Canine Distemper Virus Isolates Research Articles

Phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from giant panda and raccoon dogs in China

BackgroundCanine distemper virus (CDV) infects a variety of carnivores, including wild and domestic Canidae. In this study, we sequenced and phylogenetic analyses of the hemagglutinin (H) genes from eight canine distemper virus (CDV) isolates obtained from seven raccoon dogs (Nyctereutes procyonoides) and a giant panda (Ailuropoda melanoleuca) in China.ResultsPhylogenetic analysis of the partial hemagglutinin gene sequences showed close clustering for geographic lineages, clearly distinct from vaccine strains and other wild-type foreign CDV strains, all the CDV strains were characterized as Asia-1 genotype and were highly similar to each other (91.5-99.8% nt and 94.4-99.8% aa). The giant panda and raccoon dogs all were 549Y on the HA protein in this study, irrespective of the host species.ConclusionsThese findings enhance our knowledge of the genetic characteristics of Chinese CDV isolates, and may facilitate the development of effective strategies for monitoring and controlling CDV for wild canids and non-cainds in China.

Phylogenetic analyses of the hemagglutinin gene of wild-type strains of canine distemper virus in southern Brazil

This study examined the phylogenetic relationship of strains of canine distemper virus (CDV) collected from Paraná State, Brazil, based on the hemagglutinin gene. Urine samples were collected from 4 dogs from northern Paraná State that demonstrated clinical manifestations of canine distemper. The participation of CDV was initially confirmed by RT-PCR targeting the nucleocapsid protein, after which the complete hemagglutinin gene was sequenced from each sample. Sequences were deposited in and compared with those already in GenBank. Phylogenetic analyses, using amino acid and nucleotide sequences based on the hemagglutinin gene, demonstrated that these strains of CDV are closely related to those from the Europe 1 lineage of CDV, with marked differences from other recognized geographical clusters of CDV isolates and from the vaccine strains. The strains of CDV from this region of southern Brazil appear to be related to those from Europe 1.

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

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.

Detecção molecular e análise filogenética do gene H de amostras do vírus da cinomose canina em circulação no município de Campinas, São Paulo

O vírus da cinomose canina (CDV), um Morbillivirus da família Paramyxoviridae, é o agente etiológico de doença neurológica e sistêmica em cães. O diagnóstico laboratorial da infecção requer o isolamento viral ou detecção do material genético do vírus em secreções ou tecidos de cães com suspeita clínica da doença. A diversidade genética entre os isolados de CDV pode ser aferida pelo sequenciamento efilogenia molecular do gene que codifica a hemaglutinina viral (gene H), havendo atualmente um especial interesse em comparar as amostras circulantes a campo com o genogrupo América-1, que abrange as cepas presentes nas vacinas disponíveis no mercado. No presente estudo, foi realizada a detecção molecular do gene H de CDV a partir de amostras biológicas colhidas ante- e post- -mortem de 15 cães com sinais clínicos sugestivos de cinomose na região metropolitana de Campinas, São Paulo. Dez dos 15 cães analisados tiveram ao menos um órgão positivo na detecção molecular e os amplicons obtidos foram submetidos ao sequenciamento nucleotídico seguido de análise filogenética molecular. De forma semelhante ao que já foi reportado para estudo analisando a diversidade do gene H em outros países, a reconstrução filogenética obtida para as amostras de casos de cinomose da região de Campinas demonstrou as mesmas foram agrupadas junto a amostras norte-americanas, europeias e japonesas recentes, em um grupo genético distinto do grupo de amostras clássicas de CDV, nomeado America-1, o qual engloba as estirpes vacinais Snyder Hill, Onderstepoort e Lederle.

