Development Of Models For Pathogenesis In Fur Bearing Animals To Study The Mechanisms Of High Virulence Of Canine Distemper Virus Variant Strain

Canine distemper virus(CDV) infects a broad range of carnivores and causes a highly contagious disease with severe immunosuppression. The disease severity markedly varies in different species. The viral Hemagglutinin(H) glycoprotein plays a key role in viral virulence and host cell entry through its use of cellular receptors(SLAM and Nectin-4) and the triggering of fusion(F) protein to elicit virus-cell membranes fusion. Genetic/antigenic heterogeneity has been observed in the H gene among the various CDV strains, which appears as a good target to gather epidemiological information. Recently, several outbreaks of canine distemper(CD) occurred in breeding facilities located in China for fur animal including foxes(Vulpes vulpes), raccoon dogs(Nyctereutes procyonoides) and minks(Mustela lutreola).Compared to previous local CD outbreaks, these episodes were characterized by unusually high morbidity and mortality.To generate models suitable for the evaluation of the pathogenesis of CDV in raccoon dog, fox and mink species, three groups of CDV sero-negative animals were infected with CDV strain LN(10)1. This CDV strain belongs to the Asia-1 genotype, which is epidemiologically prevalent in the carnivores of China. CD like diseases was reproduced with different severity among the three species upon CDV infection. Raccoon dogs developed fever, severe conjunctivitis, pathological lesions, and high viral RNA loads within 15 days post infection(dpi), culminating in 100%(5/5) mortality. In infected foxes,the onset of the disease was delayed, with 40%(2/5) mortality by 21 dpi. Infected minks developed only mild clinical symptoms, minor pathological lesions, and no observed mortality. Raccoon dogs and foxes showed more severe immune suppression(lymphopenia, decreased lymphocyte proliferation, viremia and low-level virus neutralizing antibodies) than minks. We also observed a distinct pattern of cytokine mRNA transcripts at different times after infection. Decreased IFN-γ and IL-4 mRNA responses were evident in the animals with fatal disease, while increased levels of these cytokines were observed in the animals surviving infection. Increased TNF-α responses were detected in animals with mild or severe clinical signs. Based on these results, we could distinguish three different patterns of disease following experimental CDV infection, e.g. a mild form in minks, a moderate form in foxes and severe disease in raccoon dogs. The observed differences in susceptibility to CDV could reflect distinct host cytokine profiles.To determine the cause for the recent emergence of CD in Chinese fur bearing animals, a total of the sixteen strains of canine distemper virus(CDV) were isolated from foxes, minks, and raccoon dogs in four provinces of Northeastern China between the end of 2011 and 2013. Upon sequence analysis and comparison with wild-type CDV from different species in the same geographical areas, two amino acid changes, 542 isoleucine(I) to asparagines(N) and 549 tyrosine(Y) to histidine(H) were identified in key regions of the H protein of ten CDV strains from fur bearing animals. The two substitutios occurred within SLAM receptor binding sites(RBS). Interestingly, the change at residue 542 generated a novel potential N-glycosylation site. Herein, we named the viral strains containing a CDV-H gene with I542 Nand Y549H mutations, as CDV variant strains.Based on the CDV pathogenesis model developed for fur bearing animals in the present study, we charactered the CDV variant isolate(SD(14)7). It showed higher pathogenicity and increased mortility compared to the classical isolate(LN(10)1) in fur bearing animals, particularly in minks, as demonstrated through an in vivo inoculation test. These findings prompted us to hypothesize that amino acid substitutions at residues 542 and 549 might have contributed to an increase in viral pathogenicity.In order to characterize the effects of these two substitutions on viral pathogenicity, we performed site-directed mutagenesis on the CDV-H gene. The effects of these mutations onCDV-H-mSLAM interactions, and the membranefusion activity mediated by CDV-H/F, was determined in BHK-mSLAM cells. The results showed that the I542 N and Y549 H substitutions in CDV-H protein did not introduce a novel N-glycosylation site, or increase protein expression. Instead they increased mSLAM-binding activity without affecting F dependent cell fusion. Taken together, these mutations may be the reason for recent CD outbreaks in fur bearing animals in China. They could partially explain the high virulence of the CDV variant strains in mink.