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.

Fibropapillomatosis Associated with Chelonid alphaherpesvirus 5 (ChHV5) in a Green Turtle Chelonia mydas in Argentine Waters.

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.

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.

Argentinian small ruminant lentivirus (SRLV) p55gag antigen fused to maltose binding protein to use in SRLV serological confirmatory diagnosis.

The complete gag gene from small ruminant lentiviruses (SRLV) encodes for a polyprotein of 55 kDa, known as p55gag. p55gag presents multiple antigenic epitopes, which can be recognized by antibodies, increasing the opportunity to detect SRLV-positive animals. Therefore, this polyprotein is considered an excellent candidate to use in diagnostic tests to detect antibodies against SRLV. Different studies have suggested that the selection of the recombinant antigen, which must be representative of the virus strains circulating in the test population, is crucial to avoid false negative results. Thus, the use of proteins from different viral strains isolated from goats or sheep of a given region or country may be a useful strategy to increase the ability to detect SRLV-infected animals. In the present study, the pMAL-p5X vector was used to express and purify p55gag (now called rp55gag for recombinant polyprotein 55 gag). The cloned gene was inserted downstream from the malE gene of Escherichia coli, which encodes a maltose-binding protein (MBP), resulting in the expression of an MBP fusion protein. The complete gag gene was amplified by RT-PCR. Finally, after digestion, the product was cloned into the pMAL-p5X vector and used to transform E. coli ER2325 cells. After the purification of MBP-rp55gag by affinity chromatography, the eluted fraction was observed by SDS-PAGE and Western Blot (WB). The WB was carried out with 85 serum samples from small ruminants previously analysed and compared by two commercial ELISAs. The results show that 76 of the serum samples were concordant with those by both ELISAs. Regarding the other nine serum samples, which showed discordant results between both ELISAs, were positive by WB. The results thus show that the rp55gag could be considered as an antigen in a confirmatory diagnostic assay to detect SRLV by WB. For this purpose, a future study with a high number of sera to determine the test specificity and sensitivity, using the p55gag of the circulating strain in Argentina will be necessary.

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.

Host range of Triatoma virus does not extend to Aedes aegypti and Apis mellifera.

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.

Study of horn flies as vectors of bovine leukemia virus.

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.

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.

Can Triatoma virus inhibit infection of Trypanosoma cruzi (Chagas, 1909) in Triatoma infestans (Klug)? A cross infection and co-infection study.

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.

First isolation and nucleotide comparison of the gag gene of the caprine arthritis encephalitis virus circulating in naturally infected goats from Argentina.

Caprine arthritis encephalitis virus (CAEV) has been reported in different countries worldwide, based on serological and molecular detection. In Argentina, the prevalence of CAEV infections is increasing, with goats showing symptoms associated mostly with cachexia and arthritis. Although in Argentina the virus has been detected by serology, it has never been isolated or characterized. Thus, the objectives of this work were to isolate and analyze the nucleotide sequences of the gag gene of Argentine CAEV strains and compare them with those of other SRLVs previously reported. Nucleotide sequence comparison showed homology with CAEV-Co, the CAEV prototype. Phylogenetic analyses showed that the Argentine strains clustered with genotype B, subtype B1. Because the molecular characterization of the gag region is suitable for phylogenetic studies and may be applied to monitor the control of SRLV, molecularly characterizing the Argentine CAEV strains may help develop a proper plan of eradication of CAEV infections.

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.

Intrinsic, extrinsic and endoplasmic reticulum stress-induced apoptosis in RK13 cells infected with equine arteritis virus.

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.

Modelling the potential geographic distribution of triatomines infected by Triatoma virus in the southern cone of South America.

BACKGROUND: Triatoma virus (TrV) is the only entomopathogenous virus identified in triatomines. We estimated the potential geographic distribution of triatomine species naturally infected by TrV, using remotely sensed and meteorological environmental variables, to predict new potential areas where triatomines infected with TrV may be found. METHODS: Detection of TrV infection in samples was performed with RT-PCR. Ecological niche models (ENM) were constructed using the MaxEnt software. We used 42 environmental variables derived from remotely sensed imagery (AVHRR) and 19 bioclimatic variables (Bioclim). The MaxEnt Jackknife procedure was used to minimize the number of environmental variables that showed an influence on final models. The goodness of fit of the model predictions was evaluated by the mean area under the curve (AUC). RESULTS: We obtained 37 samples of 7 species of triatomines naturally infected with TrV. Of the TrV positive samples, 32% were from sylvatic habitat, 46% came from peridomicile habitats and 22% from domicile habitats. Five of the seven infected species were found only in the sylvatic habitat, one species only in the domicile and only Triatoma infestans was found in the three habitats. The MaxEnt model estimated with the Bioclim dataset identified five environmental variables as best predictors: temperature annual range, mean diurnal range, mean temperature of coldest quarter, temperature seasonality and annual mean temperature. The model using the AVHRR dataset identified six environmental variables: minimum Land Surface Temperature (LST), minimum Middle Infrared Radiation (MIR), LST annual amplitude, MIR annual amplitude annual, LST variance and MIR variance. The potential geographic distribution of triatomine species infected by TrV coincides with the Chaco and the Monte ecoregions either modelled by AVHRR or Bioclim environmental datasets. CONCLUSIONS: Our results show that the conditions of the Dry Chaco ecoregion in Argentina are favourable for the infection of triatomine species with TrV, and open the possibility of its use as a potential agent for the biological control of peridomestic and/or sylvatic triatomine species. Results identify areas of potential occurrence that should be verified in the field.

Phylogenetics based on partial ORF2 of triatoma virus in triatomines collected over a decade from domiciliary habitats.

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.

Detection of triatomine infection by Triatoma virus and horizontal transmission: protecting insectaries and prospects for biological control.

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.

First study of different insect cells to triatoma virus infection.

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.

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.

First description of hemagglutination by a virus belonging to the family Dicistroviridae.

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.