The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious.
Hepatitis E virus (HEV) is an important etiological agent of epidemic and sporadic hepatitis, which is endemic to the Indian subcontinent and prevalent in most of the developing parts of the world. The infection is often associated with acute liver failure and high mortality, particularly in pregnant women. In order to develop methods of intervention, it is essential to understand the biology of the virus. This is particularly important as no reliable in vitro culture system is available. We have constructed a cDNA clone encompassing the complete HEV genome from independently characterized subgenomic fragments of an Indian epidemic isolate. Transfection studies were carried out with HepG2 cells using in vitro-transcribed RNA from this full-length HEV cDNA clone. The presence of negative-sense RNA, indicative of viral replication, was demonstrated in the transfected cells by strand-specific reverse transcription-PCR and slot blot hybridization. The viral proteins pORF2 and pORF3 and processed components of the pORF1 polyprotein (putative methyltransferase, helicase, and RNA-dependent RNA polymerase) were identified in the transfected cells by metabolic pulse-labeling with [(35)S]methionine-cysteine, followed by immunoprecipitation with respective antibodies. The expression of viral proteins in the transfected cells was also demonstrated by immunofluorescence microscopy. Viral replication was detected in the transfected cells up to 33 days posttransfection (six passages). The culture supernatant from the transfected cells was able to produce HEV infection in a rhesus monkey (Macaca mulatta) following intravenous injection, indicating the generation of viable HEV particles following transfection of cells with in vitro-synthesized genomic RNA. This transient cell culture model using in vitro-transcribed RNA should facilitate our understanding of HEV biology.
Panda
S K
SK
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India. skpanda@medinst.ernet.in
Ansari
I H
IH
Durgapal
H
H
Agrawal
S
S
Jameel
S
S
D000818:Animals; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000235:genetics; D005455:Fluorescent Antibody Technique; D016751:Hepatitis E / Q000097:blood / Q000821:virology*; D016752:Hepatitis E virus / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity*; D007075:Immunoglobulin M / Q000097:blood; D066298:In Vitro Techniques; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016372:RNA, Antisense / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013047:Specific Pathogen-Free Organisms; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics
D018076:DNA, Complementary; D007075:Immunoglobulin M; D016372:RNA, Antisense; D012367:RNA, Viral; D014764:Viral Proteins