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Julio Reyesleyva - One of the best experts on this subject based on the ideXlab platform.

  • neuraminidase activity of blue eye disease Porcine Rubulavirus specificity affinity and inhibition studies
    Research in Veterinary Science, 2017
    Co-Authors: G Santoslopez, Humberto Ramirezmendoza, Julio Reyesleyva, Maria Del Transito Borrazarguello, Veronica Vallejoruiz, Luis Marquezdominguez, Juan Carlos Floresalonso, Bernard Priem, Sebastien Fort, Irma Herreracamacho
    Abstract:

    Abstract Porcine Rubulavirus (PorPV), also known as La Piedad Michoacan Virus (LPMV) causes encephalitis and reproductive failure in newborn and adult pigs, respectively. The hemagglutinin–neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PorPV infection; i.e. , it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid releasing) activity has been proposed as a virulence factor. This work describes the purification and characterization of PorPV HN protein (isolate PAC1). The specificity of neuraminidase is restricted to sialyl(α2,3)lactose (3SL). HN showed typical Michaelis-Menten kinetics with fetuin as substrate (km = 0.029 μM, Vmax = 522.8 nmol min − 1  mg − 1 ). When 3SL was used as substrate, typical cooperative kinetics were found (S 50  = 0.15 μM, Vmax = 154.3 nmol min − 1  mg − 1 ). The influenza inhibitor zanamivir inhibited the PorPV neuraminidase with IC 50 of 0.24 μM. PorPV neuraminidase was activated by Ca 2 + and inhibited by nucleoside triphosphates with the level of inhibition depending on phosphorylation level. The present results open possibilities to study the role of neuraminidase in the pathogenicity of PorPV infection and its potential inhibitors.

  • production of an enzymatically active and immunogenic form of ectodomain of Porcine Rubulavirus hemagglutinin neuraminidase in the yeast pichia pastoris
    Journal of Biotechnology, 2016
    Co-Authors: Julio Reyesleyva, G Santoslopez, Sandra Cuevasromero, Nora Rosasmurrieta, Jose Luis Cerritenosanchez, Irma Herreracamacho
    Abstract:

    Blue-eye disease (BED) of swine is a viral disease endemic in Mexico. The etiological agent is a paramyxovirus classified as Porcine Rubulavirus (PoRV-LPMV), which exhibits in its envelope the hemagglutinin-neuraminidase (HN) glycoprotein, the most immunogenic and a major target for vaccine development. We report in this study the obtaining of ectodomain of PoRV HN (eHN) through the Pichia pastoris expression system. The expression vector (pPICZαB-HN) was integrated by displacement into the yeast chromosome and resulted in a Mut+ phenotype. Expressed eHN in the P. pastoris X33 strain was recovered from cell-free medium, featuring up to 67 nmol/min/mg after 6 days of expression. eHN was recognized by the serum of infected pigs with strains currently circulating in the Mexican Bajio region. eHN induces antibodies in mice after 28 days of immunization with specific recognition in ELISA test. These antibodies were able to inhibit >80% replication by viral neutralization assays in cell culture. These studies show the obtaining of a protein with similar characteristics to the native HN and which may be a candidate to propose a vaccine or to use the antigen in a serologic diagnostic test.

  • co infection of classic swine h1n1 influenza virus in pigs persistently infected with Porcine Rubulavirus
    Veterinary Microbiology, 2016
    Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Atalo Martinezlara, Armando Pereztorres, Jazmin De La Luzarmendariz, Manuel Saavedramontanez, Miguel Angel Jassoescutia, Ivan Sanchezbetancourt, Humberto Ramirezmendoza
    Abstract:

