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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. [ABSTRACT FROM AUTHOR]

Background: Aujeszky's disease is mainly a swine disease, still endemic worldwide. It can infect other mammalians, including human beings, and it is usually fatal with nervous symptoms. Ever since the disease was detected in 1988 in Argentina, many outbreaks have been reported involving both feral swine and dogs., Aim: At present, in Argentina, Pseudorabies virus (PRV) cases are sporadically reported; however, clinical cases are informed. This study aims to obtain information about the seroprevalence of PRV in wild boars and to isolate and characterize PRV from clinical samples., Methods: From 2018 to 2019, 78 wild boars' serum samples from Bahía de Samborombón natural reserve were analyzed for antibodies to PRV using a virus neutralization test. Clinical samples from 17 pigs, 2 wild boars, 1 dog, and 1 cat were collected from 2013 to 2019 for viral isolation and detection of the presence of the gD gene by PCR. For sequence analysis, the gC partial gene was amplified., Results: Five strains were isolated from the dog, cat, and swine samples. The new PRV strains identified were confirmed by BLAST analysis, which revealed between 99.74% and 100% of similarity to the NIA-3 strain and phylogenetic analysis of the partial gene encoding the gC protein revealed that the PRV strains have divided into two main clades, clade 1 and clade 2., Conclusion: This report informed that most new cases of PRV were detected in the central regions of Argentina, where pig production is concentrated. The study in Bahía de Samborombón revealed a high percentage of detection but, the sampling is not representative of that of the rest of the country. Therefore, a systematic sampling effort of wild boar throughout the country should be included in the national program control. Although in Argentina only the inactivated Bartha vaccine is allowed, recombination risk should not be ignored if attenuated vaccines are incorporated into the National control plan. The two strains, one from the cat and one from the dog sample, are directly related to infected swine. The information about clinical cases and molecular characterization of new strains is important for a better understanding of the dynamics of PRV and to promote preventive measures., Competing Interests: The authors declare that there is no competing interest regarding the publication of this paper.

Fibropapillomatosis is a debilitating neoplastic disease associated with Chelonid alphaherpesvirus 5 (ChHV5) infection. We detected the Atlantic variant of ChHV5 associated with a fibropapilloma in a green turtle (Chelonia mydas) found stranded on the western coast of Rio de la Plata, Argentina. This is the southernmost registered case for the southwestern Atlantic., (© Wildlife Disease Association 2023.)

Porcine circovirus type 3 (PCV3) is considered a new circovirus and since it first description has been widely reported in most of the swine-producing countries. Multisystemic inflammation and reproductive failure are consistent and concerning issues associated with PCV3 infection. This report describes the clinical and pathological features of a chronic reproductive disorder in a swine herd in Argentina associated with the presence of PCV3. Mummified (n = 42) and stillborn piglets (n = 20) from a case of chronic reproductive disorder (Study A) and mummified and stillborn piglets (n = 141) from normal deliveries (Study B) were retrospectively assessed for the presence of multiple reproductive pathogens (PCV3, PCV2, ADV, PPV, Leptospira spp. and Brucella spp). On study, A PCV3 and PPV were detected in 15 and 8 pools, respectively, with a coinfection rate of 100% in all PPV-positive cases. Three out of 131 foetuses from three different sows from Study B were positive only for PCV3. Histological evaluation of hearts from stillborn also showed lesions similar to those previously described in the literature for PCV3-reproductive disease. Partial genome of PCV3 was amplified and phylogenetic analysis showed that strains of Study A and B clustered within the PCV3a and PCV3b clades, respectively. This study demonstrates, for the first time, the PCV3 has been circulating in Argentina at least since 2016 and its potential role in reproductive disorders. Further studies are warranted to determine the role of PCV3 in the reproductive disease complex and its prevalence in the swine industry in Argentina., (© 2020 Wiley-VCH GmbH.)

