The detection of HMPV sequences in the clinical sample does not prove conclusively that this virus was the sole cause of the pneumonia; reports indicate that viral sequences may persist for long periods of time in murine lung tissue3
. Detected HMPV sequences could represent either viable or non-viable virus particles. However, we detected signals in supernatant as well as in pellets, implying that both virus capsids and free viral nucleic acid were present. The presence of readily-detectable HMPV sequences in BAL fluid using the generic DOP assay as well as two independent PCRs for different regions of the genome imply a high probability of very recent or ongoing HMPV infection in this individual.
We identified different but overlapping DOP-PCR products in the HMPV G-gene, and did not detect other HMPV genes. The HMPV sequence is not biased in a way that would support preferential DOP-PCR amplification of the G-gene; thus, it seems more likely that the sample contained more RNA from the G-gene than from other genes. This may have been related to the refrigerator storage of the sample for five days prior to analysis—conditions that may have caused a significant amount of RNA degradation.
Although identification of HMPV in clinical samples is usually not a major diagnostic challenge, genetic heterogeneity among HMPV strains can lead to false negative RT-PCR assays, leading some investigators to seek further improvement in these assays 4,5
. Our findings imply that the DOP-PCR based universal virus assay is useful for finding viruses in patient samples where other methods have failed to identify a pathogen. This can be particularly important when sample quantity is limited, making it difficult to perform many additional tests. A single generic assay that is capable of detecting all DNA and RNA viral genomes, paired with an appropriate purification technique, thus has significant advantages. The major strength of the DOP assay is its ability to amplify viral sequences without prior knowledge of those sequences, increasing the likelihood of finding mutated or novel viruses or viruses with substantial sequence heterogeneity, including as-yet undiscovered, unexpected, or non-human viruses. This is of major importance because most (about 75%) emerging infectious diseases and 80% of select agents are of zoonotic origin6
Unlike many subtraction-based generic approaches, the non-subtractive DOP assay does not require a non-infected but otherwise genetically identical control. As compared with amplification methodologies using primers based on consensus sequences shared among related viruses7,8,9
(e.g., those that were used to discover new herpes viruses10
and to identify West Nile virus as the etiologic agent of human encephalitis in the New York outbreak11
), the DOP assay does not require the performance of multiple assays directed at different virus families, which may be impractical when sample is limited. The DOP assay also does not require accurate sequence information for each virus included in the screening12
in order to establish the assay, as do consensus primer and microarray-based techniques. Other recently described alternatives to classical virological methods used to identify a new arenavirus13
, a virus linked to colony collapse disorder of bees14
and a polyomavirus playing a role in human Merkel cell carcinoma15
use a combination of degenerate PCR and high-throughput sequencing, an approach that can be expensive and is not readily available. Enrichment of viral nucleic acids in the present approach permits identification of viral sequences even by traditional sequencing, making this approach accessible to laboratories with access to PCR, cloning, and standard sequencing. The DOP-PCR assay requires three days from sample receipt to sequence.
The DOP-PCR approach is most promising in situations where sample is limited, suspicion of viral disease is high, and standard viral diagnostic tests are negative. Viruses with substantial genetic heterogeneity and those that are not initially suspected based on the clinical presentation would be particularly amenable to detection using this method.