Phylogenetic analysis of papillomaviruses in dogs from southern Brazil: molecular epidemiology and investigation of mixed infections and spillover events.

Papillomaviruses (PVs) have been identified in several animal species, including dogs (canine papillomaviruses, CPVs) and cattle (bovine papillomaviruses, BPVs). Although some BPVs may occasionally infect species other than cattle, to the best of our knowledge, BPVs have not been reported in dogs to date. Herein, we carried out a retrospective phylogenetic study of PVs circulating in dogs from southern Brazil between 2017 and 2022, also investigating possible mixed infections and spillover events. For this, we screened 32 canine papilloma samples by PCR using the degenerate primers FAP59/64 and/or MY09/11, which amplify different regions of the L1 gene; the genomic target often used for PV classification/typing. Out these, 23 PV DNA samples were successfully amplified and sequenced. All PVs amplified by FAP59/64 (n = 22) were classified as CPV-1. On the other hand, PVs amplified by MY09/11 (n = 4) were classified as putative BPV-1. Among these, three samples showed mixed infection by CPV-1 and putative BPV-1. One of the putative BPV-1 detected in co-infected samples had the L1 gene full-sequenced, confirming the gene identity. Furthermore, the phylogenetic classifications from the FAP59/64 and/or MY09/11 amplicons were supported by a careful in silico analysis, which demonstrated that the analysis based on them matches to the classification from the complete L1 gene. Overall, we described CPV-1 circulation in southern Brazil over the years and the potencial BPV infection in dogs (potential spillover event), as well as possible CPV/1/BPV-1 co-infections. Finally, we suggest the analysis of the complete genome of the putative BPVs detected in dogs in order to deepen the knowledge about the PV-host interactions.

Novel high-coverage primers for detection of canine morbillivirus by end-point and real-time RT-PCR assays.

Canine distemper virus (CDV) is a major threat to domestic dogs and wildlife worldwide. Molecular assays are the most sensitive and specific tests to diagnose the disease, however, the high CDV genetic variability may compromise laboratory diagnosis. Herein, we designed a high-coverage primer set for end-point (RT-PCR) and real-time (RT-qPCR) for CDV detection. Initially, we collected 194 complete/near-complete CDV genomes (GenBank) and analyzed them for highly conserved regions for primer design. We then assessed the in silico coverage, analytical sensitivity, specificity and diagnostic performance of RT-PCR/RT-qPCR reactions based on our primers. Furthermore, the coverage of our primers, as well as their analytical sensitivity and diagnostic performance, were compared to a commonly used primer set for CDV detection (named PP-I). Our forward (F) and reverse (R) primers fully matched 100 % (194/194) and 99 % (192/194) of the analyzed sequences, whereas the PP-I F and R primers fully matched 15 % (29/194) and 9 % (18/194) sequences, respectively. The detection limit of our RT-PCR and RT-qPCR was equivalent to that of PP-I primers (0.001 TCID50/mL). Out of 70 clinical samples tested, 38 were positive by our RT-PCR/RT-qPCR assays, whereas reactions with primers PP-I failed to detect 9/28 (32 %) positive samples selected for comparison purposes. In addition, our assays did not amplify other canine viruses associated with respiratory and neurological diseases: canine adenovirus 2, canine parainfluenza virus 2, canine herpesvirus 1 and rabies virus. Overall, we describe a high-coverage primer set for CDV detection, which represents an attractive tool for laboratory diagnosis of canine distemper.

Phylogeny and amino acid analysis in single and mixed bovine papillomavirus infections in Southern Brazil, 2016-2020.

Bovine papillomaviruses (BPVs) exhibit a high degree of genetic variability, and several viral types have been identified based on analysis of the L1 gene. The L1 is the main capsid protein and the main target for neutralizing antibodies. We performed a retrospective study on BPVs circulating in Rio Grande do Sul state, Southern Brazil, in 2016-2020. DNA from 43 bovine papilloma samples were amplified using two degenerate primer sets - FAP59/64 and MY09/11 - targeting the L1 region, and analyzed for phylogeny, mixed BPV infections (coinfections) and amino acid (aa) sequences. We also performed an in silico analysis with 114 BPV L1 sequences from the GenBank database to assess the agreement between the phylogeny obtained based on complete L1 sequences versus that based on the region amplified using the FAP59/64 and MY09/11 primer sets. Considering single and coinfections, we identified 31 BPV-1 (31/43; 72.1%), 27 BPV-2 (27/43; 62.8%) and 4 BPV-6 (4/43; 9.3%). Coinfections with BPV-1 and BPV-2 were observed in 61.3% of the samples. Our results are supported by in silico analyses that demonstrate that the classification using FAP59/64 or MY09/11 matches the complete L1 results, except for BPV-17 and -18, which may be mistakenly classified depending on the primers used. Furthermore, we found unique or rare amino acids in at least one L1 sequence of each BPV type identified in our study, some of which have been identified previously in papillomavirus epitopes, suggesting immune-mediated selection. Finally, our study provides an overview of BPVs circulating in Southern Brazil over the last five years and point to the combined use of primers FAP59/64 and MY09/11 for analysis of BPV coinfections and putative epitopes.

Detection of Equus caballus papillomavirus-2 in equine penile/preputial papillomas and squamous cell carcinomas in southern Brazil.

