Characterization of Virus Isolates by Particle-Associated Nucleic Acid PCR

Abstract

Diagnostic virus isolation is still frequently used, particularly from respiratory tract secretions. Testing positive virus cultures for all possible viruses is time-consuming, and unexpected or unknown viruses may escape detection. Therefore, a novel random PCR approach was developed that allows sequence-independent amplification of viral nucleic acids from virus isolation-positive cultures. Selectivity for viral sequences is obtained by preferential isolation of nucleic acids that are particle associated and resistant to nucleases. Using primers with a degenerated 3 end, the isolated nucleic acids are amplified and the randomly amplified PCR products are cloned and sequenced. As proof of the concept, the PAN-PCR approach was applied to supernatants of coxsackievirus B3 and murine adenovirus type 1-infected cells. Enterovirus and adenovirus sequences were obtained, demonstrating that the random PCR approach allows detection of RNA and DNA viruses. As a first application of this PAN-PCR approach, we characterized a virus isolate from mouth-washing material of a patient with chronic fatigue syndrome and high antibody titers to coxsackievirus B2. The virus isolate had tested negative for enteroviruses and respiratory viruses (influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus, and adenovirus) by immunofluorescence and PCR. Particleassociated, nuclease-resistant RNA and DNA were prepared from the supernatant of infected cells. The DNA and the reverse-transcribed RNA were randomly amplified, and PCR products were cloned and sequenced. Of 25 sequences obtained from the DNA preparation, 24 contained herpes simplex virus type 1 (HSV-1) sequences from 14 different loci spread over the HSV-1 genome. This result was confirmed by using a standard diagnostic HSV-PCR, demonstrating that the PAN-PCR correctly identified the virus isolate. Although the identification of HSV-1 in mouth-washing material is not surprising in retrospect, it clearly demonstrates the applicability of the PAN-PCR approach. This method should be particularly useful for characterizing virus isolates that have tested negative for all expected viruses and for identifying unknown viruses.Recent identification of human metapneumovirus (13), the severe acute respiratory syndrome (SARS) coronavirus (4), and HCoNL63 coronavirus (5, 14) suggests that other viruses pathogenic to humans have either not yet been discovered or might newly emerge. This notion is further supported by the observation that in ca. 30 to 45% of patients with clinical signs of lower respiratory tract infections an etiological agent cannot be identified by laboratory diagnosis even if sensitive PCR methods are used (9, 10). In the recent past, one successful approach to identify novel viruses has been virus isolation. After exclusion of known viruses, the virus isolates were subsequently characterized by molecular biological approaches, leading to the identification of genomic fragments of the virus. In addition to the use of degenerate primers from h...

Gouléako Virus Isolated from West African Mosquitoes Constitutes a Proposed Novel Genus in the Family Bunyaviridae

“…Initial sequences were obtained by adaptor-based random amplification (Fig. 1e to g) (13,32). Two hundred eighty-one clones with inserts between 500 and 1,500 nucleotides (nt) were sequenced and compared to GenBank sequences, showing distant relationships on the amino acid (aa) level with members of the genus Phlebovirus.…”

Metagenomic Analyses of Viruses in Stool Samples from Children with Acute Flaccid Paralysis

“…PCR amplification of nucleic acids was then performed using primers consisting of the fixed portions of the random primers (38). Primers used for downstream plasmid subcloning were based on primer K (GAC CAT CTA GCG ACC TCC ACN NNN NNN N) (35) or RA01 (GCC GGA GCT CTG CAG ATA TCN NNN NNN NNN) (19). For the 10 samples submitted for 454 pyrosequencing, the following primers were used in the PCR and corresponding primers containing an additional eight N residues at the 3Ј end were used for priming during reverse transcription: 454 -A, ATC GTC GTC GTA GGC TGC TC; 454-B, GTA TCG CTG GAC ACT GGA CC; 454-C, CGC ATT GGT CGG CAC TTG GT; 454-D, CGT AGA TAA GCG GTC GGC TC; 454-E, CAT CAC ATA GGC GTC CGC TG; 454-F, CGC AGG ACC TCT GAT ACA GG; 454-G, CGC ACT CGA CTC GTA ACA GG; 454-H, CGT CCA GGC ACA ATC CAG TC; 454-I, CCG AGG TTC AAG CGA GGT TG; and 454-J, ACG GTG TGT TAC CGA CGT CC. Either amplification products were subcloned into bacterial plasmids and Sanger sequenced, or PCR products were sequenced directly using a GS-FLX 454 pyrosequencing system (Roche).…”

“…The use of sequence-independent amplification of viral nucleic acids (2,19,35,38) for viral metagenomics avoids the potential limitations of traditional methods, including the failure of virus to replicate in cell cultures, unsuccessful PCR amplification or microarray hybridization due to high-level genetic divergence from known viruses, and the failure of antibodies to known viruses to cross-react. The application of viral metagenomics may also be useful for the study of diseases with unexplained etiologies possibly involving uncharacterized viruses or combinatorial viral infections (13,21).…”

A Novel Cardiotropic Murine Adenovirus Representing a Distinct Species of Mastadenoviruses

“…In a first approach, cloning of subgenomic fragments from the unknown virus was done by particle-associated nucleic acid-PCR (PAN-PCR) as described before (28). Briefly, isolation of particles was done by centrifuging supernatant at 4,000 ϫ g for 30 min, passing it through a 0.22-m-pore sterile filter, and further purifying it by ultracentrifugation through a 30% sucrosephosphate-buffered saline (PBS) cushion for 3 h at 30,000 rpm.…”

“…Products were visualized on an agarose gel and used directly for construction of a library cloned into pCRII (Invitrogen). In both experiments, colonies were picked and tested directly by PCR as described previously (28). Different PCR products were sequenced and analyzed for homologies to viral sequences by a nucleotide-nucleotide (BLAST) and a translated BLAST search (BLASTx) at the NCBI website.…”