Characterization of new strains of Pseudorabies virus in Argentina: Detection of interspecies transmission.

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.

Clinical and molecular aspects of veterinary coronaviruses.

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.

Evidence of porcine circovirus type 2 and co-infection with ungulate protoparvovirus 1 (porcine parvovirus) in mummies and stillborn piglets in subclinically infected farm.

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.

First detection and genetic characterization of porcine circovirus type 3 (PCV3) in Argentina and its association with reproductive failure.

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.

First isolation and molecular characterization of Suid herpesvirus type 1 from a domestic dog in Argentina.

Since Aujeszky`s disease (pseudorabies), which is caused by Suid herpesvirus type 1 (SuHV-1), was first notified in Argentina in 1978, many SuHV-1 strains have been isolated from swine. However, this disease can affect other vertebrates, such as dogs (secondary hosts), and lead to fatal neurological disease. The objective of the current work is to report the first isolation and molecular characterization of SuHV-1 from a dead domestic dog from Santa Fe Province (Argentina), which had had nervous signs compatible with pseudorabies. Samples of brain and trigeminal ganglia from this dog were obtained and fixed in formol for histopathology, and virology studies were conducted after cell disruption. Supernatants of both samples were inoculated onto RK13 cells and, after 72 h, DNA was extracted with phenol-chloroform. Purified DNA was cut with a restriction enzyme and subjected to agarose gel and an aliquot was used to amplify the gD and gC genes by PCR. The gC sequence was compared with other public sequences. The strain isolated from the dog was similar to other Argentinean swine strains.

Production of pseudorabies virus recombinant glycoprotein B and its use in an agar gel immunodiffusion (AGID) test for detection of antibodies with sensitivity and specificity equal to the virus neutralization assay.

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.

Development of a peptide ELISA for the diagnosis of Equine arteritis virus.

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.

Equine arteritis virus gP5 protein induces apoptosis in cultured insect cells.

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.

Expression and purification of Suid Herpesvirus-1 glycoprotein E in the baculovirus system and its use to diagnose Aujeszky's disease in infected pigs.

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.

Analysis of the pX region of bovine leukemia virus in different clinical stages of Enzootic Bovine Leukemia in Argentine Holstein cattle.

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.

Expression of p24 gag protein of bovine leukemia virus in insect cells and its use in immunodetection of the disease.

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.

New Triatoma virus hosts in wild habitats of Argentina.

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.

A differential ELISA based on recombinant immunodominant epitopes of the gE gene of SHV-1 in a baculovirus-insect cell system to discriminate between pigs infected naturally with pseudorabies and vaccinated pigs.

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.

Extended phylogeny of the equine arteritis virus sequences including South American sequences.

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.

Genetic typing of equine arteritis virus isolates from Argentina.

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.