For approximately one decade, a novel papillomavirus termed Equus caballus papillomavirus-2 (EcPV-2) has been associated with equine penile/preputial papillomas and squamous cell carcinomas (SCCs). It is currently believed that the virus has a carcinogenic activity, being able to induce such neoplastic lesions. After being first described, EcPV-2 has been detected in many countries from North America, Europe, and Asia; however, to date, it has not been reported in Brazil. The aim of this research was to investigate the presence of EcPV-2 in penile/preputial papillomas and SCCs of Brazilian horses. Forty samples diagnosed as equine penile and/or preputial papillomas, carcinomas in situ (CIS), or SCCs in two veterinary anatomic pathology services from southern Brazil were investigated. Histologic evaluation and immunohistochemistry (IHC) using a BPV-1 antibody were performed. Posteriorly, the samples were submitted to polymerase chain reaction using two broad-spectrum (MY09/11 and FAP) and one EcPV-2-specific primer sets. Positive samples were sequenced. PV antigen expression was detected in one papilloma, one CIS, and two SCCs by IHC. Five SCCs, one papilloma, and one CIS were PV-positive on PCR. Sequencing of the seven PCR products revealed homology with EcPV-2. This study confirms the occurrence of EcPV-2 infection in Brazilian horses. Moreover, the results presented here provide useful information concerning the phylogeny from the viruses detected in our samples. We hope to encourage further studies on this novel agent, contributing to its characterization, and, possibly, to the eventual development of preventive measurements, including a possible vaccine.

Virus viability in spiked swine bone marrow tissue during above-ground burial method and under in vitro conditions.

The emergence of high consequence animal diseases usually requires managing significant mortality. A desirable aspect of any carcass management method is the ability to contain and inactivate the target pathogen. The above-ground burial (AGB) technique was recently developed and proposed as an alternative carcass management method. Here, we investigate the tenacity of swinepox virus (SwPV), as a surrogate model for African swine fever virus (ASFV) in swine carcasses during the AGB process. For this, SwPV was inoculated intrafemorally in 90 adult swine carcasses, which were subsequently disposed under AGB conditions. Bone marrow samples were recovered periodically throughout 12 months and virus viability was assessed by virus isolation (VI), whereas the presence of SwPV DNA was evaluated by quantitative polymerase chain reaction (qPCR). Additionally, an in vitro study assessed the inactivation rate of SwPV, Senecavirus A (SVA), and bovine viral diarrhoea virus (BVDV). Viral suspensions were mixed with bone marrow material and maintained at 21-23°C for 30 days. Virus viability was assessed by VI and viral titration. In the field study, SwPV remained viable only in 11 (55%) bone marrow samples collected on day 7; only viral DNA (and not infectivity) was detected afterwards. SwPV inactivation was estimated to have occurred by day 11. The in vitro testing revealed a variable tenacity of the studied viruses. The viability period was estimated in 28, 80, and 118 days, respectively, for BVDV, SwPV, and SVA. Overall, these findings indicate that the AGB technique was effective in quickly inactivating SwPV. Additionally, the SwPV inactivation rate is comparable to ASFV under field studies and poses a potential model for preliminary ASFV inactivation studies with reduced biosecurity requirements. Moreover, this study contributes to understanding the inactivation kinetics of viruses under specific conditions, which is critical when designing and applying countermeasures in case of biosecurity breaches in sites managing animal mortality.

The Participation of a Malignant Catarrhal Fever Virus and Mycoplasma bovis in the Development of Single and Mixed Infections in Beef and Dairy Cattle With Bovine Respiratory Disease.

The bovine respiratory disease (BRD) complex is a multietiological and multifactorial disease associated with a wide range of viral and bacterial pathogens. This study evaluated the contribution of specific infectious disease agents in the development of BRD in cattle from Brazil and determined if a virus within the malignant catarrhal fever virus (MCFV) group and Mycoplasma bovis, acting individually or in conjunction, can be associated with the development of BRD. Formalin-fixed paraffin-embedded pulmonary sections were used in immunohistochemical assays to determine the intralesional presence of six antigens associated with BRD: bovine alphaherpesvirus 1 (BoHV-1), bovine parainfluenza virus 3 (BPIV-3), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), MCFV, and M. bovis. Pneumonia was diagnosed in 82.7% (120/145) of all cattle evaluated. Interstitial pneumonia (60%, 72/120) and suppurative bronchopneumonia (25.8%, 31/120) were the most frequent patterns of pneumonia identified. Intralesional antigens of MCFV (53.3%, 64/120) were the most frequently associated with BRD, followed by M. bovis (47.5%, 57/120), BVDV (42.5%, 51/120), BoHV-1 (28.3%, 34/120), BRSV (24.2%, 29/120), and BPIV-3 (8.3%, 10/120). Additionally, antigens of BVDV, MCFV, and M. bovis were the most frequently identified agents associated with singular and concomitant infections. The MCFV identified during this study is more likely to be ovine gammaherpesvirus 2 (OvHV-2), since OvHV-2 is the only MCFV identified within the geographical region of this study. Interstitial pneumonia with proliferative vascular lesions may be a useful histologic feature to differentiate MCFV-induced pneumonia from other viral pneumonias of cattle. These results demonstrate that MCFV and M. bovis, in single or mixed infections, can produce pneumonia in cattle and should therefore be considered as primary agents in the development of BRD.

Sequence analysis of the DA domain of glycoprotein E2 of pestiviruses isolated from beef cattle in Southern Brazil.

The envelope glycoprotein E2 of pestiviruses is a major target for neutralizing antibodies. In this study, we analyzed the E2 DA domain of 43 pestiviruses from Southern Brazil. The isolates were identified as Bovine viral diarrhea virus (BVDV) subtypes 1a and 1b or BVDV-2b. Compared to reference strains, the BVDV-1 and -2 isolates had four and two mutations in the DA domain, respectively. All BVDV-2 isolates had a deletion of residues 724 and 725. All mutated amino acids in the BVDV isolates had the same aa substitution, and all were in previously identified antibody binding sites. It is possible that an immunity-mediated selection is acting on the pestiviruses circulating in Southern Brazil.