    Porcine Rubulavirus (PorPV) and swine influenza virus infection causes respiratory disease in pigs. PorPV persistent infection could facilitate the establishment of secondary infections. The aim of this study was to analyse the pathogenicity of classic swine H1N1 influenza virus (swH1N1) in growing pigs persistently infected with Porcine Rubulavirus. Conventional six-week-old pigs were intranasally inoculated with PorPV, swH1N1, or PorPV/swH1N1. A mock-infected group was included. The co-infection with swH1N1 was at 44 days post-infection (DPI), right after clinical signs of PorPV infection had stopped. The pigs of the co-infection group presented an increase of clinical signs compared to the simple infection groups. In all infected groups, the most recurrent lung lesion was hyperplasia of the bronchiolar-associated lymphoid tissue and interstitial pneumonia. By means of immunohistochemical evaluation it was possible to demonstrate the presence of the two viral agents infecting simultaneously the bronchiolar epithelium. Viral excretion of PorPV in nasal and oral fluid was recorded at 28 and 52 DPI, respectively. PorPV persisted in several samples from respiratory tissues (RT), secondary lymphoid organs (SLO), and bronchoalveolar lavage fluid (BALF). For swH1N1, the viral excretion in nasal fluids was significantly higher in single-infected swH1N1 pigs than in the co-infected group. However, the co-infection group exhibited an increase in the presence of swH1N1 in RT, SLO, and BALF at two days after co-infection. In conclusion, the results obtained confirm an increase in the clinical signs of infection, and PorPV was observed to impact the spread of swH1N1 in analysed tissues in the early stage of co-infection, although viral shedding was not enhanced. In the present study, the interaction of swH1N1 infection is demonstrated in pigs persistently infected with PorPV.

  • respiratory disease in growing pigs after Porcine Rubulavirus experimental infection
    Virus Research, 2013
    Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Armando Pereztorres, Sandra Cuevasromero, Humberto Ramirezmendoza
    Abstract:

    Abstract The aim of this study was to analyze the pathogenicity and distribution of Porcine Rubulavirus (PorPV) in the respiratory tract of experimentally infected pigs. Nine 6-week-old pigs were infected with PorPV and examined clinically. Blood, nasal swab, and tissue samples were collected on different days post-infection (DPI). The humoral immune responses and viral loads were evaluated. The infected pigs exhibited an increase in the respiratory clinical signs. In addition, the excretion of PorPV was extended to 23 DPI in the nasal fluid. The distribution of PorPV in the respiratory tract tissues was extended until the end of the experiment; soft palate tonsil and lymph nodes exhibited high viral loads. The major microscopic lesions observed in the lungs corresponded to interstitial pneumonia and hyperplasia of the associated lymphoid tissue. In conclusion, PorPV infection causes a pneumonic disease characterized by a prolonged virus excretion and high viral load in the lymphoid tissues.

  • persistence of Porcine Rubulavirus in experimentally infected boars
    Veterinary Microbiology, 2013
    Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Armando Pereztorres, Rebeca Martinezbautista, A C Garciacontreras, Humberto Ramirezmendoza
    Abstract:

    Porcine Rubulavirus is the etiological agent of blue eye disease in pigs. In boars, this virus causes orchitis and epididymitis and reduces seminal quality. The objective of this study was to determine the persistence of Porcine Rubulavirus in experimentally infected boars. Nine 12-month-old boars were infected with 5 ml of the PAC-3 strain of Porcine Rubulavirus at 1 × 10(5) TCID(50)/ml and held for 142 days post infection (DPI) to evaluate humoral immune response. The virus was isolated in cell cultures and detected by RT-PCR. Infection with Porcine Rubulavirus produced clinical signs beginning at 5 DPI. Necropsy results showed that 3 boars had lesions in the testicles and epididymes. Histological analysis showed the characteristic lesions in all infected boars. Porcine Rubulavirus antibodies were detected in the second week post infection and increased significantly (P<0.05) over time. Isolation of the virus from semen was achieved between 5 DPI and 48 DPI and from the testicles and epididymes between 64 DPI and 142 DPI. Viral RNA was detected in the serum between 2 DPI and 64 DPI and in the semen until 142 DPI. These results confirm that the RNA of the Porcine Rubulavirus persists in the semen and that this virus remains in the reproductive tract for prolonged periods of infection. Semen of persistently infected boars, therefore, represents an important source of the virus and a risk factor for the spread of blue eye disease in swine populations.

Edgar Zenteno - One of the best experts on this subject based on the ideXlab platform.

  • identification of potential b cell epitope determinants by computer techniques in hemagglutinin neuraminidase from the Porcine Rubulavirus la piedad michoacan
    Viral Immunology, 2007
    Co-Authors: Roberto Zentenocuevas, Humberto Ramirezmendoza, Pablo Hernandezjauregui, Julio Reyesleyva, Sara Huertayepez, Cesar Gonzalezbonilla, Concepcion Agundis, Edgar Zenteno
    Abstract:

    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...

