Detecção e caracterização de papilomavírus em caninos e bovinos

A papilomatose é uma doença causada por um grande grupo de vírus epiteliotrópicos pertencentes a família Papillomaviridae que infectam virtualmente todos os animais amniotas. Os papilomavírus são compostos por um capsídeo formado pelas proteínas L1 e L2, do qual abriga uma molécula de DNA dupla fita e circular em seu interior, não contendo envelope lipoproteico. Eles são denominados vírus oncogênicos por causarem lesões benignas e malignas na epiderme e mucosas de seus hospedeiros. Ao longo dos últimos anos, há uma crescente identificação de diversos tipos de papilomavírus causando ampla variedade de lesões tanto em animais domésticos, quanto em animais selvagens. Isso se deve a difusão e maior facilidade ao acesso às ferramentas de sequenciamento convencional e de alto desempenho das quais permitem a identificação, a diferenciação e a quantificação viral. Grande parte das lesões papilomatosas podem ter significativo impacto na saúde animal, e também importantes perdas econômicas diretas e indiretas na pecuária. Esse estudo tem por objetivo compreender a diversidade genética de papilomavírus encontrados em cães e em bovinos leiteiros, provendo a comunidade científica e veterinária uma concisa atualização sobre este campo. Dessa maneira, foi realizada a detecção dos papilomavírus presentes em lesões associadas a papilomas, utilizando análise patológica, ferramentas de biologia molecular, sequenciamento pelo método Sanger e de alto desempenho, permitindo a classificação e a inferência filogenética das sequências obtidas. Este trabalho está dividido em dois capítulos. O primeiro capítulo apresenta o relato de um caso clínico de uma cadela com lesões papilomatosas incomuns que progrediram para uma neoplasia maligna. Foi possível descrever através da caracterização patológica das lesões, utilizando imunohistoquímica e hibridização in situ, revelando fortes sinais de associação entre o CPV16 com a neoplasia maligna. Foi possível recuperar o genoma viral presente na lesão, identificar o envolvimento do CPV16, construir uma inferência filogenética e comparar suas oncoproteínas. No segundo capítulo, descrevemos o projeto que teve como objetivo utilizar a estratégia de sequenciamento de alto desempenho afim de identificar coinfecções, possibilitar a montagem completa dos genomas virais, a caracterização e inferência filogenética dos tipos de papilomavírus bovino envolvidos nas lesões de teto de vacas leiteiras. Foi observado 23,5% de coinfecções, destacando a técnica de PCR convencional seguida por sequenciamento SANGER não demonstra a real totalidade de BPVs contidos na lesão. Além disso, foram caracterizados 17 novos prováveis novos tipos de BPVs, dentre eles um novo gênero e uma nova espécie. Devido a diversidade de BPVs encontrada nas lesões não se pode associar algum tipo a determinada localização anatômica. Esse estudo destaca a importância da oncogênese induzida pelo CPV e a diversidade de tipos de BPVs encontrada em tetos de vacas leiteiras, expandindo o atual conhecimento genético da família Papillomaviridae.The papillomatosis is a disease caused by a large group epitheliotropic viruses belonging to the family Papillomaviridae that infect virtually all amniote animals. They are non-enveloped viruses composed of a capsid that is structured by the L1 and L2 proteins, which harbor a circular double-stranded DNA molecule. PVs are oncogenic viruses that cause benign and malignant lesions in the epidermis and mucosa of their hosts. Over the last few years, there is increasing identification of various types of papillomavirus causing wide range of lesions in both domestic and wild animals. This is due to the diffusion and easier access to the conventional and high throughput sequencing tools that allow identification, differentiation and viral quantification. Much of the papillomatous lesions can have a significant impact on animal health, as well as significant direct and indirect economic losses on livestock. This study aims to understand the genetic diversity of papillomavirus found in dogs and dairy cattle, providing the scientific and veterinary community a concise update on this field. Hence, the detection of papillomavirus present in papillomatous-like lesions was performed using molecular biology tools, Sanger and high yield sequencing, allowing the classification and phylogenetic inference of the sequences obtained. This work is divided into two chapters. The first chapter report a clinical case of a female dog with uncommon papillomatous lesions that progressed to a malignant neoplasm. Among them, we were able to describe the associations of a CPV16 strain with invasive SCC, build a phylogenetic inference, identify and compare the oncoprotein genes of the CPV16 strain. In the second chapter, is was applied an unbiased molecular tool for the detection and characterization of BPV in samples regarding teats warts lesion. We observed 23.5% of coinfections, highlighting that PCR followed by sanger sequencing cannot represet the totality of BPVs presents in the sample. Moreover, we described fifteen putative new BPV tipes, among them one new genus and one new species. Due to the diversity of BPVs found in the lesions, no type can be associated with a specific anatomical location. This study highlights the importance of CPV-induced oncogenesis and the diversity of BPV types found in dairy cow teats warts, expanding the current genetic knowledge of the Papillomaviridae family

