A new rat cytomegalovirus (RCMV) isolated from the placenta/uterus of a house rat (Rattus rattus diardii) was found to productively infect rat embryo fibroblast (REF) cells. The virus produced typical herpesvirus-like cytopathic effects characterized by a lytic infection. The well-known herpesvirus morphology was confirmed by electron microscopy. Its slow growth in cell culture indicated that the virus is belonging to subfamily Betaherpesvirinae. Electron microscopy techniques and immunohistochemistry confirmed the presence of herpesviral inclusion bodies and virus related particles in the cytoplasm and nucleus of infected cells. Hyperimmune serum against the Maastricht strain of RCMV revealed the virus identity in neutralization test, immunoperoxidase and immunofluorescence techniques. Despite typical characteristics of CMV, the viral genome is significantly different from that of Maastricht, English, UPM/Sg and UPM/Kn strains. The dissimilarities, which have not been reported before, had been confirmed by mean of restriction endonuclease analysis. The new RCMV strain, a virus that infects placenta and uterus of rats, has been named as ALL-03.
Endothelial cells have been implicated as key cells in promoting the pathogenesis and spread of cytomegalovirus (CMV) infection. This study describes the isolation and culture of rat brain endothelial cells (RBEC) and further evaluates the infectious potential of a Malaysian rat CMV (RCMV ALL-03) in these cultured cells. Brain tissues were mechanically fragmented, exposed to enzymatic digestion, purified by gradient density centrifugation, and cultured in vitro. Morphological characteristics and expression of von Willebrand factor (factor VIII-related antigen) verified the cells were of endothelial origin. RBEC were found to be permissive to the virus by cytopathic effects with detectable plaques formed within 7 d of infection. This was confirmed by electron microscopy examination which proved the existence of the viral particles in the infected cells. The susceptibility of the virus to these target cells under the experimental conditions described in this report provides a platform for developing a cell-culture-based experimental model for studies of RCMV pathogenesis and allows stimulation of further studies on host cell responses imposed by congenital viral infections.
The present study described the kinetics of Rat cytomegalovirus (RCMV) infection in newborn rats by monitoring infectious virus and viral antigens in various organs, viral DNA in the blood (DNAemia) and antibody response. These parameters were evaluated quantitatively using double-antibody sandwich ELISA (DAS-ELISA), real-time PCR, indirect ELISA and virus infectivity assay. For the first time DAS-ELISA was used for detection of RCMV antigen directly from organ samples. The relationships between the presence of viral antigens in the infected organs and antibody levels were established by the Spearman's rank test. It was found that the virus was present in the blood, spleen, liver, lungs, and kidneys earlier than in the salivary glands. Furthermore, the early immunity of the newborn rats led to a delayed seroconversion. We suggested that the prolonged presence of the virus in salivary glands could augment the antibody response that conversely might be responsible for a reduction of viremia. This study expanded our understanding of RCMV pathogenesis leading to improved therapeutic and preventive treatment regimens particularly for the neonatal Human cytomegalovirus (HCMV) infections. Additionally, the detection procedures developed in this study such as DAS-ELISA and real-time PCR could serve as alternative techniques for rapid screening of large number of samples.