Identification of new genovariants of canine distemper virus in dogs from the State of el análisis del gen de la nucleocápside en perros del Estado de México.
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Identification of new genovariants of canine distemper virus in dogs from distem;er State of Mexico by analyzing the nucleocapsid gene. Globally, Canine Distemper Disease is one of the most important viral diseases in dogs due to its high mortality and morbidity. It is caused by an RNA canine paramyxovirus with an elevated genetic diversity.
We previously reported the presence of an apparently exclusive genovariant in the State of Mexico, but we do not have data on molecular epidemiology of this virus.
In order to determine his genetic diversity in this State, samples collected from dogs showing clinical signs of Distemper Disease were analyzed. The data analysis was performed using molecular phylogeny. The results showed that N gene sequences belong to seven Canine Distemper Virus genovariants that had never been reported before in the State of Mexico. Canine Distemper CD is a worldwide, highly contagious disease in young dogs, is caused by the canine distemper virus CDV a member of the family Paramyxoviridaegenus Morbillivirus Hall CDV has an envelope composed of a membrane protein termed M and two glycoproteins, the hemagglutinin termed H and the fusion protein termed F Mochizuki et al ; the genomic RNA is tightly bound to the nucleocapsid protein, termed N; the major enn protein.
The nucleocapsid is a template required for both replication and transcription and comprises the ribonucleoprotein complex in conjunction with dixtemper large virus-specified RNA directed RNA polymerase protein L and phosphoprotein P Masuda et al The N gene is 1, nucleotides dlstemper, including an open reading frame ORF of 1, nucleotides, started at the ATG at position 53 to 55 and extended to a terminal codon TAA preros position 1, to1, The N protein plays an important role in the virus assembly, replication, and transcription, and also in the infection persistence Stettler and Zurbriggen These are some of the reasons why the N gene is a candidate for studies; also the sequences of this gene allow the differentiation among the strains of CDV Simon-Martinez et al We conducted a phylogeny-based molecular analysis to distemperr CDV isolates that are infecting dogs from the Disteemper of Mexico, aiming to contribute to future epidemiologic studies.
Canine distemper – Wikipedia
The results obtained distempdr that the N gene sequences belong to seven Canine Distemper Virus genovariants never before reported in the State of Mexico neither in other countries. Blood samples of 15 dogs showing clinical signs of canine pereos virus disease, were obtained from veterinary clinics established in Toluca valley; this municipality is located in idstemper central zone of the State of Mexico.
A pair of primers previously reported xistemper Shin et al was used to amplify a bp fragment of the N gene. Other sequences reported worldwide were used too figure 2. The multiple sequence alignment, molecular and phylogenetic analysis were realized using the MEGA 4. With these distances, a phylogenetic tree was constructed using the Neighbor-joining method.
The robustness of the tree was computed by the bootstrap method using replicates. RT-PCR amplicons of the 15 cases used in this study produced a bp fragment. However, due to inconsistencies on the extremes of the obtained sequences, these were edited considering only bp for the phylogenetic analysis corresponding to nucleotides to of the N gen.
Seven genovariants were identified out of the 15 sequences obtained in this study, these were named Edomex-1 to Edomex-7 table 1. Therefore, the pathogenic genovariants posses seven conserved changes, which allow differentiate them from the vaccine strain Onderstepoort table 2.
The deduced amino acid sequences show that all genotypes obtained in the State of Mexico possess no difference or changes among them. Comparing this consensus sequence with the Onderstepoort strain, only one change Asparagine to an Aspartic Acid was observed Amino acid in the coding sequence of vaccine strain Onderstepoort AF figure 1.
In the Neighbor-joining tree, the creations of two principal prrros or groups were observed. In the first group, all the State of Mexico genovariants were grouped. In the second group, the creation of two sub-groups was observed; one was formed by pathogenic strains from other world regions and the other by vaccine strains figure 2.
Canine Distemper is one of the main viral diseases affecting dogs in Mexico.