Characterization of a new dog isolate of canine distemper virus from China

Canine distemper virus (CDV) is a highly contagious pathogen of dogs. Vaccination is an effective way to protect dogs from CDV infection, but occasionally fails. In the present study, a wild type (wt) CDV, named XJ2, was isolated from a dead vaccinated dog. The hemagglutinin (H) gene of the XJ2 was amplified and analyzed for the molecular characteristics including N-glycosylation sites, phylogenesis, hydrophobicity and epitopes. The data indicated that XJ2 was a genetic variant strain of CDV. CDV-sero-negative dogs were inoculated intranasally with XJ2, developed severe clinical symptoms and died, suggesting high virulence.

Pathogenesis and phylogenetic analyses of canine distemper virus strain ZJ7 isolate from domestic dogs in China

A new isolate of canine distemper virus (CDV), named ZJ7, was isolated from lung tissues of a dog suspected with CDV infection using MDCK cells. The ZJ7 isolate induced cytopathogenic effects of syncytia in MDCK cell after six passages. In order to evaluate pathogenesis of ZJ7 strain, three CDV sero-negative dogs were intranasally inoculated with its virus suspension. All infected dogs developed clinical signs of severe bloody diarrhea, conjunctivitis, ocular discharge, nasal discharge and coughing, fever and weight loss at 21 dpi, whereas the mock group infected with DMEM were normal. The results demonstrated that CDV-ZJ7 strain isolated by MDCK cell was virulent, and the nucleotide and amino acid sequences of strain ZJ7 had no change after isolation by MDCK cell when compared with the original virus from the fresh tissues. Molecular and phylogenetic analyses for the nucleocapsid (N), phosphoprotein (P) and receptor binding haemagglutinin (H) gene of the ZJ7 isolate clearly showed it is joins to the Asia 1 group cluster of CDV strains, the predominant genotype in China.

Genetic variant of canine distemper virus from clinical cases in vaccinated dogs in South Africa

Canine distemper virus (CDV) is a highly contagious viral pathogen of worldwide distribution that can cause lethal disease in domestic dogs and other members of the family Canidae. Genetic diversity is found among reference strains and isolates of CDV, mainly in the haemagglutinin (H) protein, and this may be associated with the increasing incidence of distemper in dogs. CDV was isolated in Vero cells expressing canine signalling lymphocyte activation molecule (Vero.DogSLAM) from peripheralblood mononuclear cells and spleen of clinically diseased, previously vaccinated South African dogs. Direct fluorescence antibody test and electronmicroscopy were used to confirm the isolation procedure. Subsequently, RT-PCR was performed on the cell culture isolates, the amplified products were purified and the complete H gene wassequenced and phylogenetically analysed. The H gene of vaccines in use in South Africa was also sequenced and comparative analyses performed. However, the sequences obtained from the sick dogs showed 100% nucleotide identity and was different to that found in virus strains used in vaccines and in isolates reported from other parts of the world in GenBank. The results suggest that a novel CDV lineage may be present in South Africa and we conclude that a recent reversion of vaccine virus to virulence was not the cause of the clinical signs seen in dogs with a previoushistory of vaccination.

Isolation and molecular characterization of canine distemper virus from India

Ocular swabs from canine distemper virus (CDV) suspected live or brain tissue from dead dogs were tested for the presence of CDV nucleoprotein (N) gene using reverse transcriptase polymerase chain reaction (RT-PCR). Partial "N" gene sequencing of the RT-PCR-positive samples and the local vaccine virus revealed that the Ind/Andaman 01/07 virus was highly divergent from the rest of the CDV isolates and from the vaccine strain. Quantitative real-time PCR (qRT-PCR) using SYBR Green I chemistry for CDV haemagglutinin "H" gene quantification showed C(t) values ranging from 29.76-30.67 in the RT-PCR-positive samples. Two of the positive samples, designated Ind/TN 01/07 and Ind/Andaman 01/07 were used for virus isolation in B95a cell line. Characteristic cytopathic changes such as rounding of cells, syncytia formation, and ballooning were seen from the first passage onwards. Specific cytoplasmic fluorescence was seen in infected cells with a commercial reference serum against CDV. To the best of our knowledge, this is the first report of CDV isolation from clinical cases in India.

