*2.15. Plaque Assay of MCMV in Mouse Organs*

The organ homogenates were prepared by sonication and stored in 10% skim milk at −80 ◦C. The concentrations of all homogenates were adjusted to 10% (100 mg/mL). The presence of infectious viruses in the livers, lungs, spleens, kidneys, and salivary glands were determined by titrating organ homogenates. Plaque assays were performed by virus titration assay. The values given were calculated as PFUs per mg of tissue.

#### *2.16. Quantitative Real-Time Polymerase Chain Reaction (qPCR) Analysis for DNA Copy Number*

The total DNAs were harvested in 200 μL organ homogenates (100 mg/mL) in mice tissue using a Blood and Tissue DNeasy kit. Total DNA was dissolved in 50 μL Buffer AE (Qiagen, USA).

For the generation of a standard curve, the total DNA products were amplified by PCR using MCMV *ie-1* forward and reverse primers and the total MCMV DNA as a template, under the conditions of 500 nM for each primer and 1X Hotstar Taq polymerase master mix (Qiagen, USA) in a 50 μL mixture. The thermal cycling conditions were 95 ◦C for 15 min followed by 43 cycles of 94 ◦C for 30 s, 60 ◦C for 30 s, 72 ◦C for 1 min, and 72 ◦C for 10 min. The MCMV *ie-1* DNA PCR product (100 bp, nucleotide no.:181091-181190 in MCMV genome) was isolated to create the DNA dilution standard using the QIAquick Gel Kit Protocol. For absolute quantification of the MCMV DNA copy number in the organs, a standard curve was generated by serial dilutions of MCMV *ie-1* DNA PCR products, such that 1 <sup>μ</sup>L of the standard curve template contained 5 <sup>×</sup> <sup>10</sup>1, 5 <sup>×</sup> 102, 5 <sup>×</sup> <sup>10</sup>3, 5 <sup>×</sup> 104, 5 <sup>×</sup> <sup>10</sup>5, 5 <sup>×</sup> 106 for *ie-1* DNA copies. The standard curve was obtained by plotting the average threshold cycle (Ct) values against the logarithm of the target template molecules eluted from the MCMV *ie-1* DNA PCR products, followed by a sum of least squares regression analysis. Results were expressed as DNA copies/ mg of tissue. Since DNA yield per mg of tissue differs from tissue to tissue, DNA was extracted from 20 mg

of each tissue and all extractions were done in triplicate and the average was used to determine the DNA yield/mg of each tissue type.

All qPCRs were performed with the TaqMan gene expression master mix (2× Hotstar Taq polymerase, Qiagen, USA) using the standard curve assay. Each sample was analyzed in triplicate at a 20 μL volume. For the *ie-1* DNA copy number assay, reaction mixtures contained 150 nM of each MCMV *ie-1* primer and 100 nM of the TaqMan probe. The amplification conditions were 50 ◦C for 2 min, 95 ◦C for 10 min, followed by 43 cycles of 95 ◦C for 15 s and 60 ◦C for 1 min. Values were calculated as copies per mg of tissue [28].
