Sub-typing of PRRSV isolates by means of measurement of cross-neutralization reactions

The degree of similitude or closeness between two different isolates or strains of PRRSV is very important for deciding which strains should be used for immunization, by either vaccinating with commercial vaccines or attempting to stabilize a herd with planned wt PRRSV infection. As previously known for certain important viral diseases, such as in Foot and Mouth Disease, the reciprocal (cross-) neutralization titers between two strains may be of utmost importance to establish the degree of similarity or difference between these. There is currently a void of methods that would allow distinguishing or grouping strains of PRRSV in a manner that would appropriately reflect immunogenic relatedness and cross protection. Such approach would be a better way of grouping or subtyping isolates than the mere comparison of genetic sequences used nowadays. In this project, the generation of master reference hyperimmune swine antisera against certain selected reference strains of PRRSV allowed us grouping PRRSV strains in some distinct clusters and making inferences about their cross-reactivity and cross-protection by cross-neutralization. These experiments indicate that a high degree of complexity exists in the reciprocal neutralization among strains, suggesting that an increase of the battery of reference serums may be required in order to achieve a complete typing of the universe of PRRSV strains circulating in the U.S. Most importantly, these experiments have provided evidence of the occurrence of unexpected cross- reactivity between otherwise distinct types of PRRSV, thus indicating either the existence of groups of strains of highly atypical (yet undefined) reactivity, or the existence of strains that may emerge from recombination between the two main types of PRRSV

Identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones

AbstractIn order to determine virulence associated genes in porcine reproductive and respiratory syndrome virus (PRRSV), a series of chimeric viruses were generated where specific genomic regions of a highly virulent PRRSV infectious clone (FL12) were replaced with their counterparts of an attenuated vaccine strain (Prime Pac). Initial genome-wide scanning using a sow reproductive failure model indicated that non-structural (ORF 1a and 1b) and structural (ORF2-7) genomic regions appear to be sites where virulence determinants of PRRSV may reside. These results thus confirm the multigenic character of PRRSV virulence. Additional chimeras containing each individual structural ORFs (2 through 7) of Prime Pac and ORF5 of Neb-1 (parental strain of Prime Pac) within the FL12 backbone were generated and tested individually for further mapping of virulence determinants. Our results allow to conclude that NSP3–8 and ORF5 are the location of major virulence determinants, while other virulence determinants may also be contained in NSP1–3, NSP10–12 and ORF2

Complete Genome Sequence of Highly Virulent Porcine Reproductive and Respiratory Syndrome Virus Variants That Recently Emerged in the United States

A recent outbreak of particularly virulent disease caused by porcine reproductive and respiratory syndrome virus has occurred in swine herds across the United States. We report here the complete genome sequence of eight viral isolates from four Nebraska herds experiencing an outbreak of severe disease in 2016

Amino acid residues in the non-structural protein 1 of porcine reproductive and respiratory syndrome virus involved in down-regulation of TNF-cx expression in vitro and attenuation in vivo

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses tumor necrosis factor-alpha (TNF-α) production at both transcriptional and post-transcriptional levels by its non-structural proteins 1α and 1β (Nsp1α and Nsp1β). To identifY the amino acid residues responsible for this activity, we generated several alanine substitution mutants of Nsp1α and Nsp1β. Examination of the mutant proteins revealed that Nsp1α residues Gly90, Asn91 , Arg97, Argl 00 and Arg124 were necessary for TNF-α promoter suppression, whereas several amino acids spanning the entire Nsp1β ~ were found to be required for this activity. Two mutant viruses, with mutations at Nsp1α Gly90 or Nsp1β residues 70-74, generated from infectious cDNA clones, exhibited attenuated viral replication in vitro and TNF-α was found to be up regulated in infected macrophages. In infected pigs, the Nsp1β mutant virus was attenuated in growth. These studies provide insights into how PRRSV evades the effector mechanisms of innate immunity dUling infection

Development of a broadly protective modified-live virus vaccine candidate against porcine reproductive and respiratory syndrome virus