Diphtheric aspergillosis tracheitis with gastrointestinal dissemination secondary to viral infections in a dairy calf.

Diphtheric aspergillosis tracheitis is an uncommon syndrome described in human pathology, usually associated with immunosuppression in the affected individuals. Interestingly, no comparative/equivalent cases were found in domestic animals. This report describes the pathological and mycological findings associated with diphtheric aspergillosis tracheitis in an immunocompromised calf. The main pathological findings were diphtheric tracheitis and rhinitis, and necrotizing ruminitis associated with intralesional septate, acute branching fungal hyphae consistent with Aspergillus spp. Mycological culture and isolation confirmed the fungal hyphae as A. fumigatus due to characteristic features. Immunohistochemistry (IHC) assays identified intralesional antigens of bovine viral diarrhea virus (BVDV) and malignant catarrhal fever virus (MCFV) at the trachea and small intestine; IHC detected intralesional antigens of bovine alphaherpesvirus 1 (BoHV-1) only at the trachea. These findings confirmed the simultaneous occurrence of A. fumigatus with concomitant infections due to BVDV, MCFV, and BoHV-1 in this calf. Since ovine gammaherpesvirus-2 (OvHV-2) is the cause of MCF in Brail, it is likely that the intralesional MCFV antigens identified were those of OvHV-2. In this case, disseminated aspergillosis was probably associated with the undeveloped immunological status of the calf that was further impaired due to the combined immunodepressive effects of BVDV and BoHV-1 infections. Although BVDV and BoHV-1 are infectious disease pathogens frequently associated with the development of bovine respiratory disease (BRD) in feedlot and dairy cattle, the identification of intralesional OvHV-2-like antigens in several parts of the lungs suggest that this MCFV also played a role in the BRD-associated lesions identified in this calf.

Water-soluble tetra-cationic porphyrins display virucidal activity against Bovine adenovirus and Bovine alphaherpesvirus 1.

Porphyrins are photoactive compounds that can absorb the energy of light and transfer it to oxygen molecules, producing reactive oxygen species (ROS). Once produced, ROS may alter biological molecules and cellular mechanisms, leading to cell apoptosis or inactivation of microorganisms, such as bacteria, fungi, and viruses. Therefore, the objective of this study was to evaluate the in vitro virucidal activity of six tetra-cationic porphyrins against two bovine viruses (Bovine alphaherpesvirus 1, BoHV-1, enveloped; and Bovine adenovirus, BAV, non-enveloped). For this, viral suspensions were incubated with each porphyrin (H2TMeP, ZnTMeP, and CuTMeP at 4.0 µM, NiTMeP at 5.0 µM, and CoClTMeP and MnClTMeP at 1.0 µM) and exposed to white-light irradiation for 0, 60, 120, and 180 min (BAV) or 0, 30, 60, 90, and 120 min (BoHV-1). Then, the remaining viral titers were determined by limiting dilution and compared with the control (virus + porphyrins without light exposition). The porphyrins H2TMeP and ZnTMeP showed the highest virucidal activity against both viruses. ZnTMeP inactivated BoHV-1 after 30 min of photoactivation and H2TMeP after 60 min. The BAV was photo-inactivated by both porphyrins at 180 min of white-light exposition. CuTMeP, NiTMeP, and CoClTMeP porphyrins had weak virucidal activity against BoHV-1 and MnClTMeP showed no virucidal activity against both viruses. These results indicated that free-base H2TMeP and ZnTMeP porphyrins present virucidal activity against non-enveloped and enveloped viruses, opening the possibility for their use to inactivate viruses on surfaces, biological substrates, and solutions.

Bovine upper alimentary squamous cell carcinoma associated with bracken fern poisoning: Clinical-pathological aspects and etiopathogenesis of 100 cases.

Upper digestive tract (UDT) cancer is rare in cattle, however in Southern Brazil, the UDT squamous cell carcinomas (SCCs) are relatively common and have been associated with bracken fern consumption and the presence of papillomas. Although a theory of pathogenesis considers bovine papillomavirus type 4 (BPV-4) as a cofactor in the development of these SCCs, some aspects of the etiopathogenesis of this disease need to be more investigated. In fact, detection of BPV-4 in UDT papillomas is scarce in other regions of the world and has not been performed in Brazil. Therefore, this study had two aims: 1) to analyze the epidemiological, clinical and pathological aspects of 100 natural cases of SCCs in the UDT of cattle grazing on bracken fern (Pteridium arachnoideum) highly contaminated areas, investigating the associations between these parameters; and 2) to investigate the presence of papillomavirus DNA by polymerase chain reaction (PCR) in the UDT papillomas (n = 47) from 30 cattle that also had UDT SCCs. There were statistically significant associations between clinical signs and tumor localization in the UDT; between histological grade of differentiation and tumor localization; and a trend towards significant association between histological grade of differentiation and presence of metastases. The average age of cattle with oropharyngeal SCCs was 7.39 years, with statistically significant difference comparing to cattle with esophageal SCCs (8.6 years). No statistical association was observed among other clinical-pathological parameters (growth pattern and primary site of the tumor) analyzed. No BPV DNA was detected in papillomas by PCR. Therefore, these results suggest the possibility that papillomas of the UDT are not necessarily associated with BPV infection.