  • caracteristicas de la respuesta inmune de cerdos infectados con el Rubulavirus porcino characteristics of the immune response of pigs infected with Porcine Rubulavirus
    2004
    Co-Authors: Jesús Hernández, Julio Reyesleyva, Humberto Ramirez, Olivia Valenzuela, Edgar Zenteno
    Abstract:

    Porcine Rubulavirus is responsible for blue eye disease in Porcine, which is characterized by neurological signs, corneal opacity and high mortality in young pigs. Reproductive disorders such as epididymitis and testicular atrophy are also common in sexually mature pigs. Since its identification at the beginning of 1980´s, many researches have identified the main molecular, pathological, biological and immunological characteristics of the disease. This review discusses the recent advances related to the humoral and cellular immune response to Porcine Rubulavirus. The importance of the HemagglutininNeuraminidase (HN) protein as an inducer of specific antibodies during infection and the importance of CD4+CD8- lymphocytes during early immune responses and CD4+CD8+ lymphocytes in memory response are also discussed.

  • comparative evaluation of the cd4 cd8 and cd4 cd8 lymphocytes in the immune response to Porcine Rubulavirus
    Veterinary Immunology and Immunopathology, 2001
    Co-Authors: Jesús Hernández, Yonathan Garfias, Alejandro Nieto, Carmen Mercado, Luis F Montano, Edgar Zenteno
    Abstract:

    Abstract The Porcine immune system is unique in the expression of CD4+CD8+ (double-positive, DP) lymphocytes. These cells have been associated with immunological memory due to their gradual increase with age, the expression of memory phenotype and their ability to respond to recall viral antigen. This work analyzes the biological function of CD4+CD8− and CD4+CD8+ lymphocytes in the immune response to Porcine Rubulavirus (PRv). CD4+CD8− cells isolated from pigs 3 weeks after infection with Porcine Rubulavirus proliferated in response to homologous virus and generated lymphoblasts which were predominantly of the CD4+CD8+ phenotype, whereas stimulation with mitogen induced proliferation but did not switch the phenotype. CD4+CD8− lymphocytes isolated after 10 weeks of infection proliferated in response to phytohemagglutinin (PHA) but did not proliferate in response to homologous virus and did not change their phenotype, whereas CD4+CD8+ lymphocytes proliferated in response to PHA and to viral antigen. The cytokine profile of both lymphocyte populations showed the presence of IL-2 and IL-10 transcripts, quantitation demonstrated that CD4+CD8+ cells expressed mainly IL-10, whereas CD4+CD8− lymphocytes expressed primarily IL-2. Our results show that CD4+CD8− lymphocytes in the early phase of Porcine Rubulavirus infection can be converted to double-positive cells expressing IL-10 in an antigen-dependent manner, and that CD4+CD8− T-cells late in infection do not acquire CD8.

  • Purification and characterization of the Hemagglutinin-Neuraminidase of Porcine Rubulavirus LPMV
    Glycoconjugate Journal, 1999
    Co-Authors: Julio Reyes-leyva, Jesús Hernández, Blanca Espinosa, Roberto Zenteno, Veronica Vallejo, Gerardo Santos, Edgar Zenteno
    Abstract:

    The Hemagglutinin-Neuraminidase (HN) from the LPMV strain of Porcine Rubulavirus was purified from virions by ultracentrifugation in a continuous 20–60% sucrose gradient and by ion exchange chromatography. The HN is a glycoprotein of 66 kDa constituted by 50.5, 13.3 and 13.6% of non polar, uncharged polar, and charged polar amino acids, respectively. The HN contains 4% of carbohydrates, its glycannic portion is constituted by Man, Gal, GlcNAc, GalBAc, and Neu5Ac in 3:3:4:1:1 molar ratios. The HN possesses hemagglutinating activity in the presence of erythrocytes from several animal species, including human ABO, and treating the erythrocytes with neuraminidase or pronase abolishes this activity. The binding specificity of the purified HN was determined by hapten inhibition assays, indicating that the hemagglutinating activity of the HN is specific for sialic acid and Neu5Acα2,3Gal-containing structures.