Coronaviruses are a large group of RNA viruses that infect a wide range of animal species. The replication strategy of coronaviruses involves recombination and mutation events that lead to the possibility of cross-species transmission. The high plasticity of the viral receptor due to a continuous modification of the host species habitat may be the cause of cross-species transmission that can turn into a threat to other species including the human population. The successive emergence of highly pathogenic coronaviruses such as the Severe Acute Respiratory Syndrome (SARS) in 2003, the Middle East Respiratory Syndrome Coronavirus in 2012, and the recent SARS-CoV-2 has incentivized a number of studies on the molecular basis of the coronavirus and its pathogenesis. The high degree of interrelatedness between humans and wild and domestic animals and the modification of animal habitats by human urbanization, has favored new viral spreads. Hence, knowledge on the main clinical signs of coronavirus infection in the different hosts and the distinctive molecular characteristics of each coronavirus is essential to prevent the emergence of new coronavirus diseases. The coronavirus infections routinely studied in veterinary medicine must be properly recognized and diagnosed not only to prevent animal disease but also to promote public health., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Porcine circovirus type 2 (PCV2) and protoparvovirus 1 (PPV) were detected as single infection (6/131) and (11/131) respectively, or co-infection (6/131) in fetuses and stillborn piglets from normal deliveries in a farm without reproductive problems. Twenty in twenty-three positive samples were over 70 days of gestation, which is when the fetus becomes immunocompetent, and the presence of a NADL-2 PPV strain suggests fetal immune system impairment. Phylogenetic analysis of sequences obtained showed that 8/9 sequences are related to cluster 13 and the remaining is grouped into cluster 11 sequences. An increase in variability in ORF2 sequences in Argentina was observed. It is not clear whether the detection of fetuses positive to PPV and PCV2 is of epidemiological importance in a subclinically affected farm. However, the results of this study showed that currently used vaccines and vaccine protocols do not fully protect against PPV or PCV2 fetus infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Vector control is the most effective method to prevent transmission of Chagas disease. Control is mostly made through chemical insecticides although they have negative impact on wild pollinators, such as bees. Reducing pesticide use through biological alternatives could minimize the damage to these beneficial insects. Triatoma virus (TrV) is a pathogen able to kill triatomines and thus a valid candidate to be used as biological control agent. In this study we evaluate the capacity of TrV to infect an important beneficial insect (Apis mellifera) as well as a plague insect (Aedes aegypti). Results indicate that TrV does not infect the bees or mosquitoes tested in this study. The possible specificity of TrV for kissing bugs reinforces the possible use of TrV as a biological control agent for triatomines., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Canine distemper virus (CDV) is a pathogen which affects members of the Canidae family, causing an acute, often fatal, systemic disease. CDV is an RNA virus of the family Paramyxoviridae that contains two envelope glycoproteins: F and HA. In this study, we focused on the envelope glycoprotein F as the main target for neutralizing antibodies produced after infection or vaccination. The complete coding region of the protein (60 kDa) was expressed in the methylotrophic yeast Pichia pastoris, obtained in a recombinant form and secreted to the culture medium. Later, to analyze its immunogenicity, the protein was combined with an oily adjuvant and used to inoculate mice. The results provide evidence supporting a potential application of this recombinant protein as a subunit vaccine., Competing Interests: Declaration of competing interest The authors declare that they have no conflicto of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (© 2019 Published by Elsevier Ltd.)

Bovine leukemia virus (BLV) is the agent responsible for enzootic bovine leukosis, the most common neoplastic disease in cattle. The horn fly, a major hematophagous pest of cattle, is able to transmit different diseases in cattle. However, its implication in BLV transmission under a natural environment is still discussed. The objectives of this work were to determine the presence of BLV in horn flies (by sequencing) and to evaluate the ability of horn flies to transmit BLV to cattle (through an experimental assay under a natural environment). To demonstrate the presence of BLV in the flies, 40 horn flies were collected from a BLV-positive cow with a sweep net and 10 pools with four horn-fly mouthparts each were prepared. The presence of BLV was determined by nested polymerase chain reaction and sequencing. To demonstrate BLV transmission, other 40 flies were collected from the same BLV-positive cow with a sweep net. Eight homogenates containing five horn-fly mouthparts each were prepared and injected to eight cows of different breeds, and blood samples were collected every 21 days. Then, to evaluate the ability of horn flies to transmit BLV to grazing cattle under natural conditions, both infected and uninfected cattle from the experimental transmission assay were kept together in the same paddock with more than 200 horn flies per animal for 120 days. Blood samples were collected every 20 days and the number of flies was determined. The sequencing results confirmed the presence of the provirus in horn flies. The results also confirmed that BLV transmission is a possible event, at least experimentally. However, the role of horn flies as vectors of BLV under a natural grazing system is still discussed., Competing Interests: The authors declare that there is no conflict of interest.

