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.

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), nonstructural (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.

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. Hepatitis E virus (HEV) is the major etiological agent of waterborne epidemics in India and of a significant proportion of sporadic hepatitis cases in adults. The genome of HEV is a singlestranded, positive-sense RNA molecule of ϳ7,200 bases. Due to the lack of a cell culture system, HEV replicons are being used in studies of virus replication (4,6,7,8,10). HEV replication proceeds via a negative-sense intermediate (12,13,14) that acts as a template for synthesis of new positive-sense genomic RNA and subgenomic RNA molecules (8, 10). Detection of negative-sense RNA has been taken as evidence of viral replication. Several studies in HEV have used strand-specific assays (3,11,13,14,16,17,19,20); however, except one (11), none have addressed the issue of the specificity and accuracy of correct strand detection. This report deals with various factors which might be responsible for false-positive results in detecting negative-sense RNA.Generation of synthetic transcripts. Partial HEV open reading frame 1 (ORF1) regions (nucleotides [nt] 2987 to 5698; 2,712 bp) cloned in pGEMT-EASY vector (Promega) and an HEV fulllength cDNA clone (pGEMT-EASY-HEVT1FG) were linearized and used as templates for in vitro transcription (Ribomax in vitro transcription kit; Promega). Template DNA was removed by two rounds of RNase-free DNase treatment. Concentrations of purified RNA were determined using a NanoDrop ND-1000 spectrophotometer at 260 nm.RT-PCR. Primers used in the present study are listed in Table 1 and Table 2. RNA was denatured at 65°C for 5 min in the presence of 10 pmol of primer, and reverse transcription (RT) reactions were performed in a 20-l volume containing 625 M deoxynucleoside triphosphates (dNTPs), 40 U of RNase inhibitor, and either AMV-RT (Promega) or Superscript RT-III (Invitrogen) under the respective buffer conditions. Where mentioned, after RT, cDNA was incubated with 50 U of exonuclease I (USB) at 37°C for 30 min and purified (QIAquick PCR purification kit; Qiagen). First-round and second-round PCRs were carried out for 40 and 30 cycles, respectively.Lack of strand specificity in detecting negative-sense RNA due to false priming during cDNA synthesis. HEV diagnostic RT-PCR (2) was initially tested for strand specificity using synthetic HEV full-genome RNA (gRNA) as a template for cDNA synthesis, in two sets of reaction mixtures containing (i) forward primer (HEVF1; negative-sense RNA detection) and (ii) reverse primer (HEVR1; positive-sense RNA detection). For PCR, nested HEV-specific primer pair HEVF1 and HEVR1 (PCR I; 718...