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

Abstract

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.

Positive Regulation of Hepatitis E Virus Replication by MicroRNA-122

“…The presence of a highly conserved target site (RdRp c ) in HEV-1 genomes prompted us to examine whether HEV has the ability to modulate miR-122 expression in HepG2/C3A and S10-3 cells. For that purpose, HEV infection was initiated by transfecting cells with SAR55 full-genome replicon (pSK-HEV-2) and virus replication was confirmed by negativestrand RNA detection and immunofluorescence assay as described previously (22) (Fig. 3Ai and ii).…”

“…Negative-strand-specific RT-PCR. Total cellular RNA was extracted from cells using a Ribopure RNA extraction kit (Ambion, Life Technologies, USA), and detection of negative-sense RNA (replicative intermediate) was done by using a tagged-primer-based reverse-transcription PCR protocol as described previously (22).…”

“…Resuspended beads were split into two equal parts. One part of each bead was processed for RNA extraction using a viral RNA kit (Qiagen, Hilden, Germany), and HEV genomic copies were quantitated by real-time PCR as described previously (22). The other part was processed for miRNA isolation (using a mirVana miRNA isolation kit) followed by miR-122 quantitation.…”

Zoonotic Hepatitis E Virus: Classification, Animal Reservoirs and Transmission Routes

“…This difference raises the question of species barrier determinants. Several studies have suggested that genetic elements present in HEV ORF1 are involved in species barrier crossing [118,119,120,121]. …”

Hepatitis E virus isolated from chronic hepatitis B patients in Malaysia: Sequences analysis and genetic diversity suggest zoonotic origin

“…[4][5][6] Zoonotic potential of HEV has been reported in a previous study in which HEV antibodies was found in animal serum. 7,8 For instance, some HEV cases in Japan were found to be directly associated with eating raw or under cooked deer meat, 9 while several cases of HEV were epidemiologically linked to consumption of poorly cooked wild boar meat or pork. [10][11][12] HEV was reported to be successfully transmitted from animal to human, 1 as it causes acute infection as well as aggravates any existing chronic hepatitis condition.…”