Cloning, expression and characterization of potential immunogenic recombinant hemagglutinin-neuraminidase protein of Porcine rubulavirus.

Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P < 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease.

Antigenic differentiation of classical swine fever vaccinal strain PAV-250 from other strains, including field strains from Mexico.

Twenty-nine classical swine fever virus (CSFv) strains were grown in the PK15 or SK6 cell lines. Antigenic differentiation studies were performed using monoclonal antibodies (McAbs), produced at Lelystad (CDI-DLO), The Netherlands. The monoclonals which were classified numerically as monoclonals 2-13. Epitope map patterns that resulted from the reactivity with McAbs were found to be unrelated to the pathogenicity of the viruses studied. Antigenic determinants were recognized by McAbs 5 and 8, were not detected in some Mexican strains; however, sites for McAb 6 were absent in all strains. The PAV-250 vaccine strain was recognized by all MAbs, except by MAb 6. Furthermore, the Chinese C-S vaccine strain was found to be very similar to the GPE(-) vaccine. None of the studied Mexican vaccines or field strains was found to be similar to the PAV-250 vaccine strain.

Use of reverse transcription-polymerase chain reaction to determine the stability of rabies virus genome in brains kept at room temperature.

In tropical and subtropical climates, the shipment of animal brains for rabies diagnosis may be a problem because brain specimens sometimes arrive decomposed at the diagnostic laboratory. In this situation, reverse transcription-polymerase chain reaction (RT-PCR) may serve as a potential solution because of its high sensitivity. However, little is known about the stability of rabies viral RNA in decomposed brain tissue. To determine the stability of rabies virus genomic RNA in brain samples, 72 mice were inoculated with the challenge virus strain-11 of rabies virus. After incubation period, mice were euthanized to obtain their brains. These were categorized in 2 different groups. In the first group, 36 brains were kept at room temperature (25-27 degrees C) immediately after euthanasia. In the second group, the other 36 inoculated brains were frozen at -70 degrees C and later maintained at room temperature. In both groups, RT-PCR was performed at days 0, 1, 2, 3, 4, 7, 10, 12, 16, 18, 23, and 26 by using primers previously described in the literature and a primer set specifically designed for a Mexican variant of vampire-bat rabies. Reverse-transcriptase PCR experiments were performed in 3 different inoculated brains, in which the direct fluorescent antibody (DFA) test was previously conducted to detect rabies viral antigen in the brains kept at room temperature and in the frozen brains. The DFA test resulted positive in both groups up to day 7. In brain samples stored at ambient temperature (25-27 degrees C), the intensity of the RT-PCR band started to diminish by day 12; however, rabies virus genome could be successfully amplified by RT-PCR up to 23 days. These results indicate that brain samples kept at ambient temperature (up to 27 degrees C) may reach a reference laboratory in an adequate state for rabies diagnosis by RT-PCR.

Caracterización antigénica de cepas del virus del ectima contagioso (orf), sus interacciones y sus relaciones con parapoxvirus bovinos de México / Antigenic characterization of contagious ecthyma virus (orf) in Mexico, their relationships and interactions with bovine parapoxvirus samples

Treinta y tres muestras de costra positivas a ectima contagioso (orf) (19 de caprino y 14 ovinas) y dos de parapoxvirus bovinos (una de estomatitis papular y otra de seudoviruela) fueron analizadas antigénicamente y comparadas por doble inmunodifusión (DD) y contrainmunoelectroforesis (CIE). Las muestras bovinas fueron obtenidas de animales que convivían con rebaños de borregos y cabras. La presencia de partículas virales fue confirmada por microscopía electrónica en todos los casos. Las pruebas fueron corridas usando un suero policlonal antiectima preparado en conejo. En la prueba de DD la muestra homóloga produjo tres líneas de precipitación, las otras muestras variaron entre dos y una líneas de precipitación y algunas muestras no formaron ninguna, se observaron diferentes resultados con las pruebas de DD y CIE. Las cepas bovinas formaron dos líneas de precipitación, una de identidad entre ellas y otra en identidad con la muestra homóloga de orf caprino y con una de orf ovino, por DD. La composición de 15 muestras de orf (6 ovinas y 9 caprinas) y dos bovinas fueron examinadas por electroforesis, y 7 de ellas evaluadas antigénicamente por inmunotransferencia, usando el suero policlonal de conejo. La muestra homóloga fue más compleja, pues mostró 12 proteínas. La mayor variación en los corrimientos electroforéticos de las muestras se observó entre las proteínas de los pesos de 37 kd a 44 kd. Una proteína de 55 kd se identificó en todas las muestras, mientras que una de 9.5 kd se evidenció en 13 muestras y otra de 17 kd en 12, incluyendo las bovinas. Por inmunotransferencia fueron reconocidas dos proteínas comunes en las siete muestras analizadas con pesos de 55 y 54 kd, otras dos proteínas de 45 y 35 kd fueron reconocidas por el suero de conejo en cinco de las muestras. Estas proteínas pueden explicar las reacciones cruzadas observadas entre las muestras, en las pruebas de DD y CIE y ser de importancia en el reconocimiento inmune de estos virus.

Lesiones por parapoxvirus en veterinarios de México / Parapoxvirus infection in veterinary practitioners of México

Se describen tres casos de lesiones en las manos médicos de veterinarios, producidas por parapoxvirus. En dos de los casos, las lesiones pudieron atribuirse al virus del ectima contagioso, tomando en cuenta la historia clínica. En todos los casos el diagnóstico se confirmó mediante la observación por microscopía electrónica de las partículas virales características (Tipo 2 o C)

Biological properties of three mexican isolates of aujeszky's disease virus

Four strains of Aujeszky's Disease virus (ADV) were included in this study; three Mexican field isolates (215,145 nad C-8) in conjunction with the shope reference strain of ADV, which has known pathogeic characteristics. All four strains were included in each treatment, which consisted of heat treatment, trypsin treatment and passed ten times on chicken embryo fibroblasts. Both virus titer and plaque size were determined on the first and tenth passage and on treated and untreated strains. On each of the treatments, the plaque size had significant differences (p=0.001) which had relation to the two factors studied, namely strains and passage level. There was no significant variation related to the type of treatment between strains. With the strains under study, the authors also made rabbit pathogenicity tests, and it was found that on passage one, the strains caused clear nerovus symptoms and death, while on the tenth passage elvel, the Mexican strains produced slight pruritus, few nervous symptoms and allowed the rabbits to survive. The mouse test revealed an increased median death time after the treatments, as well as a large increase in standard deviations. These data are interpreted as an increased heterogeneity of the strains in all of the treatments to the strains of viruses