  • immunity to Porcine Rubulavirus infection in adult swine
    Veterinary Immunology and Immunopathology, 1998
    Co-Authors: Jesús Hernández, Pablo Hernandezjauregui, Julio Reyesleyva, Humberto Ramirez, Roberto Zenteno, Edgar Zenteno
    Abstract:

    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. Virusinduced lymphoblasts expressed CD4 a CD8 a phenotype, whereas lectin-induced lymphoblasts were mainly identified as CD4 a CD8 ˇ cells. Phenotype analysis of freshly prepared PBMC revealed increased number of both monocytes (PoM1 a ) and total T lymphocytes (CD2 a ) early during infection, with reduced values of B lymphocytes at 4 weeks post-infection. Decrease in CD4 a CD8 ˇ blood cells was observed at 3 weeks post-infection, whereas both CD4 ˇ CD8 a and CD4 a CD8 a cells increased 1 and 4 weeks post-infection, respectively. This work discusses the relevance of CD4 a CD8 a T cells in the control of Porcine Rubulavirus infection. # 1998 Elsevier Science B.V.

Humberto Ramirezmendoza - One of the best experts on this subject based on the ideXlab platform.

  • neuraminidase activity of blue eye disease Porcine Rubulavirus specificity affinity and inhibition studies
    Research in Veterinary Science, 2017
    Co-Authors: G Santoslopez, Humberto Ramirezmendoza, Julio Reyesleyva, Maria Del Transito Borrazarguello, Veronica Vallejoruiz, Luis Marquezdominguez, Juan Carlos Floresalonso, Bernard Priem, Sebastien Fort, Irma Herreracamacho
    Abstract:

    Abstract Porcine Rubulavirus (PorPV), also known as La Piedad Michoacan Virus (LPMV) causes encephalitis and reproductive failure in newborn and adult pigs, respectively. The hemagglutinin–neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PorPV infection; i.e. , it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid releasing) activity has been proposed as a virulence factor. This work describes the purification and characterization of PorPV HN protein (isolate PAC1). The specificity of neuraminidase is restricted to sialyl(α2,3)lactose (3SL). HN showed typical Michaelis-Menten kinetics with fetuin as substrate (km = 0.029 μM, Vmax = 522.8 nmol min − 1  mg − 1 ). When 3SL was used as substrate, typical cooperative kinetics were found (S 50  = 0.15 μM, Vmax = 154.3 nmol min − 1  mg − 1 ). The influenza inhibitor zanamivir inhibited the PorPV neuraminidase with IC 50 of 0.24 μM. PorPV neuraminidase was activated by Ca 2 + and inhibited by nucleoside triphosphates with the level of inhibition depending on phosphorylation level. The present results open possibilities to study the role of neuraminidase in the pathogenicity of PorPV infection and its potential inhibitors.

  • acute neurologic disease in Porcine Rubulavirus experimentally infected piglets
    Virus Research, 2017
    Co-Authors: Jenifer Herrera, Humberto Ramirezmendoza, Luis Gomeznunez, Rocio Lararomero, Fernando Diosdado, Atalo Martinezlara, Miguel Jasso, Armando Pereztorres, Jose Francisco Riverabenitez
    Abstract:

    Abstract The objective of this study was to evaluate the clinical disease, humoral response and viral distribution of recent Porcine Rubulavirus (PorPV) isolates in experimentally infected pigs. Four, 6-piglet (5-days old) groups were employed (G1-84, G2-93, G3-147, and G4-T). Three viral strains were used for the experimental infection: the reference strain LPMV-1984 (Michoacan 1984) and two other strains isolated in 2013, one in Queretaro (Qro/93/2013) and the other in Michoacan (Mich/147/2013). Each strain was genetically characterized by amplification and sequencing of the gene encoding hemagglutinin-neuroamidase (HN). The inoculation was performed through the oronasal and ocular routes, at a dose of 1 × 10 6  TCID 50 /ml. Subsequently, the signs were evaluated daily and necropsies were performed on 3 different days post infection (dpi). We recorded all micro- and macroscopic lesions. Organs from the nervous, lymphatic, and respiratory system were analyzed by quantifying the viral RNA load and the presence of the infectious virus. The presence of the viral antigen in organs was evidenced through immunohistochemistry. Seroconversion was evaluated through the use of a hemagglutination inhibition test. In the characterization of gene HN, only three substitutions were identified in strain Mich/147/2013, two in strain LPMV/1984 (fourth passage) and one in strain Qro/93/2013, with respect to reference strain LPMV-84, these changes had not been identified as virulence factors in previously reported strains. Neurological alterations associated with the infection were found in all three experimental groups starting from 3 dpi. Groups G1-84 and G3-147 presented the most exacerbated nervous signs. Group G2-93 only presented milder signs including slight motor incoordination, and an increased rectal temperature starting from day 5 post infection (PI). The main histopathological findings were the presence of a mononuclear inflammatory infiltrate (lymphocytic/monocytic) surrounding the ventricles in the brain and focal interstitial pneumonitis with distention of the alveolar sacs in the lungs. PorPV and RNA distribution were identified in the organs of the nervous, lymphatic, and respiratory systems of the piglets analyzed at different times (days 5, 10, and 15 PI). The viral antigen was detected in the brain and lungs in most of the assessed groups. Seroconversion was evident in groups G1-84 and G2-93. Groups G1-84 and G3-147 were the most clinically affected by the experimental infection. Both strains were isolated in the state of Michoacan. The virulence of the new isolates maintains similar characteristics to those reported more than 30 years ago.

