RESUMO
The rabies virus (RABV) is characterized by a history dominated by host shifts within and among bats and carnivores. One of the main outcomes of long-term RABV maintenance in dogs was the establishment of variants in a wide variety of mesocarnivores. In this study, we present the most comprehensive phylogenetic and phylogeographic analysis, contributing to a better understanding of the origins, diversification, and the role of different host species in the evolution and diffusion of a dog-related variant endemic of South America. A total of 237 complete Nucleoprotein gene sequences were studied, corresponding to wild and domestic species, performing selection analyses, ancestral states reconstructions, and recombination analyses. This variant originated in Brazil and disseminated through Argentina and Paraguay, where a previously unknown lineage was found. A single host shift was identified in the phylogeny, from dog to the crab-eating fox (Cerdocyon thous) in the Northeast of Brazil. Although this process occurred in a background of purifying selection, there is evidence of adaptive evolution -or selection of sub-consensus sequences- in internal branches after the host shift. The interaction of domestic and wild cycles persisted after host switching, as revealed by spillover and putative recombination events.
Assuntos
Vírus da Raiva/genética , Vírus da Raiva/isolamento & purificação , Raiva/veterinária , Animais , Animais Domésticos/virologia , Animais Selvagens/virologia , Doenças do Cão/epidemiologia , Doenças do Cão/virologia , Cães , Evolução Molecular , Raposas/virologia , Nucleoproteínas/genética , Filogenia , Raiva/epidemiologia , Raiva/virologia , Vírus da Raiva/classificação , Recombinação Genética , América do Sul/epidemiologiaRESUMO
We report an outbreak of bovine paralytic rabies in a feed lot in a disease-free area in southern Santa Fe, Argentina. Six of 122 unvaccinated steers died after showing neurological signs (morbimortality = 4.9%) for 24-72 hours, 40-75 days after being transported to the feed lot from a farm located in Formosa. Pathologic examination of the brain in 3 steers revealed gliosis, lymphoplasmacytic meningoencephalitis, and intracytoplasmic inclusion bodies in neurons, along with intralesional detection of the rabies virus antigen by immunohistochemistry in 2 cases. Rabies virus was confirmed by direct immunofluorescence in the brain, and further identified as variant 3a, typical of hematogenous bats, by inoculation in mice followed by indirect immunofluorescence. This represents the first communication of bovine paralytic rabies described in the Pampas plain in Argentina, and evidences that the transport of cattle from endemic to disease-free areas could represent a mechanism of dissemination of this communicable zoonotic disease.
Assuntos
Quirópteros , Vírus da Raiva , Raiva , Animais , Argentina/epidemiologia , Bovinos , Surtos de Doenças , Camundongos , Raiva/epidemiologia , Raiva/veterináriaRESUMO
We developed a fast, rabies virus-free, in vitro method, based on a blocking ELISA (bELISA), to detect and accurately quantify anti-rabies glycoprotein antibodies in serum of several animal species. In this method, purified rabies virus-like particles (VLPs) are used as antigen to coat the plates, while the presence of specific rabies immunoglobulins is revealed through blocking the recognition of these VLPs by a biotinylated monoclonal antibody. A quality by design approach was carried out in order to optimize the method performance, improving the sensitivity and, thereby, reducing the limit of detection of this assay. After the method validation, we confirmed that the bELISA method is able to detect a concentration of 0.06 IU/mL rabies immunoglobulins, titer lower than the 0.5 IU/mL cutoff value established as indication for correct vaccination. Further, we assessed the correlation between bELISA, the MNT, and the Platelia methods, confirming the accuracy of this new assay. On the other hand, precision was evaluated, obtaining acceptable repeatability and intermediate precision values, showing that this bELISA could be proposed as a potential alternative method, replacing the gold standard techniques in vaccination schemes and becoming a routine control technique within regional rabies surveillance programs.
Assuntos
Anticorpos Bloqueadores/imunologia , Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Raiva/sangue , Raiva/diagnóstico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/sangue , Gatos , Bovinos , Cães , Humanos , Limite de Detecção , Panthera , Raiva/imunologia , Vírus da Raiva/imunologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate. In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 IU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 IU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary interest.
