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.

Development of Glycyrrhizinic Acid-Based Lipid Nanoparticle (LNP-GA) as An Adjuvant That Improves the Immune Response to Porcine Epidemic Diarrhea Virus Spike Recombinant Protein.

Porcine epidemic diarrhea virus (PEDV) has affected the pork industry worldwide and during outbreaks the mortality of piglets has reached 100%. Lipid nanocarriers are commonly used in the development of immunostimulatory particles due to their biocompatibility and slow-release delivery properties. In this study, we developed a lipid nanoparticle (LNP) complex based on glycyrrhizinic acid (GA) and tested its efficacy as an adjuvant in mice immunized with the recombinant N-terminal domain (NTD) of porcine epidemic diarrhea virus (PEDV) spike (S) protein (rNTD-S). The dispersion stability analysis (Z-potential -27.6 mV) confirmed the size and charge stability of the LNP-GA, demonstrating that the particles were homogeneously dispersed and strongly anionic, which favors nanoparticles binding with the rNTD-S protein, which showed a slightly positive charge (2.11 mV) by in silico analysis. TEM image of LNP-GA revealed nanostructures with a spherical-bilayer lipid vesicle (~100 nm). The immunogenicity of the LNP-GA-rNTD-S complex induced an efficient humoral response 14 days after the first immunization (p < 0.05) as well as an influence on the cellular immune response by decreasing serum TNF-α and IL-1ß concentrations, which was associated with an anti-inflammatory effect.

Immunogenicity of a recombinant hemagglutinin neuraminidase-Porcine rubulavirus produced by Escherichia coli of Porcine rubulavirus gives protective immunity of litter after challenge.

Porcine rubulavirus (PRV) is a contagious virus that affects the Mexican swine industry. This work aimed to evaluate the immunogenicity of an recombinant hemagglutinin neuraminidase-Porcine rubulavirus (rHN-PorPV) candidate vaccine on pregnant sows, and the protective efficacy afforded to their 7-day-old suckling piglets against PRV lethal challenge. Three sows were immunized with rHN-PorPV formulated with immune-stimulating complex (ISCOMs) and two sows with rHN-PorPV protein alone as well as a mock-immunized pregnant sow (negative control). Quantitative ELISA detected a high concentration of anti-rHN-PorPV Immunoglobulin G (IgG) antibodies in sow sera after the second dose of vaccine administered on day 14 until farrowing, showing viral-neutralizing and cross-neutralization activity against different variants of PRV. Sera samples from piglets of immunized sows (with or without adjuvant), showed high concentrations of IgG antibodies. As expected, piglets from the negative control sow (n=5), exhibited severe signs of disease and 100% of mortality after PRV challenge study. Conversely, 75% and 87.5% of the piglets born from the rHN-PorPV and the rHN-PorPV-ISCOMs-immunized sows (n=8), survived, respectively, showing milder PRV clinical signs. Our data indicate that rHN-PorPV candidate vaccine produced in Escherichia coli induces efficient humoral response in pregnant sows and that the maternally derived immunity provides high protection to suckling piglets against PRV lethal challenge.

Development of Novel Recombinant Antigens of Nucleoprotein and Matrix Proteins of Porcine orthorubulavirus: Antigenicity and Structural Prediction.

Blue eye disease (BED) is a swine viral infection that affects the pork industry of Mexico. Porcine orthorubulavirus (PRV) is the etiological agent, and the hemagglutinin-neuraminidase protein (HN) is characterized as the best antigen for serological tests, although other structural proteins, including the nucleoprotein (NP) and the matrix (M) protein, have been investigated during the infection of members of the Paramyxoviridae family, generating promising results. Herein, for the first time, we successfully produced and characterized both the NP and M proteins of PRV by using a recombinant strategy in the E. coli heterologous system. The ORF of the NP and M genes were cloned in-frame with the pET-SUMO expression vector. Recombinant proteins proved to be a sensitive target to detect seroconversion at 7 days until 28 days in vaccinated mice (BALB/c) by indirect ELISAs. Immunoreactivity was also tested using porcine serum samples, in which antibodies were recognized from early stages to a persistence of PRV infection, which is indicative that these proteins contain properties similar to native antigens. The predicted tertiary structure showed that both proteins have a conserved structure that resembles those found in others Paramyxovirus. Our results pave the way for developing biotechnological tools based on these proteins for the control and prevention of BED.

Acute neurologic disease in Porcine rubulavirus experimentally infected piglets.

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 (Michoacán 1984) and two other strains isolated in 2013, one in Queretaro (Qro/93/2013) and the other in Michoacán (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 1×106TCID50/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 3dpi. 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 Michoacán. The virulence of the new isolates maintains similar characteristics to those reported more than 30 years ago.