  • co infection of classic swine h1n1 influenza virus in pigs persistently infected with Porcine Rubulavirus
    Veterinary Microbiology, 2016
    Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Atalo Martinezlara, Armando Pereztorres, Jazmin De La Luzarmendariz, Manuel Saavedramontanez, Miguel Angel Jassoescutia, Ivan Sanchezbetancourt, Humberto Ramirezmendoza
    Abstract:

    Porcine Rubulavirus (PorPV) and swine influenza virus infection causes respiratory disease in pigs. PorPV persistent infection could facilitate the establishment of secondary infections. The aim of this study was to analyse the pathogenicity of classic swine H1N1 influenza virus (swH1N1) in growing pigs persistently infected with Porcine Rubulavirus. Conventional six-week-old pigs were intranasally inoculated with PorPV, swH1N1, or PorPV/swH1N1. A mock-infected group was included. The co-infection with swH1N1 was at 44 days post-infection (DPI), right after clinical signs of PorPV infection had stopped. The pigs of the co-infection group presented an increase of clinical signs compared to the simple infection groups. In all infected groups, the most recurrent lung lesion was hyperplasia of the bronchiolar-associated lymphoid tissue and interstitial pneumonia. By means of immunohistochemical evaluation it was possible to demonstrate the presence of the two viral agents infecting simultaneously the bronchiolar epithelium. Viral excretion of PorPV in nasal and oral fluid was recorded at 28 and 52 DPI, respectively. PorPV persisted in several samples from respiratory tissues (RT), secondary lymphoid organs (SLO), and bronchoalveolar lavage fluid (BALF). For swH1N1, the viral excretion in nasal fluids was significantly higher in single-infected swH1N1 pigs than in the co-infected group. However, the co-infection group exhibited an increase in the presence of swH1N1 in RT, SLO, and BALF at two days after co-infection. In conclusion, the results obtained confirm an increase in the clinical signs of infection, and PorPV was observed to impact the spread of swH1N1 in analysed tissues in the early stage of co-infection, although viral shedding was not enhanced. In the present study, the interaction of swH1N1 infection is demonstrated in pigs persistently infected with PorPV.

  • respiratory disease in growing pigs after Porcine Rubulavirus experimental infection
    Virus Research, 2013
    Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Armando Pereztorres, Sandra Cuevasromero, Humberto Ramirezmendoza
    Abstract:

    Abstract The aim of this study was to analyze the pathogenicity and distribution of Porcine Rubulavirus (PorPV) in the respiratory tract of experimentally infected pigs. Nine 6-week-old pigs were infected with PorPV and examined clinically. Blood, nasal swab, and tissue samples were collected on different days post-infection (DPI). The humoral immune responses and viral loads were evaluated. The infected pigs exhibited an increase in the respiratory clinical signs. In addition, the excretion of PorPV was extended to 23 DPI in the nasal fluid. The distribution of PorPV in the respiratory tract tissues was extended until the end of the experiment; soft palate tonsil and lymph nodes exhibited high viral loads. The major microscopic lesions observed in the lungs corresponded to interstitial pneumonia and hyperplasia of the associated lymphoid tissue. In conclusion, PorPV infection causes a pneumonic disease characterized by a prolonged virus excretion and high viral load in the lymphoid tissues.