Assuntos
Antígenos Virais/imunologia , Vírus da Varíola dos Canários/imunologia , Glicoproteínas/imunologia , Vacina Antirrábica , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Vírus da Varíola dos Canários/genética , Vírus da Varíola dos Canários/crescimento & desenvolvimento , Vírus da Varíola dos Canários/isolamento & purificação , Linhagem Celular/virologia , Embrião de Galinha , Chlorocebus aethiops , Cricetinae , Fibroblastos/virologia , Glicoproteínas/genética , Rim , Mesocricetus , Camundongos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Raiva/prevenção & controle , Vacina Antirrábica/imunologia , Organismos Livres de Patógenos Específicos , Vacinas Sintéticas/imunologia , Células Vero/virologia , Proteínas do Envelope Viral/genética , Cultura de VírusRESUMO
En la Argentina, la rabia está circunscripta a algunas provincias del norte. La disponibilidad de nuevas vacunas que eliminen la manipulación del virus rábico y que permitan el control de la enfermedad es de importancia estratégica nacional y regional. Las vacunas basadas en poxvirus recombinantes se han utilizado con éxito como vacunas antirrábicas a nivel mundial. SI bien estos sistemas no están disponibles comercialmente, la plataforma de obtención de virus canarypox (CNPV) recombinantes ya ha sido implementada en nuestro laboratorio. El objetivo de este trabajo fue obtener y evaluar un candidato a vacuna antirrábica basado en CNPV recombinantes que expresan la glicoproteína G (RG) del virus rábico (RV). Se construyó un virus recombinante que expresa la secuencia codificante de RG (CNPV-RG). La inoculación de ratones con este virus indujo altos títulos de anticuerpos seroneutralizantes de RV (3,58 y 9,76 Ul/ml después de una o dos inmunizaciones, respectivamente) y protegió al 78 % de los animales desafiados intracerebralmente con RV. Además, se determinó que el CNPV-RG posee una potencia relativa de 3,5 Ul/ml. Los resultados obtenidos constituyen la primera etapa en la evaluación del CNPV-RG como candidato a vacuna antirrábica. Se requerirán nuevos ensayos para confirmar su utilidad en especies de interés veterinario.
In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 lU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 lU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary Interest.
Assuntos
Animais , Embrião de Galinha , Cricetinae , Camundongos , Antígenos Virais/imunologia , Vírus da Varíola dos Canários/imunologia , Glicoproteínas/imunologia , Vacina Antirrábica , Proteínas do Envelope Viral/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Chlorocebus aethiops , Vírus da Varíola dos Canários/genética , Vírus da Varíola dos Canários/crescimento & desenvolvimento , Vírus da Varíola dos Canários/isolamento & purificação , Linhagem Celular/virologia , Fibroblastos/virologia , Glicoproteínas/genética , Rim , Mesocricetus , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Vacina Antirrábica/imunologia , Raiva/prevenção & controle , Organismos Livres de Patógenos Específicos , Cultura de Vírus , Vacinas Sintéticas/imunologia , Células Vero/virologia , Proteínas do Envelope Viral/genéticaRESUMO
En la Argentina, la rabia está circunscripta a algunas provincias del norte. La disponibilidad de nuevas vacunas que eliminen la manipulación del virus rábico y que permitan el control de la enfermedad es de importancia estratégica nacional y regional. Las vacunas basadas en poxvirus recombinantes se han utilizado con éxito como vacunas antirrábicas a nivel mundial. SI bien estos sistemas no están disponibles comercialmente, la plataforma de obtención de virus canarypox (CNPV) recombinantes ya ha sido implementada en nuestro laboratorio. El objetivo de este trabajo fue obtener y evaluar un candidato a vacuna antirrábica basado en CNPV recombinantes que expresan la glicoproteína G (RG) del virus rábico (RV). Se construyó un virus recombinante que expresa la secuencia codificante de RG (CNPV-RG). La inoculación de ratones con este virus indujo altos títulos de anticuerpos seroneutralizantes de RV (3,58 y 9,76 Ul/ml después de una o dos inmunizaciones, respectivamente) y protegió al 78 % de los animales desafiados intracerebralmente con RV. Además, se determinó que el CNPV-RG posee una potencia relativa de 3,5 Ul/ml. Los resultados obtenidos constituyen la primera etapa en la evaluación del CNPV-RG como candidato a vacuna antirrábica. Se requerirán nuevos ensayos para confirmar su utilidad en especies de interés veterinario.(AU)
In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 lU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 lU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary Interest.(AU)
Assuntos
Animais , Embrião de Galinha , Cricetinae , Camundongos , Antígenos Virais/imunologia , Vírus da Varíola dos Canários/imunologia , Glicoproteínas/imunologia , Vacina Antirrábica , Proteínas do Envelope Viral/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Vírus da Varíola dos Canários/genética , Vírus da Varíola dos Canários/crescimento & desenvolvimento , Vírus da Varíola dos Canários/isolamento & purificação , Linhagem Celular/virologia , Chlorocebus aethiops , Fibroblastos/virologia , Glicoproteínas/genética , Rim , Mesocricetus , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Raiva/prevenção & controle , Vacina Antirrábica/imunologia , Organismos Livres de Patógenos Específicos , Vacinas Sintéticas/imunologia , Células Vero/virologia , Proteínas do Envelope Viral/genética , Cultura de VírusRESUMO
BACKGROUND: Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites. Rabies virus (RABV) circulates through two different epidemiological cycles: terrestrial and aerial, where dogs, foxes or skunks and bats, respectively, act as the most relevant reservoirs and/or vectors. It is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory. METHODS: We studied the positivity rate of RABV detection in different areas of the country, and the antigenic and genetic diversity of 99 rabies virus (RABV) strains obtained from 14 species of insectivorous bats collected in Argentina between 1991 and 2008. RESULTS: Based on the analysis of bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats ranged from 3.1 to 5.4%, depending on the geographic location. The findings were distributed among an extensive area of the Argentinean territory. The 99 strains of insectivorous bat-related sequences were divided into six distinct lineages associated with Tadarida brasiliensis, Myotis spp, Eptesicus spp, Histiotus montanus, Lasiurus blosseviilli and Lasiurus cinereus. Comparison with RABV sequences obtained from insectivorous bats of the Americas revealed co-circulation of similar genetic variants in several countries. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples. CONCLUSIONS: This study demonstrates the presence of several independent enzootics of rabies in insectivorous bats of Argentina. This information is relevant to identify potential areas at risk for human and animal infection.