Triatoma virus occurs infecting Triatominae in the wild (Argentina) and in insectaries (Brazil). Pathogenicity of Triatoma virus has been demonstrated in laboratory; accidental infections in insectaries produce high insect mortality. When more than one microorganism enters the same host, the biological interaction among them differs greatly depending on the nature and the infection order of the co-existing species of microorganisms. We studied the possible interactions between Triatoma virus (TrV) and Trypanosoma cruzi (the etiological agent of Chagas disease) in three different situations: (i) when Triatoma virus is inoculated into an insect host (Triatoma infestans) previously infected with T. cruzi, (ii) when T. cruzi is inoculated into T. infestans previously infected with TrV, and (iii) when TrV and T. cruzi are inoculated simultaneously into the same T. infestans individual. Trypanosoma cruzi infection was found in 57% of insects in the control group for T. cruzi, whereas 85% of insects with previous TrV infection were infected with T. cruzi. TrV infection was found in 78.7% of insects in the control group for TrV, whereas insects previously infected with T. cruzi showed 90% infection with TrV. A total of 67.9% of insects presented simultaneous infection with both types of microorganism. Our results suggest that TrV infection could increase adhesion of T. cruzi to the intestinal cells of triatomines, but presence of T. cruzi in intestinal cells would not increase the possibility of entry of TrV into cells. Although this study cannot explain the mechanism through which TrV facilitates the infection of triatomines with T. cruzi, we conclude that after TrV replication, changes at cellular level should occur that increase the adhesion of T. cruzi., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The family Dicistroviridae comprises three genera and about twenty species of RNA virus, most of them with health or agricultural importance. The Triatoma virus (TrV) is the only entomopathogenic virus identified in triatomine bugs up to the present. TrV replicates within the intestinal epithelial cells, causing high mortality rate and delayed development of the molt of these bugs. TrV has been proposed as a biological control agent for vectors of Chagas disease. Viral particles were purified from feces of 1, 5 and 10 insects from an experimental colony of Triatoma infestans infected with TrV. Viral concentration and infectivity were corroborated using polyacrylamide gels and RT-PCR, respectively. In this work we report a method of viral purification that allows to reduce necessary reagents and time, using a very small amount of fecal matter., (Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Pseudorabies virus (PrV) causes Aujeszky's disease (AD), which affects mainly swine, but also cattle, sheep, and wild animals, resulting in substantial economic losses due to animal mortality and lost productivity worldwide. To combat PrV, eradication programs using PrV strains lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable, easy-to-use, and sensitive tests that can detect PrV infection in pigs infected with either wild-type virus or vaccine strain (gE-deleted) virus. To meet this demand, we used the baculovirus-insect cell system to produce recombinant glycoprotein B (gB) as antigen for an immune assay. The high GC-content (70% average) of the gB gene from the Argentinian PrV CL15 strain necessitated the use of betaine as a PCR enhancer to amplify the extracellular domain. Recombinant gB was expressed at high levels and reacted strongly with sera from PrV infected pigs. We used the recombinant gB to develop an agar gel immunodiffusion (AGID) test for detection of PrV antibodies. Compared to the gold standard virus neutralization (VN) assay, the AGID sensitivity and specificity were 95% and 96.6% respectively. Thus, recombinant gB produced in the baculovirus-insect cell system is a viable source of antigen for the detection of PrV antibodies in AGID tests. Considering its relatively lower cost, simplicity of use and result interpretation, our AGID is a valuable alternative tool to the VN assay., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The modulation of the expression of caspases by viruses influences the cell survival of different cell types. Equine arteritis virus (EAV) induces apoptosis of BHK21 and Vero cell lines, but it is not known whether EAV induces apoptosis in RK13 cells, a common cell line routinely used in EAV diagnosis and research. In this study, we determined that caspase-3 expression was triggered after infection of RK13 cells with EAV in a time- and dose-dependent manner. We also detected caspase-8 and caspase-9 activation, indicating the stimulation of both extrinsic and intrinsic apoptosis pathways. Finally, we found caspase-12 activation, an indicator of endoplasmic reticulum stress-induced apoptosis. The variability observed in the apoptotic response in the different cell lines demonstrates that apoptosis depends on the distinctive sensitivity of each cell line used for investigation., (Published by Elsevier B.V.)

