ABSENCE OF PARVOVIRUS SHEDDING IN FECES OF THREATENED CARNIVORES FROM MISIONES, ARGENTINA.

Since its emergence in the 1970s, canine parvovirus (CPV) has spread worldwide and infects a wide variety of mammalian hosts, including domestic and nondomestic carnivores. Today it is one of the most important pathogenic viruses associated with high morbidity and mortality in domestic dogs ( Canis familiaris). In South America, the range of wild hosts has been scarcely studied and the epidemiology of CPV in wildlife is still unclear. In 2011, feces from five wild carnivores (bush dog [ Speothos venaticus] , jaguar [ Panthera onca], puma [ Puma concolor], oncilla [ Leopardus guttulus], and ocelot [ Leopardus pardalis]) were collected in Misiones, Argentina, using a detection dog. Of the 289 feces collected, 209 (72.3%) had sufficient sample remaining to be used in this study and the majority of these were genetically confirmed to individual (81.3%) and sex (78.4%) level. In fact, these samples represent a minimum of 115 individuals (10 jaguars, 13 pumas, 33 ocelots, 38 oncillas, and 21 bush dogs). Through polymerase chain reaction, a 583-bp fragment in the VP2 gene of CPV was amplified in these samples. While no samples showed evidence of infection, this does not exclude the occurrence of CPV in wild carnivores in the area, as intermittent viral shedding could condition the diagnosis of CPV in feces of infected wild mammals. Locally, it is recommended that long-term monitoring of parvovirus be continued in wildlife and expanded to domestic carnivores. Internationally, this study provides a useful contribution to the approach to the sylvatic cycle of parvovirus in wild carnivores.

Efeito do extrato aquoso de própolis marrom sobre a produção de IFN- Após imunização contra parvovírus canino (CPV) e coronavírus canino (CCoV) / Effect of water extract from brown propolis on production of IFN- After immunization against canine parvovirus (CPV) and canine coronavirus (CCoV)

The purpose of this study was to evaluate the imunnostimulatory adjuvant capacity of water extract from brown propolis (WEBP) when added to a vaccine against canine parvovirus (CPV) and canine coronavirus (CCoV), regarding the production of IFN-?. Mice were vaccinated with CPV and CCoV (3.0 x 106 TCID50) with or without 400 µg/dose of WEBP. Thirty days after the third dose, splenocytes were cultured to measure the expression levels of IFN-? mRNA in the immunized animals. Increased levels of IFN-? mRNA expression for CCoV were evidenced by RT-PCR, in the splenocytes of mice inoculated with the vaccine containing 400 ?g/dose of WEBP, demonstrating the ability of propolis to stimulate cellular immune responses against the antigens of this virus. In contrast, the levels of IFN-? to CPV were not influenced by the presence of WEBP.

O objetivo deste estudo foi avaliar a capacidade adjuvante imunoestimulatória do extrato aquoso de própolis marrom (EAPM) quando associado a uma vacina contra parvovírus canino (CPV) e coronavírus canino (CCoV), com relação à produção de IFN-?. Camundongos foram vacinados com CPV e CCoV (3,0x106 TCDI50) em associação ou não com 400 ?g/dose de EAPM. Trinta dias após a terceira dose foi realizado cultivo de esplenócitos para mensuração dos níveis de expressão de mRNA para IFN-? nos animais imunizados. O aumento nos níveis de expressão de mRNA para IFN-? para CCoV nos esplenócitos dos camundongos inoculados com a vacina contendo 400 ?g/dose de EAPM foi evidenciado por RT-PCR, demonstrando a capacidade da própolis em estimular a resposta imune celular contra os antígenos desse vírus. Ao contrário, os níveis de IFN-? para CPV não sofreram influência da presença do EAPM.

Efeito do extrato aquoso de própolis marrom sobre a produção de IFN- Após imunização contra parvovírus canino (CPV) e coronavírus canino (CCoV) / Effect of water extract from brown propolis on production of IFN- After immunization against canine parvovirus (CPV) and canine coronavirus (CCoV)

The purpose of this study was to evaluate the imunnostimulatory adjuvant capacity of water extract from brown propolis (WEBP) when added to a vaccine against canine parvovirus (CPV) and canine coronavirus (CCoV), regarding the production of IFN-?. Mice were vaccinated with CPV and CCoV (3.0 x 106 TCID50) with or without 400 µg/dose of WEBP. Thirty days after the third dose, splenocytes were cultured to measure the expression levels of IFN-? mRNA in the immunized animals. Increased levels of IFN-? mRNA expression for CCoV were evidenced by RT-PCR, in the splenocytes of mice inoculated with the vaccine containing 400 ?g/dose of WEBP, demonstrating the ability of propolis to stimulate cellular immune responses against the antigens of this virus. In contrast, the levels of IFN-? to CPV were not influenced by the presence of WEBP.(AU)

