Reinfection Of Murine Cytomegalovirus Aggravating The Diseases Of The Mice

ObjectivesTo observe the influence of reinfection of murine cytomegalovirus (MCMV) on the pathological changes and virus loads of the organs of the disseminated infection model and explore its immunological mechanisms preliminarily. Methods1. The establishment of reinfection models and the collection of specimens:Ninety-six four-week-old female BALB/c mice were randomly divided into four groups:normal control group (abbreviated as M, DMEM/H+DMEM/H), once infection group (abbreviated as S, DMEM/H+MCMV Smith), reinfection with the same strain group (abbreviated as SS, MCMV Smith+MCMV Smith), reinfection with different strains group (abbreviated as SR, MCMV Smith+MCMV RM461). The procedure in detail was as following:the mice of each group were inoculated intraperitoneally with DMEM/H or MCMV Smith for establishing the disseminated infected model, the second infection was carried out with DMEM/H or MCMV Smith or MCMV RM461on the fifth day after the first injection. The salivary glands, lungs, hearts, livers, kidneys and spleens were collected aseptically on3d,7d,14d,28d post the second infection for testing.2. Assessing the degree of histopathology:Taking each paraffin section of different specimen for routine hematoxylin-eosin staining and assessing the degree of histopathology according to corresponding tissue semi-quantitation criteria.3. Measuring the infection virus titers of multiple visceral organs:The infectious virus titers of various organs were detected by standard plaque assay. Calculating the median virus loads in accordance with the following formula: Infectious virus titers (PFU/g)=mean plaque forming units×dilution times/inoculation amount (g)4. Detecting the percentage of Thl and Th2by flow cytometry:Collection each group spleens whose cells were isolated sterilely.The spleen cells were co-stimulated by inactivated MCMV and PMA/Ionomycin in vitro for6h. Detecting the percentages of CD4IFN-y+cells and CD4+IL-4+cells in spleen cells by flow cytometry at different time points.Results1. Apparent inflammatory changes of organ of infected mice were observed at3d post infection. At7d, all organs except for kidney which experienced the most serious damage at14d suffered from the most severe inflammatory response. The histological changes slowly mitigated over time. In general, degree of inflammatory changes of repeated infection mice with the same strain and different strains was more severe than that of once infection group.The following were the histopathological features of various organs. The histological changes of lung were composed of widened alveolar septa, intra-alveolar edema and hemorrhage, neutrophil infiltration, the shedding of epithelial cells of terminal bronchioles, capillary congestion, inclusion bodies within the terminal bronchioles epithelial cells. The histological changes of liver consist of ballooning degeneration, acidophilic bodies, piecemeal necrosis, portal inflammation cells, viral inclusions in hepatocytes. The histological changes of kidney included cells increased in glomerular, glomerular swelling, narrowed Bowman’s capsule, renal interstitial a small amount of inflammatory cells infiltration, necrosis and viral inclusions of renal tubular epithelial cell. The heart were myocardial cell edema, lysis and necrosis, neutrophil infiltration of myocardial interstitial, inclusion bodies. Histological evaluation of spleen wae based on proliferation of secondary follicles, phagocytic cells infiltration, pathological hyperplasia of white pulp and necrosis. Salivary glands were found organized infiltrates foci and destruction of the organizational structure.2.3days post infection, infectious virus was detected in mainly disseminated visceral organs. The virus loads of salivary glands were the highest at the same time point among all organs, followed by lungs, which reached the peak at7d post infection and then gradually decreased. The virus loads of mice’s organs of SS and SR were higher than that of organs of once infection mice model.Only in the early acute infection (3d), the virus titers of salivary gland in the mice of SR is mild higher compared to SS. The difference was statistically significant (P<0.05). Infectious virus in the kidney of once infected mice was just detected at7d post-infection, in addition, was significantly lower than that of secondary infections. Infectious virus loads of kidney of the secondary infection mice were gradually increased over time, reached the peak at14days. Liver tissue detected low levels of virus titers at3d, no significant differences between each infection group. Infectious virus of spleen was only occasionally detected in the secondary infection at3d. Infectious virus of heart was not detected at any time point. Infectious virus can not detect in various organs of infected mice at28days after infection.3. Percentages of Thl (CD4+IFN-γ+) and Th2(CD4+IL-4+) cells in spleen lymphocytes:at the3d and7d, percentages of Thl (CD4+IFN-γ+) in spleen lymphocytes stimulated with appropriate induction method were significantly higher in MCMV-infected mice than that in control mice. The level of Thl of secondary infection mice with the same strain and different strains were statistically significant compared with those in once infected mice (P<0.05). The percentages of Thl in infected mice decreased rapidly and were roughly analogous with mock-infected mice at14d and28d. The proportion of Th2(CD4+IL-4+) in infected mice were relatively low at3d,7d and14d. Until28d, the rate of Th2in repeated infected mice with different strains was markedly increased over those in control mice and other infected ones.Conclusions1. Repeated infection animal model of murine cytomegalovirus can be successfully established in immunocompetent BALB/c mice by sequential intraperitoneal injection of Smith and RM461strains.2. In the acute phase of infection, MCMV repeated infections not only increase the amount of infectious virus of organs but also aggravate the histopathological changes, in the early of acute infection, repeated infection with different MCMV strains can lead to higher viral loads compared with that of repeated infection with same MCMV strain.3. The adverse effect of repeated infection on the host may be closely related to high level Th1(CD4+IFN-γ+) leading to inflammatory damage in the early and abnormally increased Th2(CD4+IL-4+) level causing immunosuppression.