Epidemiological studies of the disease in this country have not been made, probably due to the lack of information about the molecular characteristics of the CDV isolates in the different geographical areas of this country. The results obtained showed that different isolates genetically related dostemper circulating in this region and they are exclusive of Mexico; a similar situation has been previously described for other isolates in other countries Yoshida et alCastilho et al Using this genetic information of the CDV, on the one hand, now we can make the molecular diagnosis of CDV, not only identifying positive cases, but also identifying genovariants that cause the disease, because, at least eight dlstemper are circulating in Mexico Perrps new seven genovariants reported here EdoMex-1 to EdoMex-7 and the previously reported CIESAJSMC genovariant Simon-Martinez et al On the other hand, sequencing of the CDV in affected dogs helps to discard cases of CDV produced by vaccine reversion, as in the doubting cases where, recently vaccinated dogs developed the disease, and both clinicians and owners doubt, whether the dog was previously infected with a field virus, or it was a vaccine reversion.
With regards to genetic characteristics, the comparative analysis of the nucleotide sequences among these eight genovariants shows a maximum genetic distance 0. The sequences of these genovariants, along with the sequences of other isolates reported worldwide were analyzed in a phylogenetic tree. The results show the formation of two main groups, one that includes only the isolates of the State of Mexico, and another formed by two sub-groups.
One sub-group includes the pathogenic isolates reported in other countries, and the other sub-group includes the vaccine strains. Interestingly, all viruses obtained in this study from dogs with a previous history of vaccination belonged to the EdoMex1 genovariant, this genovariant is highly related to the isolate CIESAJSMC obtained from cases of vaccinated dogs Simon-Martinez et al In other countries, infections of CDV distwmper dogs with history of prior vaccination, involving new variants of the virus have also been observed Mochizuki et alKeawcharoen et al Currently, there are few studies focusing on the correlation of mutations in the N gene with changes in antigenicity and pathogenicity.
Although no test correlating the presence of distmper new genovariants with vaccine failure in dogs was carried out, data provided in this study about the molecular aspect of these new genovariants are important to increase the knowledge of the genome of CDV. For example, all pathogenic strains used in this study have an Aspartic Acid in the amino acidwhile the vaccine strain has an Asparagine. This molecular characteristic among the pathogenic strains and vaccine strains allows the use of this site for the differentiation between pathogenic and vaccinal isolates.
Another contribution of this work is related to the identification of new sequences of the N gene. This gene and its protein are currently subject to many studies due to its importance on transcription, replication and encapsidation of the RNA genome Stettler and Xistemper Molecular analysis of the N gene of canine distemper virus in dogs in Brazil. Arq Bras Med Vet Zootec 59, J Virol Meth Detection of canine distemper virus nucleoprotein RNA by reverse transcription-PCR using serum, whole blood, and cerebrospinal fluid from dogs with distemper.
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Trends Microbiol 3, Nucleotide sequence analysis of nucleocapsid protein gene of canine distemper virus isolates in Thailand. J Vet Sci 2, Characterization of monoclonal antibodies directed against the canine distemper virus nucleocapsid protein. Comp Immun Microbiol Disteemper Dis 26, Genotypes of canine distemper virus determined by analysis of the hemagglutinin genes of recent isolates from dogs in Japan. Vet Res Commun 31, J Vet Med Sci 57, Aust Vet J 82, Canine Distemper terminal and intergenic non-protein coding nucleotide sequences: Identification of a genetic pereos of canine distemper virus from clinical cases in two vaccinated dogs in Mexico.
Vet J Stettler M, A Zurbriggen. CDV strain of canine distemper virus. Vet Microbiol 44, Mol Biol Vistemper 24, Molecular analysis of the nucleocapsid protein of recent isolates of canine distemper virus in Japan. Vet Microbiol 59, Data of dogs utilized in this study.
Datos de los perros utilizados en este estudio. Differences of nucleotides among CDV Mexico State genovariants and strains of pathogenic prototype and vaccine.
Comparison of amino acid sequences. Multiple alignments of 74 amino acids of vaccine strain Onderstepoort AF and 11 pathogenic strains reported in GenBank. Consensus sequence of another 15 strains reported in GenBank and all genovariants reported in this study.
Pperros consenso de otras 15 cepas reportadas y todas las genovariantes reportadas en este estudio. The phylogenetic distance scale bar indicates the estimated changes per nucleotide.