Molecular characterisation of Porcine rubulavirus (PorPV) isolates from different outbreaks in Mexico.

Since the report of the initial outbreak of Porcine rubulavirus (PorPV) infection in pigs, only one full-length genome from 1984 (PorPV-LPMV/1984) has been characterised. To investigate the overall genetic variation, full-length gene nucleotide sequences of current PorPV isolates were obtained from different clinical cases of infected swine. Genome organisation and sequence analysis of the encoded proteins (NP, P, F, M, HN and L) revealed high sequence conservation of the NP protein and the expression of the P and V proteins in all PorPV isolates. The V protein of one isolate displayed a mutation that has been implicated to antagonise the antiviral immune responses of the host. The M protein indicated a variation in a short region that could affect the electrostatic charge and the interaction with the membrane. One PorPV isolate recovered from the lungs showed a mutation at the cleavage site (HRKKR) of the F protein that could represent an important factor to determine the tissue tropism and pathogenicity of this virus. The HN protein showed high sequence identity through the years (up to 2013). Additionally, a number of sequence motifs of very high amino acid conservation among the PorPV isolates important for polymerase activity of the L protein have been identified. In summary, genetic comparisons and phylogenetic analyses indicated that three different genetic variants of PorPV are currently spreading within the swine population, and a new generation of circulating virus with different characteristics has begun to emerge.

Long-term RNA persistence of porcine rubulavirus (PorPV-LPMV) after an outbreak of a natural infection: the detection of viral mRNA in sentinel pigs suggests viral transmission.

The persistence of porcine rubulavirus (PorPV-LPMV) in five pigs that had survived an outbreak of a natural infection was determined. After the resolution of the outbreak, each animal was housed in an isolation pen together with one sentinel pig. Approximately every 2 months thereafter one group of animals was euthanized and tissue samples taken for virological and serological analysis. Infectious virus was not isolated from any samples; antibodies to PorPV-LPMV were detected in convalescent pigs by virus neutralisation test and blocking ELISA but not in sentinel pigs. PorPV-LPMV mRNA of the nucleoprotein (NP) and phosphoprotein (P) genes was detected by a nested polymerase chain reaction (nPCR) in samples of trigeminal and optic nerves, cervical spinal cord, tonsils, salivary gland, lung and pancreas from convalescent pigs. mRNA was also detected in the midbrain, corpus callosum, or olfactory bulb in four out of five pigs by nRT-PCR, this result was confirmed by the sequencing of a 260bp PCR product of P gene region. The highest average viral copies/µg of total RNA occurred in the olfactory bulb and pancreas tissues of convalescent pigs and midbrain, tonsil and pancreas of sentinel pigs housed with the convalescent pigs. Satellitosis and gliosis of the midbrain, olfactory bulb, corpus callosum, medulla oblongata or choroid plexus were microscopically observed in four convalescent pigs. The control pig remained negative in all tests. The results indicate that PorPV-LPMV mRNA persists and induces a durable humoral immune response in pigs that have recovered from a natural infection. After a possible reactivation of the virus, it was transmitted to sentinel pigs in contact with the convalescent pigs.

Biodetoxification of aflatoxin-contaminated chick feed.

Two studies were done to study detoxification of aflatoxin (AF)-contaminated chick feed with Nocardia corynebacteroides (NC). In the first study, pathogenicity of the bacteria was studied; in the second, the nutritional value of detoxified feed was evaluated. Commercial corn was divided into 2 sublots, one of which was contaminated with AF. Both lots were divided into 2 parts; the first was inoculated with NC. Four corn-soybean diets were prepared from the 4 corn lots. A completely randomized design was used with 2 x 2 factorial arrangement in which the factors were AF contaminated or not and NC inoculated or not. One hundred Ross 308 chicks (1-d-old, male) were used in 4 treatments with 5 repetitions and 5 chickens per cage. Bird weight and feed consumption were recorded weekly. Each week, 1 chick per treatment repetition was killed for histopathologic analysis of liver, kidney, bursa of Fabricius, pancreas, and small intestine (duodenum, jejunum, and ileum) and for analysis by scanning electron microscopy of the 3 sections of the intestine. At 21 d (the end of both experiments), 1 chick per treatment repetition was killed, and moisture, lipid content, and residual AF in liver were detected. Results at 3 wk did not show differences between treatments (P > 0.05) in any of the variables. In the second study, the same methodology was used except that greater levels of AF were used (800 and 1,200 mug of AFB1/kg of feed). Results showed differences (P < 0.05) in body weight, lipid content, and residual AF in liver. Histopathologic studies showed statistical differences in lesion severity in liver, duodenum, and kidney. Scanning electron microscopy analysis showed severe lesions of intestinal mucosa that mainly affected tight junctions in AF treatments. It can be concluded that NC is safe for chicks and may be used to partly detoxify chicken feed contaminated with AF.

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 and C-8) in conjunction with the Shope reference strain of ADV, which has known pathogenic 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 strain 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 nervous symptoms and death, while on the tenth passage level, 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.

A plaque-purified rabies virus (strains V-319) derived from a vampire bat (Desmodus rotundus) in Mexico: vaccine potential.

