José La Torre

Molecular characterization of canine parvovirus strains in Argentina: Detection of the pathogenic variant CPV2c in vaccinated dogs.

PCR amplification with sequence-specific primers was used to detect canine parvovirus (CPV) DNA in 38 rectal swabs from Argentine domestic dogs with symptoms compatible with parvovirus disease. Twenty-seven out of 38 samples analyzed were CPV positive. The classical CPV2 strain was not detected in any of the samples, but nine samples were identified as CPV2a variant and 18 samples as CPV2b variant. Further sequence analysis revealed a mutation at amino acid 426 of the VP2 gene (Asp426Glu), characteristic of the CPV2c variant, in 14 out of 18 of the samples identified initially by PCR as CPV2b. The appearance of CPV2c variant in Argentina might be dated at least to the year 2003. Three different pathogenic CPV variants circulating currently in the Argentine domestic dog population were identified, with CPV2c being the only variant affecting vaccinated and unvaccinated dogs during the year 2008.

Updating of the correlation between lpELISA titers and protection from virus challenge for the assessment of the potency of polyvalent aphtovirus vaccines in Argentina

Routine vaccination campaigns are carried out in Argentina twice a year, involving more than 100 million doses of foot-and-mouth disease (FMD) vaccine. Although the challenge test in cattle has not been totally replaced for the assessment of FMD vaccine potency, Argentine Animal Health authorities have used an indirect alternative method based on specific correlation studies of protection against podal generalization (PPG) tests performed in cattle with a validated liquid phase blocking ELISA (lpELISA). The change of vaccine formulations that took place after the 2000-2001 outbreaks, generated a gap in the correlation between lpELISA titers and PPG for the new FMD virus strains. A reappraisal of the correlation between lpELISA titers measured at 60 dpv and virus challenge by the PPG method at 90 dpv, performed for the four virus strains presently included in the Argentine vaccine is presented in this work. The data were obtained from 40 bovine challenge trials (647 sera) performed using exclusive batches of commercial vaccine from the year 2001 to January 2008 for A24/Cruzeiro, A/Argentina/2001, O1/Campos and C3/Indaial FMD virus strains. Curves of percentage of expected protection (EPP) versus lpELISA titers were obtained by logit regression for A/Argentina/2001, O1/Campos and C3/Indaial strains, but not for A24/Cruzeiro strain. The concordance between the direct and indirect tests using an EPP cut off value of 75% (82%, kappa = 0.62), in agreement with data originating from many years of vaccine control in Argentina, remarks the relevance of the acceptance of indirect alternatives to in vivo potency testing.

Evolution of Canine Parvovirus in Argentina between years 2003 and 2010: CPV2c has become the predominant variant affecting the domestic dog population

Abstract The current frequency of Canine Parvovirus variants (CPV2a, CPV2b and CPV2c) in the Argentine dog population was investigated by PCR amplification of a 583bp fragment in the VP2 gene. From a total of 79 rectal swab samples that have been submitted to our laboratory since 2008, 55 (69.6%) resulted positive and were further analyzed by direct DNA sequencing. Fifty positives samples (91%) were characterized as CPV2c variant, which appeared in Argentina in the year 2003 and has been the prevalent type since 2008, whereas CPV2a and CPV2b, still found in Argentine dogs, were represented in 3.6% and 5.4% of the population, respectively. Considering that CPV2c is spreading worldwide, and that this variant is also affecting vaccinated dogs, efforts should be made towards the development of new matched CPV vaccines.

Evidence of two co-circulating genetic lineages of canine distemper virus in South America.