In vitro activity of six antiviral drugs against equid alphaherpesvirus type 1 indicates ganciclovir as promising drug for in vivo studies / A atividade in vitro de seis drogas antivirais contra o alfaherpesvírus equino tipo 1 indica que o ganciclovir é uma droga promissora para estudos in vivo

ABSTRACT: Equid alphaherpesvirus type 1 (EHV-1) is distributed worldwide and is a major agent of abortion, respiratory and neurological disease in horses. No specific treatment is available for EHV-1 infection, yet the potential of antiviral therapy has been explored. In this study we investigated the in vitro activity of Acyclovir, Ganciclovir, Foscarnet, Famciclovir, Vidarabina and Cidofovir against EHV-1. For this, the MTT test was performed, in which all the tested drugs showed no toxicity up to 200μg/mL. Subsequently, different drug concentrations were submitted to viral plaque reduction assays in cell culture. The selectivity index (SI) of the compounds was determined using the cytotoxic concentration for 50% of cells (CC50), obtained by MTT, and effective drug concentration to inhibit by 50% the number of viral plaques (EC50). Ganciclovir (SI: 490; EC50: 1.9 μg/mL) was the most efficient and safest drug against EHV-1, followed by Cidofovir (SI: 150, EC50: 5.7μg/mL), Acyclovir (SI: 37.4, EC50: 22.2μg/mL), Famciclovir (SI: 25.1, EC50: 24.5μg/mL), Vidarabine (SI: 12.2, EC50: 40.9μg/mL) and Foscarnet (SI: 6.9, EC50: 49.5 μg/mL), respectively. These results indicated that Ganciclovir (followed by Cidofovir), is a promising candidate for use in in vivo experiments.

RESUMO: O alfaherpesvírus equino tipo 1 (EHV-1) está amplamente distribuído nos rebanhos equinos de todo o mundo e é um dos principais agentes causadores de abortos, doença respiratória e neurológica em equinos. Ainda não há tratamento específico para a infecção pelo EHV-1 em equinos, mas o potencial da terapia antiviral tem sido investigado. Neste trabalho, foi investigada a atividade anti-herpética in vitro dos fármacos Aciclovir, Ganciclovir, Foscanet, Famciclovir, Vidarabina e Cidofovir frente ao EHV-1. Para isso, foi realizado o teste de MTT, em que todas as drogas não apresentaram citotoxicidade até a dose de 200μg/mL. A seguir, diferentes concentrações dos fármacos foram submetidas ao teste de redução de placas virais em cultivo celular. O índice de seletividade (IS) dos compostos foi determinado usando a concentração citotóxica para 50% dos cultivos celulares (CC50), obtida pelo MTT, e pela concentração dos fármacos efetiva para inibir em 50% o número de placas virais (EC50). O Ganciclovir (IS: 490; EC50: 1,9μg/mL) foi o mais eficiente e seguro frente ao EHV-1, seguido pelo Cidofovir (IS: 150; EC50: 5,7 μg/mL), Aciclovir (IS: 37,4; EC50: 22,2μg/mL), Famciclovir (IS: 25,1; EC50: 24,5μg/mL), Vidarabina (IS: 12,2; EC50: 40,9μg/mL) e Foscarnet (IS: 6,9; EC50: 49,5μg/mL). Estes resultados indicam que o Ganciclovir constitui-se em um candidato para uso em experimentos in vivo.

Antibodies against rabies virus in dogs with and without history of vaccination in Santa Maria - RS - Brazil / Anticorpos contra o vírus da raiva em cães com e sem histórico de vacinação em Santa Maria - RS - Brasil

ABSTRACT: The present study investigated the frequency and magnitude of neutralizing antibodies to rabies virus (RABV) in dogs with and without historic of vaccination in Santa Maria/RS. Group A included serum samples from 440 dogs with recent historic of vaccination against rabies, obtained during the 2015 rabies vaccination campaign. Group B included 300 serum samples from dogs submitted to the Veterinary Hospital of the Universidade Federal de Santa Maria in 2015, whose historic of rabies vaccination was unknown. Serum samples were submitted to the rapid fluorescent focus inhibition test (RFFIT) to detect neutralizing antibodies against RABV. In group A, 70.6% (310/440) of the samples had neutralizing antibody titers ≥0.5 international units per milliliter (IU mL-1), considered an indicative of protection against rabies by the World Health Organization. However, approximately 30% of the dogs did not contain antibodies in adequate levels. In group B, 42.3% (127/300) of the samples contained neutralizing antibody titers ≥0.5IU mL-1 and 57.7% (173/300) were negative or contained titers below of the value considered immunized. These results demonstrate that an important proportion of vaccinated dogs (~30%) did not develop adequate antibody levels, mainly those receiving a single vaccine dose. Serologic testing of animals with unknown historic of vaccination revealed relatively low vaccine coverage in the general dog population. Thus, reformulation of immunization strategies - especially the recommendation of a boost vaccination 30 days after the primary dose - and extension of vaccination campaigns are necessary to reach adequate levels and coverage of immunity against RABV in the canine population.

