Canine Distemper Outbreaks in Wild Carnivores in Northern Italy.

Canine distemper (CD) is a fatal, highly contagious disease of wild and domestic carnivores. In the Alpine territory, several outbreaks have occurred in the past few decades within wild populations. This study investigated the presence of canine distemper virus (CDV) infections in wild carnivores in Lombardy, relating to the different circulating genotypes. From 2018 to 2020, foxes, badgers, and martens collected during passive surveillance were subjected to necropsy and histological examination, showing classical signs and microscopic lesions related to CDV. Pools of viscera from each animal were analysed by molecular methods and immunoelectron microscopy. Total prevalences of 39.7%, 52.6%, and 14.3% were recorded in foxes, badgers, and stone martens, respectively. A phylogenetic analysis showed that the sequences obtained belonged to the European 1 lineage and were divided into two different clades (a and b) according to the geographical conformation of alpine valleys included in the study. Clade a was related to the European outbreaks originating from Germany in 2006-2010, while clade b was closely related to the CDV sequences originating from northeastern Italy during the 2011-2018 epidemic wave. Our results suggest that CDV is currently well adapted to wild carnivores, mostly circulating with subclinical manifestations and without severe impact on the dynamics of these populations.

Genetic Adaptations, Biases, and Evolutionary Analysis of Canine Distemper Virus Asia-4 Lineage in a Fatal Outbreak of Wild-Caught Civets in Thailand.

Canine morbillivirus (CDV) is a serious pathogen that can cause fatal systemic disease in a wide range of domestic and wildlife carnivores. Outbreaks of CDV in wildlife species lead to questions regarding the dispersal of the CDV origin. In the present study, we identified a fatal CDV outbreak in caged wild-caught civets in Thailand. Full-length genetic analysis revealed that CDV from the Asia-4 lineage served as the likely causative agent, which was supported by the viral localization in tissues. Evolutionary analysis based on the CDV hemagglutinin (H) gene revealed that the present civet CDV has co-evolved with CDV strains in dogs in Thailand since about 2014. The codon usage pattern of the CDV H gene revealed that the CDV genome has a selective bias of an A/U-ended codon preference. Furthermore, the codon usage pattern of the CDV Asia-4 strain from potential hosts revealed that the usage pattern was related more to the codon usage of civets than of dogs. This finding may indicate the possibility that the discovered CDV had initially adapted its virulence to infect civets. Therefore, the CDV Asia-4 strain might pose a potential risk to civets. Further epidemiological, evolutionary, and codon usage pattern analyses of other CDV-susceptible hosts are required.

The fusion protein signal-peptide-coding region of canine distemper virus: a useful tool for phylogenetic reconstruction and lineage identification.

Canine distemper virus (CDV; Paramyxoviridae, Morbillivirus) is the etiologic agent of a multisystemic infectious disease affecting all terrestrial carnivore families with high incidence and mortality in domestic dogs. Sequence analysis of the hemagglutinin (H) gene has been widely employed to characterize field strains, permitting the identification of nine CDV lineages worldwide. Recently, it has been established that the sequences of the fusion protein signal-peptide (Fsp) coding region are extremely variable, suggesting that analysis of its sequence might be useful for strain characterization studies. However, the divergence of Fsp sequences among worldwide strains and its phylogenetic resolution has not yet been evaluated. We constructed datasets containing the Fsp-coding region and H gene sequences of the same strains belonging to eight CDV lineages. Both datasets were used to evaluate their phylogenetic resolution. The phylogenetic analysis revealed that both datasets clustered the same strains into eight different branches, corresponding to CDV lineages. The inter-lineage amino acid divergence was fourfold greater for the Fsp peptide than for the H protein. The likelihood mapping revealed that both datasets display strong phylogenetic signals in the region of well-resolved topologies. These features indicate that Fsp-coding region sequence analysis is suitable for evolutionary studies as it allows for straightforward identification of CDV lineages.

Deduced sequences of the membrane fusion and attachment proteins of canine distemper viruses isolated from dogs and wild animals in Korea.

