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Humberto Ramirezmendoza - One of the best experts on this subject based on the ideXlab platform.
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neuraminidase activity of blue eye disease porcine Rubulavirus specificity affinity and inhibition studies
Research in Veterinary Science, 2017Co-Authors: Gerardo Santoslopez, Humberto Ramirezmendoza, Julio Reyesleyva, Maria Del Transito Borrazarguello, Veronica Vallejoruiz, Luis Marquezdominguez, Juan Carlos Floresalonso, Bernard Priem, Sebastien Fort, Irma HerreracamachoAbstract: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.
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acute neurologic disease in porcine Rubulavirus experimentally infected piglets
Virus Research, 2017Co-Authors: Jenifer Herrera, Humberto Ramirezmendoza, Luis Gomeznunez, Rocio Lararomero, Fernando Diosdado, Atalo Martinezlara, Miguel Jasso, Armando Pereztorres, Jose Francisco RiverabenitezAbstract: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.
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co infection of classic swine h1n1 influenza virus in pigs persistently infected with porcine Rubulavirus
Veterinary Microbiology, 2016Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Atalo Martinezlara, Armando Pereztorres, Jazmin De La Luzarmendariz, Manuel Saavedramontanez, Miguel Angel Jassoescutia, Ivan Sanchezbetancourt, Humberto RamirezmendozaAbstract: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.
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respiratory disease in growing pigs after porcine Rubulavirus experimental infection
Virus Research, 2013Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Armando Pereztorres, Sandra Cuevasromero, Humberto RamirezmendozaAbstract: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.
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development of a real time rt pcr method for detection of porcine Rubulavirus porv lpmv
Journal of Virological Methods, 2013Co-Authors: Sandra Cuevasromero, Humberto Ramirezmendoza, Pablo Hernandezjauregui, Anne Lie Blomstrom, Arcelia Alvarado, Francisco Riverabenitez, Mikael BergAbstract: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.
Julio Reyesleyva - One of the best experts on this subject based on the ideXlab platform.
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neuraminidase activity of blue eye disease porcine Rubulavirus specificity affinity and inhibition studies
Research in Veterinary Science, 2017Co-Authors: Gerardo Santoslopez, Humberto Ramirezmendoza, Julio Reyesleyva, Maria Del Transito Borrazarguello, Veronica Vallejoruiz, Luis Marquezdominguez, Juan Carlos Floresalonso, Bernard Priem, Sebastien Fort, Irma HerreracamachoAbstract: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.
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production of an enzymatically active and immunogenic form of ectodomain of porcine Rubulavirus hemagglutinin neuraminidase in the yeast pichia pastoris
Journal of Biotechnology, 2016Co-Authors: Julio Reyesleyva, Gerardo Santoslopez, Sandra Cuevasromero, Nora Rosasmurrieta, Jose Luis Cerritenosanchez, Irma HerreracamachoAbstract: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.
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co infection of classic swine h1n1 influenza virus in pigs persistently infected with porcine Rubulavirus
Veterinary Microbiology, 2016Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Atalo Martinezlara, Armando Pereztorres, Jazmin De La Luzarmendariz, Manuel Saavedramontanez, Miguel Angel Jassoescutia, Ivan Sanchezbetancourt, Humberto RamirezmendozaAbstract: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.
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respiratory disease in growing pigs after porcine Rubulavirus experimental infection
Virus Research, 2013Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Armando Pereztorres, Sandra Cuevasromero, Humberto RamirezmendozaAbstract: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.
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genetic and antigenic changes in porcine Rubulavirus
Canadian Journal of Veterinary Research-revue Canadienne De Recherche Veterinaire, 2012Co-Authors: Jose Ivan Sanchezbetancourt, Julio Reyesleyva, Maria Elena Trujillo, Susana E Mendoza, Rogelio A AlonsoAbstract:Blue eye disease, caused by a porcine Rubulavirus (PoRV), is an emergent viral swine disease that has been endemic in Mexico since 1980. Atypical outbreaks were detected in 1990 and 2003. Growing and adult pigs presented neurological signs, mild neurological signs were observed in piglets, and severe reproductive problems were observed in adults. Amino acid sequence comparisons and phylogenetic analysis of the hemagglutinin-neuraminidase (HN) protein revealed genetically different lineages. We used cross-neutralization assays, with homologous and heterologous antisera, to determine the antigenic relatedness values for the PoRV isolates. We found antigenic changes among several strains and identified a highly divergent one, making up a new serogroup. It seems that genetically and antigenically different PoRV strains are circulating simultaneously in the swine population in the geographical region studied. The cross neutralization studies suggest that the HN is not the only antigenic determinant participating in the antigenic changes among the different PoRV strains.
