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.

Development of a real-time RT-PCR method for detection of porcine rubulavirus (PoRV-LPMV).

In order to provide a rapid and sensitive method for detection of the Porcine rubulavirus La Piedad-Michoacan-Mexico Virus (PoRV-LPMV), we have developed a specific real-time reverse transcriptase polymerase chain reaction assay. The detection of PoRV-LPMV, represents a diagnostic challenge due to the viral RNA being present in very small amounts in tissue samples. In this study, a TaqMan(®) real-time PCR assay was designed based on the phosphoprotein gene of PoRV-LPMV, to allow specific amplification and detection of viral RNA in clinical samples. Assay conditions for the primers and probe were optimized using infected PK15 cells and ten-fold serial dilutions of a plasmid containing the whole P-gene. The sensitivity of the developed TaqMan(®) assay was approximately 10 plasmid copies per reaction, and was shown to be 1000 fold better than a conventional nested RT-PCR. The performance of this real-time RT-PCR method enables studies of various aspects of PoRV-LPMV infection. Finally, the assay detects all current known variants of the virus.

Investigation of T-cell responses and viral mRNA persistence in lymph nodes of pigs infected with porcine rubulavirus.

Selected lymphocyte subpopulations were studied and the distribution of viral mRNA were investigated during acute and persistent porcine rubulavirus (PoRV-LPMV) infection in Vietnamese pot-bellied pigs. Six pigs infected with PoRV-LPMV at 17 days of age exhibited clinical signs 7-10 days post-inoculation (pi). One infected piglet died 11 days pi while the other five recovered around day 13 pi and survived until euthanasia on day 277 pi. Increased numbers of CD8+, CD4+ and CD2+ T cells were detected during the acute phase of infection while CD8+ cells were elevated throughout the infection, including during the persistent stage. Specific antibodies against the haemagglutinin-neuraminidase protein of PoRV-LPMV were detected during persistent infection. Although infectious virus could not be recovered from tissues from any of the infected pigs at necropsy 277 days pi, PoRV-LPMV mRNA was detected in lymph nodes, pancreas and central nervous system using a nested polymerase chain reaction technique. Continued lymphocyte interaction with viral RNA may be an important factor in promoting cellular and humoral responses during persistent PoRV-LPMV infection.

Identification of potential B cell epitope determinants by computer techniques, in hemagglutinin-neuraminidase from the porcine rubulavirus La Piedad Michoacan.

Hemagglutinin-neuraminidase (HN) from porcine rubulavirus La Piedad Michoacan (RvpLPM) is one of the most antigenic proteins known, and is responsible for virus-host cell interaction. We analyzed the amino acid sequence of HN, using computer-assisted techniques to identify B cell epitopes. From a pool of 18 possible antigenic peptides, we evaluated the antigenicity of the 2 peptides with the highest scores and the 1 with lowest score. Antibodies from RvpLPM-infected pigs recognized the synthesized HN-A, HN-B, and HN-R peptides (optical density [OD]: 0.33 +/- 0.02 for HN-A, 0.20 +/- 0.02 for HN-B, and 0.07 +/- 0.01 for HN-R); bovine serum albumin-coupled HN-A and HN-B induced rabbit anti-RvpLPM antibodies (OD: 0.39 +/- 0.01 for HN-A and 0.35 +/- 0.02 for HN-B). Loop 5 from the outer membrane protein, OmpC, from Salmonella typhi was replaced with HN-B; this protein was then expressed in Escherichia coli UH302. BALB/c mice were challenged intraperitoneally or orogastrically with the fusion protein expressed in E. coli and murine antibodies obtained from both types of administration inhibited virus-hemagglutinating activity, as did the antibodies from RvpLPM-infected swine. These results suggest that HN-A and HN-B are peptides involved in RvpLPM cell carbohydrate recognition, and could therefore be considered potential targets for vaccine and diagnostic procedures development.

Estudio comparativo de prevalencia de hiperplasia epitelial focal en tres grupos poblacionales del estado de Puebla / Comparative study of focal epithelial hyperplasia prevalence among 3 populations in Puebla State

Se presenta un trabajo de investigación de prevalencia epidemiológica que describe la cantidad de desórdenes existentes de HEF en una población particular. Los resultados obtenidos mostraron niveles bajos de dicha alteración, con lo que se concluyó que dicha alteración no constituye un problema de salud pública en la zona estudiada

Localización con inmunoperoxidasa de antígenos rábicos en la superficie celular en cultivos celulares infectados a baja multiplicidad / Immunoperoxidase cell surface localization of rabies virus antigen in tissue cultures at a low viral multiplicity

Con el propósito de observar los antígenos del virus rábico en cultivos celulares se utilizaron las inmunoglobulinas provenientes de una yegua hiperinmunizada, se acoplaron los anticuerpos antivirus rábico a peroxidasa tipo VI, se incubaron con células 13 SCL infectadas con virus rábico a las 24, 48 y 72 horas, y se oservaron a los microscopios de luz y electrónico. Con el primero no se vieron sitios de reconocimiento de antígeno; sin embargo, con el microscopio electrónico fue posible observar el precipitado del complejo de inmunoperoxidasa en la superficie de los virus y de la membrana celular de células infectadas. El sistema permite evaluar cuantitativamente y con resolución ultraestructural los sitios de reconocimiento de superficie de antígenos del virus rábico

Microanastomosis arterial por invaginación: estudio arteriográfico y de microscopias de luz y electrónica de barrido / Microarterial anstomosis by invagination: light microscopy and scanning electron microscopy

Se estudiaron las imágenes angiográficas y los cambios histológicos de microanastomosis arteriales en las que se utilizaron dos puntos de sutura de monofilamento de nailon del número 10-0 e invaginación del cabo proximal dentro del cabo distal de la arteria en 18 carótidas de rata Sprague Dawley. Los animales se sacrificaron a los tres minutos, una hora, un dia, una semana, un mes y tres meses después del procedimiento. Antes del sacrificio se hizo angiografia, y después los especímenes se estudiaron con microscopias de luz y electrónica de barrido. Inicialmente se observó plegamiento longitudinal del cabo proximal invaginado dentro del distal, con estrechamiento anular a nivel de la anastomosis sin oclusión arterial por trombosis. Desde el mes después del procedimiento hubo recuperación total del diámetro interior de la arterial al nivel de las anastomosis