RT-PCR Assay for Detection of Enterotropic Strains of Mouse Hepatitis Virus in Faecal Samples

Background: Mouse Hepatitis Virus (MHV) is one of the most important and common viral infections of laboratory mice, due to its highly contagious and subclinical nature, posing threat to the research outcomes. Periodic screening of laboratory mice for MHV is mandatory. Hence, this study was intended to develop sensitive faecal based RT-PCR assays to detect active infection of enterotropic MHV in laboratory mice. Methods: Primers targeting N-gene of MHV were selected and their sensitivity was analysed in tenfold serially diluted gene template in the presence of negative mouse faecal cDNA. Thirty-six weaned mice at the age of 6 to 18 weeks were randomly selected at different periods and the blood and faecal sample were collected for serology and RT-PCR assay respectively. RT-PCR assay in colon samples was carried out for comparison. Result: PCR assay of MHV detected as low as 4 fg of plasmid DNA. Seroprevalence of MHV is very high than the prevalence by RT-PCR assay showing its retrospective nature and also seroprevalence includes both enterotropic and polytropic strain. RT-PCR results in faecal samples are analogous with that of the colon samples, showing the reliability of antemortem testing of mice for enterotropic strain of MHV.

Non-Invasive Fecal Based PCR Assays for Detection of Mouse Parvo Virus and Minute Virus of Mice in Laboratory Mice

Background: Murine parvoviruses are a few of the most common pathogens of laboratory mice posing potential threats to mice colonies and experiments conducted in mice. We initiated this study to develop the fecal based PCR assays as an alternative to serology and to check its feasibility to detect the infections in mice caused by Mouse Parvo Virus (MPV) and Minute Virus of mice (MVM).Methods: Primers targeting the VP2 gene of MPV and MVM were selected and their sensitivity was analysed in tenfold serially diluted gene template in the presence of negative mouse fecal DNA. Selected thirty-seven mice at the age of 6 to 18 weeks randomly and collected blood samples for serology and fecal samples for PCR assays. Result: PCR assays of MPV and MVM detected as low as 0.4 fg of the target plasmid DNA. PCR assays in fecal samples of mice detected the presence of natural infections of MPV and MVM and their respective prevalence was 24% and 30%. Diagnostic sensitivity of MPV and MVM were 74% and 76% respectively. Our findings indicate that the fecal-PCR assay may be a useful, non-invasive and sensitive diagnostic tool in the ante-mortem detection of MPV and MVM in the health monitoring program.

The influence of whole-arm trisomy on gene expression in Drosophila.

The biochemical consequences of extensive aneuploidy in Drosophila have been examined by measuring the levels of specific proteins in larvae trisomic for entire chromosome arms. By far the most common effect is a reduction in gene product levels (per gene template) by one-third from the diploid quantity, consistent with the model that concentration-dependent repressors of these loci reside on the duplicated chromosome arms. Most loci appear sensitive to such repression in one or more of the trisomies examined, suggesting that such regulatory loci might be quite common. Repression of gene-product levels in trisomies may significantly contribute to their inviability. Few loci are activated in trisomies implying that most factors necessary for gene expression are in excess. While autosomal trisomies can repress the expression of both X-linked and autosomal loci, X-chromosomal trisomies have little effect on most autosomal genes. A family of genes coding for larval serum proteins do not respond similarly in trisomies, suggesting that regulation operates on a process which is not common to their coordinate regulation. Finally, Adh genes transposed to new chromosomal positions maintain their ability to be repressed in 3L trisomies suggesting that this response to regulation involves a closely linked cis-acting regulatory element.

Packing of a specific gene into higher order structures following repression of RNA synthesis.

Transcription of the Balbiani ring (BR) genes of the dipteran Chironomus tentans was inhibited by teh nucleoside analogue DRB (5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole). The BR genes were emptied of RNA polymerases and the subsequent packing of the genes was monitored by transmission electron microscopy. The thin chromatin axis of the transcriptionally active genes condensed into a thick (20-25 nm) chromatin fiber, which was recorded as a linear structure, an open loop or a supercoiled loop. The compacted genes were finally packed into dense clumps of chromatin. It was proposed that upon repression of RNA synthesis the BR gene template attains the following consecutive stages with increasing compaction: transcription loop----linear thick fiber----open thick fiber loop----supercoiled thick fiber loop----dense chromatin. Within the chromatin blocks structures that resembled the supercoiled loops were discerned, suggesting that the final packing of the template might be accomplished by a close alignment of supercoiled loops.