Pablo Hernández-Jáuregui

The structural proteins of a porcine paramyxovirus (LPMV).

The porcine paramyxovirus is a newly identified agent of a fatal disease in piglets, endemic in Mexico since 1980, where it was seen around the town of La Piedad, Michoacan, Mexico (hence LPM virus). At least six [35S]methionine-labelled proteins could be resolved by SDS-PAGE and five of them were clearly immunoprecipitated. Selective labelling of LPMV-infected cells with [3H]glucosamine revealed two bands with an Mr of about 66K and 59K, corresponding to the two viral glycoproteins, the haemagglutinin-neuraminidase protein and the fusion protein. Labelling of virus with [32P]orthophosphate disclosed one band with an Mr of 52K, corresponding to the phosphoprotein. Analysis of nucleocapsids obtained from purified virus or from a permanently infected cell line revealed one major band with an Mr of 68K, the nucleoprotein. Two other proteins were also identified, the large protein and the matrix protein, with apparent Mr of about 200K and 40K, respectively. The protein migration pattern of LPMV was compared, by SDS-PAGE, with that of Newcastle disease virus, bovine parainfluenza 3 virus and Sendai virus. Differences in the Mr of LPMV proteins and the proteins of these paramyxoviruses were observed. We propose that LPMV should be classified as a novel member of the genus Paramyxovirus.

Immunity to porcine rubulavirus infection in adult swine

The immune response against the porcine rubulavirus was analyzed in experimentally infected adult pigs. High titers of virus neutralizing and hemagglutinating inhibitory antibodies were identified in infected animals. The antibody specificity was directed towards HN, M, and NP rubula virion proteins; immunodominance of HN proteins was demonstrated. Peripheral blood mononuclear cells from infected, but not from non-infected pigs proliferated in vitro in response to virus antigenic stimuli, showing a bell-shaped plot with the highest peak at 5 weeks post-infection. Virus-induced lymphoblasts expressed CD4+ CD8+ phenotype, whereas lectin-induced lymphoblasts were mainly identified as CD4+ CD8- cells. Phenotype analysis of freshly prepared PBMC revealed increased number of both monocytes (PoM1+) and total T lymphocytes (CD2+) early during infection, with reduced values of B lymphocytes at 4 weeks post-infection. Decrease in CD4+ CD8- blood cells was observed at 3 weeks post-infection, whereas both CD4- CD8+ and CD4+ CD8+ cells increased 1 and 4 weeks post-infection, respectively. This work discusses the relevance of CD4+ CD8+ T cells in the control of porcine rubulavirus infection.

Human papillomavirus type 13 DNA in focal epithelial hyperplasia among Mexicans

SummaryHuman papillomavirus (HPV) type 13 DNA was detected in focal epithelial hyperplasia lesions of the oral mucosa in seven half-caste mexicans. The lesions contained intracellular papillomavirus-like particles with a diameter of about 50 nm. DNA extracted from biopsies contained unintegrated HPV type 13 DNA genomes as revealed by Southern blot hybridization. The HPV 13 DNA that was isolated in the present study had the same restriction enzyme cleavage map as HPV 13 DNA, previously described by others. It was moreover confirmed that HPV type 13 genome is related to the genomes of HPV types 6 and 11.

The porcine paramyxovirus LPM specifically recognizes sialyl (α 2,3) lactose-containing structures

SummaryThe porcine paramyxovirus LPM recognizes α, but not β, anomers of sialic acid containing structures, specifically sialyl (α 2,3) lactose. The virus specificity is directed to the sialyl residue and to the C′ 4 axial OH and the C′ 6 CH2OH of the galactose present in this structure.

NeuAcα2,3Gal-Glycoconjugate Expression Determines Cell Susceptibility to the Porcine Rubulavirus LPMV

Relevance of membrane sialoglycoconjugates as receptors for infection by the porcine rubulavirus has been determined in vitro by sugar and lectin competition assays and by inhibition of glycosylation. Our results show that NeuAc alpha 2,3Gal but not NeuAc alpha 2,6Gal inhibits the virus infectivity of Vero cells, and the virus was effectively blocked with the lectin Maackia amurensis, specific for NeuAc alpha 2,3Gal. Inhibition of the cellular glycosylation with tunicamycin, deoxinojirimycin as well as neuraminidase treatment diminishes the viral capacity to bind and infect this cell line. Dexamethasone, which promotes the activity of sialyl alpha 2,6 glycosyltransferase, also diminishes the cell susceptibility for infection. This is the first report confirming that NeuAc alpha-2,3Gal recognition is determinant in the pathogenesis of the porcine rubulavirus.

