Canine Distemper Virus Vaccine Strains Research Articles

Development of a monoclonal antibody recognizing novel linear neutralizing epitope on H protein of canine distemper virus vaccine strains (America-1 genotype)

Canine distemper virus (CDV) is an economically important virus responsible for canine distemper (CD), a highly contagious disease that afflicts various animal species worldwide. The hemagglutinin (H) protein is the major neutralizing target of virus. Therefore, it is often considered as immunogen to prepare neutralizing antibodies. The accurate identification of neutralizing epitope will provide important antigenic information and extend the knowledge of mechanisms of virus neutralization. In this study, we generated a neutralizing monoclonal antibody (mAb) 4C6 against CDV H protein, and defined the minimal linear epitope 238DIEREFDT245, which was highly conserved in America-1 genotype of CDV strains (vaccines). The mAb 4C6 could not react with a CDV strain that had two substitutions of D238Y and R241G in the epitope, which appeared in most CDV strains of the other genotypes. Besides, a few different amino acid mutations in the epitope were also included. Collectively, the epitope 238DIEREFDT245 was variable in the other genotypes of CDV strains. The epitope 238DIEREFDT245 was exposed to the surface of CDV H protein, showing good antigenicity. These data will provide insights into structure, function and antigenicity of H protein and lay the foundation for the development of diagnostic technologies and vaccine design for CDV.

Establishment of Multiplex RT-PCR for Differentiating between the CD1901 and Lederle Strains of Canine Distemper Virus

Canine distemper virus (CDV) is a life-threatening pathogen in dogs. Clinical, pathological and molecular methods are required to diagnose CDV infection, and it is important to differentiate between the Korean CDV strain CD1901 and Lederle CDV vaccine strain. Therefore, in this study, we used multiplex reverse- transcription polymerase chain reaction (RT-PCR) to differentiate between the CD1901 and Lederle strains. A primer set was designed based on the CDV nucleoprotein gene and nucleotide sequence variation in the fusion (F) gene. First, 224-bp DNA bands were amplified from viral RNA of the CD1901 and Lederle strains. Then, 428- and 326-bp DNA bands were amplified in the CD1901 and Lederle strain, respectively. The multiplex RT-PCR detection limits were 2.53 and 0.8 median tissue culture infectious dose/reaction for the CD1901 and Lederle strains, respectively. No cross-reactions were detected in non-CDV reference viruses, including rabies virus, parvovirus, canine adenovirus types 1 and 2, and parainfluenza virus. The results indicate that our one-step multiplex RT-PCR is useful for differentiating between wildtype and vaccine CDV strains.

Canine distemper virus induces early stages of apoptosis in in vitro culture of canine peripheral blood mononuclear cells

Canine distemper virus (CDV) has been described to induce apoptosis in lymphoid tissues, lymphoid cells, and the cerebellum of naturally infected dogs. The immunosuppression stage established from CDV infection is not always associated with virus detection in damaged cells. Examining the host response at the early stages of immunosuppression, especially the expression of activators and/or inhibitors of apoptosis processes, can provide an important understanding of how the immune responses to CDV are orchestrated. For this purpose, canine peripheral blood mononuclear cells (PBMC) collected from healthy dogs were cultured and infected by the CDV vaccine strain (Onderstepoort). After 24 h post-infection (p.i.), early events of apoptosis were detected by search for annexin V™ and ApoTrace™ markers. The expression of mRNA of Bax, BCl-2, caspase-3, 8 and 9, and also survivin genes was performed in infected and uninfected canine PBMC at 24 h p.i. by real-time polymerase chain reaction. The vaccine strain induced loss of PBMC viability with the expression of annexin™ V FITC and Apo-Trace™ FITC in more than 80% of infected cells compared to the control group (P < 0.001) at 24 h p.i. The initiation of early apoptosis after 24h post-infection contrasts with higher expression of mRNA of survivin and BCl-2, two major anti-apoptotic proteins (p < 0.0002). PBMC infected by CDV increased survivin and BCL-2 gene transcription, concomitant to a reduced level of Bax, caspase 3, 8 and 9. This is the first description of survivin expression, an anti-apoptotic mediator, induced by CDV infection on in vitro cultured canine PBMC.