  • development of a real time rt pcr method for detection of Porcine Rubulavirus porv lpmv
    Journal of Virological Methods, 2013
    Co-Authors: Sandra Cuevasromero, Pablo Hernandezjauregui, Anne Lie Blomstrom, Arcelia Alvarado, Francisco Riverabenitez, Humberto Ramirezmendoza
    Abstract:

    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.

Jesús Hernández - One of the best experts on this subject based on the ideXlab platform.

  • co infection of classic swine h1n1 influenza virus in pigs persistently infected with Porcine Rubulavirus
    Veterinary Microbiology, 2016
    Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Atalo Martinezlara, Armando Pereztorres, Jazmin De La Luzarmendariz, Manuel Saavedramontanez, Miguel Angel Jassoescutia, Ivan Sanchezbetancourt, Humberto Ramirezmendoza
    Abstract:

    Porcine Rubulavirus (PorPV) and swine influenza virus infection causes respiratory disease in pigs. PorPV persistent infection could facilitate the establishment of secondary infections. The aim of this study was to analyse the pathogenicity of classic swine H1N1 influenza virus (swH1N1) in growing pigs persistently infected with Porcine Rubulavirus. Conventional six-week-old pigs were intranasally inoculated with PorPV, swH1N1, or PorPV/swH1N1. A mock-infected group was included. The co-infection with swH1N1 was at 44 days post-infection (DPI), right after clinical signs of PorPV infection had stopped. The pigs of the co-infection group presented an increase of clinical signs compared to the simple infection groups. In all infected groups, the most recurrent lung lesion was hyperplasia of the bronchiolar-associated lymphoid tissue and interstitial pneumonia. By means of immunohistochemical evaluation it was possible to demonstrate the presence of the two viral agents infecting simultaneously the bronchiolar epithelium. Viral excretion of PorPV in nasal and oral fluid was recorded at 28 and 52 DPI, respectively. PorPV persisted in several samples from respiratory tissues (RT), secondary lymphoid organs (SLO), and bronchoalveolar lavage fluid (BALF). For swH1N1, the viral excretion in nasal fluids was significantly higher in single-infected swH1N1 pigs than in the co-infected group. However, the co-infection group exhibited an increase in the presence of swH1N1 in RT, SLO, and BALF at two days after co-infection. In conclusion, the results obtained confirm an increase in the clinical signs of infection, and PorPV was observed to impact the spread of swH1N1 in analysed tissues in the early stage of co-infection, although viral shedding was not enhanced. In the present study, the interaction of swH1N1 infection is demonstrated in pigs persistently infected with PorPV.

  • respiratory disease in growing pigs after Porcine Rubulavirus experimental infection
    Virus Research, 2013
    Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Armando Pereztorres, Sandra Cuevasromero, Humberto Ramirezmendoza
    Abstract:

    Abstract The aim of this study was to analyze the pathogenicity and distribution of Porcine Rubulavirus (PorPV) in the respiratory tract of experimentally infected pigs. Nine 6-week-old pigs were infected with PorPV and examined clinically. Blood, nasal swab, and tissue samples were collected on different days post-infection (DPI). The humoral immune responses and viral loads were evaluated. The infected pigs exhibited an increase in the respiratory clinical signs. In addition, the excretion of PorPV was extended to 23 DPI in the nasal fluid. The distribution of PorPV in the respiratory tract tissues was extended until the end of the experiment; soft palate tonsil and lymph nodes exhibited high viral loads. The major microscopic lesions observed in the lungs corresponded to interstitial pneumonia and hyperplasia of the associated lymphoid tissue. In conclusion, PorPV infection causes a pneumonic disease characterized by a prolonged virus excretion and high viral load in the lymphoid tissues.