Modified-live virus (MLV) vaccines are widely used to protect pigs against porcine reproductive and respiratory syndrome virus (PRRSV). However, current MLV vaccines do not confer adequate levels of heterologous protection, presumably due to the substantial genetic diversity of PRRSV isolates circulating in the field. To overcome this genetic variation challenge, we recently generated a synthetic PRRSV strain containing a consensus genomic sequence of PRRSV-2. We demonstrated that our synthetic PRRSV strain confers unprecedented levels of heterologous protection. However, the synthetic PRRSV strain at passage 1 (hereafter designated CON-P1) is highly virulent and therefore, is not suitable to be used as a vaccine in pigs. In the present study, we attenuated CON-P1 by continuously passaging the virus in MARC-145 cells, a non-natural host cell line. Using a young pig model, we demonstrated that the synthetic virus at passages 90 and 122 (designated as CON-P90 and CON-P122, respectively) were fully attenuated, as evidenced by the significantly reduced viral loads in serum and tissues and the absence of lung lesion in the infected pigs. Most importantly, CON-P90 confers similar levels of heterologous protection as its parental strain CON-P1. Taken together, the results indicate that CON-P90 is an excellent candidate for the formulation of next generation of PRRSV MLV vaccines with improved levels of heterologous protection

Characterization of a serologic marker candidate for development of a live-attenuated DIVA vaccine against porcine reproductive and respiratory syndrome virus

DIVA (differentiating infected from vaccinated animals) vaccines have proven extremely useful for control and eradication of infectious diseases in livestock. We describe here the characterization of a serologic marker epitope, so-called epitope-M201, which can be a potential target for development of a live-attenuated DIVA vaccine against porcine reproductive and respiratory syndrome virus (PRRSV). Epitope-M201 is located at the carboxyl terminus (residues 161-174) of the viral M protein. The epitope is highly immunodominant and well-conserved among type-II PRRSV isolates. Rabbit polyclonal antibodies prepared against this epitope are non-neutralizing; thus, the epitope does not seem to contribute to the protective immunity against PRRSV infection. Importantly, the immunogenicity of epitope-M201 can be disrupted through the introduction of a single amino acid mutation which does not adversely affect the viral replication. All together, our results provide an important starting point for the development of a liveattenuated DIVA vaccine against type-II PRRSV

Porcine Reproductive and Respiratory Syndrome Virus Infects Mature Porcine Dendritic Cells and Up-Regulates Interleukin-10 Production

Porcine reproductive and respiratory syndrome virus (PRRSV) infects mature dendritic cells (mDCs) derived from porcine monocytes and matured with lipopolysaccharide. The infection of mDCs induced apoptosis, reduced the expression of CD80/86 and major histocompatibility complex class II molecules, and increased the expression of interleukin-10, thus suggesting that such mDC modulation results in the impairment of T-cell activation

Identification of amino acid residues important for anti-IFN activity of porcine reproductive and respiratory syndrome virus non-structural protein 1

The non-structural protein 1 (nsp1) of porcine reproductive and respiratory syndrome virus is partly responsible for inhibition of type I interferon (IFN) response by the infected host. By performing alanine-scanning mutagenesis, we have identified amino acid residues in nsp1α and nsp1β~ (the proteolytic products of nsp1) that when substituted with alanine(s) exhibited significant relief of IFNsuppression. A mutant virus (16-SA, in which residues 16-20 of nsp1β were substituted with alanines) encoding mutant nsp1β recovered from infectious cDNA clone was shown to be attenuated for growth in vitro and induced significantly higher amount of type I IFN transcripts in infected macrophages. In infected pigs, the 16-SA virus exhibited reduced growth at early times after infection but quickly regained wild type growth properties as a result of substitutions within the mutated sequences. The results indicate a strong selection pressure towards maintaining the IFN-inhibitory property of the virus for successful propagation in pigs

Curvas de crecimiento predestete en terneros del sistema de doble propósito.

Existen pocos estudios sobre curvas de crecimiento predestete en bovinos de doble propósito (DP), generalmente éstos son cruces de Bos taurus x Bos indicus. La curva de crecimiento debe señalar los puntos críticos en esta etapa de cría para aplicar los correctivos necesarios, básicamente en aspectos de manejo y alimentación, su importancia se da en términos técnico-económicos. El trabajo se realizó en el Centro de Investigación El Nus del ICA, ubicado en zona de ladera y clima medio de Antioquia. Los pesos mensuales, obtenidos desde 1986 hasta 1992 de 479 terneros criados bajo el sistema de DP, se examinaron mediante análisis de regresión y correlación para 48 animales, según sexo y grupo genético. El peso promedio al nacimiento fue de 31.14 más o menos 4.82, y al destete, de 149.84 más o menos 36.91. En estas fechas los machos superaron a las hembras en 0.95 y 13.40 kg, respectivamente. Se determinaron curvas de crecimiento desde el nacimiento hasta el destete (9 meses) para cada una de las composiciones genéticas, encontrándose r al cuadrado superiores al 69 por ciento para las ecuaciones lineal, cuadrática y cúbica. La ecuación lineal (Y igual 31.980 más 0.411x con r al cuadrado igual 0.720) fue la más utilizada por su fácil manejo y baja variabilida