Modulation of cellular autophagy by genotype 1 hepatitis E virus ORF3 protein.

Hepatitis E virus (HEV) egresses from infected hepatocytes as quasienveloped particles containing open reading frame 3 (ORF3) protein. HEV ORF3 (small phosphoprotein) interacts with host proteins to establish a favourable environment for virus replication. It is a functional viroporin that plays an important role during virus release. Our study provides evidence that pORF3 plays a pivotal role in inducing Beclin1-mediated autophagy that helps HEV-1 replication as well as its exit from cells. The ORF3 interacts with host proteins involved in regulation of transcriptional activity, immune response, cellular and molecular processes, and modulation of autophagy, by interacting with proteins, DAPK1, ATG2B, ATG16L2 and also several histone deacetylases (HDACs). For autophagy induction, the ORF3 utilizes non-canonical NF-κB2 pathway and sequesters p52NF-κB and HDAC2 to upregulate DAPK1 expression, leading to enhanced Beclin1 phosphorylation. By sequestering several HDACs, HEV may prevent histone deacetylation to maintain overall cellular transcription intact to promote cell survival. Our findings highlight a novel crosstalk between cell survival pathways participating in ORF3-mediated autophagy.

Transcriptome analysis of hepatoma cells transfected with Basal Core Promoter (BCP) and Pre-Core (PC) mutant hepatitis B virus full genome construct.

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.

Declining trends in Hepatitis A seroprevalence over the past two decades, 1998-2017, in Pune, Western India.

Reduction in seroprevalence of Hepatitis A virus (HAV) is known to be associated with improvements in socioeconomic conditions of the community. National Institute of Virology, Pune has been studying seroprevalence of hepatitis viruses in Pune region over the past four decades. In total, 1438 samples were collected from urban general (UGEN), urban lower socioeconomic stratum (ULSES) and rural (RURAL) populations of the Pune district. Based on estimates in previous studies, subjects were enrolled from age groups '6-10', '15-25' and '40 + ' years. HAV seroprevalence in younger population showed a significant decline. A significant decline in HAV seroprevalence in '15-25' years age group in UGEN (from 85.9% to 73.9%; OR = 0.46, 95% CI: 0.25-0.86) and RURAL (from 98.6% to 91.4%; OR = 0.15, 95% CI: 0.05-0.45) populations suggested that the trend probably started more than a decade ago. Seroprevalence of HAV among ULSES '6-10' children was found to be significantly higher (70.4%) than that among the RURAL children (44.2%; OR = 3.0, 95%CI: 1.7-5.2) and UGEN children (40.4%; OR = 3.5, 95%CI: 1.8-6.7). In view of increasing rates of urbanisation in India, ULSES population needs special consideration while designing future studies and viral hepatitis vaccination/elimination strategies. Our findings call for robust population-based studies that consider heterogeneity within populations and dynamics of socio-economic parameters in various regions of a country.

Investigation of a large waterborne acute gastroenteritis outbreak caused by group B rotavirus in Maharashtra state, India.

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.

A virus precipitation method for concentration & detection of avian influenza viruses from environmental water resources & its possible application in outbreak investigations.

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.

Hepatitis E virus RNA-dependent RNA polymerase: RNA template specificities, recruitment and synthesis.

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)-1 harbouring HEV-4 non-structural protein (ORF1) replicates in transfected porcine kidney cells.

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 ORF1 encoded macro domain protein interacts with light chain subunit of human ferritin and inhibits its secretion.

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.

Determination of Species Identity and Genetic Diversity of Aedes aegypti and Other Medically Important Mosquitoes of India Using DNA Barcoding.