O objetivo deste estudo foi avaliar a capacidade adjuvante imunoestimulatória do extrato aquoso de própolis marrom (EAPM) quando associado a uma vacina contra parvovírus canino (CPV) e coronavírus canino (CCoV), com relação à produção de IFN-?. Camundongos foram vacinados com CPV e CCoV (3,0x106 TCDI50) em associação ou não com 400 ?g/dose de EAPM. Trinta dias após a terceira dose foi realizado cultivo de esplenócitos para mensuração dos níveis de expressão de mRNA para IFN-? nos animais imunizados. O aumento nos níveis de expressão de mRNA para IFN-? para CCoV nos esplenócitos dos camundongos inoculados com a vacina contendo 400 ?g/dose de EAPM foi evidenciado por RT-PCR, demonstrando a capacidade da própolis em estimular a resposta imune celular contra os antígenos desse vírus. Ao contrário, os níveis de IFN-? para CPV não sofreram influência da presença do EAPM.(AU)

Selective regimen shift and demographic growth increase associated with the emergence of high-fitness variants of canine parvovirus.

The natural evolution of Canine parvovirus (CPV) is characterized by a variety of mutations, mainly in the VP1/VP2 gene. Although positive selection has been previously reported in CPV, little is known about its overall contribution to viral adaptation in the canine population. Herein, the influences of the evolutive constraints on CPV during a period of viral adaptation into a previously uninfected population are more clearly investigated. To do this, 31 sequences of VP1/VP2 gene obtained from symptomatic domestic dogs in Brazil were used, sampled from 1980 to 2000. Marked evolutionary changes in CPV associated with a process of fine-tuning adaptation were observed. Specifically, sequences from the 1980s revealed two distinct antigenic types (i.e. 2a and 2b) cocirculating in Brazil. Moreover, analysis of the selective regimen showed that 90% of the VP2 sites were conserved (d(N)/d(S)=0). In contrast, sequences from the 1990s were composed solely of CPV-2a with 96% of VP2 sites under purifying selection (d(N)/d(S)<1) and site 297 under strong positive selection (omega=4.9). Important features regarding the demographic history of CPV in Brazil were also observed. The viral population size passed through a short period of explosive growth that declined and then stabilized into a constant rate of spread. Remarkably, the explosive growth coincided with the appearance of CPV variants that presented a unique repertoire of mutations never before seen in other worldwide strains. The analysis also showed that the estimated nucleotide substitution was similar to those commonly observed in fast evolving RNA viruses. The present results demonstrated the adaptive potential of CPV to acquire, in short interval of 10 years, key mutations in the VP1/VP 2 gene that increased viral fitness and enabled the virus to disseminate even in vaccinated dogs.

Characterization of canine parvovirus (CPV) interactions with 3201 T cells: involvement of GPI-anchored protein(s) in binding and infection.

Binding of canine parvovirus (CPV) to the susceptible feline T cell line 3201 was quantitated by fluorescence-activated cell sorter (FACS) analysis. CPV bound to the cells in a dose-dependent manner, while no binding to the non-permissive MSB-1 avian lymphoma cell line was detected. Binding could be competitively inhibited by addition of excess unlabeled empty capsids, or by pre-incubation of virus with a CPV-specific monoclonal antibody. To characterize the biochemical nature of this binding, live cells were treated with a variety of enzymes prior to use in the binding assay. Treatment with neuraminidase removed a significant proportion of the wild-type virus binding activity, while both proteinase K and phosphatidylinositol-specific phospholipase C (PI-PLC) prevented binding of a non-hemagglutinating (non-HA), non-sialic acid binding mutant to 3201 cells. This suggests that CPV binds to sialic acid expressed on host cells as well as erythrocyte membranes, and that it also binds a protein moiety which is glycosylphosphatidylinositol (GPI)-anchored. The role of these components in CPV infection was also examined by pretreating cells with neuraminidase or PI-PLC prior to inoculating them with either wild-type CPV or the non-hemagglutinating mutant. Neuraminidase treatment had no effect on the ability of CPV to infect the cells, while infectivity was severely compromised by pretreating the cells with either proteinase K or PI-PLC. GPI-anchored proteins on 3201 cells were further characterized by Triton X-114 extraction and reactivity to anti-CRD after PI-PLC treatment.