Year: 2010
Type: article
Abstract: Background: Detection and identification of known and unknown viruses can be challenging, especially for those with substantial genetic divergence, e.g. caliciviruses. To address this issue, we developed a universal virus detection assay combining virus capsid enrichment with a generic PCR. We analyzed stool and cell culture samples with our degenerate oligonucleotide primer (DOP) PCR. Caliciviruses are small, non-enveloped (+)ssRNA viruses. The family Caliciviridae is comprised of four genera, Norovirus, Sapovirus, Vesivirus and Lagovirus. Vesiviruses and lagoviruses infect a wide range of animal hosts; noroviruses and sapoviruses are recognized as human pathogens, causing acute gastroenteritis. Human noroviruses and sapoviruses cannot be cultured which impedes the research of these viruses considerably. Methods: The first step of the assay is the physical and biochemical purification by targeted digestion of contaminating host nucleic acids followed by DOP PCR. The primer population is optimized for the detection of virus-sized genomes. Products can be identified by cloning and sequencing or by high throughput sequencing. Various DNA viruses (including HSV, VZV, SV40, AAV, EBV, parvoviruses, and hepatitis B) and RNA viruses (including HTLV-1, HTLV-2, several animal retroviruses, poliovirus, hepatitis A, human corona virus, human metapneumovirus, and influenza virus) were detected in previous studies in cell cultures and clinical samples. Results: Human norovirus (stool) and feline calicivirus (vesivirus, cell culture) were identified with our universal assay. Approximately 35% of the virus genomes were obtained with a single assay. We also identified enterovirus sequences from an asymptomatic individual. Conclusion: The findings presented here demonstrate the ability of the DOP-PCR assay to not only detect and identify viruses in clinical and cell culture samples but to also provide a large portion of the sequence information with a single assay. Human diarrheal diseases cause a significant disease burden; an estimated 1.8 million deaths in children under the age of five are caused by gastroenteritis annually. Gastroenteritis is the third leading cause of death due to infection, yet, about 40% of cases are of unknown etiology. Universal detection of viruses with an assay as it was described here could lead to the detection of known yet unsuspected viruses or the discovery of novel viruses. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,900
OpenAlex ID: W2166356332