Identification of enteric viruses circulating in a dog population with low vaccine coverage

Although the use of vaccines has controlled enteric diseases in dogs in many developedcountries, vaccine coverage is still under optimal situation in Brazil. There is a large popula-tion of nonimmunized dogs and few studies about the identification of the viruses associated with diarrhea. To address this situation, stool samples from 325 dogs were analyzed bypolymerase chain reaction for the detection of common enteric viruses such as Canine ade-novirus (CAdV), Canine coronavirus (CCoV), Canine distemper virus (CDV), Canine rotavirus (CRV)and Carnivorous protoparvovirus 1 (canine parvovirus 2; CPV-2). At least one of these specieswas detected in 56.6% (184/325) of the samples. The viruses detected most frequently ineither diarrheic or nondiarrheic dog feces were CPV-2 (54.3% of the positive samples), CDV(45.1%) and CCoV (30.4%), followed by CRV (8.2%) and CAdV (4.9%). Only one agent wasdetected in the majority of the positive samples (63%), but co-infections were present in 37%of the positive samples and mainly included CDV and CPV-2. The data presented herein canimprove the clinical knowledge in regions with low vaccine coverage and highlight the needto improve the methods used to control these infectious diseases in domestic dogs

The genetic diversity of “papillomavirome” in bovine teat papilloma lesions

Background: Papillomaviruses are small nonenveloped, circular double-stranded DNA viruses that belong to the Papillomaviridae family. To date, 29 Bos taurus papillomavirus (BPV) types have been described. Studies involving mixed BPV infections have rarely been reported in contrast to human papillomavirus (HPV), which is commonly described in numerous studies showing coinfections. Moreover, previous studies had shown that HPV coinfections increase the risk of carcinogenesis. In the present study, we used rolling-circle amplification followed by a high-throughput sequencing (RCA-HTS) approach in 23 teat papillomas from southern Brazil. Results: Eleven well-characterized BPV types and 14 putative new BPV types were genetically characterized into the Xi, Epsilon and Dyoxipapillomavirus genera according to phylogenetic analysis of the L1 gene, which expands the previous 29 BPV types to 43. Moreover, BPV coinfections were detected in the majority (56.3%) of the papilloma lesions analyzed, suggesting a genetic diverse “papillomavirome” in bovine teat warts. Conclusions: The data generated in this study support the possibility that a wide range of BPV is probably underdetected by conventional molecular detection tools, and that BPV coinfections are underestimated and probably genetic diverse. Additionally, 14 new BPV types were characterized, increasing the knowledge regarding BPV genetic diversity

