Use of quantitative real-time PCR (qRT-PCR) to measure cytokine transcription and viral load in murine cytomegalovirus infection.
A quantitative real-time PCR (qRT-PCR) assay was developed to measure cytokine transcription profiles and viral load during sub-clinical and clinical infection with murine cytomegalovirus (MCMV). Primers/fluorogenic probes specific for mouse cytokines and for the immediate early gene 1 (IE1) of MCMV were used to quantitate cytokine responses and viral load in various organs of MCMV infected mice. Increased mRNA levels of TNF-alpha, INF-gamma and IL-10 were detected in the spleens, lungs and livers of clinically infected mice at 5 days post-infection. Transcription of these cytokines was 2-5-fold lower (p=0.07 for each cytokine) in the spleens and 10-100-fold lower in the lungs (p=0.03 for INFgamma, not significant for IL-10 and TNFalpha) and livers (p<0.05 for each cytokine) of sub-clinically infected mice. Clinical MCMV infection induced high levels of IL-6 in the lungs and spleens of infected animals, while no significant transcription of IL-6 was detected in any organ during sub-clinical infection (p<0.05). The timing of peak amounts of INF-gamma, IL-10 and IL-6 observed in the spleens of clinically infected mice correlated with high viral loads in these organs. Cytokine expression rose in the salivary glands later, at day 15, corresponding to the increase in salivary gland viral load. The qRT-PCR demonstrates that infection with MCMV induces an organ-specific cytokine response characterized by the production of TNF-alpha, INF-gamma, IL-6 and IL-10 which correlates with severity of the disease (sub-clinical versus clinical) and with viral load. In summary, qRT-PCR is a sensitive and accurate method to study MCMV infection and host responses to the virus.