Resurgence of canine parvovirus 2a strain in the domestic dog population from Argentina.

Ninety-three rectal swab samples were taken, from dogs suspected of canine parvovirus (CPV) infection and analyzed by PCR. A fragment of the VP2 gene, was amplified in 41 (44%) of them, resulting CPV positive samples. Sequencing analysis of these PCR products showed that 37 samples (90.2%) belonged to the CPV2c type, whereas four samples (9.8%) were identified as CPV2a, which has not been found since 2008. It was also found that 24 out of 37 CPV2c samples (65%), carried the mutation Thr440Ala, whereas this mutation was absent in the four CPV2a strains reported herein. Using phylogenetic analysis of the full length VP2 gene, which was amplified by PCR in six local samples, it was seen that CPV2a Argentine strains reported in this study, were genetically closer to a previous local CPV2a isolate (year 2003) and to a South African CPV2a strain, than to any of the recently reported Uruguayan CPV2a strains. The results obtained in this work, together with those reported previously in Uruguay strongly suggest that, in spite of the geographical proximity, wild type CPV strains undergo different evolutive pathways in each country, resulting in the prevalence of different strains in related dog populations. Further extensive epidemiological studies are needed in order to improve the understanding of CPV evolution.

Dam methylation regulates the expression of SPI-5-encoded sopB gene in Salmonella enterica serovar Typhimurium.

DNA adenine methylation is an essential factor in Salmonella virulence. Here, we investigate the involvement of DNA adenine methylase (Dam) in the expression and translocation of a SPI-5-encoded effector of S. Typhimurium. SopB expression and secretion were determined using SopB-FLAG-tagged wild type and dam strains of S. Typhimurium. Western blot and quantitative reverse transcriptase PCR analysis showed that the dam mutant expresses lower levels of SopB protein and sopB mRNA than the wild type strain under SPI-1 and SPI-2 inducing conditions in vitro. SopB secretion was also considerably impaired in the absence of dam. In agreement with in vitro experiments, SopB synthesis in dam mutants recovered from infected epithelial cells and from murine mesenteric lymph nodes was reduced by 40% respect to the wild type strain (p < 0.05). SopB translocation was neither detected in the cytosol of epithelial cells nor in the cytosol of cells isolated from mesenteric lymph nodes infected with the dam mutant. Taken together, our results demonstrate that, in S. Typhimurium, Dam methylation modulates the expression and translocation of SPI-5-encoded SopB effector.

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.

Evidence of two co-circulating genetic lineages of canine distemper virus in South America.

Canine distemper virus (CDV) is the etiological agent of a multisystemic infection that affects different species of carnivores and is responsible for one of the main diseases suffered by dogs. Recent data have shown a worldwide increase in the incidence of the disease, including in vaccinated dog populations, which necessitates the analysis of circulating strains. The hemagglutinin (H) gene, which encodes the major antigenic viral protein, has been widely used to determine the degree of genetic variability and to associate CDVs in different worldwide circulating lineages. Here, we obtained the sequence of the first full-length H gene of field South American CDV strains and compared it with sequences of worldwide circulating field strains and vaccine viruses. In South America, we detect two co-circulating lineages with different prevalences: the Europe 1 lineage and a new South America 2 lineage. The Europe 1 lineage was the most prevalent in South America, and we suggest renaming it the Europe 1/South America 1 lineage. The South America 2 lineage was found only in Argentina and appears related to wild CDV strains. All South American CDV strains showed high amino-acid divergence from vaccine strains. This genetic variability may be a possible factor leading to the resurgence of distemper cases in vaccinated dog populations.

Evolution of canine parvovirus in Argentina between years 2003 and 2010: CPV2c has become the predominant variant affecting the domestic dog population.

The current frequency of Canine Parvovirus variants (CPV2a, CPV2b and CPV2c) in the Argentine dog population was investigated by PCR amplification of a 583 bp fragment in the VP2 gene. From a total of 79 rectal swab samples that have been submitted to our laboratory since 2008, 55 (69.6%) resulted positive and were further analyzed by direct DNA sequencing. Fifty positives samples (91%) were characterized as CPV2c variant, which appeared in Argentina in the year 2003 and has been the prevalent type since 2008, whereas CPV2a and CPV2b, still found in Argentine dogs, were represented in 3.6% and 5.4% of the population, respectively. Considering that CPV2c is spreading worldwide, and that this variant is also affecting vaccinated dogs, efforts should be made towards the development of new matched CPV vaccines.

Molecular characterization of canine parvovirus strains in Argentina: Detection of the pathogenic variant CPV2c in vaccinated dogs.

PCR amplification with sequence-specific primers was used to detect canine parvovirus (CPV) DNA in 38 rectal swabs from Argentine domestic dogs with symptoms compatible with parvovirus disease. Twenty-seven out of 38 samples analyzed were CPV positive. The classical CPV2 strain was not detected in any of the samples, but nine samples were identified as CPV2a variant and 18 samples as CPV2b variant. Further sequence analysis revealed a mutation at amino acid 426 of the VP2 gene (Asp426Glu), characteristic of the CPV2c variant, in 14 out of 18 of the samples identified initially by PCR as CPV2b. The appearance of CPV2c variant in Argentina might be dated at least to the year 2003. Three different pathogenic CPV variants circulating currently in the Argentine domestic dog population were identified, with CPV2c being the only variant affecting vaccinated and unvaccinated dogs during the year 2008.

Detection by RT-PCR and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in Argentina.

RT-PCR was used to detect canine distemper virus (CDV) RNA in clotted blood from Argentine domestic dogs. The NP gene was detected in 73 out of 99 blood samples analyzed. The deduced amino acid sequence of these gene fragments showed 100% identity with the sequence of other wild-type and vaccine strains. A fragment of the hemagglutinin gene was amplified from 24 (32.9%) of the NP-RNA-positive clinical specimens. These H fragments were further analyzed by restriction fragment length polymorphism (RFLP) and sequencing. A single NdeI site was detected in all 24 wild-type strains but was absent in the vaccine strains. Phylogenetic analysis of the partial hemagglutinin amino acid sequences showed close clustering for local strains, clearly distinct from vaccine strains and other wild-type foreign CDV strains. One of the local strains, Arg 23, branched out of the root of the Argentine clade, close to the European strains, suggesting that two different pathogenic CDV genotypes are currently circulating in Argentina, one of them clearly predominant.

Characterization of infectious bursal disease viruses from Argentina.

Infectious bursal disease (IBD) viruses detected in commercial flocks of different regions of Argentina were analyzed by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RFLP) of a VP2 gene fragment, followed by sequence analysis. Two out of eight IBD viruses presented an SspI restriction site, typical of the very virulent phenotype. Three IBD viruses presented a SacI restriction site, typical of classic virulent strains, and one isolate presented restriction sites for both enzymes. The Argentine IBD viruses showed three different molecular patterns by RFLP with the restriction endonuclease BstNI and five different patterns with MboI. By comparison of nucleotide and deduced amino acid sequences of the hypervariable region of the VP2 protein, four Argentine viruses were found to be closely related to Brazilian subclinical strains and two isolates were found to be related to vaccine IBDV strains in use in Argentina. Strain LD9569 was genetically characterized as a very virulent strain and was found to be closely related to international and regional vvIBDV strains. This is the first report on variability of IBDV strains circulating in Argentina.