ATTENUATION OF A REACTIVATED CARDIOVIRULENT COXSACKIEVIRUS-B3 - THE 5'-NONTRANSLATED REGION DOES NOT CONTAIN MAJOR ATTENUATION DETERMINANTS

To investigate the molecular basis of pathogenicity of Coxsackieviruse s, a virus was reactivated by transfection from a full-length cDNA clo ne derived from cardiovirulent Coxsackievirus B3 (CVB3). The reactivat ed virus, rCVB3, was passaged serially in human dermatofibroblasts (HD F). No cytopathic effect was observed up to 12 days after inoculation with rCVB3 or early-passage virus, although disintegration of the mono layers was observed with late-passage virus (10th to 14th passages). A pproximately 10% of HDF inoculated with rCVB3 were positive for viral antigens by immunofluorescence using enterovirus- or CVB3-specific mon oclonal antibodies. These observations, together with the low infectiv ity titre of rCVB3 in HDF, suggest that HDF initially support only car rier state infection. After the 14th passage, the cardiovirulence of p assaged virus (p14V) in mice was attenuated by a factor of > 10(4). Ph enotypic changes of plaque size were also noticed in p14V: An attenuat ed variant (p14V-1) that produced larger plaques than rCVB3 in Vero ce lls has been plaque purified. The 5'-terminus of the genome of attenua nt p14V-1 was amplified by polymerase chain reaction (PCR) and its seq uence determined. Only one point mutation was found within the 5'-nont ranslated region (5'NTR) at position 690 (A to U) compared to the vira l RNA sequence obtained for rCVB3. An intertypic chimeric virus was re activated from a cDNA clone after replacing the 5'-terminal 891 nucleo tides of the wild-type genome with the corresponding region of the att enuant p14V-1. This chimeric virus, CB3/p14V-1/1, produced wild-type p laques in Vero cells and showed cardiovirulence similar to that of rCV B3 in mice. It is concluded that, unlike polioviruses, the 5'NTR of p1 4V-1 does not contain major determinants of attenuation. (C) 1993 Wile y-Liss, Inc.