Experimental porcine rubulavirus (La Piedad-Michoacan virus) infection in pregnant gilts.

Porcine rubulavirus (La Piedad-Michoacan virus) (PoRV-LPMV) is a member of the Paramyxoviridae family that causes encephalitis in young piglets and infertility in adult sows and boars. Infertility in sows naturally infected by PoRV-LPMV is characterized by an increased number of returns to oestrus, stillbirths and mummified fetuses. In this study, nine seronegative gilts were inoculated intranasally with the PAC-3 strain of PoRV-LPMV at week 6 or 10 of gestation. These animals were then killed at weeks 8 or 15 of gestation (seven gilts) or after natural parturition (two gilts). Four control gilts were mock-infected at gestation week 6 or 10 and killed between 2 and 4 weeks later. Gross lesions of focal congestion and haemorrhage were seen in the placenta and endometrium of one gilt infected at gestation week 6 and one infected at gestation week 10. PoRV-LPMV was isolated, at 2-6 weeks post-inoculation (pi), from lung, tonsils, ovary, placenta, uterus and lymph nodes of three of the gilts infected at gestation week 6 and at 2-3 weeks pi from lung, tonsil and ovary of two gilts infected at gestation week 10. Many of the fetuses of eight infected gilts were smaller than normal and had dermal ecchymoses. Dehydrated or mummified fetuses were present in six of the infected gilts but not in any control animal. PoRV-LPMV was isolated from brain, lung and liver of fetuses from two gilts infected at gestation week 6, and from two infected at gestation week 10. These results indicate that, after experimental infection, PoRV can replicate in tissues of seronegative pregnant gilts, cross the placenta, and cause fetal death and mummification.

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.

Uptake of Porcine Rubulavirus (LPMV) by PK-15 Cells

BACKGROUND The porcine virus denominated La Piedad Michoacan Virus (LPMV) is a member of the family Paramyxoviridae and is the cause of a disease in pigs present only in Mexico. The disease is characterized by meningoencephalitis and respiratory distress in young pigs, epididymitis and orchitis in boars, and reproductive failure and abortion in sows. METHODS The cytopathology, morphology, and distribution of the hemagglutination neuraminidase (HN) and nucleoprotein (NP) proteins of LPMV were investigated following inoculation into PK-15 cells. The cytopathic effect was characterized by cytoplasmic vacuolation and the formation of syncytia and cytoplasmic inclusion bodies. RESULTS In immunofluorescence assays using a monoclonal antibody (MAb) against the HN protein at 5-60 min post-infection (early infection), a diffuse immunofluorescence was observed near the cell membrane and adjacent to the nuclear membrane. At 24 h post-infection (late infection), a dust-like immunofluorescence was observed throughout the cytoplasm. LPMV-infected cells incubated with the MAb against the NP protein showed punctate cytoplasmic fluorescence during the early stages of infection. At the late infection stage, these fluorescent particles became larger and were seen predominantly in the cytoplasm of syncytia. This pattern was also apparent by immunohistochemical labeling and immunogold electron microscopy. The latter technique revealed that HN protein was diffusely distributed throughout the cytoplasm. When using the MAb against the NP protein, nucleocapsid organization was the most prominent feature and resulted in the formation of cytoplasmic inclusion bodies visible by light and electron microscopy. Immunogold labeling of purified nucleocapsids was shown by electron microscopy. Virus particles and nucleocapsids were morphologically similar to members of the Paramyxoviridae family. CONCLUSIONS The morphologic characteristics of the virions and the distribution patterns of the HN and NP proteins in PK-15 infected cells indicate that the mechanisms of LPMV replication are generally similar to those of the members of the Paramyxoviridae family.

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.

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

Abstract 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.