The use of viruses for biological control is a new option to be considered. The family Dicistroviridae, which affects only invertebrates, is one of the families that have been proposed for this purpose. The Triatoma virus (TrV), a member of this family, affects triatomine transmitters of Chagas disease, which is endemic in Latin America but also expanding its worldwide distribution. To this end, we attempted virus replication in Diptera, Aedes albopictus (clone C6/36) and Lepidoptera Spodoptera frugiperda (SF9, SF21) and High Five (H5) cell lines. The methodologies used were transfection process, direct inoculation (purified virus), and inoculation of purified virus with trypsin. Results were confirmed by SDS-PAGE, Western blotting, RT-PCR, electron microscopy, and immunofluorescence. According to the results obtained, further analysis of susceptibility/infection of H5 cells to TrV required to be studied.

The only virus sequenced and studied in triatomines is the Triatoma virus, from the Dicistroviridae family, which causes delayed development, reduced oviposition, and premature death of infected insects. With the goal of expanding the sequences already obtained in previous years and verifying if any changes occurred in their genomic sequences, 68 samples of triatomines from several provinces of Argentina were analyzed. Sixteen positive samples were obtained by Reverse Transcription (RT)-polymerase chain reaction using the VP3-VP1 subregion of open reading frame-2 as a diagnostic method; after sequencing, 11 samples were obtained from Triatoma infestans. These new sequences showed no significant differences in the analyzed regions, which were not grouped by species or habitat or geographical distribution. There were no differences when compared with the sequences found during 2002-2012, all obtained from the wild. We conclude that despite being an RNA virus, the different sequences show high homology., (© The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.)

Background: Chagas disease is caused by Trypanosoma cruzi, and humans acquire the parasite by exposure to contaminated feces from hematophagous insect vectors known as triatomines. Triatoma virus (TrV) is the sole viral pathogen of triatomines, and is transmitted among insects through the fecal-oral route and, as it happens with T. cruzi, the infected insects release the virus when defecating during or after blood uptake., Methods: In this work, we analysed the occurrence of anti-TrV antibodies in human sera from Chagas disease endemic and non-endemic countries, and developed a mathematical model to estimate the transmission probability of TrV from insects to man, which ranged between 0.00053 and 0.0015., Results: Our results confirm that people with Chagas disease living in Bolivia, Argentina and Mexico have been exposed to TrV, and that TrV is unable to replicate in human hosts., Conclusions: We presented the first experimental evidence of antibodies against TrV structural proteins in human sera.

