RAPID SUBGENUS IDENTIFICATION OF HUMAN ADENOVIRUS ISOLATES BY A GENERAL PCR

In most clinical situations involving adenovirus infection, subgenus ( subgroup) identification of an adenovirus isolate is as informative as a finer identification by serotype. A PCR method which allows the ide ntification of human adenovirus isolates as members of subgenera A, B: 1, B:2, C, D, E, or F is described. It is based on a simple (nonnested ) PCR using primers which bind to regions immediately flanking the VA RNA-encoding regions of human adenovirus genomes. The PCR allows ampli fication of DNA from all 49 human adenovirus prototype strains so far described. Since there are differences in the lengths of the VA RNA-en coding regions in adenoviruses of different subgenera, it is possible to differentiate some subgenera according to the size of the PCR produ ct determined by electrophoresis. This forms the basis of an initial b road categorization of isolates as belonging to either (i) subgenus B: 1, C, D, or E or (ii) subgenus A, B:2, or F, Subgenus identification i s completed by a one-step restriction enzyme digestion and gel electro phoresis, The method was assessed by blind subgenus identification of 200 miscellaneous primate adenovirus isolates prepared by the referenc e laboratory at Bilthoven, The Netherlands. Identification at the subg enus level by PCR correlated 91.5% with the results of serotyping. A f urther 5.5% of isolates were correctly identified as belonging to one of two specified subgenera. Six of the 200 identifications (3%) were u nsuccessful for various reasons, including weak PCR products, intermed iate strains, and mistaken primate host. The method should serve as a rapid means of confirming adenovirus cytopathic effects in laboratorie s performing virus culture, with simultaneous subgenus identification of the isolate. It will also have relevance as an aid to conventional serotyping for epidemiological purposes, since for all adenoviruses ex cept those belonging to subgenus D, neutralization tests need only inv olve a maximum of four type-specific antisera.