Detection and molecular characterization of porcine parvovirus in fetal tissues from sows without reproductive failure in Argentina.

Porcine parvovirus (PPV) is one of many pathogens responsible for reproductive failure in pregnant sows. Several studies have reported the appearance of new PPV strains that differ from previous isolates both genetically and antigenically. Thus, the protective effects of commercially inactivated vaccines could not be complete. In South America, the information about PPV is limited. Thus, the aim of the present study was to detect and characterize the PPV strains present in 131 mummies or stillbirths from normal deliveries in sows from a commercial swine farm of Argentina that uses the commercial vaccine. PCR results showed that 17/131 were positive to PPV. Ten of these viruses were isolated and sequenced. All viruses were related to the PPV1 sequence (NADL-2), maintaining the amino acid differences in positions 436 (S-P) and 565 (R-K). This study is the first to report the isolation of PPV in Argentina and the results suggest that PPV can cross the placenta even in vaccinated sows, thus affecting some of the fetuses and being able to cause fetal death in sows without reproductive failure. The results also suggest that vaccination only reduces clinical signs and reproductive disorders and may thus not be a perfect tool to manage PPV infection. This study provides information that needs to be studied in depth to improve strategies to prevent and control PPV infection in swine farms.

Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus.

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.

The equine arteritis virus isolate from the 2010 Argentinian outbreak.

A semen sample from a stallion infected during the 2010 equine arteritis virus (EAV) outbreak was received for viral isolation prior to castration of the animal. The virus was identified using a polyclonal antibody immunofluorescence test. Reverse-transcription polymerase chain reaction (RT-PCR) was used to amplify a region of the GP5 gene with primers GL105F and GL673R. The PCR products were purified and sequences of both strands were determined in a MegaBACE™1000 with inner primers CR2 and EAV32. A phylogenetic dataset was built with the previously reported sequences of five strains isolated in Argentina, together with a group of selected sequences obtained from GenBank. The unrooted neighbour-joining tree was constructed using molecular evolutionary genetic analysis (MEGA) and bootstrap analyses were conducted using 1,000 replicate datasets. Evolutionary distances were computed using the maximum composite likelihood method. A NetNGlyc server analysis at the Technical University of Denmark (www.cbs.dtu.dk/services/NetNGlyc/) was used to predict N-glycosylation in GP5 sequences. The phylogenetic analysis revealed that the new strain GLD-LP-ARG), together with other strains previously isolated, belongs to the European group EU-1 but in a different branch. The new strain shows 99% nucleotide identity with strain Al1and 98.1% with the Belgian strain 08P178. Persistently infected stallions and their cryopreserved semen constitute a reservoir of EAV, which ensures its persistence in the horse population around the world. These findings reinforce the importance of careful monitoring of persistently infected stallions, as well as semen straws, by RT-PCR or test mating, in accordance with national regulations.

ELA-DRA polymorphisms are not associated with Equine Arteritis Virus infection in horses from Argentina.

Polymorphisms at Major Histocompatibility Complex (MHC) genes have been associated with resistance/susceptibility to infectious diseases in domestic animals. The aim of this investigation was to evaluate whether polymorphisms of the DRA gene the Equine Lymphocyte Antigen is associated with susceptibility to Equine Arteritis Virus (EAV) infection in horses in Argentina. The equine DRA gene was screened for polymorphisms using Pyrosequencing® Technology which allowed the detection of three ELA-DRA exon 2 alleles. Neither allele frequencies nor genotypic differentiation exhibited any statistically significant (P-values=0.788 and 0.745) differences between the EAV-infected and no-infected horses. Fisher's exact test and OR calculations did not show any significant association. As a consequence, no association could be established between the serological condition and ELA-DRA.

Eca20 microsatellite polymorphisms in equine viral arteritis-infected horses from Argentina.

