Establishment Of A Model For Coxsackievirus Infection In Macaques And Study On Its Pathogenicity

In our previous study, He et al. found that a female Sichuan snub-nosed monkeydied from viral myocarditis caused by CVB3. This CVB3strain was derived from thisdead Sichuan snub-nosed monkey and was named SSM-CVB3. Because CVB3infection were considered as a significant risk factor that could cause serious diseasein susceptible animals, primates, and even in humans, and fatalities have frequentlybeen associated with myocarditis or DCM. Thus, an experimental model usingmacaques infected with SSM-CVB3was developed for studying the pathogenicityand pathogenesis of SSM-CVB3. Such study may have clinical importance whenmonitoring the progress of CVB3infection, including evaluation of efficacy oftreatment in this infection.First, BALB/c mice were orally or intraperitoneally inoculated with SSM-CVB3,then observed and compared the clinical signs and pathologic changes. The resultsshowed that the mice after i.p. infection fell ill earlier, and the mortality (84%,25/30)was higher compared with the oral group (47%,14/30); the virus titres were highestin sera in i.p. infected mice at day5and were highest in sera in orally infected mice atday7; the levels of RBC, WBC in infected mice had the significant decrease, but thelevels of RBC, WBC in i.p. infected mice decreased more significantly compared withthe orally infected mice; the levels of HGB also had a decrease, but not significant;the percentage of LYM and MON obviously increased, and the percentage of NEUTin infected mice decreased a lot; the levels of CK, CK-MB, AST, ALT, BUN, CRE,AMY were higher in sera in infected mice compared with the control mice, but thelevels of GLU in sera were lower; different degrees of pathological changes could bedefinitely observed in various organs, except for obvious cardic damage, the hepaticand renal damage were more obvious; the pathological changes in small intestine inorally infected mice were more severe compared with the i.p. infected mice; themRNA expression of SSM-CVB3in the heart, liver, kidney in mice were higher thanother tissues during the course of infection (P<0.05). An experimental model usingmice infected with SSM-CVB3was established successfully. Based on the results from the study of mice, an experimental model wasestablished. Each macaque was inoculated, intraperitoneally (ip), with5ml (5×106TCID50/ml) of SSM-CVB3virus. During the course of infection, the clinicalsymptoms of macaques were recorded; hematological, biochemical andhistopathological evaluations were completed; viral titers and neutralization titers(NT-titers) in sera were tested; and the mRNA of SSM-CVB3were determined. AfterSSM-CVB3infection, the macaques showed a lack of activity, a poor appetite, ahigher body temperature, and severe diarrhea. The macaques also developedhematuria and albuminuria at4to10days post-inoculation. Virus titers were higher at6to10days post-inoculation, and NT-titers reached plateaus at8to14dayspost-inoculation. The infected macaques developed serious anemia with decreasedRBC and WBC, but the percentages of LYM were increased. The levels of CK,CK-MB, AST and ALT in the sera were84~169U/L,87.6~271.1U/L,43~87U/Land43~82U/L, respectively, and all of those were higher than normal. Histologicalanalysis showed obvious cardiac, hepatic and renal damage in the infected macaquesand the mRNA of SSM-CVB3were determined in the heart, liver, spleen, lung,kidney and brain by RT-PCR analysis. These results indicated that an experimentalmodel using nacaques infected with SSM-CVB3was established successfully, andsuggested that SSM-CVB3caused damage to a variety of tissues, with more severedamage observed in cardiac, hepatic and renal structures in macaques.To better evaluate the distribution of SSM-CVB3, coxsackievirus and adenovirusreceptor (CAR) and decay accelerating factor (DAF) during the course of infection,the immunofluorescence or immunohistochemical analysis was first conducted, thenthe mRNA levels of SSM-CVB3, CAR and DAF were determined by quantitativeRT-PCR analysis. Moreover, using CAR antibody and DAF antibody effected on Verocells,then testing the function of them to protect cells during CVB3infection,we canindirectly found the function of CAR and DAF during CVB3invading into cells. Theresults showed that the antigens of SSM-CVB3, CAR and DAF could be detected inthe various tissues including the heart, liver, spleen, lung, kidney in infectedmacaques at15days post-inoculation or30days post-inoculation, and that much morecells staining positive for antigens in these selected tissues from infected macaqueswere found at15days post-inoculation than those at30days post-inoculation; asignificant positive correlation was found between the mRNA content of SSM-CVB3 and the mRNA content of CAR or DAF during CVB3infection, and the mRNAcontents of SSM-CVB3, CAR and DAF in the heart, liver and kidneys of infectedmacaques were higher (P<0.05). After using CAR antibody and DAF antibodyeffected on Vero cells, the chance of SSM-CVB3attacking on the cells were reducedand the cell survival were improved. These results indicated that the cellular receptorsare essential for virus entry and infection, and SSM-CVB3directly caused damage oftarget cells and target tissues with the participation of CAR, DAF.To better evaluate the distribution of SSM-CVB3, cytokine and chemokineduring the course of infection, SSM-CVB3in serum and organs was measured usingby the Spearman-Karber's50%tissue culture infectious dose (TCID50) method orquantitative RT-PCR analysis, and the levels of10cytotines and chemokines in serumand organs were measured by indirect-ELISA. The results showed that the cytokinesand chemokines including IL-1β, IL-2, IL-6, IL-12p40, IL-17α, IFN-γ, TNF-α,MCP-1, MIP-1β, except for IL-10, were detected on all days in serum, and peaked onday6to10, consistant with virus levels; in addition, higher levels of virus titers andrelative viral mRNA were associated with higher levels of cytokines and chemokinesin selected tissues including heart, liver, spleen, lung, kidney and brain in infectedmacaques during the course of infection. These results indicated that the immunesystem of macaques was activated by SSM-CVB3infection, resulted in the changes ofa large number of the immune-related cytokines and chemokines, speeded up theoccurrence and development of inflammation.