Triatoma virus (TrV) is the only triatomine entomopathogenic virus identified so far. Propagation of TrV in insectaries depends on handling procedures and triatomine population dynamics. The effects of propagation can be devastating and entire colonies must often be sacrificed to prevent spread of the virus throughout the insectary. This study found that after 41.3 days from TrV ingestion of human blood with 0.04 mg of viral protein by 5th instar Triatomainfestans, viral particles could be detected by RT-PCR; in a second horizontal transmission experiment time to detection resulted in a mean of 42.5 days. These results should rise awareness of TrV dynamics in nature, help estimate the spread of this virus when TrV-infected field-collected insects are incorporated into an insectary, and provide a base for the consideration of TrV as an agent of biological control of some species of triatomines., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A peptide-based indirect ELISA was developed to detect antibodies against Equine arteritis virus (EAV). Two peptides for epitope C of protein GP5 and fragment E of protein M were designed, synthesized, purified and used as antigens either alone or combined. Ninety-two serum samples obtained from the 2010 Equine viral arteritis outbreak, analyzed previously by virus neutralization, were evaluated by the ELISA here developed. The best resolution was obtained using peptide GP5. The analysis of the inter- and intraplate variability showed that the assay was robust. The results allow concluding that this peptide-based ELISA is a good alternative to the OIE-prescribed virus neutralization test because it can be standardized between laboratories, can serve as rapid screening, can improve the speed of diagnosis of EAV-negative horses and can be particularly useful for routine surveillance in large populations., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Equine Arteritis Virus (EAV) has been shown to induce apoptosis in vitro but the induction of this mechanism has not been previously associated with any viral gene product. In this work, we found a cytotoxicity effect of the EAV gP5 protein on baculovirus-insect cells and a low yield of protein recovery. Besides, different morphological features by electron transmission microscopy, DNA fragmentation in agarose gel, TUNEL analysis and caspase 3 activity were found. All these findings indicate that the EAV gP5 protein induces apoptosis in insect cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Triatoma virus is the only virus whose genome has been sequenced and studied in triatomines. It belongs to the family Dicistroviridae. In order to detect whether TrV has the ability to agglutinate erythrocytes of domestic and laboratory animals, we performed a hemagglutination assay. Positive hemagglutination was found for red blood cells of guinea pigs. The HA assay could be used as a titration method, at least for purified viral particles obtained from triatomine stool. This is the first record of hemagglutinating properties for Dicistroviridae.

Enzootic Bovine Leukosis is a chronic disease caused by the bovine leukemia virus (BLV). Smudge cells, also known as Gumprecht shadows, are not simple artifacts of slide preparation, but ragged lymphoid cells found mainly in peripheral blood smears from human patients with chronic lymphocytic leukemia. In this study, we report the presence of Gumprecht shadows in peripheral blood from BLV-positive cattle., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Suid Herpesvirus 1 (SHV-1) is the etiological agent of Aujeszky's disease (AD), which affects swine herds worldwide and causes substantial economic losses due to animal mortality and lost productivity. In order to eradicate SHV-1, vaccination programs using viruses lacking the gene encoding glycoprotein E (gE) are ongoing in several countries. These eradication programs have generated a currently unmet demand for affordable and sensitive tests that can detect SHV-1 infection, yet distinguish between infected and vaccinated pigs. To meet this demand, we used the baculovirus-insect cell system to produce immunologically authentic full-length recombinant gE protein for use in a serum ELISA assay. As previous efforts to clone the gE gene had failed due to its extremely high GC-content (75% average), we used betaine as a PCR enhancer to facilitate amplification of the entire gE gene from the Argentinian CL15 strain of SHV-1. The cloned gE gene was expressed at high levels in recombinant baculovirus-infected insect cells and reacted strongly with sera from SHV-1 infected pigs. We used the recombinant gE protein to develop a local indirect ELISA test with sensitivity and specificity comparable to currently available commercial tests. Thus, recombinant gE produced in baculovirus-infected insect cells is a viable source of antigen for the detection of SHV-1 in ELISA tests. We also provide evidence supporting a potential application of this recombinant form of gE as a SHV-1 subunit vaccine., (Copyright © 2013. Published by Elsevier Inc.)

Bovine leukemia is a common retroviral infection of cattle. The disease is characterized by a strong immunological response to several viral proteins, but the antibodies against p24 and gp51 are predominant. In this study, a recombinant baculovirus containing the gag gene p24 was constructed and the protein, used as antigen, analyzed by western blot and an indirect in-house rp24-ELISA test. This allowed detecting the presence of antibodies for bovine leukemia virus in a panel of cattle sera. The authentication of the protein expands its potential use for different medical applications, from improved diagnosis of the disease to source of antigens to be included in a subunit vaccine.

