Case reportUse of a universal virus detection assay to identify human metapneumovirus in a hematopoietic stem cell transplant recipient with pneumonia of unknown origin
Introduction
Detection of unknown viruses remains one of the most challenging problems in virology. When classical methods fail to detect viruses, development of molecular tests depends upon the availability of accurate sequence information. Generic virus detection tests allow for the simultaneous detection of multiple (if not all) virus sequences. We previously developed and optimized an improved universal virus detection method that sensitively and broadly detects DNA and RNA viruses in infected cell cultures and spiked cells with limiting amounts of nucleic acid or viruses.1 This technique requires no prior sequence information from the virus being detected, implying promise for virus discovery and detection of virus in samples for which other methods have failed.
A case of pulmonary infiltrates and fever of unknown origin occurring in a hematopoietic stem cell transplant (HSCT) recipient was investigated using a modified version of the generic virus detection assay after conventional methods failed to identify a pathogen. Preceding studies of bronchoalveolar lavage (BAL) fluids were negative for typical respiratory pathogens using standard microbiological techniques, including bacteria (including nocardia, legionella, mycoplasma, chlamydophila and mycobacteria), fungi, and pneumocystis. Routine viral studies for influenza A and B, adenovirus, parainfluenza, herpes simplex virus, cytomegalovirus, and respiratory syncytial virus were also negative. An aliquot of the previously tested BAL was obtained after it was stored at refrigerator temperatures for several days. Because the storage conditions might have damaged virus capsids (and thus made the viral nucleic acid vulnerable to nuclease digestion) we modified the previously described purification procedure1 by eliminating the use of nucleases.
This method is based on physical separation of host and virus nucleic acids followed by a degenerate oligonucleotide primer PCR (DOP-PCR) that is optimized for virus-sized genomes and permits non-specific nucleic acid amplification at a high degree of sensitivity (100–1000 copies of viral nucleic acid). The PCR uses a single primer population with a short conserved 3′ sequence (TGTGG) and is several logs more sensitive than a previously described non-specific viral PCR assay.2 The DOP-PCR was experimentally shown to amplify a range of viral genomes, including the DNA viruses SV40, HSV, VZV, EBV, and adeno-associated virus, and the RNA viruses poliovirus, influenza virus, HTLV-1, and HTLV-2.1
Section snippets
Materials and methods
Research was deemed exempt by the Offices of Human Subjects Research at NIH and performed in accordance with the ethical standards of FDA and NIH institutional review boards, and with the Helsinki Declaration of 1975, as modified in 1983. Two 200 μL BAL aliquots were centrifuged at 18,000 × g for 2 h at 4 °C. DNA and RNA were separately extracted from 100 μL of each supernatant and each pellet (AllPrep DNA/RNA kit, Qiagen, Valencia, CA). The cDNA syntheses were carried out using a first strand kit
Results
We sequenced DNA from four bands representing DOP-PCR products derived from cDNA. Five out of 63 clones from the DOP-PCR yielded sequence that matched the known sequence of the G-gene from human metapneumovirus (Fig. 1B). The largest band (∼620 bp) yielded human mitochondrial sequence, the second band (∼480 bp) yielded two human mitochondrial sequences and two HMPV sequences, the third band (∼420 bp) yielded one human sequence, and the smallest band (∼250 bp) yielded four human and three HMPV
Discussion
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 tissue.3 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
Acknowledgements
This study was supported by the intramural research programs of the National Institute of Allergy and Infectious Diseases and the National Institutes of Health Clinical Center. We gratefully acknowledge the FDA/CBER core facility for sequencing. We also thank Dr. Shuang Tang, Dr. Shasta McClenahan and Amita Patel for helpful comments.
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