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Detection of newly recognized rodent parvoviruses by PCR
D G Besselsen1, C L Besch-Williford, D J Pintel
1Department of Veterinary Pathology, University of Missouri, Columbia 65211, USA.
Abstract:
Several autonomous parvovirus isolates distinct from the prototypic rodent parvoviruses have recently been identified. These include variants of a mouse orphan parvovirus (MOPV) and a hamster isolate designated hamster orphan parvovirus (HOPV). In this study, a PCR primer set specific for these newly identified rodent parvoviruses was designed on the basis of DNA sequence comparisons of these isolates with other autonomous parvoviruses. The specificity of the primer set was determined by testing viral preparations of seven different parvoviruses and eight other viruses known to infect rodents. The PCR assay amplified the expected 260-bp product only in the presence of DNA from MOPV, HOPV, or LuIII a parvovirus of unknown species origin. The assay was able to detect as little as 10 pg of MOPV viral DNA or 1 pg of HOPV viral DNA, and it was able to detect MOPV in tissues from naturally infected mice and HOPV in tissues from experimentally infected hamsters. In contrast, the 260-bp product was not amplified from tissues of MOPV-negative mice or mock-infected hamsters. Our findings indicate that this PCR assay provides a rapid, specific, and sensitive method for the detection of MOPV in mice, HOPV in hamsters, and MOPV and HOPV in cell culture systems and that it may also be useful for the detection of LuIII contamination of cell culture systems.
Insights
A new PCR assay effectively detects novel rodent parvoviruses, mouse orphan parvovirus (MOPV) and hamster orphan parvovirus (HOPV). This sensitive method aids in identifying these viruses in infected animals and cell cultures.
Area of Science:
- Virology
- Molecular Biology
Background:
- Recent identification of novel autonomous parvovirus isolates, including mouse orphan parvovirus (MOPV) and hamster orphan parvovirus (HOPV).
- Need for specific diagnostic tools to differentiate these new rodent parvoviruses from prototypic strains.
Purpose of the Study:
- To design and validate a Polymerase Chain Reaction (PCR) primer set for the specific detection of MOPV and HOPV.
- To assess the sensitivity and specificity of the developed PCR assay.
Main Methods:
- DNA sequence comparison of novel parvovirus isolates with known autonomous parvoviruses.
- Design of a specific PCR primer set based on sequence data.
- Testing primer set specificity against seven different parvoviruses and eight other rodent viruses.
- Determination of assay sensitivity using known quantities of MOPV and HOPV DNA.
- Application of the PCR assay to detect viruses in infected animal tissues and cell cultures.
Main Results:
- The PCR assay specifically amplified a 260-bp product from MOPV, HOPV, and LuIII parvovirus.
- The assay demonstrated high sensitivity, detecting as little as 10 pg of MOPV DNA or 1 pg of HOPV DNA.
- MOPV was detected in naturally infected mice tissues, and HOPV in experimentally infected hamster tissues.
- No amplification was observed in tissues from MOPV-negative mice or mock-infected hamsters.
Conclusions:
- The developed PCR assay is a rapid, specific, and sensitive method for detecting MOPV in mice and HOPV in hamsters.
- The assay is effective for identifying these viruses in cell culture systems.
- The PCR assay may also be valuable for detecting LuIII contamination in cell cultures.