Aedes aegypti-borne viruses (i.e., dengue, chikungunya, and Zika) have become endemic to India, posing a severe threat to public health. Vector control remains the mainstay of disease management due to nonavailability of licensed vaccines/therapeutics. Conventional morpho-taxonomical methods cannot differentiate between closely related sibling species or species complexes, and hence we evaluated two molecular markers, mitochondrial cytochrome c oxidase subunit 1 (Cox1) and nuclear DNA internal transcribed spacer 2 (-2) gene sequences, to characterize seven populations of Ae. aegypti and four medically important mosquito species (Aedes albopictus, Anopheles stephensi, Culex tritaeniorhyncus, and Culex murrelli). DNA extracted from the 11 mosquito populations (two mosquitoes per population) was polymerase chain reaction amplified, sequenced, and analyzed. Molecular characterization was found to be congruent with morphological identification, suggesting no variants or cryptic species exist in Ae. aegypti and the other mosquitoes studied. Phylogenetic analysis with sequences obtained with Cox1 gene of Ae. aegypti and other Aedes and non-Aedes mosquito species showed clustering of sequences from different species representing different clades, distinctly separating one taxon from the other, whereas ITS-2 sequences of Aedes aegypti from across the world clustered tightly. Nucleotide divergence values revealed a low percentage of intraspecies variation and a higher percentage of interspecies variation. The present study authenticates the applicability of Cox1 and ITS-2 in the precise identification of Ae. aegypti mosquitoes against cryptic or sibling species. Cox1 appeared to be a more reliable marker because it showed distinct clustering of mosquito species, and some sequence variations to represent genetic diversity.

Circulation of a single hepatitis A virus genotype IIIA with two distinct clusters in different states of India.

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 â€‹= â€‹280) were screened for HAV RNA and sequences for 5'non-coding and VP3 regions were generated from positive samples (n â€‹= â€‹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.

Genomic Characterization of Mosquito Isolates of Chikungunya Virus (Outbreak Strains 2022) Using Next-Generation Sequencing.

Background: A massive outbreak of dengue-like illness was reported from Pune district of Maharashtra, India during May-June 2022. Isolation and characterization of the etiological agent at genomic level for possible mutations that led to higher transmissibility is the topic of the study. Methods: Entomological investigations were carried out by ICMR-National Institute of Virology (Pune, India); Aedes aegypti mosquitoes were collected and processed for virus detection by molecular techniques. Positive mosquito pools were processed for virus isolation in cell culture. Sanger sequencing and whole-genome sequencing (WGS) using Oxford Nanopore Technology platform were used for genomic characterization. Results: Reverse transcriptase RT-PCR and qRT-PCR analysis detected chikungunya virus (CHIKV) in mosquito samples. Six CHIKV isolates were obtained. WGS revealed four nonsynonymous mutations in the structural polyprotein region, and five in the nonstructural polyprotein encoding region when compared with Yawat-2000 and Shivane-2016 strains. Sixty-four nucleotide changes in the nonstructural polyprotein region and 35 in the structural polyprotein region were detected. One isolate had an exclusive amino acid change, T1123I, in the nsP2 (protease) region. Conclusion: Abundant Ae. aegypti breeding and detection of CHIKV RNA in mosquitoes confirmed it as a chikungunya outbreak. Novel mutations detected in the epidemic strain warrants investigations to address their role in disease severity, transmission, and fitness.

Detection of negative-sense RNA in packaged hepatitis E virions by use of an improved strand-specific reverse transcription-PCR method.

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.

Deubiquitination activity associated with hepatitis E virus putative papain-like cysteine protease.

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.

RNA 5'-triphosphatase activity of the hepatitis E virus helicase domain.

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'-[gamma-(32)P]RNA and 5'-[alpha-(32)P]RNA, HEV helicase released (32)P only from 5'-[gamma-(32)P]RNA, showing specificity for the gamma-beta-triphosphate bond. Removal of gamma-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.

NTPase and 5' to 3' RNA duplex-unwinding activities of the hepatitis E virus helicase domain.

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.

Detection of human parvovirus 4 DNA in the patients with acute encephalitis syndrome during seasonal outbreaks of the disease in Gorakhpur, India.

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.

