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.

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.

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.

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.

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.

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.

Evaluation of Liposome, Heat-Killed Mycobacterium w, and Alum Adjuvants in the Protection Offered by Different Combinations of Recombinant HA, NP proteins, and M2e Against Homologous H5N1 Virus.

Continued evolution of highly pathogenic H5N1 viruses causing high mortality in humans obviates need for broadly cross-reactive vaccines. For this, hemagglutinin (HA) inducing specific protective antibodies, highly conserved nucleoprotein (NP), and ectodomain of matrix (M2e) protein, either singly or in combination, were evaluated in BALB/c mice. Recombinant HA and NP (baculovirus system) and M2e (synthetic peptide) and 3 adjuvants, that is, liposomes, Mw (heat killed Mycobacterium w), and alum were utilized for the homologous virus challenge. Additional immunogens included liposome-encapsulated HA/NP proteins and corresponding DNAs. Mice groups received two doses of respective formulations given at 3-week intervals and challenged intranasally with 100LD50 of H5N1 virus strain. Dynamics of weight loss, lung viral load, titres of IgG-anti-HA, NP, and M2e antibodies (ELISA), and IgG-subtype analysis was done. Two doses of all the formulations led to 100% seroconversion against the immunogens evaluated (100% seroconversion after the first dose in majority). Antibody titres against the components were dependent on the adjuvant and combination. HA-driven Th2 response with all the adjuvants, balanced Th1/Th2 response to NP protein, and Th2-bias with alum were noted. Low anti-M2e antibody titres did not allow subtype analysis. On challenge, complete protection was observed with Mw-HA, alum-HA+NP, Lipo-HA+NP+M2e, alum-HA+NP+M2e, and HA-DP formulations with 12-fold, 8-fold, 720-fold, 17-fold, and no reduction, respectively, in lung viral load. In conclusion, the results identify several adjuvant-immunogen combinations conferring 100% protection in mice that need further evaluation in higher animals.

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 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.

Innate immune responses in human hepatocyte-derived cell lines alter genotype 1 hepatitis E virus replication efficiencies.

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)-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 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.

Mutagenesis of hepatitis E virus helicase motifs: effects on enzyme activity.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is a non-enveloped RNA virus. The open reading frame 1 encoded non-structural polyprotein has putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase, however processing of this polyprotein is still uncertain. HEV helicase belongs to superfamily 1 and has all seven conserved motifs typical of the family. NTPase and RNA duplex unwinding activities of HEV helicase domain were recently demonstrated by us. A non-radioactive RNA unwinding assay was developed using biotin and digoxigenin labeled duplex RNA substrate with 5' overhangs for measuring strand displacement activity of the helicase. A series of deletion mutants were constructed to investigate role of individual motifs in the enzymatic activities. Deletion mutants for motif M I and M IV showed increase in ATPase activity. Deletion mutant M VI retained ATPase activity comparable to wild type protein. Mutant M II showed reduced ATPase activity (P=0.003) with no significant decrease in unwinding activity while mutants M Ia and M III showed major reduction of both ATPase and unwinding activities indicating crucial role of these motifs in the helicase function. Overall analysis of deletion mutants showed that Motif I, IV, V and VI have alternative motifs to carry out enzymatic functions of the protein while motifs Ia and III are critical as well as unique motifs in the protein. Knowing the important role of helicase protein during positive sense RNA virus replication, these unique motifs could be good antiviral targets.

Analysis of antiviral response in human epithelial cells infected with hepatitis E 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.

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.

NTPase and 5'-RNA triphosphatase activities of Chikungunya virus nsP2 protein.

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.

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.