Differentiation of canine distemper virus isolates in fur animals from various vaccine strains by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations in china

In order to effectively identify the vaccine and field strains of Canine distemper virus (CDV), a new differential diagnostic test has been developed based on reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). We selected an 829 bp fragment of the nucleoprotein (N) gene of CDV. By RFLP analysis using BamHI, field isolates were distinguishable from the vaccine strains. Two fragments were obtained from the vaccine strains by RT-PCR-RFLP analysis while three were observed in the field strains. An 829 nucleotide region of the CDV N gene was analyzed in 19 CDV field strains isolated from minks, raccoon dogs and foxes in China between 2005 and 2007. The results suggest this method is precise, accurate and efficient. It was also determined that three different genotypes exist in CDV field strains in fur animal herds of the north of China, most of which belong to Asian type. Mutated field strains, JSY06-R1, JSY06-R2 and JDH07-F1 also exist in Northern China, but are most closely related to the standard virulent strain A75/17, designated in Arctic and America-2 genetype in the present study, respectively.

Propagation of Asian isolates of canine distemper virus (CDV) in hamster cell lines

BackgroundsThe aim of this study was to confirm the propagation of various canine distemper viruses (CDV) in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance.MethodsThe growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST) cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains) and by observing the development of cytopathic effect (CPE) in infected cultures of hamster cell lines.ResultsEleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively.ConclusionThe present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically.

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

BackgroundAlthough 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.ResultsThe 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.ConclusionAsia 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.

EPIDEMIOLOGY, PATHOLOGY, AND GENETIC ANALYSIS OF A CANINE DISTEMPER EPIDEMIC IN NAMIBIA

Severe population declines have resulted from the spillover of canine distemper virus (CDV) into susceptible wildlife, with both domestic and wild canids being involved in the maintenance and transmission of the virus. This study (March 2001 to October 2003) collated case data, serologic, pathologic, and molecular data to describe the spillover of CDV from domestic dogs (Canis familiaris) to black-backed jackals (Canis mesomelas) during an epidemic on the Namibian coast. Antibody prevalence in jackals peaked at 74.1%, and the clinical signs and histopathologic observations closely resembled those observed in domestic dog cases. Viral RNA was isolated from the brain of a domestic dog from the outbreak area. Sequence data from the phosphoprotein (P) gene and the hemagglutinin (H) genes were used for phylogenetic analyses. The P gene sequence from the domestic dog shared 98% identity with the sequence data available for other CDV isolates of African carnivores. For the H gene, the two sequences available from the outbreak that decimated the lion population in Tanzania in 1994 were the closest match with the Namibian sample, being 94% identical across 1,122 base pairs (bp). Phylogenetic analyses based on this region clustered the Namibian sample with the CDV that is within the morbilliviruses. This is the first description of an epidemic involving black-backed jackals in Namibia, demonstrating that this species has the capacity for rapid and large-scale dissemination of CDV. This work highlights the threat posed to endangered wildlife in Namibia by the spillover of CDV from domestic dog populations. Very few sequence data are currently available for CDV isolates from African carnivores, and this work provides the first sequence data from a Namibian CDV isolate.

Relationship Between Growth Behavior in Vero Cells and the Molecular Characteristics of Recent Isolated Classified in the Asia 1 and 2 Groups of Canine Distemper Virus

Ten recent isolates of canine distemper virus (CDV) strains were classified according to the growth ability and development of syncytial cytopathic effects (CPE) in Vero cells. Strains P94S, Ac96I, S124C, MD231, MS232, MSA5 and 095Cr were classified as Type 1 and exhibited hardly and did not develop CPE in Vero cells. Strains 007Lm, 009L and 011C were classified as Type 2 as grew well but failed to develop a syncytial CPE in Vero cells. A comparison of the phylogenetic trees of the H and P genes showed that all Type 1 strains belonged to the Asia 1 group and all Type 2 strains belonged to the Asia 2 group. Our findings suggest that the recent Asia 2 isolated of CDV in Japan, but not Asia 1 may grow in Vero cells, and their growth ability may be related with their molecular characteristics.

