title: Attenuation of a reactivated cardiovirulent coxsackievirus B3: The 5'-nontranslated region does not contain major attenuation determinants.
abstract: To investigate the molecular basis of pathogenicity of Coxsackieviruses, a virus was reactivated by transfection from a full-length cDNA clone derived from cardiovirulent Coxsackievirus B3 (CVB3). The reactivated virus, rCVB3, was passaged serially in human dermatofibroblasts (HDF). No cytopathic effect was observed up to 12 days after inoculation with rCVB3 or early-passage virus, although disintegration of the monolayers was observed with late-passage virus (10th to 14th passages). Approximately 10% of HDF inoculated with rCVB3 were positive for viral antigens by immunofluorescence using enterovirus- or CVB3-specific monoclonal antibodies. These observations, together with the low infectivity titre of rCVB3 in HDF, suggests that HDF initially support only carrier state infection. After the 14th passage, the cardiovirulence of passaged virus (p14V) in mice was attenuated by a factor of > 10(4). Phenotypic changes of plaque size were also noticed in p14V: An attenuated variant (p14V-1) that produced larger plaques than rCVB3 in Vero cells has been plaque purified. The 5'-terminus of the genome of attenuant p14V-1 was amplified by polymerase chain reaction (PCR) and its sequence determined. Only one point mutation was found within the 5'-nontranslated region (5'NTR) at position 690 (A to U) compared to the viral RNA sequence obtained for rCVB3. An intertypic chimeric virus was reactivated from a cDNA clone after replacing the 5'-terminal 891 nucleotides of the wild-type genome with the corresponding region of the attenuant p14V-1. This chimeric virus, CB3/p14V-1/1, produced wild-type plaques in Vero cells and showed cardiovirulence similar to that of rCVB3 in mice.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, Charing Cross and Westminister Medical School, London, United Kingdom.
authors.forename: H Y
authors.initials: HY
authors.lastname: Zhang
authors.forename: G E
authors.initials: GE
authors.lastname: Yousef
authors.forename: L
authors.initials: L
authors.lastname: Cunningham
authors.forename: N W
authors.initials: NW
authors.lastname: Blake
authors.forename: X
authors.initials: X
authors.lastname: OuYang
authors.forename: T A
authors.initials: TA
authors.lastname: Bayston
authors.forename: R
authors.initials: R
authors.lastname: Kandolf
authors.forename: L C
authors.initials: LC
authors.lastname: Archard
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004279:DNA, Viral; D004195:Disease Models, Animal; D029822:Enterovirus B, Human / Q000235:genetics* / Q000472:pathogenicity*; D005347:Fibroblasts / Q000166:cytology / Q000382:microbiology; D006801:Humans; D007438:Introns; D008297:Male; D008969:Molecular Sequence Data; D009205:Myocarditis / Q000382:microbiology; D010641:Phenotype; D016133:Polymerase Chain Reaction; D014162:Transfection; D014709:Vero Cells; D014774:Virulence / Q000235:genetics
chemical_list: D004279:DNA, Viral