Canine distemper virus (CDV) causes highly contagious respiratory, gastrointestinal, and neurological diseases in wild and domestic animal species. Despite a broad vaccination campaign, the disease is still a serious problem worldwide. In this study, six field CDV strains were isolated from three dogs, two raccoon dogs, and one badger in Korea. The full sequence of the genes encoding fusion (F) and hemagglutinin (H) proteins were compared with those of other CDVs including field and vaccine strains. The phylogenetic analysis for the F and H genes indicated that the two CDV strains isolated from dogs were most closely related to Chinese strains in the Asia-1 genotype. Another four strains were closely related to Japanese strains in the Asia-2 genotype. The six currently isolated strains shared 90.2-92.1% and 88.2-91.8% identities with eight commercial vaccine strains in their nucleotide and amino acid sequences of the F protein, respectively. They also showed 90.1-91.4% and 87.8-90.7% identities with the same vaccine strains in their nucleotide and deduced amino acid sequences of the H protein, respectively. Different N-linked glycosylation sites were identified in the F and H genes of the six isolates from the prototype vaccine strain Onderstepoort. Collectively, these results demonstrate that at least two different CDV genotypes currently exist in Korea. The considerable genetic differences between the vaccine strains and wild-type isolates would be a major factor of the incomplete protection of dogs from CDV infections.

Development of a combined canine distemper virus specific RT-PCR protocol for the differentiation of infected and vaccinated animals (DIVA) and genetic characterization of the hemagglutinin gene of seven Chinese strains demonstrated in dogs.

A combined reverse-transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of the canine distemper virus (CDV). A pair of primers (P1/P2) was used to detect both CDV wild-type strains and vaccines. Another pair (P3/P4) was used to detect only CDV wild-type strains. A 335bp fragment was amplified from the genomic RNA of the vaccine and wild-type strains. A 555bp fragment was amplified specifically from the genomic RNA of the wild-type strains. No amplification was achieved for the uninfected cells, cells infected with canine parvovirus, canine coronavirus, or canine adenovirus. The combined RT-PCR method detected effectively and differentiated the CDV wild-type and vaccine strains by two separate RT-PCRs. The method can be used for clinical detection and epidemiological surveillance. The phylogenetic analysis of the hemagglutinin gene of the local wild-type CDV strains revealed that the seven local isolates all belonged to the Asia-1 lineage, and were clustered closely with one another at the same location. These results suggested that the CDV genotype Asia-1 is circulating currently in domestic dogs in China.

A multiplex reverse transcription-nested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus.

A multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR) method was developed for the detection and differentiation of wild-type and vaccine strains of canine distemper virus (CDV). A pair of primers (P1 and P4) specific for CDV corresponding to the highly conserved region of the CDV genome were used as a common primer pair in the first-round PCR of the nested PCR. Primers P2 specific for CDV wild-type strains, were used as the forward primer together with the common reverse primer P4 in the second round of nested PCR. Primers P3, P5 specific for CDV wild-type strain or vaccine strain, were used as the forward primer together with the common reverse primer P4+P6 in the second round of nested PCR. A fragment of 177 bp was amplified from vaccine strain genomic RNA, and a fragment of 247 bp from wild-type strain genomic RNA in the RT-nPCR, and two fragments of 247 bp and 177 bp were amplified from the mixed samples of vaccine and wild-type strains. No amplification was achieved for uninfected cells, or cells infected with Newcastle disease virus (NDV), canine parvovirus (CPV), canine coronavirus (CCV), rabies virus (RV), or canine adenovirus (CAV). The RT-nPCR method was used to detect 30 field samples suspected of canine distemper from Heilongjiang and Jilin Provinces, and 51 samples in Shandong province. As a result of 30 samples, were found to be wild-type-like, and 5 to be vaccine-strain-like. The RT-nPCR method can be used to effectively detect and differentiate wild-type CDV-infected dogs from dogs vaccinated with CDV vaccine, and thus can be used in clinical detection and epidemiological surveillance.

The identification of frequent variations in the fusion protein of canine distemper virus.