First Isolation and Characterization of Canine Distemper Virus in Vietnam with the Immunohistochemical Examination of the Dog

Canine distemper caused by canine distemper virus (CDV) is a contagious, incurable, often fatal, multisystemic viral disease that affects the respiratory gastrointestinal and central nervous system. Strains Vn86 and Vn99 of CDV were isolated, we believe for the first time, in Vietnam from two 4-month-old autopsied dogs pathologically showing non-suppurative encephalitis with pneumonia, lymphoid depletion and severe gastroenteritis. These strains caused syncytium cytopathic effect in Vero cells and Vero cells expressing canine signaling lymphocyte activation molecules. The titers of cell-associated viruses of both strains were higher than for released viruses. Molecular analysis showed that both new isolates of CDV joined to the group of classic type that is far from the Asia 1 and Asia 2 groups. These results indicated that first isolation and characterization of canine distemper virus in Vietnam with the immunohistochemical examination of the dog.

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.

Identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus

Canine distemper (CD) is a widely distributed disease of dogs, caused by the canine distemper virus (CDV). In the present study, the gene encoding the hemagglutinin (H) protein of a CDV isolate from central Taiwan was sequenced and compared with other strains. Sequence variations were noticed in the H gene from the field CDV strain that had previously been implicated in the increasing incidence of CD. To establish a serology-based diagnostic test, the full-length H protein, as well as five deletion mutants of a recombinant H protein of the local isolate, were produced using an E. coli expression system. Three truncated recombinant proteins with relatively high expression levels, designated HM3, HM4 and HM5, were used as antigens to examine their reactivity with canine sera. By using three negative sera and 17 CD-positive sera, the high specificity of recombinant H proteins was observed by ELISA. In addition, immunoblotting demonstrated that all three purified recombinant proteins exhibit an antigenic property recognized by the serum of a CD-suspected dog.

Pathological and phylogenetic features of prevalent canine distemper viruses in wild masked palm civets in Japan

Ten wild masked palm civets infected with canine distemper virus (CDV), captured in Japan from 2005 to 2007, were histopathologically and phylogenetically analyzed. Phylogenetic analysis based on the amino acid sequences of the H protein of two CDV isolates from masked palm civets revealed that the two isolates were classified into the clade of recent isolates in Japan. Histopathologically marked lesions of virus encephalitis were present in the brain, whereas gastrointestinal lesions were absent or at a mild degree. The distribution of the lesions resembles that of recent CDV cases in dogs. Therefore, recent CDV infections in masked palm civets could be caused by recently prevalent CDV in dogs. The possibility of the masked palm civet as a spreader of CDV among wildlife is also discussed.

Establishment of canine and feline cells expressing canine signaling lymphocyte activation molecule for canine distemper virus study

Signaling lymphocyte activation molecule (SLAM) is one of the receptors for canine distemper virus (CDV). In this study, canine and feline cells expressing canine SLAM, designated A-72/cSLAM and CRFK/cSLAM, were established for the in vitro study of canine distemper. Recent CDV isolates, KDK-1 and 246, which belong to genotypes Asia/H1 and Asia/H2, respectively, rapidly grew and produced distinct syncytia in both the SLAM-expressing cells. The virus-neutralizing (VN) test was successfully performed using these cells, and the results indicated that sera from dogs experimentally infected with KDK-1 had higher VN titers for homologous strain KDK-1 than for heterologous strain 246 and the vaccine Onderstepoort. These newly established cells expressing canine SLAM would help virological and serological analyses of canine distemper.