Jesús Hernández - One of the best experts on this subject based on the ideXlab platform.
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co infection of classic swine h1n1 influenza virus in pigs persistently infected with porcine Rubulavirus
Veterinary Microbiology, 2016Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Atalo Martinezlara, Armando Pereztorres, Jazmin De La Luzarmendariz, Manuel Saavedramontanez, Miguel Angel Jassoescutia, Ivan Sanchezbetancourt, Humberto RamirezmendozaAbstract: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.
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respiratory disease in growing pigs after porcine Rubulavirus experimental infection
Virus Research, 2013Co-Authors: Jose Francisco Riverabenitez, Jesús Hernández, Julio Reyesleyva, Armando Pereztorres, Sandra Cuevasromero, Humberto RamirezmendozaAbstract: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.
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Efficacy of quantitative RT-PCR for detection of the nucleoprotein gene from different porcine Rubulavirus strains
Archives of Virology, 2013Co-Authors: José Francisco Rivera-benitez, Adelfa Del Carmen García-contreras, Julio Reyes-leyva, Jesús Hernández, José Iván Sánchez-betancourt, Humberto Ramírez-mendozaAbstract: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.
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Persistence of porcine Rubulavirus in experimentally infected boars
Veterinary Microbiology, 2013Co-Authors: José Francisco Rivera-benitez, Rebeca Martínez-bautista, Adelfa Del Carmen García-contreras, Julio Reyes-leyva, A. Pérez-torres, Jesús Hernández, Humberto Ramírez-mendozaAbstract: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 5ml of the PAC-3 strain of porcine Rubulavirus at 1??105 TCID50/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
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molecular characterization of the hemagglutinin neuraminidase gene of porcine Rubulavirus isolates associated with neurological disorders in fattening and adult pigs
Research in Veterinary Science, 2008Co-Authors: Jose Ivan Sanchezbetancourt, Jesús Hernández, Humberto Ramirezmendoza, Julio Reyesleyva, Rogelio A Alonso, Gerardo Santoslopez, J M Doporto, S Mendoza, Maria Elena TrujilloAbstract: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.
Mikael Berg - One of the best experts on this subject based on the ideXlab platform.
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cloning expression and characterization of potential immunogenic recombinant hemagglutinin neuraminidase protein of porcine Rubulavirus
Protein Expression and Purification, 2016Co-Authors: Julieta Sandra Cuevasromero, Pablo Hernandezjauregui, Jose Francisco Riverabenitez, Anne Lie Blomstrom, Mikael Berg, Eliseo Hernandezbaumgarten, Marco Vega, Claudia BauleAbstract:Abstract 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 ( r HN-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 r HN-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 r HN-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 r HN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the r HN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease.
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molecular and epidemiological studies of porcine Rubulavirus infection an overview
Infection ecology & epidemiology, 2015Co-Authors: Julieta Sandra Cuevasromero, Anne Lie Blomstrom, Mikael BergAbstract:Porcine Rubulavirus -La Piedad-Michoacan-Mexico virus (PorPV-LPMV) was identified as the causative agent of a viral disease that emerged spontaneously in Mexican swine in the 1980s. Since the report of the initial outbreak of the disease, only one full-length genome from a strain isolated in 1984 (PorPV-LPMV/1984) has been sequenced; sequence data are scarce from other isolates. The genetic variation of this virus that has spread throughout the main endemic region of Mexico is almost a complete mystery. The development of molecular techniques for improved diagnostics and to investigate the persistence, molecular epidemiology, and the possible reservoirs of PorPV are needed. Together, this will provide greater knowledge regarding the molecular genetic changes and useful data to establish new strategies in the control of this virus in Mexico. Keywords: Porcine Rubulavirus; PorPV; PorPV-LPMV; bats; epidemiology; persistent infection; RNA (Published: 18 November 2015) Citation: Infection Ecology and Epidemiology 2015, 5: 29602 - http://dx.doi.org/10.3402/iee.v5.29602
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development of a real time rt pcr method for detection of porcine Rubulavirus porv lpmv
Journal of Virological Methods, 2013Co-Authors: Sandra Cuevasromero, Humberto Ramirezmendoza, Pablo Hernandezjauregui, Anne Lie Blomstrom, Arcelia Alvarado, Francisco Riverabenitez, Mikael BergAbstract: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.
