Human wild-type (wt) hepatitis A virus (HAV), the causative agent of acute hepatitis, barely grows in cell culture and in the process accumulates attenuating and cell culture-adapting mutations. This genetic instability of wt HAV in cell culture is a major roadblock to studying HAV pathogenesis and producing live vaccines that are not overly attenuated for humans. To develop a robust cell culture system capable of supporting the efficient growth of wt HAV, we transfected different cell lines with in vitro RNA transcripts of wt HAV containing the blasticidin resistance gene. Blasticidin-resistant colonies grew only in transfected Huh7 cells and produced infectious virus. HAV was genetically stable in Huh7 cells for at least nine serial passages and did not accumulate attenuating or cell culture-adapting mutations. Treatment with alpha interferon A/D cured the blasticidin-resistant Huh7 cells of the HAV infection. The cured cells, termed Huh7-A-I cells, did not contain virus or HAV antigens and were sensitive to blasticidin. Huh7-A-I cells were more permissive than parental cells for wt HAV infection, including a natural isolate from a human stool sample, and produced 10-fold-more infectious particles. This is the first report of a cell line that allows the genetically stable growth of human wt HAV. The viral vectors and cells described here should allow better insight into the pathogenesis of HAV and the development of attenuated vaccines. The cell lines susceptible to wt HAV growth may also be used to detect and isolate infectious virus from patient and environmental samples.Hepatitis A virus (HAV) is a member of the Picornaviridae that causes acute hepatitis in humans, a worldwide preventable infectious disease. In the United States, approximately 25,000 cases of HAV are reported each year, and an estimated average of 263,000 HAV cases occur annually when corrected for underreporting and asymptomatic infections (17). HAV is a nonenveloped virus that contains a 7.5-kb single-stranded positive-sense genomic RNA encapsidated in an icosahedral 27-to 32-nm particle. The viral RNA has a 5Ј-end nontranslated region of approximately 750 bases containing an internal ribosome entry site (IRES) (39), and a 3Ј-end short nontranslated region followed by a poly(A) tail. The viral protease 3C pro cleaves the HAV polyprotein into smaller structural (VP0, VP3, and VP1-2A) and nonstructural (2B, 2C, 3A, 3B, 3C, and 3D) proteins (references 25, 27, and 31 and references therein), and a cellular protease cleaves the VP1-2A precursor (24).Human wild-type (wt) HAV rarely grows in cell culture, is genetically unstable, and requires several weeks to months in culture before it can be detected (4,7,14,19). HAV has been adapted to a variety of primate (3, 9, 18, 34, 38) and nonprimate (10, 16) cell lines, but this process results in the accumulation of cell culture-adapting and attenuating mutations (4,5,9,11,20,34). During adaptation to cell culture, two main hot-spot mutation areas in the HAV genome were identified, one located in the...