José Francisco Rivera-Benitez

Identification of Antigenic Variants of the Porcine Rubulavirus in Sera of Field Swine and their Seroprevalence

We sampled sera from 1013 non-vaccinated swine from four states in Mexico, Guanajuato, Jalisco, Michoacán and the Estado de Mexico, to analyse anti-porcine rubulavirus antibody titres against three different porcine rubulavirus isolates (PAC-4/1993, PAC-6/2001, and PAC-9/2003) using a hemagglutination inhibition assay. The results revealed that there were antigenic differences among the isolates assessed. In particular, the estimated correlation between the PAC-4/1993 and PAC-6/2001 (0.50) isolates and between the PAC-4/1993 and PAC-9/2003 isolates (0.56) displayed a moderate positive correlation. In contrast, there was a strong positive correlation between the PAC-6/2001 and PAC-9/2003 isolates (0.73). We also found that in the state of Guanajuato, PAC-4/1993 was the isolate that was most frequently identified; in Jalisco, the isolate was PAC-6/2001; and in Michoacán, the isolate was PAC-9/2003. By contrast, in the Estado de Mexico, all three isolates appeared to circulate with a low seroprevalence. In general, the analysed sera from the four states displayed a porcine rubulavirus serological prevalence ranging from 9% to 23.7%. These data indicate that there is not complete antibody cross-antigenicity among the three isolates, and the antigenic variations in the antibody response found in this study implies that the use of a monovalent vaccine would not generate complete protection against the different antigenic subtypes.

Persistence of porcine rubulavirus in experimentally infected boars

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.

Evidence of transmission and risk factors for influenza A virus in household dogs and their owners

The possible transmission of influenza A virus between dogs and humans is important, as in Mexico City there are approximately 1·2 million dogs. We present the first evidence of influenza A virus infection in household dogs in Mexico.

Respiratory disease in growing pigs after Porcine rubulavirus experimental infection.

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.

Serological Survey of Veterinarians to Assess the Zoonotic Potential of Three Emerging Swine Diseases in Mexico

We conducted an immunological assay of blood samples taken from 85 swine‐specialist veterinarians attending the Congress of the Mexican Association of Swine Specialist Veterinarians in Mexico in 2011. Serum samples were assayed for Porcine rubulavirus (PorPV), Encephalomyocarditis virus (EMCV) and Leptospira spp. antibodies. Using a hemagglutination inhibition test, we registered 2.3% and 27% seropositivity for PorPV and EMCV, respectively. Using viral neutralization tests, we registered 5.8% and 47% seropositivity for PorPV and EMCV, respectively. For Leptospira spp., we registered a seropositivity of 38.8%. The variables (sex, age, years of exposure, number of visited farms, biosecurity level and region) showed no significant effect (P > 0.05) on the seropositivity for EMCV, PorPV and Leptospira spp. except for number of visited farms on HI seropositivity for EMCV (P < 0.05; odds ratio: 1.38). The data obtained provide information on the epidemiology of emerging diseases with zoonotic potential in occupational risk groups.

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

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

Molecular detection and isolation of avian metapneumovirus in Mexico.

We conducted a longitudinal study to detect and isolate avian metapneumovirus (aMPV) in two highly productive poultry areas in Mexico. A total of 968 breeder hens and pullets from 2 to 73 weeks of age were analysed. Serology was performed to detect aMPV antibodies and 105 samples of tracheal tissue were collected, pooled by age, and used for attempted virus isolation and aMPV nested reverse transcriptase-polymerase chain reaction (nRT-PCR). The serological analysis indicated that 100% of the sampled chickens showed aMPV antibodies by 12 weeks of age. Five pools of pullet samples collected at 3 to 8 weeks of age were positive by nRT-PCR and the sequences obtained indicated 98 to 99% similarity with the reported sequences for aMPV subtype A. Virus isolation of nRT-PCR-positive samples was successfully attempted using chicken embryo lung and trachea mixed cultures with subsequent adaptation to Vero cells. This is the first report of detection and isolation of aMPV in Mexico.

Co-infection of classic swine H1N1 influenza virus in pigs persistently infected with porcine rubulavirus.

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.

Molecular characterization of the spike gene of the porcine epidemic diarrhea virus in Mexico, 2013–2016

In Mexico, the first outbreaks suggestive of the circulation of the porcine epidemic diarrhea virus (PEDV) were identified at the beginning of July 2013. To identify the molecular characteristics of the PEDV Spike (S) gene in Mexico, 116 samples of the intestine and diarrhea of piglets with clinical signs of porcine epidemic diarrhea (PED) were obtained. Samples were collected from 14 farms located in six states of Mexico (Jalisco, Puebla, Sonora, Veracruz, Guanajuato, and Michoacán) from 2013 to 2016. To identify PEDV, we used real-time RT-PCR to discriminate between non-INDEL and INDEL strains. We chose samples according to state and year to characterize the S gene. After amplification of the S gene, the obtained products were sequenced and assembled. The complete amino acid sequences of the spike protein were used to perform an epitope analysis, which was used to determine null mutations in regions SS2, SS6, and 2C10 compared to the sequences of G2. A phylogenetic analysis determined the circulation of G2b and INDEL strains in Mexico. However, several mutations were recorded in the collagenase equivalent (COE) region that were related to the change in polarity and charge of the amino acid residues. The PEDV strain circulating in Jalisco in 2016 has an insertion of three amino acids (232LGL234) and one change in the antigenic site of the COE region, and strains from the years 2015 and 2016 changed the index of the surface probability, which could be related to the re-emergence of disease outbreaks.

Serological study of influenza viruses in veterinarians working with swine in Mexico

Humans and swine are both affected by influenza viruses, and swine are considered a potential source of new influenza viruses. Transmission of influenza viruses across species is well documented. The aim of this study was to evaluate the seroprevalence of different influenza virus subtypes in veterinarians working for the Mexican swine industry, using a hemagglutination inhibition test. All sera tested were collected in July 2011. The data were analysed using a generalized linear model and a linear model to study the possible association of seroprevalence with the age of the veterinarian, vaccination status, and biosecurity level of the farm where they work. The observed seroprevalence was 12.3%, 76.5%, 46.9%, and 11.1% for the human subtypes of pandemic influenza virus (pH1N1), seasonal human influenza virus (hH1N1), the swine subtypes of classical swine influenza virus (swH1N1), and triple-reassortant swine influenza virus (swH3N2), respectively. Statistical analysis indicated that age was associated with hH1N1 seroprevalence (P < 0.05). Similarly, age and vaccination were associated with pH1N1 seroprevalence (P < 0.05). On the other hand, none of the studied factors were associated with swH1N1 and swH3N2 seroprevalence. All of the pH1N1-positive sera were from vaccinated veterinarians, whereas all of those not vaccinated tested negative for this subtype. Our findings suggest that, between the onset of the 2009 pandemic and July 2011, the Mexican veterinarians working in the swine industry did not have immunity to the pH1N1 virus; hence, they would have been at risk for infection with this virus if this subtype had been circulating in swine in Mexico prior to 2011.