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investigation of t cell responses and viral mrna persistence in lymph nodes of pigs infected with porcine Rubulavirus
Veterinary Immunology and Immunopathology, 2009Co-Authors: Sandra J Cuevas, Seamus Kennedy, Jorge Morenolopez, Mikael Berg, A Rodriguezropon, Pablo HernandezjaureguiAbstract: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.
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both the p and v proteins of the porcine Rubulavirus lpmv interact with the np protein via their respective c terminal unique parts
Virus Research, 2002Co-Authors: Martin Svenda, Tommy Linne, Bernt Hjertner, Mikael BergAbstract:Abstract In this paper we show that the porcine Rubulavirus LPMV phosphoprotein (P) and V protein (V) both interact with the nucleoprotein (NP). There are also indications for an interaction between P and V with L protein. Further analysis of the domains of the P and V which are necessary for interaction with the NP protein demonstrates that the interaction is not mediated from their common part but instead from their unique C-terminal parts, respectively. The common N-terminus of P and V appear to mediate the interaction with L. We also map the regions of NP that are necessary for interaction with P and V, respectively. Both P and V interact with regions of NP, which reside in the N-terminal part but appear not to overlap.
Machiko Nishio - One of the best experts on this subject based on the ideXlab platform.
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Human parainfluenza virus type 2 polymerase complex recognizes leader promoters of other species belonging to the genus Rubulavirus
Medical Microbiology and Immunology, 2017Co-Authors: Yusuke Matsumoto, Keisuke Ohta, Machiko NishioAbstract:Leader sequence, located at the 3′ terminus of paramyxovirus genomes, determines the degree of viral transcription and replication. The essential nucleotides in the leader sequence that influence viral propagation, however, have not been investigated in detail. In the present study, we show that polymerase complex of human parainfluenza virus type 2 (hPIV2) uses a luciferase-encoding hPIV2 mini-genome possessing the leader sequence from other closely related viruses as a template. Furthermore, we demonstrate that although hPIV2 polymerase complex can recognize the leader sequence of hPIV4B, mumps virus (MuV) and PIV5 as well as Newcastle disease virus (NDV), it cannot recognize measles virus, hPIV1, Sendai virus (SeV) or hPIV3. We could obtain the chimeric hPIV2 possessing the leader sequence from hPIV4B, MuV and PIV5, but not from other species, including NDV and SeV. These results reveal that although hPIV2 polymerase complex can recognize the leader sequence from Rubulaviruses to achieve efficient viral infection, this does not apply to viruses belonging to other genus. A comparison of leader sequence nucleotides among paramyxoviruses highlights the importance of the conservation in the first 13 nucleotides for infectious hPIV2 growth.
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Tetherin antagonism by V proteins is a common trait among the genus Rubulavirus
Medical Microbiology and Immunology, 2017Co-Authors: Keisuke Ohta, Yusuke Matsumoto, Machiko NishioAbstract:Tetherin (BST-2/CD317/HM1.24) is an anti-viral factor that restricts the budding of several enveloped viruses. Most of these viruses have evolved to encode tetherin antagonists. Our previous study demonstrated that the growth of human parainfluenza virus type 2 (hPIV-2), a member of the genus Rubulavirus in the family Paramyxoviridae , was inhibited by tetherin, and its V protein decreases the amount of cell surface tetherin by the interaction. In the present study, we investigated whether tetherin inhibits the growth of other Rubulaviruses including PIV-5, mumps virus (MuV), simian virus 41, and hPIV-4, and whether their V proteins act as tetherin antagonists. Plaque assay demonstrated that the growth of PIV-5 and MuV was inhibited by tetherin. Flow cytometry and immunoblot analyses revealed that the infection of PIV-5 and MuV caused reduction of cell surface tetherin without affecting total amount of tetherin. Immunoprecipitation analysis showed that all V proteins of Rubulaviruses tested bound to tetherin. These results suggest that tetherin antagonism by V proteins is common among the genus Rubulavirus .