RESUMO: O presente estudo investigou a frequência e a magnitude dos anticorpos neutralizantes do vírus da raiva (RABV) em cães com e sem histórico de vacinação em Santa Maria/RS. O Grupo A incluiu amostras de soro de 440 cães com histórico recente de vacinação contra a raiva, obtidos durante a campanha de vacinação contra a raiva de 2015. O Grupo B incluiu 300 amostras de cães submetidos ao Hospital Veterinário da Universidade Federal de Santa Maria em 2015, cujo histórico de vacinação antirrábica era desconhecido. As amostras de soro foram submetidas ao teste rápido de inibição de focos fluorescentes para detecção de anticorpos neutralizantes do RABV. No grupo A, 70,6% (310/440) das amostras possuíam títulos de anticorpos neutralizantes ≥0,5 unidades internacionais por mililitro (UI mL-1), considerado um indicador de proteção contra a raiva pela Organização Mundial da Saúde. No entanto, aproximadamente 30% dos animais não continham anticorpos em níveis adequados. No grupo B, 42,3% (127/300) das amostras continham títulos de anticorpos neutralizantes ≥0,5UI mL-1 e 57,7% (173/300) eram negativas ou continham títulos abaixo do valor considerado imunizado. Estes resultados demonstram que uma proporção importante de cães vacinados (~30%) não desenvolveu níveis adequados de anticorpos, principalmente aqueles que receberam uma única dose de vacina. O teste sorológico de animais com histórico de vacinação desconhecido revelou uma cobertura vacinal relativamente baixa na população geral de cães. Assim, a reformulação das estratégias de imunização - especialmente a recomendação de uma vacinação de reforço 30 dias após a primeira dose - e a extensão das campanhas de vacinação são necessárias para atingir níveis e cobertura adequados de imunidade contra o RABV na população canina.

Serological response to rabies virus induced by commercial vaccines in cattle / Resposta sorológica ao vírus da raiva induzida por vacinas comerciais em bovinos

ABSTRACT: The antibody response to rabies virus (RABV) induced by commercial vaccines in heifers was investigated. For this, 84 heifers were vaccinated twice (30 days interval) with each of four vaccines (G1 = 14 animals; G2 = 24; G3 = 22 and G4 = 24) and received a booster vaccination 360 days later. Serum samples collected at different intervals after vaccination and 30 days after booster were submitted to a virus neutralizing (VN) assay for RABV antibodies. Thirty days after the second vaccine dose, 92% of the immunized animals presented VN titers ≥0.5UI/mL (geometric medium titers [GMT] 1.7 to 3.8UI/mL). At the day of the booster (360 days post-vaccination); however, the percentage of animals harboring antibody titers ≥0.5UI/mL had dropped to 31% (0-80% of the animals, depending on the vaccine), resulting in lower GMT (0.1 to 0.6UI/mL). Booster vaccination at day 360 resulted in a detectable anamnestic response in all groups, resulting in 83% of animals (65 to 100%) harboring VN titers ≥0.5UI/mL thirty days later (GMT 0.6 to 4.3UI/mL). These results indicated that these vaccines were able to induce an adequate anti-RABV response in all animals after prime vaccination (and after booster as well). However, the titers decreased, reaching titers <0.5UI/mL in approximately 70% of animals within the interval before the recommended booster. Thus, booster vaccination for rabies in cattle using the current vaccines should be performed before the recommended one-year interval, as to maintain neutralizing antibodies levels in most vaccinated animals.

RESUMO: A resposta sorológica contra o vírus da raiva (RABV) induzida por vacinas comerciais foi investigada em bovinos. Para isso, 84 novilhas foram vacinadas duas vezes (30 dias de intervalo) com cada vacina (G1 = 14 animais; G2 = 24; G3 = 22 e G4 = 24) e receberam uma vacinação de reforço 360 dias depois. Amostras de soro coletadas em diferentes momentos após a vacinação e após o reforço vacinal foram submetidas ao teste de vírus neutralização (VN) para detecção de anticorpos contra o RABV. Trinta dias após a segunda dose vacinal, 92% dos animais apresentaram títulos neutralizantes ≥0,5UI/mL (GMT 1,7 a 3,8UI/mL). Porém, no dia do reforço (360 dias pós-vacinação), a porcentagem de animais que ainda apresentava títulos ≥0,5UI/mL havia se reduzido a 31% dos animais (0 a 80%, dependendo da vacina), resultando em baixos TMGs (0,1 a 0,6UI/mL). A vacinação de reforço no dia 360 resultou em resposta anamnéstica em todos os grupos, resultando em 83% (65 a 100%) de animais com títulos VN ≥0,5UI/mL trinta dias após (GMT 0,6 a 4,3UI mL-1). Esses resultados indicam que as vacinas avaliadas induzem uma resposta adequada de anticorpos anti-RABV após a vacinação (e também após o reforço). No entanto, os títulos reduzem-se, atingindo níveis <0,5UI/mL em 70% dos animais durante o intervalo antes do reforço. Assim, vacinação de reforço contra a raiva em bovinos, utilizando-se as vacinas atuais, deve ser realizada em intervalo inferior a um ano, de forma a manter os níveis de anticorpos neutralizantes na maioria dos animais.

Detection of respiratory viruses in shelter dogs maintained under varying environmental conditions

Abstract Three dog shelters in Rio Grande do Sul were investigated for associations between the occurrence of respiratory viruses and shelter environmental conditions. Nasal secretions randomly collected during the cold season were tested via PCR, and this data collection was followed by nucleotide sequencing of the amplicons. In shelter #1 (poor sanitary and nutritional conditions, high animal density and constant contact between dogs), 78% (58/74) of the nasal samples were positive, 35% (26/74) of which were in single infections and 44% (32/74) of which were in coinfections. Shelters #2 and #3 had satisfactory sanitary and nutritional conditions, outdoors exercise areas (#2) and animal clustering by groups (#3). In shelter #2, 9% (3/35) of the samples were positive for Canine parainfluenza virus (CPIV), and 6% (2/35) were positive for Canid herpesvirus 1 (CaHV-1). In shelter #3, 9% (7/77) of the samples were positive for Canine adenovirus type 2 (CAdV-2), and 1% (1/77) were positive for Canine distemper virus (CDV). The amplicon sequences (CPIV and CDV nucleoprotein gene; CAdV-2 E3 gene; CaHV-1 glycoprotein B gene) showed 94-100% nucleotide identity with GenBank sequences. Our results demonstrate that CPIV, CAdV-2 and CDV are common in dog shelters and that their frequencies appear to be related with environmental and nutritional conditions. These results indicate the need for control/prevention measures, including vaccination and environmental management, to minimize these infections and improve dog health.

