Rescue of hepatitis A virus from cDNA-transfected but not virion RNA-transfected mouse Ltk- cells.
Hepatitis A virus (HAV) has stringent replication requirements and a restricted host-range. Mouse Ltk- cells do not support growth of HAV upon infection or transfection of virion RNA. However, low levels of HAV were rescued from Ltk- cells transiently transfected with its infectious cDNA. Ltk- stable transfectants that expressed HAV antigens and produced infectious HAV were selected and termed Ltk-pJH15 cells. After a few serial passages, HAV became undetectable in the Ltk-pJH15 cells. Multiple rounds of single cell cloning of HAV antigen positive Ltk-pJH15 cells resulted in the isolation of clone E8 that produced higher levels of HAV for at least 5 passages. HAV produced in E8 cells was similar to the parental virus as shown by infectivity assays. Luciferase assays using a bi-cistronic construct containing the HAV 5' noncoding region showed similar levels of HAV IRES-dependent translation in Ltk- and Ltk-pJH15 cells, which suggested that HAV IRES-dependent translation was not a limiting factor for HAV growth in these cells. The availability of the Ltk-pHJ15 cells will allow the identification of cellular factors required for HAV growth, which could lead to the development of a mouse model to study pathogenesis of HAV.
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Laboratory of Hepatitis, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, USA.
Dveksler
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G
Kaplan
G G
GG
D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D017957:Hepatitis A Virus, Human / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D051379:Mice; D012367:RNA, Viral / Q000378:metabolism; D011971:Receptors, Immunologic / Q000172:deficiency / Q000235:genetics; D013045:Species Specificity; D014162:Transfection; D014776:Virus Cultivation; D014779:Virus Replication
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral; D011971:Receptors, Immunologic