Adaptation Mechanism Of Canine Distemper Virus To Human SLAM Receptor And Its Replication In Human SLAM Transgenic Mice

Canine distemper(CD)is a kind of infectious disease caused by Canine distemper virus(CDV).Signalling lymphocyte activation molecular(SLAM)is the main receptor for CDV and other members of the Morbillivirus genus to infect the host,which plays an important role in the interspecific transmission of virus and the pathogenicity of the host.CDV H protein plays an important role in recognizing SLAM receptors,mediating viral invasion and regulating viral force.In recent years,with the change of ecological environment and the mutation of virus,the natural host of CDV has expanded from canines,cats and raccoons to macaques of primates.Therefore,the scientific issue of whether CDV can break the species barrier and spread to humans through the human SLAM receptor deserves our attention.Based on the previously cloned CDV human SLAM(hSLAM)receptor adaptor strain5804Pe/H-VHS,we confirmed that the mutation of CDV-H protein D540G amino acid can mediate the effective infection of the virus to Vero cells expressing hSLAM receptor(Vero-hSLAM).In this study,we further studied the molecular mechanism of 5804Pe/H-VHS strain adapting to hSLAM receptor through hSLAM receptor block test,H/hSLAM interaction three-dimensional model,western blotting,H/hSLAM immunoprecipitation,human PBMC infection in vitro and serum antibody cross neutralization protection experiments.The results showed that:(1)D540G mutation did not change the expression of CDV H protein on the cell membrane(p>0.05);(2)Anti-hSLAM monoclonal antibody could significantly block CDV infection of Vero-hSLAM cells(p<0.05);(3)CDV H protein D540G may interforce with hSLAM 38(Q)and 41(S)amino acids,respectively;(4)D540G mutation did not significantly enhance the interaction between H/hSLAM proteins(p>0.05);(5)5804Pe/H-VHS strain could effectively infect human PBMC in vitro(viral load up to 10^4.50 TCID₅₀/m L)(p<0.01),and significantly inhibited the proliferation activity of PBMC(p<0.05);(6)CDV vaccine immunized serum could produce cross immunity against 5804Pe/H-VHS strain(p<0.01).To further clarify the pathogenicity in vivo,hSLAM^(+/+)/STAT1^(-/-)transgenic mice were nasally infected with 5804Pe/H-VHS and 5804Pe/H strains(10^6.0TCID₅₀),respectively.The mice were anesthetized and examined 7 days after infection.RT-PCR and Vero-hSLAM cells were used to detect viral nucleic acid,viral load and viremia in tissues,respectively.Laser confocal technique was used to study the co-localization of the virus with hSLAM receptors in tissues.The above results showed that(1)compared with the 5804Pe/H-VHS infection group,the spleen in the 5804Pe/H-VHS infection group was significantly enlarged and increased in weight(p<0.01);(2)In the 5804Pe/H-VHS infection group,CDV RNA was strongly positive in spleen and lung,weakly positive in lymph node,thymus and kidney,and negative in brain.In the5804Pe/H infection group,CDV RNA was negative in all tissues except thymus;(3)Viral load of5804Pe/H-VHS infection group was significantly higher than that of 5804Pe/H-VHS infection group in immune cells and organs(PBMC,spleen,lymph node,thymus)and epithelial group(p<0.01);(4)Virus co-localization with hSLAM occurred in spleen,lung and lymph nodes of5804Pe/H-VHS infection group,while no e GFP fluorescence was observed in 5804Pe/H infection group.In conclusion,CDV 5804Pe/H-VHS strain can effectively infect human PBMC in vitro and inhibit its cell proliferation activity.The virus replicates in immune organs and epithelial tissues of transgenic mice infected with hSLAM,leading to the production of viremia.In this study,the molecular mechanism of CDV adaptation to hSLAM receptor was preliminarily revealed,and the pathogenicity model of CDV infection in mice was established,which provided a research basis for further evaluation of the risk of CDV infection in humans.