title: Alternative cell lines to improve the rescue of infectious human astrovirus from a cDNA clone.
abstract: A reverse genetics system for human astrovirus (HAstV) was established previously; however, it has not been exploited mainly because cells used for virus packaging are not permissive, requiring several rounds of replication to obtain acceptable infectious virus. In this work, in the search for alternative permissive cell lines to be used as packaging cells, Hek-293 and Huh7.5.1 were tested. Given that HAstV infection in Hek-293 showed differences with that in Caco-2, the gold standard for HAstV growth but scarcely transfectable, and it was more similar to that observed in the hepatoma Huh7.5.1 cell line, these last cells were further used to transfect viral RNA. Virus titers near to 10(8) infectious particles per ml (ffu/ml) were obtained at 16-20 h after transfection with RNA from infected cells. However, virus titers close to 10(6) ffu/ml were obtained by using in vitro transcribed RNA from a cDNA HAstV-1 clone. In contrast, virus recovery in BHK-21, reported previously as the packaging cells, from this RNA was of about 10(4) ffu/ml, two logarithms less than in Huh7.5.1. Apparently, the 5'-end modification of the viral RNA determined its specific infectivity, since virus recovery was abolished when the total RNA was treated with proteinase-K, probably by removing a protein-linked genome protein, but it increased when capping of the in vitro transcribed RNA was more efficient. Thus, an alternative and more efficient reverse genetics system for HAstV was established by using Huh7.5.1 cells.
authors: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, Mexico.
authors: Rodrigo
authors: R
authors: Velázquez-Moctezuma
authors: Ma del Rocío
authors: Mdel R
authors: Baños-Lara
authors: Yunuén
authors: Y
authors: Acevedo
authors: Ernesto
authors: E
authors: Méndez
mesh_terms: D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D001257:Mamastrovirus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D059386:Reverse Genetics / Q000379:methods*; D014162:Transfection; D019562:Viral Load; D014773:Virology / Q000379:methods*; D019065:Virus Assembly; D014776:Virus Cultivation / Q000379:methods
chemical_list: D018076:DNA, Complementary; D012367:RNA, Viral