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV) strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3). The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

Safety and immunogenicity of a glycoprotein E gene-deleted bovine herpesvirus 1 strain as a candidate vaccine strain / Atenuação e imunogenicidade de uma cepa recombinante do herpesvírus bovino tipo 1 defectiva na glicoproteína E

A glycoprotein E-deleted Brazilian bovine herpesvirus 1 (BoHV-1gEΔ) was tested regarding to safety and immunogenicity. Intramuscular inoculation of young calves with a high virus dose did not result in clinical signs or virus shedding during acute infection or after dexamethasone administration. Calves vaccinated once IM (group I) or subcutaneously (group II) with live BoHV-1gEΔ or twice with inactivated virus plus aluminum hydroxide (group IV) or Montanide™ (group V) developed VN titers of 2 to 8 (GMT:2); 2 to 4 (GMT:1.65); 2 to 16 (GMT:2.45) and 2 to 128 (GMT:3.9), respectively. All BoHV-1gEΔ vaccinated calves remained negative in an anti-gE ELISA. Lastly, six young calves vaccinated with live BoHV-1gEΔ and subsequently challenged with a virulent BoHV-1 strain shed less virus and developed only mild and transient nasal signs comparing to unvaccinated calves. Thus, the recombinant BoHV-1gEΔ is safe and immunogenic for calves and allows for serological differentiation by a gE-ELISA test.(AU)

Um isolado brasileiro de herpesvírus bovino tipo 1, contendo uma deleção na glicoproteína E (gE - BoHV-1gEΔ) foi submetido a testes para avaliar a sua segurança e imunogenicidade em bovinos. Bezerros foram submetidos à inoculação intramuscular com uma alta dose viral e não demonstraram sinais clínicos ou excreção viral durante a fase aguda ou após tentativa de reativação viral pela administração de dexametasona. Bezerros que receberam uma dose do vírus vivo, contendo a deleção na gE, pela via intramuscular (grupo I) ou pela via subcutânea (grupo II) ou duas doses do vírus inativado utilizando o adjuvante hidróxido de alumínio (grupo IV) ou Montanide™ (grupo V), desenvolveram títulos de anticorpos neutralizantes de 2 a 8 (GMT: 2); 2 a 4 (GMT: 1,65); 2 a 16 (GMT: 2,25) e de 2 a 128 (GMT: 3,9), respectivamente. Todos os bezerros vacinados se mantiveram soronegativos quando utilizado um kit ELISA específico para a gE. Para o teste de segurança, seis bezerros foram vacinados com o vírus vivo BoHV-1gEΔ, sendo estes posteriormente desafiados com uma cepa virulenta de BoHV-1. Estes bezerros excretaram menos vírus e desenvolveram apenas sinais clínicos moderados e transitórios quando comparados com dados coletados de quatro animais não-vacinados. Com base nestes resultados, podemos confirmar que o vírus do BoHV-1 que contém a deleção na gE (BoHV-1gEΔ) é seguro e suficientemente imunogênico para bezerros e permite a diferenciação sorológica entre os animais vacinados e infectados perante a a um teste ELISA commercial específico para a gE.(AU)

Detection of respiratory viruses in shelter dogs maintained under varying environmental conditions.

Three dog shelters in Rio Grande do Sul were investigated for associations between the occurrence of respiratory viruses and shelter environmental conditions. Nasal secretions randomly collected during the cold season were tested via PCR, and this data collection was followed by nucleotide sequencing of the amplicons. In shelter #1 (poor sanitary and nutritional conditions, high animal density and constant contact between dogs), 78% (58/74) of the nasal samples were positive, 35% (26/74) of which were in single infections and 44% (32/74) of which were in coinfections. Shelters #2 and #3 had satisfactory sanitary and nutritional conditions, outdoors exercise areas (#2) and animal clustering by groups (#3). In shelter #2, 9% (3/35) of the samples were positive for Canine parainfluenza virus (CPIV), and 6% (2/35) were positive for Canid herpesvirus 1 (CaHV-1). In shelter #3, 9% (7/77) of the samples were positive for Canine adenovirus type 2 (CAdV-2), and 1% (1/77) were positive for Canine distemper virus (CDV). The amplicon sequences (CPIV and CDV nucleoprotein gene; CAdV-2 E3 gene; CaHV-1 glycoprotein B gene) showed 94-100% nucleotide identity with GenBank sequences. Our results demonstrate that CPIV, CAdV-2 and CDV are common in dog shelters and that their frequencies appear to be related with environmental and nutritional conditions. These results indicate the need for control/prevention measures, including vaccination and environmental management, to minimize these infections and improve dog health.