Quantitation of hepatitis B virus DNA by real-time PCR using internal amplification control and dual TaqMan MGB probes.

Hepatitis B virus (HBV) DNA quantitation is used extensively for monitoring of antiviral treatment of HBV infection. A real-time PCR assay was developed using a TaqMan minor groove binder probe and primers corresponding to HBV pre-core region for HBV DNA quantitation. A 228 bp fragment from this genomic region of HBV was cloned and serial dilutions of plasmid DNA were used as an external DNA standard. Comparison of the real-time PCR quantitation results from 35 clinical serum samples with those obtained by COBAS Amplicor HBV DNA monitor kit (Roche Diagnostics) revealed a significant correlation (r = 0.92) for all the samples. The assay showed wide dynamic linear range between 2.5 x 10(2) and 2.5 x 10(10) copies/ml serum. Sera from 25 healthy individuals tested negative indicating the high specificity of the assay. The median coefficients of variation for both intra- and inter-experimental variability were 4.9% and 10.6%, respectively, which indicated remarkable reproducibility. An internal amplification control (IC) was developed to detect the presence of PCR inhibitors in the samples to avoid false negative results. The IC had the same primer binding sites but different internal sequence and it competed with the virus-derived target. The optimum concentration of IC was found to be 100 copies/reaction. The assay was validated by testing serial dilutions of the WHO international HBV DNA standard. Since conserved regions were considered during primer and probe design, the assay should be applicable to all HBV genotypes. The real-time assay will be useful for monitoring HBV-infected patients in routine diagnostic laboratories and in clinical practice enabling analysis of a wide dynamic range of HBV DNA in a single, undiluted sample.

Full genome sequence and analysis of Indian swine hepatitis E virus isolate of genotype 4.

The full-length genomic sequence of an Indian swine hepatitis E virus (HEV) isolate (IND-SW-00-01) recovered from feces of a pig experimentally infected with swine HEV pool from western India was determined. The genome consisted of 7,240 nucleotides, excluding the poly (A) tail of at least 22 residues and contained three open reading frames (ORFs), ORF-1 encoding 1,707 amino acids, ORF-2 encoding 674 amino acids and ORF-3 encoding 114 amino acids. Comparative full-length genome sequence and phylogenetic analyses suggested that the Indian swine HEV represents a distinct variant among the genotype 4 isolates with a divergence of 15-16.6%. Analyses based on ORF-1, 2 and 3 as well as partial ORF-2 (227 nucleotides) yielded similar results. As compared to type 4 HEV isolates, 26 unique amino acid substitutions were recorded, 16 in ORF-1, 8 in ORF-2 and 2 in ORF-3. IND-SW-00-01 showed insertion of 'C' at 5159 position while all other type 4 isolates have insertion of 'U' at the same position. Whether these changes contribute towards observed absence of type 4 HEV infections in Indian patients needs to be determined.

Hepatitis E virus ORF1 encoded non structural protein-host protein interaction network.

Hepatitis E virus ORF1 encoded non-structural polyprotein (nsP) consist of multiple domains, namely: Methyltransferase, Y-domain, Protease, X-domain, Helicase and RNA dependent RNA polymerase. We have attempted to identify human liver cell proteins that are interacting with HEV ORF1 encoded functional domains by using Y2H screening. A total of 155 protein-protein interactions between HEV-ORF1 and human proteins were identified. Comparative analysis of the HEV-ORF1-Human interaction network with reconstructed human interactome showed that the cellular proteins interacting with HEV-ORF1 are central and interconnected. Enrichment analysis of Gene Ontology and cellular pathways showed that the viral proteins preferentially interacted with the proteins of metabolism and energy generation along with host immune response and ubiquitin proteasomal pathways. The mTOR and focal adhesion pathways were also targeted by the virus. These interactions suggest that HEV probably utilizes important proteins in carbohydrate metabolism, energy generation and iron homoeostasis in the host cells during its establishment.