Canine distemper virus (CDV) is the etiological agent of a multisystemic infection that affects different species of carnivores and is responsible for one of the main diseases suffered by dogs. Recent data have shown a worldwide increase in the incidence of the disease, including in vaccinated dog populations, which necessitates the analysis of circulating strains. The hemagglutinin (H) gene, which encodes the major antigenic viral protein, has been widely used to determine the degree of genetic variability and to associate CDVs in different worldwide circulating lineages. Here, we obtained the sequence of the first full-length H gene of field South American CDV strains and compared it with sequences of worldwide circulating field strains and vaccine viruses. In South America, we detect two co-circulating lineages with different prevalences: the Europe 1 lineage and a new South America 2 lineage. The Europe 1 lineage was the most prevalent in South America, and we suggest renaming it the Europe 1/South America 1 lineage. The South America 2 lineage was found only in Argentina and appears related to wild CDV strains. All South American CDV strains showed high amino-acid divergence from vaccine strains. This genetic variability may be a possible factor leading to the resurgence of distemper cases in vaccinated dog populations.

Confidence in indirect assessment of foot-and-mouth disease vaccine potency and vaccine matching carried out by liquid phase ELISA and virus neutralization tests.

The necessity of avoiding the use of animals in vaccine potency testing has been widely recognized. The repeatability and reproducibility of the Expected Percentage of Protection (EPP) as a serological potency surrogate for A24 Cruzeiro foot-and-mouth disease virus (FMDV) strain was assessed, and compared with the results obtained with challenge in the Protection against Podal Generalization (PPG) test. To determine the EPPs, the serum titers obtained by liquid phase blocking competitive ELISA (lpELISA) and virus neutralization (VNT) in 10 potency trials using the same A24 Cruzeiro vaccine, were interpolated into previously validated logit transformation curves that correlate PPG with serology. Indirect serological assessment of vaccine matching between the serotype A FMDV strains A24 Cruzeiro and A/Argentina/01 was also carried out by lpELISA and VNT. The results obtained in this study strongly support the replacement of challenge tests for vaccine potency by indirect serological assays, at least for A24 Cruzeiro FMDV strain. While determination of EPPs by lpELISA titers showed an excellent repeatability, reproducibility and concordance with PPG for vaccine potency, assessments of cross-protection by VNT titers were more consistent with the PPG outcome.

Biochemical characterization of an aphthovirus type C3 strain resende attenuated for cattle by serial passages in chicken embryos

We have compared several aspects of an aphthovirus strain attenuated for cattle (C3R-O/E) with the original strain (C3Res) from which it was derived after serial passages in chicken embryos. Biochemical differences detected by protein analysis in regular polyacrylamide gels (SDS-PAGE) and on electrofocusing gels (NEPHGE) suggest the presence of mutations throughout the genome. Changes were located in coat proteins VP1 and VP3 and in the polymerase precursor P100 (P3/ABCD). No other differences were found at the protein level by means of the techniques used. Polypeptide P100 of the attenuated strain showed a faster electrophoretic mobility in SDS-PAGE with respect to that of the wild-type strain, and the change seems to be located on its amino terminus half. Several functional differences were also found between the two viruses. Both strains grew equally well in BHK cells reaching roughly similar titers in plaque assays. However, the wild-type strain maintained its titer in cells of bovine origin (BK), whereas the titer of C3R-O/E strain decreased approximately one log in this cell system; moreover, plaques elicited by the attenuated strain were much smaller than the ones produced by C3Res. A diminution in the rate of RNA synthesis induced by C3R-O/E in BK cells compared with that of the wild-type strain was also detected; this trait was not observed in BHK cells. A delay in the kinetics of RNA synthesis was also detected in this strain. The virus yield of attenuated strain in BK cells was four times lower than in BHK cells.

Microscopic Studies in Shunts for Hydrocephalus

Valve-regulated systems, used for the treatment of hydrocephalus, were studied with the scanning electron microscope. The study was performed on unused systems of shunt replaced after different pathologies and on systems removed due to cerebrospinal fluid infection. Defects in the material, such as holes and protrusions, were systematically found in new systems. Aged shunts (1--8 years of implantation) as well as those removed in cases of cerebrospinal fluid infections showed important changes in the structure of the material too; it was also found that biological material, such as fibrin, red blood cells and bacteria, were able to adhere to the walls of the catheter tubing. These results indicate that the scanning electron microscope could be a complementary tool to be used in research, diagnosis, and quality control of silicone rubber prosthesis.