Isolation and Phylogenetic Analysis of the Canine Distemper Virus from a Naturally Infected Dog in China

Background: Canine distemper virus (CDV) can infect a variety of predators, such as Canidae, Mustelidae and Procyonidae family and cause high morbidity and mortality. Genetic/antigenic heterogeneity has been observed among various CDV strains, notably in the haemagglutinin (H) gene. Based on sequence analysis of the H gene, wild-type CDV strains can be divided into distinct genotypes. Methods: We used Vero/SLAM cells to isolate CDV strains from clinically naturally infected dog tissues. Here, the H gene sequence of the isolated strain was analyzed for the restriction enzyme polymorphism with RT-PCR-RFLP method and the method could be used to distinguish CDV vaccine strains from wild-type strains. Furthermore, the genetic and antigenic variation of the H gene was also analyzed.Result: Our results indicated that Vero cells expressing canine SLAM allow the efficient isolation of CDV wild-type strains. The isolated CDV was named Shandong strain. Phylogenetic analysis identified that Shandong strain was CDV wild-type strain, the Asian genotype I. Analysis of the potential glycosylation site of the H gene and determination of the neutralization titer PD50 showed that there were immunogenic differences between the Shandong strain and the vaccine strains.

Viral Pathogenesis, Recombinant Vaccines, and Oncolytic Virotherapy: Applications of the Canine Distemper Virus Reverse Genetics System.

Canine distemper virus (CDV) is a highly contagious pathogen transmissible to a broad range of terrestrial and aquatic carnivores. Despite the availability of attenuated vaccines against CDV, the virus remains responsible for outbreaks of canine distemper (CD) with significant morbidity and mortality in domesticated and wild carnivores worldwide. CDV uses the signaling lymphocytic activation molecule (SLAM, or CD150) and nectin-4 (PVRL4) as entry receptors, well-known tumor-associated markers for several lymphadenomas and adenocarcinomas, which are also responsible for the lysis of tumor cells and apparent tumor regression. Thus, CDV vaccine strains have emerged as a promising platform of oncolytic viruses for use in animal cancer therapy. Recent advances have revealed that use of the CDV reverse genetic system (RGS) has helped increase the understanding of viral pathogenesis and explore the development of recombinant CDV vaccines. In addition, genetic engineering of CDV based on RGS approaches also has the potential of enhancing oncolytic activity and selectively targeting tumors. Here, we reviewed the host tropism and pathogenesis of CDV, and current development of recombinant CDV-based vaccines as well as their use as oncolytic viruses against cancers.

Isolation and sequence analysis of the complete H gene of canine distemper virus from domestic dogs in Henan Province, China.

Eighteen canine distemper virus (CDV) isolates were obtained from clinical samples in Henan province, China, between 2012 and 2016. These viruses could not be recognized by 1A4, a monoclonal antibody specific for the H protein of CDV vaccine strains. The complete haemagglutinin (H) genes of all 18 isolates were sequenced, and phylogenetic analysis showed that they segregated into two clusters within the Asia-1 genotype. Moreover, the H genes of four viruses were found to lack a potential N-glycosylation site at position 309, which is the most conserved site within the Asia-1 genotype of CDV, and a novel potential N-glycosylation site (amino acids 517–519) was found in strain HL013, which has not been reported previously. These results will help in achieving a better understanding of the evolution of CDV in China.Electronic supplementary materialThe online version of this article (10.1007/s00705-019-04298-7) contains supplementary material, which is available to authorized users.

Update on canine distemper virus (CDV) strains of Arctic-like lineage detected in dogs in Italy.

Canine distemper virus (CDV) is the etiologic agent of distemper in dogs. It exhibits an elevated potential of crossing species barriers, infecting a wide range of wild and domestic carnivores. Of its encoding genes, hemagglutinin (H) shows high heterogeneity, and it was used to determine the relationship between CDV strains due to its variability and key role in determining cell tropism, host shift, and in eliciting a protective immune response. This study analysed the full-length H gene sequence of Arctic-like CDV strains collected from dogs in Italy during a period in which an increased activity of CDV diffusion was observed. The common amino acid changes and features of Arctic-like CDV strains collected from 2011 to 2016 in Europe were described, providing an updated analysis of the genomic features. A comparison with CDV vaccine strains was carried out to evaluate the increased genomic difference with CDV Arctic-like field strains. This study provides a complete and updated analysis of the current spreading strains of Arctic-like lineage and the main amino acid variations in the hemagglutinin gene sequence circulating in Italy. Moreover, it provides novel information regarding the evolution of the most recent CDV Arctic-like lineage strains collected in Europe.

Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens.