Phylogenetic characterization of canine distemper virus isolates from naturally infected dogs and a marten in Korea

We sequenced the hemagglutinin (H) genes from four canine distemper virus (CDV) isolates obtained from three dogs and a marten in Korea. These sequences were included in subsequent H gene-focused phylogenetic tree analysis of 89 CDV strains. This analysis revealed eight clades designated as EU1, EU2, EU3, NA1, NA2, Asia 1, Asia 2 and Vaccine. Three of the Korean isolates (97Jindo, 98Marten and 07D111) occurred in the Asia 2 group that also contains many Japanese CDV strains isolated in 1998. The remaining Korean strain (07Q72) fell into the Asia 1 group. The 21 H protein sequences of 25 Asia 1 strains are generally predicted to bear nine potential N-linked glycosylation sites. In contrast, the 9 H protein sequences of 12 Asia 2 strains had eight potential N-linked glycosylation sites. The remaining strains had six (98Marten and 07D111) and seven (97Jindo) potential N-linked glycosylation sites.

Phocine distemper virus: characterization of the morbillivirus causing the seal epizootic in northwestern Europe in 2002

In 2002, 14 years after the first devastating die-off in the northwestern European seal population in 1988, a second epizootic was caused by the re-emergence of phocine distemper virus (PDV) in this area. The nucleotide sequence of the surface hemagglutinin (H) gene from the PDV of 2002 and the deduced amino acid sequence were determined and aligned with those of PDV isolates from 1988. There was a 98–99% nucleotide and a 98% amino acid identity of the H protein between the PDV isolates. From the close relationship shown in this study, it was concluded that PDV seems to be a strictly seal-adapted virus with only minor genetic variability over a longer period. In 1988, a fatal phocine distemper epizootic in seals from northwestern European waters led to the death of nearly 23,000 animals [13]. After the recovery of the seal population in the following years, a second mass die-off caused by phocine distemper virus (PDV) occurred in 2002 with the loss of approximately 30,000 seals [13]. Both epizootics showed similarities regarding the clinical and pathological findings, but some distinct features regarding the time of disease outbreak and epidemiologic progress were noticed [13, 22]. Until today, the origin and geographic distribution of PDV in northwestern Europe remain unclear in many aspects. It has been hypothesized that migrating seal species from other regions may act as carriers for PDV in the European seal populations [7]. PDV is a member of the genus Morbillivirus, closely related to canine distemper virus (CDV). In contrast, two other morbilliviruses from marine mammals, i.e. the porpoise and the dolphin morbilliviruses, are more closely related to the rinderpest (RPV) and the peste des petits ruminants viruses, respectively [2]. The surface and core proteins of PDV causing the first epizootic in Europe have been characterized in detail [2, 6, 18]. Both surface proteins, the hemagglutinin (H) and fusion (F) proteins, play an important role in viral attachment to and fusion with the host cell and formation of syncytia [2]. Variation in these proteins may cause changes in virulence and host-cell tropism. Earlier studies revealed a high identity between the F proteins from different CDV and PDV isolates, whereas the H protein was more variable [18, 24]. In order to analyse whether modifications of virulence and host range had occurred between the PDV isolates from both epizootics, the nucleotide and deduced amino acid sequences of the H gene of a PDV isolate from 2002 (PDV/ 02) were determined and compared to sequences from previous isolates. O.C.T-embedded lung tissue samples from three seals that died during the seal distemper epidemic in 2002 at the German North Sea, were obtained for evaluation. The samples were shock-frozen in liquid nitrogen and stored at -80 C until use. For verification of the PDV infection, a set of universal morbillivirus primers based on a conserved sequence of the phosphoprotein P gene was used as G. Muller U. Kaim P. Wohlsein W. Baumgartner Department of Pathology, University of Veterinary Medicine Hannover, Bunteweg 17, 30559 Hannover, Germany