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Human Parainfluenza Virus Type 2 L Protein Regions Required for Interaction with Other Viral Proteins and mRNA Capping
Journal of Virology, 2010Co-Authors: Machiko Nishio, Masato Tsurudome, Tetsuya Nosaka, Hiroshi Komada, Dominique Garcin, Philippe Le Mercier, Daniel KolakofskyAbstract:The large RNA polymerase (L) protein of human parainfluenza virus type 2 (hPIV2) binds the nucleocapsid, phosphoprotein, and V protein, as well as itself, and these interactions are essential for transcription and replication of the viral RNA genome. Although all of these interactions were found to be mediated through the domains within the N terminus of L, the C terminus of the L protein was also required for minigenome reporter gene expression. We have identified a highly conserved Rubulavirus domain near the C terminus of the L protein that is required for mRNA synthesis but not for genome replication. Remarkably, this region of L shares homology with a conserved region of cellular capping enzymes that binds GTP and forms a lysyl-GMP enzyme intermediate, the first step in the cellular capping reaction. We propose that this conserved region of L also binds GTP (or GDP) to carry out the second step of the unconventional nonsegmented negative-strand virus capping reaction.
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Human parainfluenza virus type 2 V protein inhibits genome replication by binding to the L protein: possible role in promoting viral fitness.
Journal of Virology, 2008Co-Authors: Machiko Nishio, Junpei Ohtsuka, Masato Tsurudome, Tetsuya Nosaka, Daniel KolakofskyAbstract:The human parainfluenza virus type 2 (hPIV2) V protein plays important roles in inhibiting the host interferon response and promoting virus growth, but its role in hPIV2 replication and transcription is not clear. A green fluorescent protein (GFP)-expressing a negative-sense minigenomic construct of hPIV2 has been established by standard technology, with helper plasmids expressing the nucleocapsid protein (NP), phosphoprotein (P), and large RNA polymerase (L) protein, to examine the role of V protein. We found that the simultaneous expression of wild-type V protein in the minigenome system inhibited GFP expression, at least in part, by inhibiting minigenome replication. In contrast, expression of C terminally truncated or mutant hPIV2 V proteins had no effect. Moreover, the V protein of simian virus 41, the Rubulavirus most closely related virus to hPIV2, also inhibited GFP expression, whereas that of PIV5, a more distantly related Rubulavirus, did not. Using these other Rubulavirus V proteins, as well as various mutant hPIV2 V proteins, we found that the ability of V protein to inhibit GFP expression correlated with its ability to bind to L protein via its C-terminal V protein-specific region, but there was no correlation with NP binding. A possible role for this inhibition of genome replication in promoting viral fitness is discussed.
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Identification of Paramyxovirus V Protein Residues Essential for STAT Protein Degradation and Promotion of Virus Replication
Journal of Virology, 2005Co-Authors: Machiko Nishio, Masato Tsurudome, Dominique Garcin, Daniel KolakofskyAbstract:Some paramyxovirus V proteins induce STAT protein degradation, and the amino acids essential for this process in the human parainfluenza virus type 2 (hPIV2) V protein have been studied. Various recombinant hPIV2s and cell lines constitutively expressing various mutant V proteins were generated. We found that V proteins with replacement of Cys residues of the Cys cluster were still able to bind STATs but were unable to induce their degradation. The hPIV2 V protein binds STATs via a W-(X)3-W-(X)9-W Trp motif located just upstream of the Cys cluster. Replacements of two or more Trp residues in this motif resulted in a failure to form a V/STAT2 complex. We have also identified two Phe residues of the hPIV2 V protein that are essential for STAT degradation, namely, Phe207, lying within the Cys cluster, and Phe143, in the P/V common region of the protein. Interestingly, infection of BHK cells with hPIV2 led to the specific degradation of STAT1 and not STAT2. Other evidence for the cell species specificity of hPIV2-induced STAT degradation is presented. Finally, a V-minus hPIV2, which can express only the P protein from its P gene, was generated and partially characterized. In contrast to V-minus viruses of other paramyxovirus genera, this V-minus Rubulavirus was highly debilitated, and its growth even in Vero cells was very limited. The structural Rubulavirus V proteins, as expected, are thus clearly important in promoting virus growth, independent of their anti-interferon (IFN) activity. Interestingly, many of the residues that are essential for anti-IFN activity, e.g., the Cys of this cluster and Phe207 within this cluster, as well as the Trp of this motif, are also essential for promoting virus growth.
