Genotyping Based on the LTR Region of Small Ruminant Lentiviruses from Naturally Infected Sheep and Goats from Mexico.

Small ruminant lentiviruses (SRLVs) belong to the genus Lentivirus in the Retroviridae family. There are five genotypes (A, B, C, D, and E), where genotypes A and B have a global distribution and genotypes C, D, and E are limited to Europe. The presence of SRLV has been confirmed in Mexico, with genotype B detected in the central region of the country. We examined the presence of SRLVs and genotype prevalence in 1014 sheep and 1383 goats from 12 Mexican states. Using a commercial competitive ELISA (cELISA) test, we detected SRLV antibodies in 107 sheep (10.55%) and 466 goats (33.69%). We used an endpoint PCR to amplify the LTR region on seropositive animals. A total of 50 sheep and 75 goats tested positive via PCR. Positive amplicons from 11 sheep and 17 goats from ten Mexican States were cloned and sequenced. With the LTR sequence data obtained in this study, a phylogenetic analysis was performed; we also constructed a phylogenetic tree using the obtained sequences and GenBank's available sequences. All studied sequences were associated with genotype B, specifically with the FESC-752 isolate previously identified in Mexico. Highly conserved transcription factor binding sites were observed in analyzed alignments, such as AML (vis), AP-4, and TATA box. However, we identified nucleotide differences at site AP-1 that suggest function loss. Our study found that ovine and caprine genotype B SRLVs are widely distributed in Mexico; a highly conserved LTR region among the sequences evaluated in this study was also found.

Production of an enzymatically active and immunogenic form of ectodomain of Porcine rubulavirus hemagglutinin-neuraminidase in the yeast Pichia pastoris.

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.

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.

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

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.

West Nile virus infection of birds, Mexico.

West Nile virus (WNV) has caused disease in humans, equids, and birds at lower frequency in Mexico than in the United States. We hypothesized that the seemingly reduced virulence in Mexico was caused by attenuation of the Tabasco strain from southeastern Mexico, resulting in lower viremia than that caused by the Tecate strain from the more northern location of Baja California. During 2006-2008, we tested this hypothesis in candidate avian amplifying hosts: domestic chickens, rock pigeons, house sparrows, great-tailed grackles, and clay-colored thrushes. Only great-tailed grackles and house sparrows were competent amplifying hosts for both strains, and deaths occurred in each species. Tecate strain viremia levels were higher for thrushes. Both strains produced low-level viremia in pigeons and chickens. Our results suggest that certain avian hosts within Mexico are competent for efficient amplification of both northern and southern WNV strains and that both strains likely contribute to bird deaths.