Canine distemper (CD) is a highly contagious disease with a worldwide distribution. Genetic diversity in genes encoding the haemagglutinin (H) and fusion (F) virus envelope proteins have been implicated in the increasing incidence of CD. Unlike the H gene, little is known about the genetic variability of the F gene in this virus. In the present study sequence analysis of the complete coding region of the F protein from CD virus isolates from Taiwan were carried out. Phylogenetic analysis demonstrated that the majority of isolates were similar to those found in neighbouring China and Japan, but were genetically distinct from vaccine strains. Remarkable variations were found scattered throughout the pre-peptide region (residues 1-135). The sequence identity of this region between locally sourced strains and between these strains and vaccine strains was 89% and 64 to 67%, respectively. Analysis suggested a novel strain of distant genetic lineage was present in dogs in the geographically isolated city of Hualien.

The Asia 2 specific signal peptide region and other domains in fusion protein genes characterized Asia 1 and Asia 2 canine distemper viruses.

BACKGROUND: Although the presence of Asia 2 group of canine distemper virus (CDV) was known by the sequencing and phylogenetic analysis of hemagglutinin (H) gene, the fusion (F) protein gene sequence of Asia 2 group had not been identified. So, the sequence analysis of F gene was carried out to elucidate the genotypic varaitons among Asian isolates. RESULTS: The phylogenetic analysis of F and H gene sequences from fourteen CDV isolates obtained from diseased dogs in Japan and Thailand indicated that the F genes had a new initiation codon and extra 27 nucleotides upstream of the usual open reading frame (ORF) and the F proteins had extra 9 amino acids at the N-terminal position only in Asia 2 isolates. On the contrary, the Asia 1 isolates had three extra putative N-glycosylation sites (two sites in the signal peptide region and one site in the F1 region) except for two strains of Th12 and Ac96I (two sites in signal peptide region) adding to four putative N-glycosylation sites that were conserved among all Asian isolates and Onderstepoort strain. In addition to this difference in N-glycosylation sites, the signal peptide region had a great diversity between Asia 1 and Asia 2 isolates. Also, characteristic amino acids were detected for some strains. CONCLUSION: Asia 2 isolates were distinguished from other CDV lineages by the extra 27 nucleotide sequence. The signal peptide region of F gene gives a remarkable differentiation between Asia 1 and Asia 2 isolates. Strains Th12 and Ac96I were differentiated from other Asia 1 strains by the F protein glycosylation sites.

Phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in North America.

In 2004, six puppies and one adult dog from a total of four premises were subjected to necropsy evaluation. For five of the seven dogs, disease caused by canine distemper virus (CDV) infection was suspected based on clinical signs. In all of the dogs, a diagnosis of CDV infection was established by the presence of compatible gross and histologic lesions, immunohistochemical labeling for CDV antigen, and detection of CDV RNA by reverse transcription-PCR. To further characterize the CDV strains detected in the four cases, complete gene sequences were determined for the hemagglutinin (H) and fusion (F) protein genes, while partial gene sequencing was performed for the phosphoprotein gene. A total of 4,508 bases were sequenced for the CDV strains detected from each of the four cases. Two cases were found to have identical sequences except for 2 bases in the intergenic region of the F and H genes. Phylogenetic analysis strongly suggested an evolutionary relationship between sequences detected in these two cases and those of phocine distemper virus 2 and two other strains of CDV not previously detected in the continental United States. Clear phylogenetic relationships were not established for viruses detected in the two additional cases; however, one strain showed similarity to CDV strains detected in a panda from China. Importantly, the three CDV strains detected were demonstrated to be genetically distinct from known vaccine strains and strains previously reported in the continental United States.

Retrospective differentiation of canine distemper virus and phocine distemper virus in phocids.

Formalin-fixed paraffin-embedded tissues from one Caspian seal (Phoca caspica), one harp seal (Phoca groenlandica), one hooded seal (Cystophora cristata), and one harbor seal (Phoca vitulina vitulina) were used to compare the utility of immunohistochemistry (IHC) versus that of a novel seminested reverse transcriptase polymerase chain reaction (RT-PCR) to detect and differentiate canine distemper virus (CDV) and phocine distemper virus (PDV). Four antibodies made against PDV were able to detect both viruses. Two antibodies made against cetacean morbillivirus (CMV) did not label antigens from either CDV or PDV. A third anti-CMV antibody inconsistently stained CDV antigens but did not label PDV antigens. The seminested RT-PCR was able to detect RNA of the phosphoprotein gene in all positive cases. Nucleotide sequence analyses of seminested RT-PCR products were used to differentiate CDV RNA from PDV RNA. From these data, it was determined that IHC using antibodies generated against PDV provided a rapid means of detection for both CDV and PDV antigens; however, differentiation between CDV and PDV was achieved only with the RT-PCR assay.