Bovine leukemia virus (BLV) infection in cattle causes Enzootic Bovine Leukemia (EBL). About 30% of infected cattle develop persistent lymphocytosis (PL), a 0.1-5% develops tumors, and a 70% remains asymptomatic in an aleukemic stage (AL). Regulatory genes of BLV (Tax, Rex, R3 and G4) are located in a region known as pX(BLV). The variability of those genes had been postulated with the progression of the disease. The aim of this work was to compare the wild-type proviral pX(BLV) region at different stages of BLV natural infected cattle from Argentine Holstein. Pairs of primers were designed to amplify the proviral pX region of 12 cattle by PCR, and products were then sequenced, aligned and compared both with each other and with the reference sequence. Results show a divergence percentage from 0 to 6.1 for the Tax gene, from 0 to 9.4% for the Rex gene, from 0 to 12.1% for the R3 gene and finally from 0 to 6.5% for the G4 gene. Results obtained with hierarchical clustering showed two clusters well differentiated, where the members of each cluster are cattle that had tumor, PL and AL, not allowing differentiate those two cluster by clinical stage., (Copyright © 2012. Published by Elsevier B.V.)

Triatoma virus (TrV), a member of the Dicistroviridae family, replicates in intestinal epithelial cells, causing delayed development and death of infected individuals. The aims of this study were to find naturally infected species of Triatominae in the wild in the region endemic for Chagas disease and analyze and compare the sequence diversity of TrV obtained from different Triatominae. A total of 253 Triatominae belonging to 10 species were captured by active or passive collection. Three new sequences were obtained from Triatoma infestans, Triatoma delpontei and Psammolestes coreodes and the analysis revealed that these sequences were very similar. Ps. coreodes is a new host for TrV., (Copyright © 2012. Published by Elsevier Inc.)

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., (Copyright © 2011. Published by Elsevier B.V.)

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., (© 2011 John Wiley & Sons A/S.)

In the present study, the fragment corresponding to the immunodominant epitopes of the gE gene (gEpi) from the CL15 Argentinean strain of pseudorabies virus was expressed successfully in a baculovirus-insect cell system that contained the M6 gene of Bluetongue virus, which encodes the NS1 nonstructural protein. This protein has the ability to polymerize into highly immunogenic tubules inside infected cells that can be purified at large quantities by ultracentrifugation. Previously, the NS1 protein has been expressed by fusing it to sequences derived from viruses, such as human immunodeficiency virus type 1, hepatitis B virus, bovine leukemia virus, foot-and-mouth disease virus and influenza A virus. In the present study, a recombinant protein was obtained containing the gEpi fused to NS1 (NS1-gEpi) and used it as ELISA antigen for detection of anti-gE antibodies in order to discriminate between infected and vaccinated animals. This is the first report where gEpi was expressed in this particular baculovirus-insect cell system., (© 2010 Elsevier B.V. All rights reserved.)

Objective: To perform genetic analysis of the ORF5 of equine arteritis virus (EAV) may provide new insights into the genetic evolution and origin of the Argentinean EAV sequences., Methods: A total of 76 sequences were analyzed by neighbor joining (NJ), maximum parsimony and maximum likelihood algorithms. The analysis of the selective pressures was performed using the Tajima's test., Results: The trees showed similar topologies. Two clades were identified: the first clade was formed by strains isolated mainly from a donkey, whereas the second clade presented four large groups from different geographic regions exclusively from Equus caballus. In this clade, we identified a group formed by South African and another one by South American and European sequences. In the latter, the monophyletic group was formed by seven Argentinean sequences. In the NJ tree, we identified a group formed by six Argentinean sequences. The Tajima's test showed a D value of 1.73663, indicating that the sequences analyzed follow a neutral evolution model., Conclusion: We concluded that the Argentinean sequences have a paraphyletic origin and that the fixation of point mutation might follow the neutral model evolution; however, we identified purifying pressures that may be involved in the differentiation of the EAV sequences., (Copyright © 2010 S. Karger AG, Basel.)

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%.

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.

The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throughput genotyping of major histocompatibility complex genes in horses and other animal species, making this system interesting as a rapid screening method for animal genotyping of immune-related genes.

Triatoma virus (TrV) is the only entomopathogenic virus found in triatomines. TrV replicates in cells of the midgut epithelium of triatomines, causing a high mortality rate and delayed development of the infected insect. In this work, we report an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of TrV infection. For antiserum production, rabbits and hens where inoculated with purified TrV. Antiserum reactivity was checked by immunodiffusion, and its specificity was confirmed by western blot and AC-ELISA. Totally 90 fecal samples from T. infestans were analysed. AC-ELISA and RT-PCR results correlated well with transmission electron microscopy (EM) observations, which are considered the gold standard, with Kappa values of 0.73 for AC-ELISA and 0.93 for RT-PCR when compared with EM. Applications and complementary uses of the two techniques reported in this work are discussed.