Antibodies against vesicular stomatitis virus in horses from southern, midwestern and northeastern Brazilian States / Anticorpos contra o vírus da estomatite vesicular em equinos de Estados das regiões sul, centro-oeste e nordeste do Brasil

ABSTRACT: Vesicular stomatitis virus (VSV) is the agent of a vesicular disease that affects many animal species and may be clinically confounded with foot-and-mouth disease in ruminant and swine. Horses are especially susceptible to VSV and may serve as sentinels for virus circulation. The present study investigated the presence of neutralizing antibodies against VSV Indiana III (VSIV-3) in serum samples of 3,626 horses from six states in three Brazilian regions: Southern (RS, n = 1,011), Midwest (GO/DF, n = 1,767) and Northeast (PB, PE, RN and CE, n = 848) collected between 2013 and 2014. Neutralizing antibodies against VSIV-3 (titers ≥40) were detected in 641 samples (positivity of 17.7%; CI95%:16.5-19.0%), being 317 samples from CE (87.3%; CI95%: 83.4-90.5 %); 109 from RN (65.7%; CI95%: 57.8 -72.7%); 124 from PB (45.4%; CI95%: 39.4-51.5%); 78 from GO/DF (4.4%; CI95%: 3.5-5.5%) and nine samples of RS (0.9%; CI95%: 0.4-1.7%). Several samples from the Northeast and Midwest harbored high neutralizing titers, indicating a recent exposure to the virus. In contrast, samples from RS had low titers, possibly due to a past remote exposure. Several positive samples presented neutralizing activity against other VSV serotypes (Indiana I and New Jersey), yet in lower titers, indicating the specificity of the response to VSIV-3. These results demonstrated a relatively recent circulation of VSIV-3 in northeastern Brazilian States, confirming clinical findings and demonstrating the sanitary importance of this infection.

RESUMO: O vírus da estomatite vesicular (vesicular stomatitis virus, VSV) é o agente de doença vesicular que afeta várias espécies e que, em suínos e ruminantes, é clinicamente confundível com a febre aftosa. Os equinos são particularmente susceptíveis ao VSV, servindo de sentinelas para a circulação viral. O presente trabalho investigou a presença de anticorpos neutralizantes contra o VSV Indiana III (VSIV-3) em amostras de soro de 3626 equinos de seis estados das regiões Sul (RS, n=1011), Centro-oeste (GO e DF, n=1767) e Nordeste (PE, PB, RN e CE, n=848), coletadas entre 2013 e 2014. Anticorpos neutralizantes contra o VSIV-3 em títulos iguais ou superiores a 40 foram detectados em 641 amostras (17,7%; IC95%: 16,5-19,0%), sendo 317 do CE (positividade de 87,3%; IC95%: 83,4-90,5%); 109 do RN (65,7%; IC95%: 57,8-72,7%); 124 da PB (45,4%; IC95%: 39,4-51,5%); 78 de GO/DF (4,4%; IC95%: 3,5-5,5%) e em nove amostras do RS (0,9%; IC95%: 0,4-1,7%). Uma parcela das amostras dos estados do Nordeste e Centro-oeste apresentou altos títulos neutralizantes, indicando exposição recente ao vírus. Já as amostras do RS apresentaram títulos baixos de anticorpos, indicando provável exposição temporalmente remota. Quando testadas contra outros sorotipos do VSV (Indiana I e New Jersey), várias amostras apresentaram atividade neutralizante, porém em títulos muito inferiores, indicando a especificidade dos anticorpos para o VSIV-3. Esses resultados demonstram circulação relativamente recente do VSIV-3 em várias regiões do Brasil, sobretudo em estados do Nordeste, confirmando relatos clínicos e demonstrando a importância sanitária dessa infecção.

Experimental infection of horses with Vaccinia virus / Infecção experimental de equinos com o vírus Vaccínia

ABSTRACT: Vaccinia virus (VACV) is the etiologic agent of bovine vaccinia, an emerging zoonotic disease with potential health issues for dairy herds and humans. VACV may occasionally infect other species, including horses. In this sense, an outbreak of VACV disease in horses was described in Pelotas, RS, in 2008, where a co-infection with two VACV strains (named Pelotas Virus 1 [P1V] and Pelotas Virus 2 [P2V]) was detected. Considering the rare occurrence of VACV infection in horses, the objective of this study was to investigate the susceptibility and pathogenesis of VACV infection in this species. Six adult horses were inoculated with VACV P1V or P2V (106.3TCID50/ml) through scarification of the nasolabial surface and monitored for virological and clinical aspects during 28 days. Four inoculated horses (4/6) developed mild lesions in the site of inoculation. Ulcers and scabs restricted to inoculated areas were observed between days 2 and 8 post-inoculation (pi). Microscopically there were acanthosis, ballooning degeneration of the stratum spinosum, necrosis and loss of the epidermis. Infiltration of neutrophils, macrophages and lymphocytes were observed in the dermis. Intracytoplasmic eosinophilic inclusions were infrequently observed in degenerate keratinocytes from adjacent necrotic areas. Virus shedding was detected between days 4 and 8 pi by PCR and virus isolation (infectious virus) from the lesions of one horse inoculated with P2V. No neutralizing antibodies were detected in inoculated animals at day 28 pi. In summary, inoculation of horses with VACV P1V and P2V isolates resulted in a low level of replication and at low frequency, with mild cutaneous lesions, when compared with the course of infection of other susceptible species to VACV. Therefore, horses possibly have a low potential for viral maintenance and transmission to other species, albeit being susceptible to VACV infection.