Assuntos
Quirópteros/virologia , Vírus da Raiva/classificação , Vírus da Raiva/isolamento & purificação , Raiva/veterinária , Animais , Argentina/epidemiologia , Análise por Conglomerados , Epidemias , Epidemiologia Molecular , Dados de Sequência Molecular , Filogeografia , RNA Viral/genética , Raiva/epidemiologia , Raiva/virologia , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Análise de Sequência de DNA , SorotipagemRESUMO
The nucleoprotein genes of 54 human, domestic and wild animals rabies isolates obtained in Argentina between 1995 and 2002 were characterized using monoclonal antibodies and partial gene sequence analysis. The antigenic and genetic diversities of rabies virus in samples from bat and bat-related cases were studied, leading to the identification of five distinct genetic variants. Rabies viruses isolated from vampire bat related cases were very similar to each other, showing 98.9% overall similarity. Specific antigenic variants (AgV) were detected associated with different insectivorous bats species, in samples from Tadarida brasiliensis and Eumops patagonicus bats. In contrast, isolates from Myotis sp. and Histiotus sp. bats could not be matched to any antigenic type. Additionally, bat rabies cases were also detected in southern provinces previously considered rabies-free. Finally, two independent antigenic and genetic variants co-circulating in northern Argentina were found in isolates obtained from dogs and dog-related cases, suggesting two independent cycles of virus transmission. This is the first national coordinated study of antigenic as well as molecular epidemiology of rabies in Argentina. The information presented here will improve our knowledge about rabies epidemiology and therefore, will assist preventing fatal human cases.
Assuntos
Antígenos Virais/análise , Genes Virais , Vírus da Raiva/classificação , Vírus da Raiva/isolamento & purificação , Raiva/virologia , Animais , Argentina , Bovinos , Quirópteros , DNA Complementar/química , DNA Viral/química , Cães , Variação Genética , Humanos , Epidemiologia Molecular , Dados de Sequência Molecular , Nucleoproteínas/genética , Filogenia , Raiva/veterinária , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Análise de Sequência de DNA , Proteínas Virais/genéticaRESUMO
Este trabajo intenta establecer apreciaciones diagnósticas surgidas de la investigación clínica y corroborarlas con los procesos de mentalización detectados en los relatos del T.A.T. Participaron en la muestra 30 niños de 6 a 8 años, con diagnóstico médico de asma bronquial. Un equipo de trabajo analizó caso por caso el material sin T.A.T., otro profundizó el análisis cuantitativo y cualitativo del T.A.T., a ciegas. Conclusiones: 1) un mismo síntoma somático no tiene valor en sí mismo, su aparición depende de la organización y funcionamiento psíquico del niño; 2) las apreciaciones diagnósticas surgidas de la investigación clínica se corroboraron con las obtenidas en el TAT a) Niños con características neuróticas presentan mejor calidad de mentalización. El síntoma somático toma el carácter de episodio ó fenómeno reorganizativo de la homeoestasis psicosomática. Los relatos TAT muestran: existencia de conflicto intrapsíquico, vida fantasmática expresada a través de dramatizaciones, etc. b) Niños con rasgos de carácter o de comportamiento evidencian déficit en la mentalización, pobreza del mundo representacional, carente de significatividad libidinal, y poseen una organización psíquica con vulnerabilidad somática. Los relatos TAT presentan procedimientos que traducen: mecanismos de inhibición del conflicto y del pensamiento y de descarga a nivel del comportamiento o carácter; 3) El TAT aplicado a partir de los 6 años (propuesta de R. Debray) nos revela la calidad de la mentalización, de los recursos defensivos y el tipo de relaciones objetales; permitiéndonos precisar con fineza el valor funcional del asma bronquial en las diferentes organizaciones psíquicas infantiles Palabras-chave: Psicosomática; asma bronquial; mentalización; T.A.T. en ninõs; diagnóstico (AU)