  • Efficacy of quantitative RT-PCR for detection of the nucleoprotein gene from different Porcine Rubulavirus strains
    Archives of Virology, 2013
    Co-Authors: José Francisco Rivera-benitez, Adelfa Del Carmen García-contreras, Julio Reyes-leyva, Jesús Hernández, José Iván Sánchez-betancourt, Humberto Ramírez-mendoza
    Abstract:

    Blue-eye disease is an emergent viral swine infection caused by Porcine Rubulavirus (PoRV). We have developed a qRT-PCR method to detect and quantify expression of the nucleoprotein gene for different PoRV strains. The limit of detection for this assay was 10^2 copies of synthetic RNA. Viral RNA from PoRV was detectable at a TCID_50 of 0.01. Significant differences were observed between viral RNA quantification and virus titration results for nine PoRV strains. For nasal and oral swab samples that were collected from experimentally infected pigs, the qRT-PCR assay was more sensitive (87.1–83.9 %) for the detection of positive samples than methods involving isolation of virus. The implementation of highly sensitive assays that yield results quickly will be of great assistance in the eradication of PoRV from Mexico. We also believe that the newly developed qRT-PCR assay will help reduce the spread of this viral infection to other countries.

  • persistence of Porcine Rubulavirus in experimentally infected boars
    Veterinary Microbiology, 2013
    Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Armando Pereztorres, Rebeca Martinezbautista, A C Garciacontreras, Humberto Ramirezmendoza
    Abstract:

    Porcine Rubulavirus is the etiological agent of blue eye disease in pigs. In boars, this virus causes orchitis and epididymitis and reduces seminal quality. The objective of this study was to determine the persistence of Porcine Rubulavirus in experimentally infected boars. Nine 12-month-old boars were infected with 5 ml of the PAC-3 strain of Porcine Rubulavirus at 1 × 10(5) TCID(50)/ml and held for 142 days post infection (DPI) to evaluate humoral immune response. The virus was isolated in cell cultures and detected by RT-PCR. Infection with Porcine Rubulavirus produced clinical signs beginning at 5 DPI. Necropsy results showed that 3 boars had lesions in the testicles and epididymes. Histological analysis showed the characteristic lesions in all infected boars. Porcine Rubulavirus antibodies were detected in the second week post infection and increased significantly (P<0.05) over time. Isolation of the virus from semen was achieved between 5 DPI and 48 DPI and from the testicles and epididymes between 64 DPI and 142 DPI. Viral RNA was detected in the serum between 2 DPI and 64 DPI and in the semen until 142 DPI. These results confirm that the RNA of the Porcine Rubulavirus persists in the semen and that this virus remains in the reproductive tract for prolonged periods of infection. Semen of persistently infected boars, therefore, represents an important source of the virus and a risk factor for the spread of blue eye disease in swine populations.

  • molecular characterization of the hemagglutinin neuraminidase gene of Porcine Rubulavirus isolates associated with neurological disorders in fattening and adult pigs
    Research in Veterinary Science, 2008
    Co-Authors: Jose Ivan Sanchezbetancourt, Jesús Hernández, Humberto Ramirezmendoza, Julio Reyesleyva, G Santoslopez, Rogelio A Alonso, J M Doporto, S. Mendoza, Maria Elena Trujillo
    Abstract:

    Abstract “Blue eye disease” is a viral infection of swine endemic in Mexico, which produces fatal encephalitis accompanied by respiratory signs and corneal opacity in suckling piglets. An atypical blue eye disease outbreak presented high rates of neurological signs in fattening and adult pigs from 2000 to 2003. In order to identify the basis of increased neurovirulence, the hemagglutinin-neuraminidase (HN) gene of several Porcine Rubulavirus isolates were sequenced and compared with that of La Piedad Michoacan virus and other isolates that did not produce neurological disorders in weaned pigs. Nine amino acid mutations distinguished the high neurovirulent PAC6–PAC9 viruses, whereas five mutations characterized the low neurovirulent PAC2 and PAC3 viruses. HN protein three-dimensional models showed that the main conformation and functional domains were preserved, although substitutions A223T and A291D occurred in PAC2 and PAC3 viruses, as well as A511K and E514K presented in PAC6–PAC9 viruses considerably modified the properties of the HN protein surface. The increased positive charge of the HN protein of PAC6–PAC9 viruses seems to be associated with their increased neurovirulence.

Jorge Morenolopez - One of the best experts on this subject based on the ideXlab platform.

  • 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
    Virus Research, 2014
    Co-Authors: Sandra Cuevasromero, Pablo Hernandezjauregui, Eliseo Hernandezbaumgarten, S. Kennedy, Mikael Berg, Jorge Morenolopez
    Abstract:

    Abstract 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 260 bp 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.

