor, nsP1234, which is processed further into different non structural proteins (nsP1, nsP2, nsP3 and nsP4). Sequence based analyses have shown helicase domain at the N-terminus and protease domain at C-terminus of nsP2. A detailed biochemical analysis of NTPase/RNA helicase and 5'-RNA phosphatase activities of recombinant CHIKV-nsP2T protein (containing conserved NTPase/helicase motifs in the N-terminus and partial papain like protease domain at the C-terminus) was carried out. The protein could hydrolyze all NTPs except dTTP and showed better efficiency for ATP, dATP, GTP and dGTP hydrolysis. ATP was the most preferred substrate by the enzyme. CHIKV-nsP2T also showed 5'-triphosphatase (RTPase) activity that specifically removes the γ-phosphate from the 5' end of RNA. Both NTPase and RTPase activities of the protein were completely dependent on Mg(2+) ions. RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA. Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg(2+) ion binding motif (DEXX) suggesting that they have a common catalytic site.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae . The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5′ overhangs showing 5′-to-3′ polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg 2+ binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5′ overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.

BACKGROUND: An unusually high number of severe pneumonia cases with considerable mortality is being observed with the pandemic H1N1 2009 virus infections globally. In India, all mild as well as critically ill cases were admitted and treated in the government hospitals during the initial phase of the pandemic. The present study was undertaken during this early phase of the pandemic. METHODOLOGY: The role of viral load and host factors in the pathogenesis were assessed by examining 26 mild (MP), 15 critically ill patients (CIP) and 20 healthy controls from Pune, India. Sequential blood and lung aspirate samples were collected from CIP. Viral load and cytokines/chemokine levels were determined from the plasma and lung aspirates of the patients. TLR levels were determined by staining and FACS analysis. Gene profiling was done for both cells in the lung aspirates and PBMCs using TaqMan Low Density arrays. Antibody titres and isotyping was done using HA protein based ELISAs. PRINCIPAL FINDINGS: 13/15 critically ill patients expired. All plasma samples were negative for the virus irrespective of the patient's category. Sequential lung samples from CIP showed lower viral loads questioning association of viral replication with the severity. Anti-rpH1N1-09-HA-IgG titres were significantly higher in critically ill patients and both categories circulated exclusively IgG1 isotype. Critically ill patients exhibited increase in TLR-3, 4, 7 and decrease in TLR-2 expressions. The disease severity correlated with increased plasma levels of IL1RA, IL2, IL6, CCL3, CCL4 and IL10. Majority of the immune-function genes were down-regulated in the PBMCs and up-regulated in the cells from lung aspirates of critically ill patients. No distinct pattern differentiating fatal and surviving patients was observed when sequential samples were examined for various parameters. CONCLUSIONS: Disease severity was associated with pronounced impairment of host immune response.

Phylogenetic analysis of the sequences of the 5′ noncoding regions (5′NCR) of 149 samples from hepatitis C virus (HCV) RNA-positive chronic carriers representing northern, southern, eastern, and western India showed that type 3 and type 1 are the predominant genotypes circulating in India, with an overall prevalence of 53.69 and 38.25%, respectively. Type 4 viruses (6.04%) were seen only in southern India. Sequence analysis of the core region of 51 of the above isolates enabled us to classify them further into subtypes as 1b (number of isolates [ n ] = 10), 1a ( n = 6), 3a ( n = 9), 3g ( n = 14), 3f ( n = 1), and 4d ( n = 3). Three new subtypes were identified for the first time and designated as 3i ( n = 5), 3j ( n = 2), and 6l ( n = 1). Sequencing the 5′NCR could differentiate HCV types, whereas classification at the level of subtype was possible with sequence analysis of the core region.

According to World Health Organization estimates, India will have the greatest number of human immunodeficiency virus (HIV)-infected individuals of any country by the end of this decade (1, 6). High rates of sexually transmitted diseases, rapidly increasing seroprevalence in female commercial sex workers, and inadequate facilities for HIV testing, counseling, and prevention are the major contributing factors in the recent explosive increases in the numbers of HIV infections (5, 6, 24, 29). While antiretroviral drugs have reduced mortality from AIDS in developed nations, their effect will be negligible elsewhere due to their cost. For most communicable diseases, vaccines offer the most cost-effective control strategy. It is likely that development of a vaccine for HIV will require knowledge of the viral variants being transmitted in the target population. Despite India’s impending predominance in the worldwide pandemic, little is known of the genetic diversity of HIV-1 in India. The HIV-1 sequence database is growing exponentially, but the distribution of submitted sequences is not representative of the worldwide picture. Subtype C has been reported in nearly every region affected by HIV-1 (11, 23, 28) and predominates in India, and it also causes 74% of infections in southern Africa and 96% of infections in northern Africa (11, 18, 32). Given the combined population of India and the other regions affected, subtype C is likely to be the most commonly transmitted HIV-1 subtype worldwide. In contrast, 7% of the available HIV-1 sequence data is from subtype C-infected individuals (37), and of the 46 completely sequenced HIV-1 genomes (excluding multiple derivatives of HIV-1LAI), only two are of subtype C, one from a 1992 Brazilian sample and the other from a 1986 Ethiopian sample (37). In November 1997, an analysis of cross-clade epitope variation (9) excluded the C clade from evaluation of p24gag epitopes because of a lack of sequence data, whereas there was sufficient data to analyze subtypes A, B, D, F, G, and H (no HIV-1 harboring a subtype E gag gene has been found). Further sequence data from subtype C is needed, but the past approach of generating data from small subgenomic amplicons is no longer sufficient. Recent developments have made full-genome characterization of HIV-1 isolates both important and feasible. First, the recognition of intersubtype recombination in a significant proportion of HIV-1 sequences (44, 45) has led to detection of mosaic genomes in many regions of the world affected by multiple subtypes (14, 17, 31). Subtypes A, B, and C in India have been reported (4, 22, 30, 31, 59), but mosaic HIV-1 there has not been reported. The existence of such recombinants makes characterization of variants by analyzing subgenomic segments incomplete. Second, immune responses to vaccines based on single genes such as env have been limited (13), and attention is being shifted toward multivalent vaccines that incorporate other gene products. Third, interactions among discontinuous regions of the genome, such as between the long terminal repeat (LTR) and pol (26), can be detected only when such regions can be analyzed from the same template. In an effort to characterize subtype C virus genomes being transmitted currently in India, viral isolates were obtained from individuals with seroincident infections in India. Three of the isolates (collected in 1994 and 1995) were known to be non-syncytium inducing (NSI) and therefore resembled viruses transmitted through unprotected sexual contact, which account for 75 to 85% of new infections (2, 15, 61). These isolates were cloned, and nearly full-length genomic sequences were determined. Detailed sequence analysis was performed, as was an analysis of variation in characterized cytotoxic T lymphocyte (CTL) epitopes.