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. 2014 Sep 1:205:53-6.
doi: 10.1016/j.jviromet.2014.03.029. Epub 2014 Apr 18.

Rapid and sequential quantitation of salivary gland-associated mouse cytomegalovirus in oral lavage

Yosuke Kamimura  1 , Lewis L Lanier  2
Affiliations

Rapid and sequential quantitation of salivary gland-associated mouse cytomegalovirus in oral lavage

Yosuke Kamimura et al. J Virol Methods. .

Abstract

Cytomegalovirus (CMV) establishes a persistent infection in the salivary glands and transmits to other hosts. Mouse cytomegalovirus (MCMV) is a well-characterized model for studying the mechanisms of host responses against CMV. The viral load in salivary glands has been measured traditionally because it has been considered to reflect the consequence of anti-virus responses by T cells and natural killer (NK) cells. However, the standard plaque assay is cumbersome and it is impossible to monitor sequentially the viral load in same host. Hence, the goal of this study was to develop a real-time quantitative PCR (qPCR)-based procedure to measure the viral load in oral lavage. This report demonstrates that the viral load in oral lavage correlates well with viral titers in the salivary glands. This method allows sequential quantitation of viral loads without sacrificing mice and provides a technique that will facilitate kinetic studies of anti-viral immunity mediated by the innate and adaptive immune systems.

Keywords: Cytomegalovirus; PCR; Protocols.

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Figures

Fig. 1
Fig. 1
Sensitivity and specificity of qPCR MCMV viral load assay. (A) Serially diluted MCMV-IE-1 plasmids were amplified. Copy number of IE-1 amplicons was plotted against cycle of threshold. (B) Template DNA was prepared from MCMV-infected salivary glands (n=4). The melting curve was graphed by plotting −dF/dT against temperature (°C). Note that peak (Tm) for IE-1 amplicons was 81° C.
Fig. 2
Fig. 2
Detection of MCMV in oral lavage, peripheral blood, and salivary gland. Each of three samples (oral lavage, peripheral blood, and salivary gland) was collected from same donor at indicated time points. The viral load in oral lavage (A) and peripheral blood (B) was quantified by qPCR. The viral load in salivary gland was quantified by qPCR (C) and plaque assay (D). The data were combined from two independent experiments. Error bars indicate SEM (n=8 for each time point).
Fig. 3
Fig. 3
Comparison of the viral loads in oral lavage and salivary gland. The viral loads at day 7, 14, and 21, obtained as described in Fig. 2, were analyzed. Correlation between the viral loads in salivary gland quantified by qPCR and plaque assay (A), oral lavage and salivary gland quantified by qPCR (B), and oral lavage quantified by qPCR and salivary gland quantified by plaque assay (C) is shown. An equation for the line determined by regression analysis and R2 values is provided in the graph.
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