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

Abstract

Identification of previously unrecognized viral agents in serum or plasma samples is of great medical interest but remains a major challenge, primarily because of abundant host DNA. The current methods, library screening or representational difference analysis (RDA), are very laborious and require selected sample sets. We have developed a simple and reproducible method for discovering viruses in single serum samples that is based on DNase treatment of the serum followed by restriction enzyme digestion and sequence-independent single primer amplification (SISPA) of the fragments, and have evaluated its performance on known viruses. Both DNA viruses and RNA viruses at a concentration of approximately 10(6) genome equivalents per ml were reproducibly identified in 50 microl of serum. While evaluating the method, two previously unknown parvoviruses were discovered in the bovine sera used as diluent. The near complete genome sequence of each virus was determined; their classification as two species (provisionally named bovine parvoviruses 2 and 3) was confirmed by phylogenetic analysis. Both viruses were found to be frequent contaminants of commercial bovine serum. DNase treatment of serum 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 also enables rapid sequence determination of high-titer viruses.

Figure 1

The effect of DNase treatment and filtering of serum on detection of HBV by SISPA. An HBV-containing serum sample diluted to 10⁸ GE/ml was pretreated in different ways before DNA extraction and SISPA. Products were separated on an agarose gel. The six bands in lane 3 were verified to have HBV sequence. Lane 1, no treatment; Lane 2, DNase treatment only; Lane 3, filtering (0.22 μm) and DNase treatment; Lane 4, negative PCR control (no template); M, molecular weight marker PhiX 174 (HaeIII fragments).

Figure 2

Detection levels of different virus genomes by DNase-SISPA. HBV, bacteriophage M13, and GBV-B were diluted in 10-fold increments in bovine serum and subjected to the DNase-SISPA procedure. Products were separated on an agarose gel. The virus titer (GE/ml) is shown above each lane. M, molecular weight marker PhiX 174 (HaeIII fragments).

Figure 3

Overview of the BPV-2 (A) and BPV-3 (B) genomes as deposited in GenBank. The actual sizes and sequences of the transcripts and proteins of the indicated ORFs have not been investigated. NS, nonstructural gene.

Figure 4

The use of DNase-SISPA for obtaining the sequence of unknown viruses, illustrated by SISPA products of a serum containing 10⁸ GE/ml of BPV-3. (Left) Marker. (Center) SISPA based on digestion with Csp6.I. (Right) SISPA based on digestion with TaqI.

Figure 5

Phylogenetic trees of full-length nucleotide sequences (A) and truncated ORF2 amino acid sequences (B) of parvoviruses (Parvovirinae subfamily), including BPV-2 and BPV-3. Bootstrap values >70% are indicated.