We investigated the association of equine arteritis virus (EAV) infection and three short tandem repeat (STR) polymorphisms located within or in close proximity to equine lymphocyte antigen (ELA) region. We used a case-control design as a first approach before proceeding to select candidate genes. One hundred and sixty-five Silla Argentino horses were taken in 2002 from positive serological detections of EAV in Argentina, to determine whether STR genotypes were correlated to genetic susceptibility to EVA. Allele frequency distribution did not show significant differences between both groups (P = 0.0781). However, in particular alleles, Fisher exact test and odds ratio calculations showed significant values >1 for TKY08 and LEX52, and <1 for UM011, TKY08, LEX52 and VHL20. Interestingly, TKY08 STR is located in ELA class I region.

Phylogenetic analysis of Suid Herpesvirus 1 isolates from Argentina.

Argentinean Suid Herpesvirus 1 isolates were compared with reference strains and sequences available at GenBank and phylogenetically analyzed. A short fragment of the gE gene of the immunodominant epitopes was used for preliminary grouping of isolates by phylogenetic analysis. The analysis of the partial gC gene provided more precise genetic typing and segregation into the main genotypes I and II. Results confirmed that the Argentinean genotype I isolates predominate in our country. The topology of the partial gC gene was similar to that previously reported. The Argentinean type I isolates belonged to one cluster and grouped together with NIA-3 and American and Brazilian genotype I strains. However, the results obtained by the algorithms allow inferring that the Yamagata S-81 and Mer (genotype II) strains are grouped together.

Characterization of Suid herpesvirus 1 field isolates from Argentina.

The genomic characterization of Suid herpesvirus 1 (SHV-1) isolates from Argentina was accomplished by restriction pattern analysis using the BamHI, BstEII and XhoI enzymes. Type II genome has been described only once in Argentina. This study revealed considerable homogeneity of BamHI endonuclease sites in all the strains analyzed, according to the number and size of the fragments. No deletion of BamHI fragment #7 among the Argentinean isolates suggests that these strains are wild-type. In addition, the main antigenic domain of glycoprotein E of all the Argentinean strains, as well as the reference strains and sequences available in the GenBank, were characterized. The similarity percent oscillated between 99 and 100%.

Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported.

Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.

Evaluation of neutralization patterns of the five unique Argentine equine arteritis virus field strains reported / Evaluación de los patrones de neutralización de las únicas cinco cepas argentinas descritas de arteritis viral equina

Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.

La arteritis viral equina (AVE) ocasiona infecciones, en su mayoría subclínicas, pero puede causar abortos y enfermedad respiratoria. Si bien se ha descrito un solo serotipo de AVE, existen diferencias en cuanto a la antigenicidad, patogenicidad y patrones de neutralización en las cepas de campo. Los ORF5 y ORF6 del virus codifican las proteínas de envoltura GP5 y M; la interacción entre estas proteínas es crítica para la infectividad. Los cambios en las secuencias de aminoácidos en la proteína GP5, especialmente en la región V1, afectan el fenotipo neutralizante, sin tener en cuenta variaciones aminoacídicas de otras proteínas virales. En este estudio evaluamos los fenotipos neutralizantes de las 5 únicas cepas de arteritis viral equina aisladas en Argentina y los comparamos con los de la cepa de referencia EAV-UCD por virus neutralización cruzada y análisis de secuencias aminoacídicas de las proteínas M y GP5. Las cepas argentinas presentaron un patrón de neutralización similar cuando se utilizaron sueros positivos del banco de sueros, mientras que fueron neutralizadas en menor medida por los sueros policlonales de referencia anti-AVE. A excepción de la cepa LP01, las cepas argentinas tienen casi las mismas sustituciones aminoacídicas en la primera región variable V1 de la proteína GP5, específicamente en los sitios neutralizantes B y C, pero difieren en gran medida respecto de la cepa de referencia EAV-UCD. Las diferencias encontradas en los aislamientos LP02/R, LP02/C, LP02/P y LT-LP-ARG no se reflejaron en variaciones en el fenotipo neutralizante.