Emergence of Canine Distemper Virus Strains With Modified Molecular Signature and Enhanced Neuronal Tropism Leading to High Mortality in Wild Carnivores

Abstract

An ongoing canine distemper epidemic was first detected in Switzerland in the spring of 2009. Compared to previous local canine distemper outbreaks, it was characterized by unusually high morbidity and mortality, rapid spread over the country, and susceptibility of several wild carnivore species. Here, the authors describe the associated pathologic changes and phylogenetic and biological features of a multiple highly virulent canine distemper virus (CDV) strain detected in and/or isolated from red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), stone (Martes foina) and pine (Martes martes) martens, from a Eurasian lynx (Lynx lynx), and a domestic dog. The main lesions included interstitial to bronchointerstitial pneumonia and meningopolioencephalitis, whereas demyelination-the classic presentation of CDV infection-was observed in few cases only. In the brain lesions, viral inclusions were mainly in the nuclei of the neurons. Some significant differences in brain and lung lesions were observed between foxes and mustelids. Swiss CDV isolates shared together with a Hungarian CDV strain detected in 2004. In vitro analysis of the hemagglutinin protein from one of the Swiss CDV strains revealed functional and structural differences from that of the reference strain A75/17, with the Swiss strain showing increased surface expression and binding efficiency to the signaling lymphocyte activation molecule (SLAM). These features might be part of a novel molecular signature, which might have contributed to an increase in virus pathogenicity, partially explaining the high morbidity and mortality, the rapid spread, and the large host spectrum observed in this outbreak.

Identification of Amino Acid Substitutions with Compensational Effects in the Attachment Protein of Canine Distemper Virus

“…The wild-type H gene (H301F) was amplified from CDV strain W10/301F (GenBank accession number JF810106) that had been isolated from the lung of a naturally CDV-infected red fox during an outbreak that occurred in Switzerland in 2009 and 2010 (19). The sequence of the second H gene was a consensus sequence obtained from four CDV strains detected during a CDV outbreak that occurred in wild carnivores in Germany in 2008 (20).…”

“…Following the identification of the five SNPs differentiating the H gene of Swiss strain W10/301F from that of the consensus sequence obtained from the German strains, we designed 5 pairs of primers (available upon request) for site-directed mutagenesis that was carried out according to the instructions provided by Stratagene (La Jolla, CA) in the QuikChange site-directed mutagenesis kit. Whereas H301F was previously cloned into an expression vector (pCI; Promega, Madison, WI) carrying a FLAG tag located at the 3= end of the gene (pCI-H301F) (19), HCons was obtained through a series of additive sitedirected mutagenesis steps carried out on pCI-H301F. The first run of mutagenesis aimed to obtain all the single mutations, i.e., N71S, V159I, M195V, M500L, and R580Q.…”

“…All the subsequent mutations were obtained by sequential additive site-directed mutagenesis until the full mutant with the N71S, V159I, M195V, M500L, and R580Q mutations (HCons) was obtained. Every H mutant was sequenced to confirm the presence of the desired mutation(s), cloned, and expanded as previously described (19). Besides H301F, intermediate mutants, and HCons, the H gene from wildtype CDV reference strain A75/17 (Hwt), which was already available as a clone in the pCI vector, was used as an additional control in the functional studies.…”

“…Besides H301F, intermediate mutants, and HCons, the H gene from wildtype CDV reference strain A75/17 (Hwt), which was already available as a clone in the pCI vector, was used as an additional control in the functional studies. Additionally, the F-protein gene from wild-type strain A75/17 (Fwt) strain previously cloned in the pCI vector was also available (19,21).…”

“…In order to partially answer these questions, we selected two H genes from two distinct but related CDV strains that were part of the same outbreak occurring in wild carnivores (red foxes [Vulpes vulpes] and Eurasian badgers [Meles meles]) and that were detected 2 years apart in Germany (2008) and Switzerland (2010), respectively (19,20). We analyzed all the missense single nucleotide polymorphisms (SNPs) that differentiated the two H genes of the two strains, i.e., a wild type and a consensus strain, respectively.…”