Equine arteritis virus (EAV) was isolated from a testicle of the presumable first stallion infected with EAV in Argentina. This virus isolate (named LT-LP-ARG) was confirmed by GP5-specific PCR and indirect immunofluorescence assays. The PCR product was sequenced, and the phylogenetic analysis revealed that the LT-LP-ARG strain of EAV forms a monophyletic group, together with other strains previously isolated in our laboratory (LP02 group). However, all Argentinean EAV strains belong to a polyphyletic group. We believe that the virus isolate presented in this report could be the origin of EAV infection in our country.

We report the nucleotide sequence and genetic diversity of four Equine Arteritis Virus (EAV) ORF 5 and 6 from Argentina isolates, obtained from asymptomatic virus-shedding stallions. Nucleic acid recovered from the isolates were amplified by RT-PCR and sequenced. Nucleotide and deduced amino acid sequences from the Argentine isolates were compared with 17 sequences available from the GenBank. Phylogenetic analysis revealed that the Argentine isolates grouped together in a definite cluster near European strains. Despite the greater genetic variability among ORF 5 from different isolates and strains of EAV, phylogenetic trees based on ORF 5 and 6 are similar. Both trees showed that virus sequences from America and Europe segregate into distinct clades based on sequence analysis of either ORF 5 or 6. This study constitutes the first characterization of Argentine EAV isolates.

Ten Campylobacter jejuni isolates, 8 identified as C. jejuni biotype II of Lior and 2 as C. jejuni biotipe I, were recovered from aborted pig fetuses. In order to discriminate among strains, restriction fragment length polymorphism (RFLP) using Ddel of polymerase chain reaction (PCR) products of flaA gen was used. C. jejuni biotype II strains could be diferenciated in 6 by PCR-RFLP, and one subtype was obtained from C. jejunibiotype I. Although there is great variability of molecular techniques applied to the Campylobacter epidemiological studies, PCR-RFLP demonstrated to be a simple and accessible technique to discriminate Campylobacter jejuni isolates.

This paper describes the first isolation of equine arteritis virus (EAV) in Argentina. The virus was isolated from the semen of an imported seropositive stallion held in isolation at a breeding farm in Tandil in the Buenos Aires Province. In addition, viral nucleic acid was detected in seminal plasma using the reverse-transcription polymerase chain reaction. The isolated virus was propagated in cell cultures and confirmed as EAV by indirect immunofluorescence and virus neutralisation, using a serum specific for the reference Bucyrus strain of EAV. As far as the authors are aware, this is the first time that EAV has been isolated in South America. The equine industry is very important for Argentina and international movement of horses is very intensive. This finding may have effects on the international trade of horses and semen from Argentina.

Campylobacter jejuni and Campylobacter coli were isolated from aborted pig fetuses which proceeded from different animals and farms between February 2000 and March 2001. Seven Campylobacter jejuni biotype II, three biotype I and one Campylobacter coli biotype I were identified by phenotypic tests and Lior's scheme. To corroborate and compare the phenotypic results, 7.5, 10 and 12.5% polyacrilamide gel electrophoresis (SDS-PAGE) were used under reducing conditions. Characteristic bands of hypervariable dense zone within C. jejuni and C. coli species were observed in all the whole cell protein extracts with differences in mobility. It was possible to establish differences between identical phenotypic Campylobacter isolates and different protein profile from fetuses of the same litter. SDS-PAGE is a stable and reproducible method to establish differences between Campylobacter strains and is considered applicable for the differentiation of the wide variability of Campylobacter species for epidemiologic purposes.