The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains.IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that received recombinant rabies viruses carrying only the CDV attachment protein according to the same immunization scheme died. Irrespective of the CDV antigens used, all animals developed protective titers against rabies virus, illustrating that a bivalent rabies virus-based vaccine against CDV induces protective immune responses against both pathogens.

The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays

Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n=33) and 41.1% (n=23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control.

Influence of vaccine strains on the evolution of canine distemper virus

Canine distemper virus (CDV) is a major dog pathogen belonging to the genus Morbillivirus of the family Paramyxoviridae. CDV causes disease and high mortality in dogs and wild carnivores. Although homologous recombination has been demonstrated in many members of Paramyxoviridae, these events have rarely been reported for CDV. To detect potential recombination events, the complete CDV genomes available in GenBank up to June 2015 were screened using distinct algorithms to detect genetic conversions and incongruent phylogenies. Eight putative recombinant viruses derived from different CDV genotypes and different hosts were detected. The breakpoints of the recombinant strains were primarily located on fusion and hemagglutinin glycoproteins. These results suggest that homologous recombination is a frequent phenomenon in morbillivirus populations under natural replication, and CDV vaccine strains might play an important role in shaping the evolution of this virus.

Detection and differentiation of wild-type and vaccine strains of canine distemper virus by a duplex reverse transcription polymerase chain reaction.

Canine distemper virus (CDV) is the cause of canine distemper (CD) which is a severe and highly contagious disease in dogs. In the present study, a duplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the detection and differentiation of wild-type and vaccine strains of CDV. Four primers were designed to detect and discriminate the two viruses by generating 638- and 781-bp cDNA products, respectively. Furthermore, the duplex RT-PCR method was used to detect 67 field samples suspected of CD from Guangdong province in China. Results showed that, 33 samples were to be wild-type-like. The duplex RT-PCR method exhibited high specificity and sensitivity which could be used to effectively detect and differentiate wild-type and vaccine CDV, indicating its use for clinical detection and epidemiological surveillance.

Early life DNA vaccination with the H gene of Canine distemper virus induces robust protection against distemper

Young mink kits (n=8) were vaccinated with DNA plasmids encoding the viral haemagglutinin protein (H) of a vaccine strain of Canine distemper virus (CDV). Virus neutralising (VN) antibodies were induced after 2 immunisations and after the third immunisation all kits had high VN antibody titres. The VN antibody titres remained high for more than 4 months and the mink were protected against viraemia, lymphopenia, clinical disease and changes in the percentage of IFN-γ producing peripheral blood leucocytes after challenge inoculation with a recent wild type strain of CDV. Essentially, these results demonstrate that early life DNA vaccination with the H gene of a CDV vaccine strain induced robust protective immunity against a recent wild type CDV.

Detecção do vírus da cinomose canina por RT-PCR utilizando-se oligonucleotídeos para os genes da fosfoproteína, hemaglutinina e neuraminidase

Empregou-se a técnica de reação em cadeia pela polimerase precedida de transcrição reversa para detecção do vírus da cinomose canina (CC). Para a padronização da técnica foram selecionados quatro pares de oligonucleotídeos (P1, P2, N1, H1), baseados em seqüências dos genes da fosfoproteína, neuraminidase e hemaglutinina, sendo utilizadas três cepas vacinais de vírus da CC como controles positivos. Foram analisadas três amostras isoladas de cães com cinomose e quatro amostras provenientes de cães com suspeita clínica de cinomose. Não houve amplificação nas amostras com suspeita clínica da doença. Os resultados obtidos com os oligonucleotídeos P1 e N1 foram superiores aos de H1. Os oligonucleotídeos P2 foram considerados inapropriados para a detecção do vírus da CC. Os amplicons obtidos com os oligonucleotídeos P1, N1 e H1 foram clivados com endonucleases de restrição, sendo os perfis das amostras virais comparados aos da amostra vacinal Lederle, utilizada como referência. Um padrão similar de restrição foi observado em todas as amostras analisadas.

The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F WT) and attachment (H WT) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H WT determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F WT reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.

Seroprevalence of canine distemper virus in cats.

A seroepidemiological survey of canine distemper virus (CDV) infection in Asian felids revealed that the prevalence of antibodies varied depending on region and, in some cases, exposure to dogs. The serologic pattern in cats with antibodies indicated that they had likely been exposed to field strains rather than typical CDV vaccine strains.