RESUMO: O vírus Vaccinia (VACV) é o agente etiológico da vaccínia bovina, uma doença zoonótica re-emergente e de importância sanitária e econômica para rebanhos leiteiros. O VACV também pode ocasionalmente infectar outras espécies, incluindo equinos. Nesse sentido, um surto de VACV em equinos foi descrito em Pelotas, RS, em 2008, no qual uma coinfecção com dois isolados de VACV (denominados Pelotas 1 [P1V] e Pelotas 2 [P2V]) foi detectada. Considerando a rara ocorrência da infecção pelo VACV em equinos, o objetivo deste estudo foi investigar a susceptibilidade e a patogenia experimental da infecção pelo VACV nessa espécie. Para isso, seis equinos adultos foram inoculados com P1V ou P2V (106,3DICC50/ml) após escarificação na superfície nasolabial e monitorados para os aspectos virológicos, clínicos e patológicos por 28 dias. Quatro animais inoculados (4/6) desenvolveram lesões macroscópicas leves nos locais da inoculação, consistentes com aquelas causadas por VACV, caracterizadas por úlceras e crostas focais restritas à área inoculada, entre os dias 2 e 8 pós-inoculação (pi). Microscopicamente, havia acantose, degeneração hidrópica do estrato espinhoso e necrose da epiderme, com bactérias intralesionais. Infiltração moderada de neutrófilos, macrófagos e linfócitos foi observada na derme superficial. Inclusões intracitoplasmáticas eosinofílicas foram infrequentemente observadas nos queratinócitos degenerados adjacentes às áreas necróticas. Excreção viral foi detectada por PCR e isolamento viral das lesões de um equino inoculado com o P2V, entre os dias 4 e 8pi. Não foi detectada produção de anticorpos específicos para o VACV em nenhum animal inoculado. Em resumo, a inoculação de cavalos com os isolados P1V e P2V resultou em um baixo nível de replicação viral e em baixa frequência, resultando em lesões cutâneas mais discretas em comparação com outras espécies susceptíveis ao VACV. Apesar dos equinos serem susceptíveis à infecção pelo VACV, possivelmente apresentam baixo potencial de manutenção e de transmissão do vírus para outras espécies.

Resposta sorológica aos herpesvirus bovino tipos 1 e 5 e vírus da diarreia viral bovina induzida por vacinas comerciais / Serological response to bovine herpesvírus 1 and 5 and bovine viral diarrea virus induced by comercial vaccines

Este trabalho avaliou a imunogenicidade de vacinas para os herpesvírus bovino 1 e 5 (BoHV-1, BoHV-5) e vírus da diarreia viral bovina 1 e 2 (BVDV-1, BVDV-2), disponíveis no mercado brasileiro. Para isso, novilhos de raças de corte foram alocados em grupos de 10-12 animais e vacinados duas vezes, com intervalo de 30 dias, com cada uma das oito vacinas disponíveis. Amostras de soro coletadas 30 dias após a segunda dose foram submetidas ao teste de virusneutralização (VNT), frente a cepas de BoHV-1, BoHV-5, BVDV-1 e BVDV-2. Com exceção de duas vacinas que induziram soroconversão em 8/10 e 9/10 dos animais, as demais induziram anticorpos neutralizantes contra o BoHV-1 em todos os animais vacinados (títulos médios geométricos [GMTs] entre 1,7 e 4,8). Quatro vacina s induziram anticorpos reagentes com o BoHV-5 em todos os animais (GMTs de 1,0 a 4,2), enquanto três vacinas induziram soroconversão parcial em 5/10, 6/10 e 7/10 animais. Apenas uma vacina induziu resposta sorológica detectável frente ao BVDV-1 em todos os animais vacinados (GMT=6,7). Soroconversão parcial ao BVDV-1 foi detectada em quatro grupos vacinais (6/10, GMT 4,0 6/10, GMT 5,6 e 4/10, GMT 1,8). Uma vacina induziu resposta em apenas um animal (título de 40) e três vacinas não induziram anticorpos detectáveis contra o BVDV-1 em nenhum animal. Atividade neutralizante frente ao BVDV-2 foi detectada apenas em três grupos vacinais, e parcialmente (10/10, GMT 6,5; 5/10, GMT 1,6 e 2/10, GMT 1,0). Cinco vacinas não induziram atividade neutralizante detectável frente ao BVDV-2 em nenhum dos animais imunizados. Esses resultados demonstram que o componente BoHV-1 da maioria das vacinas comerciais possui imunogenicidade adequada. No entanto, o componente BVDV da grande maioria das vacinas não induz resposta neutralizante consistente frente ao BVDV-1 e, ...

This study evaluated the immunogenicity of vaccines for bovine herpesvirus 1 and 5 (BoHV-1/5) and viral diarrhea virus 1 and 2 (BVDV-1/2) available in the Brazilian market. Calves were divided into groups (10-12), vaccinated twice with a 30 days interval. Samples collected 30 days after the second dose were submitted to virus neutralization test against BoHV-1, BoHV-5, BVDV-1 and BVDV-2. With the exception of two vaccines that induced seroconversion in 8/10 and 9/10 of the animals, the other induced antibodies against BoHV-1 in all vaccinated animals (geometric mean titers [GMTs] 1.7 and 4.8) and four induced antibodies against BoHV-5 in all animals (GMTs 1.0 to 4.2), whereas three vaccines induced partial seroconversion (5/10, 6/10 and 7/10 animals). Only one vaccine induced antibody response to BVDV-1 in all vaccinated animals (GMT=6.7). Partial seroconversion to BVDV-1 was detected in four vaccine (6/10, GMT 4.0; 6/10, GMT 5.6 and 4/10, GMT 1.8). A vaccine induced response in only one animal and three vaccines did not induce antibodies against BVDV-1 in any animal. Antibodies to BVDV-2 were detected only in three vaccine groups, and partly (10/10, GMT 6.5; 5/10, GMT 1.6 and 2/10, GMT 1.0). Five vaccines did not induce antibodies to BVDV-2 in any of the animals. Results demonstrate that the BoHV-1 component of commercial vaccines seems to have acceptable immunogenicity. However, the BVDV component of most vaccines does not induce consistent response against BVDV-1, especially, to BVDV-2. It is evident that strategies for production of vaccines, particularly to BVDV must be urgently revised.