Equine herpesvirus 1 (EHV-1) is the causative agent of abortion, perinatal foal mortality, neurological and acute respiratory diseases in horses. Conventional laboratory diagnosis involving viral isolation from aborted foetuses is laborious and lengthy and requires processing of samples within 24 h of collection, which is problematic for samples that come from long distances. The aim of this study was to develop a polymerase chain reaction (PCR) assay useful in Argentina to detect DNA sequences of EHV-1 in different tissues from aborted equine foetuses with variable quality of preservation and without the use of conventional DNA fenolic extraction. Several DNA extraction protocols and primers were evaluated. The amplification method was standardized and its specificity was analysed using 38 foetal samples of variable quality of preservation. Of the 38 different foetal tissues, nine livers, six spleens and two lungs in good preservation and eight livers, one spleen and four lungs in a poor state of preservation were positive for PCR. EHV-1 was recovered only from the nine livers, five spleens and two lungs in good preservation. No virus was isolated from the samples that were poorly preserved. Viral isolation was confirmed by cytopathic effect and indirect immunofluorescence. The specificity of the PCR results was confirmed by the restriction endonuclease digestion of PCR products and hybridization.

In Argentina pseudorabies is an endemic disease. Routine diagnosis is made by virus isolation. It is a very long procedure to carry out and gives variable results depending on the quality of sample, hence the need for effective techniques, which are rapid and not dependent on the isolation of infectious virus. The polymerase chain reaction (PCR) technique has provided a sensitive, specific and rapid mean to detect DNA sequences. This study describes a PCR method for detection of pseudorabies virus sequences in swine tissues. In order to determine the presence of suid herpesvirus-1 DNA and antigens, 36 tissue samples collected from 19 dead pigs, with signs of pseudorabies infection, were examined by PCR, virus isolation and indirect immunofluorescence, respectively. Fifteen out of 19 pigs were positive at least for one tissue by PCR (15/19) while only three pseudorabies virus strains were isolated (3/19). All the amplified products were identified by digestion with Sa/l and hybridization. The method described herein circumvents tedious viral isolation and DNA purification and would be a valuable tool for rapid diagnosis, since it would take less than 5 h to reach an accurate result even in poorly preserved tissue samples.

A blocking enzyme-linked immunosorbent assay (ELISA) using a urease conjugate (U-B-ELISA) was evaluated for screening sera for antibodies to pseudorabies virus under field conditions. A total of 764 serum samples were analyzed by U-B-ELISA. Of these, 264 were evaluated by both virus neutralization and U-B-ELISA, and the results were compared. U-B-ELISA showed 98.5% and 98.9% sensitivity and specificity, respectively. This test combines the sensitivity and specificity of the blocking ELISA format while allowing visual assessment of results.

An indirect enzyme linked immunosorbent assay was developed. Infected and non infected allantoic fluids precipitated with polyetilenglycol 6000 were used as antigen and control antigen, respectively. Serum samples were diluted 1/20 and a commercial horse radish peroxidase-labelled rabbit anti-equine IgG was used as second antibody. The reaction was developed using azino-diethylbenzotyazol-sulfonate (ABTS). Cut-off was determined by ratio sample (Rs). The hemagglutination inhibition test was used as a reference test for the 391 samples analyzed. Of these, 301 sera were positive by hemagglutination inhibition test and indirect ELISA, 75 were negative by both techniques, and 15 were positive by indirect ELISA and negative by hemagglutination inhibition test. Using hemagglutination inhibition test as standard, the indirect ELISA showed a relative specificity and sensitivity of 83.3 and 100%, respectively. This indirect ELISA is useful as screening test.

The genomes of 10 equine herpesvirus 1 (EHV-1) strains isolated in Argentina from 1979 to 1991, and a Japanese HH1 reference strain were compared by restriction endonuclease analysis. Two restriction enzymes, BamHI and BglII, were used and analysis of the electropherotypes did not show significant differences among isolates obtained from horses with different clinical signs. This suggests that the EHV-1 isolates studied, which circulated in Argentina for more than 10 years, belong to a single genotype.

Various methods have been employed for the diagnosis of pseudorabies in Argentina. A large serological survey was carried out by means of enzyme-linked immunosorbent assay (blocking ELISA) and virus neutralisation (VN). An outbreak was studied by virological and immunohistochemical methods and in situ nucleic acid hybridisation.