The nucleotide and predicted amino acid sequence of the fusion protein of recent isolates of canine distemper virus in Japan.

Analysis of the molecular properties of fusion (F) proteins of field isolates of canine distemper virus (CDV) by immunoprecipitation analysis revealed an identical molecular mass of F protein of 3 field isolates as well as the Onderstepoort laboratory strain. Sequencing showed that the F gene of a field isolate (the Yanaka strain) shared 90.1% and 95.7% identities with the Onderstepoort strain at nucleotide and amino acid levels, respectively. All of the 13 cysteine residues and 4 potential asparagine-linked glycosylation sites were completely conserved amongst these strains. These results indicate that the F proteins is much less heterogeneous than that observed in the hemagglutinin proteins of CDV.

Biological properties of phocine distemper virus and canine distemper virus.

Morbilliviruses constitute a major threat to the health of animal and man. To date the Morbillivirus genus in the Paramyxoviridae family comprises five established members, namely canine distemper virus (CDV), phocine distemper virus (PDV), measles virus (MV), rinderpest virus (RPV), and peste-des-petits-ruminants virus (PPRV). In addition, morbillivirus candidates infecting aquatic mammals were recently discovered. The present review on the biology of morbilliviruses focuses on knowledge gained by our group in studies on PDV and CDV. The aims of these studies were: i) to investigate the biological properties of the recently recognized PDV, which was found to be the primary etiology of epidemics with high mortality in seals in Western Europe, ii) to extend our knowledge of the biological properties of CDV. The morbillivirus particle is enveloped. The helical nucleocapsid core contains a single-stranded, non-segmented RNA genome of negative sense of 15 to 16 kilobases in length. The genome is organized in six transcriptional units or genes. Overall, the studies of the genome of PDV revealed a genetic map principally fitting with that determined for other morbilliviruses. The nucleotide and deduced amino acid sequences have been determined for five PDV genes named in analogy with the encoded structural proteins of other morbilliviruses in the order: 3'N(1683)-P(1644)-M(1443)-F(2206)-H(1952)-L5' (The figures in brackets denote nucleotide lengths of the genes of the Danish PDV isolate). The L gene (covering approximately 8900 nucleotides) remains to be sequenced. The six genes are likely to code for at least eight distinct proteins. The nucleocapsid (N) protein was found to consist of 523 amino acids in PDV. The following gene of the transcription map encoded the P protein of 507 amino acid residues. Similar to other morbilliviruses, the P gene of PDV was shown to have additional coding capacity for two distinct proteins V (299 amino acids) and C (174 amino acids). The results presented provide evidence for editing at transcript of the PDV P gene by insertion of nontemplated G residues at a specific site. The edited version of the mRNA was found to encode the cystein-rich V protein. The three envelope-associated proteins of PDV were predicted to consist of 335 (M), 537 (F0) and 607 (H) amino acid residues. The nucleotide and deduced amino acid sequences of the N, P, M, F, and H genes of PDV were aligned with corresponding sequences of other established members of the genus Morbillivirus.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of measles virus strains and a strain of canine distemper virus by monoclonal antibodies.

Monoclonal antibodies raised against the Hallé strain of measles virus were tested for their strain specificity using other measles virus isolates (Edmonston, Leningrad, Lec and two fresh isolates JT and Fasquelle). Monoclonal antibodies to L, HA and NP polypeptides, with one exception, reacted with all the measles virus strains tested; antibody from hybrid line 25 failed to react with the NP polypeptide of JT virus by immunofluorescence or radioimmunoprecipitation. Three other monoclonal antibodies, two reacting with NP and one with HA, although giving a positive immunofluorescence reaction, did not immunoprecipitate labelled antigen from JT virus-infected cells. The immunological relationship of canine distemper and measles viruses was also investigated with monoclonal antibodies. Two of the three anti-NP and three of the four anti-HA monoclonal antibodies reacted with canine distemper virus in an immunofluorescence reaction but only one (anti-NP) reacted in a radioimmunoprecipitation test.