近年, 野外から分離された犬ジステンパーウイルスの生物学的・血清学的性状

Co-cultivation with Vero or B95a cell lines was used to study the biological and serological properties of 15 strains of canine distemper virus (CDV) isolated from dogs in Japan from 1982 to 1998. Those properties were then compared with those of the CDV vaccine strain used in Japan. The vaccine strain and some of the fieldisolates cultured in Vero cells demonstrated proliferation potency in chicken embryonic fibroblast cell culture and pock-forming capacity and neurovirulence in suckling mice. Cross-neutralization tests using 9 fieldisolates, and the vaccine strain revealed no significant antigenic difference among the field-isolates. The fieldisolates were, however, somewhat different strain from the vaccine strain in antigenicity. Since the antiserum to the vaccine dramatically neutralizes all the field-isolates, the vaccine used currently in Japan can be considered effective against recently isolated CDV strains.

Canine Distemper Virus Causes Apoptosis of Vero Cells

Apoptosis of Vero cells infected with two canine distemper virus (CDV) vaccine strains was detected using TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end-labelling (TUNEL), flow cytometric analysis, agarose gel electrophoresis and electron microscopy (EM). By TUNEL, apoptotic cells were found in CDV-Onderstepoort (CDV-Ond)-infected cells. DNA fragments isolated from infected cells were separated by agarose gel electrophoresis and a 'ladder' pattern appeared. EM observations demonstrated that the cells undergoing cytopathic effect (CPE) possessed morphological characteristics of apoptotic cells. Flow cytometric analysis indicated that CDV could induce apoptosis of Vero cells, but the percentages of the apoptotic cells were correlated with the CPE types. The strain showing the cell-rounding type of CPE produced a much higher percentage of apoptotic cells than CDV-Ond with the syncytium type of CPE (P < 0.01). It was concluded that CDV vaccine strains could induce apoptosis of Vero cells and the apoptosis was virus strain-dependent and cell-dependent. The mechanism remains to be studied.

Canine distemper virus from diseased large felids: biological properties and phylogenetic relationships

Specific pathogen free (SPF) domestic cats were inoculated with tissue homogenate obtained from a Chinese leopard (Panthera pardus japonensis) that had died in a North American zoo from a natural infection with canine distemper virus (CDV). The cats developed a transient cell-associated CDV viraemia along with pronounced lymphopenia but did not show any clinical symptoms. Plasma neutralizing-antibody titres against the homologous CDV (A92-27/4, isolated from the Chinese leopard) were consistently higher than against the CDV vaccine strain 'Bussell'. The Chinese leopard CDV isolate showed in vitro biological properties reminiscent of virulent, wild-type CDV strains. Sequence analysis of the H gene of two large felid CDV isolates from the USA (A92-27/4 and A92-6) revealed up to 10% amino acid changes including up to four additional potential N-linked glycosylation sites in the extra-cytoplasmic domain as compared to CDV vaccine strains. Phylogenetic analysis was performed using the entire coding region of the H gene and a 388 bp fragment of the P gene of several morbillivirus species. Evidence was obtained that recent CDV isolates from different species in the United States (including isolates from large felids), Europe and Africa are significantly distinct from CDV vaccine strains. All wild-type CDV isolates analysed clustered according to geographical distribution rather than to host species origin. By sequence analysis a CDV epizootic among large felids in a Californian safari park was linked to a virus which most likely originated from feral non-felid carnivores.

The biological characterization of field isolates of canine distemper virus from Japan.

Eight isolates of canine distemper virus (CDV) were obtained from seven dogs suffering from distemper by co-cultivation of their mononuclear cells with a marmoset B lymphoblastoid cell line, B95a. Six of the seven dogs had received one or more vaccinations. All of the isolates readily proliferated in B95a cells, but were not completely neutralized by anti-CDV canine plasma, which had high neutralizing activity against the Onderstepoort laboratory strain of CDV. Furthermore, different reactivities of monoclonal antibodies (MAbs) against CDV were observed between the field isolates and laboratory or vaccine strains of CDV in immunofluorescence studies. Immunoprecipitation analysis using MAbs detected the haemagglutinin protein of each new field isolate as 69K, 75K and 155K forms, and the fusion protein as 64K and 65K forms; the corresponding proteins of the Onderstepoort strain were detected as 75K and 61K proteins respectively. It is apparent from these results that the new field isolates of CDV have very different antigenic properties from the Onderstepoort vaccine strain.