Quantification of vertical transmission of Murine polyoma virus by real-time quantitative PCR

Abstract

Pathogenesis studies of viral infectionsin vivorequire sensitive assay methods. A sensitive and specific real-time quantitative PCR (RQ-PCR) assay was developed to detectMurine polyoma virus(MuPyV) DNA sequences. A quantitative assay to measure the single-copy murine wild-type p53 gene was developed to normalize viral gene copies to cell numbers. Both assays were sensitive over a seven-log dynamic range, with a reproducible detection limit of 10 copies per reaction. To determine viral loads and tissue distribution following vertical transmission of MuPyV, pregnant BALB/c mice were inoculated intraperitoneally with virus in late pregnancy. Progeny animals born to infected mothers were followed for 21 days. Viral loads in four tissues (salivary gland, kidney, liver and spleen) were highest at 7 days after birth and dropped to low levels by 14 and 21 days of age, with loads ranging from 5 to 2 million MuPyV copies per 103cells. Significant animal-to-animal variation occurred. Fourteen of 21 (67 %) progeny were virus-positive in one or more tissue samples. Transplacental transmission was observed in 6/7 (86 %) litters. Infected fetuses per positive litter ranged from 1/7 (14 %) to 5/6 (83 %) with viral loads ranging from 5 to 25 417 MuPyV copies per 1000 fetal cells. Maternal tissues and blood were frequently highly positive 2 days after inoculation, but viral loads were low by day 14. This study demonstrated the vertical transmission, including transplacental transmission, of MuPyV following acute infection of pregnant mice. It should be considered that there is a possibility that other polyomaviruses, including those in humans, may be vertically transmitted.