Viral DNA load within the hearts was evaluated by quantitative PCR (qPCR) at 3, 7, and 14 dpi. From each heart, 0.1 g tissue was homogenized, and DNA was isolated using the QiaAmp DNA extraction kit (Qiagen, Hilden, Germany). The early gene 1 (E1) was amplified by using E1-specific primers (F-5′TCGCCCATCGTTTCGAGA-3′), (R-5′TCTCGTAGGTCCACTGACGGA-3′), and probe (6FAM-ACTCGAGTCGGACGCTGCATCAGAAT-TAMRA), as previously described [15 (link),37 (link),38 (link)]. Viral copy number was determined using the pCB-E1 plasmid containing the MCMV early gene 1. E1 was cloned into a pcDNA3.1 plasmid by using the fast cloning technique [39 (link),40 (link)]. The qPCR assay was conducted using an AB7000 machine (Applied Biosystems, Foster City, CA, USA) with the following temperature profiles: 50 °C for 2 min; 95 °C for 10 min; and 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and 70 °C for 1 min.
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