A plaque-purified experimental rabies vaccine was developed from an isolate (strain V-319) made from a naturally infected vampire bat (Desmodus rotundus). Two different vaccines were prepared; one was live virus and the second was an inactivated rabies virus preparation. The live virus vaccine, as well as a betapropiolactone-inactivated vaccine, gave complete protection to challenge inoculation after 1 year. In contrast, greater than 80% of the non-vaccinated experimental control cattle died of rabies. The live virus vaccine could be given at doses as low as 10(5) PFU without loss of efficacy. It did not cause adverse reactions. More than 10,000 cattle have been vaccinated with the live virus vaccine under field conditions. No rabies deaths occurred in vaccinated cattle during a 2-year postvaccinal period. The serological responses of vaccinated cattle indicated protection that endured at least 1 year.

An ultrastructural and immunofluorescent study of rabies virus in tissue culture for antiviral drug evaluation.

With the purpose of establishing a model for antiviral drug evaluation the ERA and V319 rabies fixed virus strains were studied in tissue cultures, for studies of the rabies virus cycle. Viral incubation was made using a low viral input multiplicity and allowing substances that may alter, cell or virus morphology. Immunofluorescence and electron microscopy studies were carried out for quantitative evaluation of structures involved in rabies virus replication after a 24 hours incubation period. The number of fluorescent cells decreased with the viral dilutions. ERA rabies strain had more fluorescent cells than V319 strain. Also viral structures were commonly found on different penetration steps to the cells, with the ERA strain. Interpretation of our results was made on the basis that ERA strain showed a shorter latency period than the V319 rabies strain. Differences in the replication pattern between fixed rabies strains using similar experimental conditions were found.

Estudio ultraestructural y de inmunofluorescencia de la replicacion en cultivos celulares del virus rabico para la evaluacion de farmacos antivirales. / An ultraestructural and inmunofluorescent study of rabies virus in tissue culture for antiviral drug evaluation

Se estudiaron en cultivos celulares las cepas ERA y V319 de virus rabico para evaluar el ciclo viral. Las incubaciones se llevaron a cabo usando una baja multiplicidad de virus rabico y evitando usar substancias que pudieran alterar la interaccion entre virus y celula. Se realizaron estudios con inmunofluorescencia y con microscopia electronica para evaluar cuantitativamente las estructuras involucradas en la multiplicacion del virus despues de 24 horas de incubacion. El numero de celulas fluorescentes decrecio, como era de esperarse, con la dilucion viral. La cepa ERA mostro mayor numero de celulas fluorescentes, comparada con la cepa V319. Con la cepa ERA fue frecuente demostrar diferentes formas de penetracion del virus a la celula.Los resultados se interpretaron considerando que la cepa ERA mostro un periodo de latencia mas breve en comparacion con la cepa V319. Se encontraron diferencias en el patron de replicacion viral entre las dos cepas de virus fijo estudiadas, no obstante que las condiciones experimentales fueron semejantes

In vitro effect of isoprinosine on rabies virus.

The in vitro effect of isoprinosine, an antiviral compound, was evaluated on ERA and VA319 rabies viral strains. Isoprinosine diminished the number of immunofluorescent cells, viral titers, and structures involved in rabies virus replication. Mechanism of antiviral action was probably mediated by modification of the polyribosome conformation and function.

Adaptation, attenuation and plaque purification of a rabiesvirus isolate (V319) from a vampire bat (Desmodus rotundus).

Five rabiesvirus isolates, four of vampire bat origin and one from the brain of a rabid cow, were studied with respect to their adaptability to tissue culture. Only two strains (V319 and Mazatan strains) adapted readily. One (Mazatan) failed to yield high titers in vitro after isolation, plaque purification and further passage; however, the V319 strain routinely yielded titers of 10(9) PFU/ml. In limited pathogenicity tests in cattle and dogs, the V319 strain was found not to be excreted in the saliva of inoculated animals. Strain V319 was innocuous to cattle and dogs by the intramuscular route of inoculation, but it was still pathogenic by the intracerebral route. It also was found pathogenic for suckling and 21-day-old mice following inoculation by the intracerebral route. If given by the intramuscular route to mice of both ages, however, strain V319 was of low virulence. It was not virulent for mice when administered by the intraperitoneal route. It was concluded that strain V319 (plaque 476) possessed suitable characteristics for a potential vaccine.

Bovine papular stomatitis, first report of the disease in Mexico.

In a herd of 120 two to eight months old calves kept at a Mexican Government experimental station in Ajuchitlan, Querétaro, weight loss and ptialism were observed. Upon a clinical examination, it was found that 31 (25.8%) of the animals disclosed papules in the oral and perioral regions. Biopsies from the affected tissues were studied with the light and electron microscopes. Serological studies and isolation of the virus were also carried out. A Pox virus was identified (240 x 100 x 150 +/- 7% nm) with the electron microscope. Dermatophilus sp. was occasionally observed. Bovine kidney monolayers, inoculated with affected bovine tissues demonstrated cytopathic effect up to the 4th serial passage. Inoculation with cell cultured infectious material in the oral submucosa (cell lysate) produced typical lesions of BPS on a heifer. Infectious tissues from this experimentally inoculated animal produced cytopathic effect in tissue cultured cells after 24 hours, and this last material was infectious for a second young heifer. Virus-neutralization tests, using an hyperimmune serum, disclosed a neutralization index of 1.5 logarithms. It was concluded that bovine papular stomatitis virus was the etiological agent.

Miniature tissue culture technique with a modified plastic microplate.

Plastic microplates were modified to accommodate four glass microscope slides as surfaces for cell cultivation. Tissue cultures grown on these slides can be utilized for fluorescent-antibody and other cytological staining techniques.