Rapid methodology for antigenic profiling of FMDV field strains and for the control of identity, purity and viral integrity in commercial virus vaccines using monoclonal antibodies.

Monoclonal antibodies (MAbs) developed against different foot-and-mouth disease virus (FMDV) vaccine strains were extensively used to study any possible antigenic variations during vaccine production in Argentine facilities. Additionally, a typing ELISA using strain specific MAbs was developed to detect potential cross contaminations among FMDV strains in master and working seeds with high specificity and sensitivity and to confirm strains identity in formulated vaccines. This assay was carried out for the South American strains currently in use in production facilities in Argentina (A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial) and for the strain O/Taiwan, produced only for export to Asia. These non-cross reactive MAbs were also used to analyze the integrity of viral particles belonging to each one of the individual strains, following isolation of 140S virions by means of sucrose density gradients from the aqueous phase of commercial polyvalent vaccines. Antigenic profiles were defined for FMDV reference strains using panels of MAbs, and a coefficient of correlation of reactivity with these panels was calculated to establish consistent identity upon serial passages of master and production seeds. A comparison of vaccine and field strain antigenic profiles performed using coefficients of correlation allowed the rapid identification of two main groups of serotype A viruses collected during the last FMD epidemic in Argentina, whose reactivity matched closely to A/Argentina/2000 and A/Argentina/2001 strains.

Horizontal Transmission of Triatoma Virus Through the Fecal-Oral Route in Triatoma infestans (Hemiptera: Triatomidae)

Abstract Feces from Triatoma infestans (Klug) infected with TrV showed a large number of well-preserved viral particles when examined by electron microscopy. No viral particles were observed in suspensions of feces from uninfected insects. Fecal suspensions inoculated parenterally into uninfected triatomines killed the insects within 36 h, showing that infective TrV is present in the feces of infected insects. It also is demonstrated that T. infestans becomes infected with TrV while feeding on contaminated chickens, and all the chickens used to feed a colony of triatomines infected with TrV showed high anti-TrV titer in their sera, although no TrV replication could be demonstrated in chickens. Oral infection of T. infestans by contaminated feces probably contributes to virus dispersal in nature. This observation provides the rationale for the potential use of TrV as a biological control agent.

Immunocompetent truncated E2 glycoprotein of bovine viral diarrhea virus (BVDV) expressed in Nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines.

The bovine viral diarrhea virus (BVDV) is the etiological agent responsible for a wide spectrum of clinical diseases in cattle. The glycoprotein E2 is the major envelope protein of this virus and the strongest inductor of the immune response. There are several available commercial vaccines against bovine viral diarrhea (BVD), which show irregular performances. Here, we report the use of tobacco plants as an alternative productive platform for the expression of the truncated version of E2 glycoprotein (tE2) from the BVDV. The tE2 sequence, lacking the transmembrane domain, was cloned into the pK7WG2 Agrobacterium binary vector. The construct also carried the 2S2 Arabidopsis thaliana signal for directing the protein into the plant secretory pathway, the Kozak sequence, an hexa-histidine tag to facilitate protein purification and the KDEL endoplasmic reticulum retention signal. The resulting plasmid (pK-2S2-tE2-His-KDEL) was introduced into Agrobacterium tumefaciens strain EHA101 by electroporation. The transformed A. tumefaciens was then used to express tE2 in leaves of Nicotiana tabacum plants. Western blot and ELISA using specific monoclonal antibodies confirmed the presence of the recombinant tE2 protein in plant extracts. An estimated amount of 20 μg of tE2 per gram of fresh leaves was regularly obtained with this plant system. Injection of guinea pigs with plant extracts containing 20 μg of rtE2 induced the production of BVDV specific antibodies at equal or higher levels than those induced by whole virus vaccines. This is the first report of the production of an immunocompetent tE2 in N. tabacum plants, having the advantage to be free of any eventual animal contaminant.