A virus discovery method incorporating DNase treatment and its applicationto the identification of two bovine parvovirus species

Identification of previously unrecognized viral agents in serum or plasma s amples is of great medical interest but remains a major challenge, primaril y because of abundant host DNA. The current methods, library screening or r epresentational difference analysis (RDA), are very laborious and require s elected sample sets. We have developed a simple and reproducible method for discovering viruses in single serum samples that is based on DNase treatme nt of the serum followed by restriction enzyme digestion and sequence-indep endent single primer amplification (SISPA) of the fragments, and have evalu ated its performance on known viruses. Both DNA viruses and RNA viruses at a concentration of approximate to 10(6) genome equivalents per ml were repr oducibly identified in 50 mul of serum. While evaluating the method, two pr eviously unknown parvoviruses were discovered in the bovine sera used as di luent. The near complete genome sequence of each virus was determined; thei r classification as two species (provisionally named bovine parvoviruses 2 and 3) was confirmed by phylogenetic analysis. Both viruses were found to b e frequent contaminants of commercial bovine serum. DNase treatment of seru m samples may prove to be a very useful tool for virus discovery. The DNase -SISPA method is suitable for screening of a large number of samples and al so enables rapid sequence determination of high-titer viruses.