  • investigation of t cell responses and viral mrna persistence in lymph nodes of pigs infected with Porcine Rubulavirus
    Veterinary Immunology and Immunopathology, 2009
    Co-Authors: Sandra J Cuevas, Seamus Kennedy, Jorge Morenolopez, A Rodriguezropon, Pablo Hernandezjauregui
    Abstract:

    Abstract 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.

  • experimental Porcine Rubulavirus la piedad michoacan virus infection in pregnant gilts
    Journal of Comparative Pathology, 2004
    Co-Authors: Pablo Hernandezjauregui, Ramirez H Mendoza, Jorge Morenolopez, Mercado C Garcia, S. Kennedy
    Abstract:

    Abstract 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.

  • apoptosis in lymph nodes and changes in lymphocyte subpopulations in peripheral blood of pigs infected with Porcine Rubulavirus
    Journal of Comparative Pathology, 2003
    Co-Authors: A Rodriguezropon, Pablo Hernandezjauregui, Jorge Morenolopez, L Sancheztorres, L Favilacastillo, Sergio Estradaparra, S. Kennedy
    Abstract:

    Abstract In a first experiment, five pigs were inoculated intranasally with Porcine Rubulavirus (PoRV) at 5 days of age and killed 7 days post-infection (pi). In a second experiment, four pigs were infected with the same virus at 17 days of age and killed at 9 or 15 days pi. Control piglets in each experiment received uninfected cell culture supernate. All PoRV-infected pigs developed respiratory and nervous signs, and histological lesions of non-suppurative encephalitis and interstitial pneumonia. All control pigs remained clinically normal and did not have histological lesions. Significantly increased numbers of apoptotic cells were detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) in tonsil and lymph nodes of the pigs infected at 7 days of age and killed at 7 days pi. Significantly increased percentages of CD2 + and CD8 + T lymphocytes were also found in peripheral blood of these animals at this time, while the percentages of CD4 + and MHC class II lymphocytes were significantly reduced. Significantly increased numbers of apoptotic cells were detected in lymphoid tissues of the pigs infected at 17 days of age and killed at 9 days pi. The percentages of CD2 + , CD8 + and MHC class II lymphocytes in peripheral blood were also significantly increased at this time; the percentage of MHC class II lymphocytes remained elevated at 15 days pi. These results indicate that induction of apoptosis is an important mechanism in the pathogenesis of PoRV infection in young pigs, and that this virus induces changes in lymphocyte subpopulations in peripheral blood.

  • development of a blocking elisa for screening antibodies to Porcine Rubulavirus la piedad michoacan virus
    Journal of Veterinary Diagnostic Investigation, 1999
    Co-Authors: Ann Nordengrahn, Ann-christin Bergvall, M Svenda, Jorge Morenolopez, Francis Mcneilly, Gordon Allan, P. Hernández, Malik Merza
    Abstract:

    A blocking enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to Porcine Rubulavirus (La Piedad Michoacan Virus (LPMV)) in serum samples from pigs. The test, based on a monoclonal antibody against the LPMV hemagglutinin-neuraminidase glycoprotein, had a sensitivity of 99% and a specificity of 97%. The results of this test were in agreement with those obtained by an indirect ELISA and hemagglutination inhibition, indirect immunofluorescence, and virus neutralization tests. The blocking ELISA is considered the most suitable test for routine screening for antibodies against LPMV. The family Paramyxoviridae is classified into 4 gen- era: Paramyxovirus, Rubulavirus, Morbillivirus , and Pneumovirus. 13 These viruses have been identified as the causative agents of a variety of diseases in both humans and animals. Some of these viruses, such as Newcastle disease virus in chickens, 3 canine distemper virus in dogs, 1 and measles and mumps viruses in hu- mans, 5,15 can affect the central nervous system. The Porcine Rubulavirus was originally isolated from the brain of a piglet showing signs of a central nervous system disorder, pneumonia, and corneal opacity. 9 The disease was first observed in 1980 during an outbreak of encephalitis in piglets on farms around the town La Piedad, district of Michoacan, Mexico. Since the re- port of the initial outbreak of the disease, the La Pie- dad Michoacan virus (LPMV) has spread throughout Mexico and is now endemic in that country. 16 Because