Detecção e análise genômica do Mamastrovirus 5 em cães no Brasil

O mamastrovirus 5 (MAstV5) é classificado no gênero Mamastrovirus da família Astroviridae, sendo associado com surtos agudos de gastroenterite transitória em filhotes de cães ao redor do mundo. O objetivo desta dissertação foi detectar e analisar a variabilidade genética dos MAstV5 circulantes em cães no Brasil. Para isto, amostras de suabe retal foram coletadas de 269 cães de diferentes regiões do Brasil no período de 2008-2014, dos quais 26,39% foram positivos para MAstV5 através de RT-PCR convencional e de RT-Hemi-nested PCR, amplificando porção conservada do gene do capsídeo e do gene da polimerase, respectivamente. Quatro destas cepas tiveram seu genoma parcialmente sequenciado, caracterizado e analisado filogeneticamente. A caracterização dessas amostras revelou uma notável heterogeneidade genética entre as cepas de MAstV5. A baixa identidade entre as sequências do gene do capsídeo (<85%) indicaria uma possível nova classificação entre a espécie MAstV5 em dois genótipos. Conclue-se que o MAstV5 ocorre em cães no Brasil e as cepas circulantes possuem uma grande diversidade genética.The Mamastrovirus 5 (MAstV5) is classified in the genus Mamastrovirus of the Astroviridae family, being associated with acute episodes of transient gastroenteritis in puppies around the world. The aim of this work was to detect and analyze the genetic variability of circulating MAstV5 in dogs in Brazil. For this, rectal swab samples were collected from 269 dogs from different regions of Brazil in the 2008-2014 period, of which 26.39% were positive for MAstV5 by conventional RT-PCR and RT-Hemi-nested PCR, amplifying conserved portion of the capsid gene and polymerase gene, respectively. Four of these strains had its genome sequenced partially characterized and analyzed phylogenetically. The characterization of these samples revealed a remarkable genetic heterogeneity among strains of MAstV5. The low identity between the sequences of capsid gene (<85%) indicate a possible new classification between the two genotypes MAstV5 species. We conclude that the MAstV5 occurs in dogs in Brazil and circulating strains have a high genetic diversity

Detecção e análise genômica do Mamastrovirus 5 em cães no Brasil

O mamastrovirus 5 (MAstV5) é classificado no gênero Mamastrovirus da família Astroviridae, sendo associado com surtos agudos de gastroenterite transitória em filhotes de cães ao redor do mundo. O objetivo desta dissertação foi detectar e analisar a variabilidade genética dos MAstV5 circulantes em cães no Brasil. Para isto, amostras de suabe retal foram coletadas de 269 cães de diferentes regiões do Brasil no período de 2008-2014, dos quais 26,39% foram positivos para MAstV5 através de RT-PCR convencional e de RT-Hemi-nested PCR, amplificando porção conservada do gene do capsídeo e do gene da polimerase, respectivamente. Quatro destas cepas tiveram seu genoma parcialmente sequenciado, caracterizado e analisado filogeneticamente. A caracterização dessas amostras revelou uma notável heterogeneidade genética entre as cepas de MAstV5. A baixa identidade entre as sequências do gene do capsídeo (<85%) indicaria uma possível nova classificação entre a espécie MAstV5 em dois genótipos. Conclue-se que o MAstV5 ocorre em cães no Brasil e as cepas circulantes possuem uma grande diversidade genética.The Mamastrovirus 5 (MAstV5) is classified in the genus Mamastrovirus of the Astroviridae family, being associated with acute episodes of transient gastroenteritis in puppies around the world. The aim of this work was to detect and analyze the genetic variability of circulating MAstV5 in dogs in Brazil. For this, rectal swab samples were collected from 269 dogs from different regions of Brazil in the 2008-2014 period, of which 26.39% were positive for MAstV5 by conventional RT-PCR and RT-Hemi-nested PCR, amplifying conserved portion of the capsid gene and polymerase gene, respectively. Four of these strains had its genome sequenced partially characterized and analyzed phylogenetically. The characterization of these samples revealed a remarkable genetic heterogeneity among strains of MAstV5. The low identity between the sequences of capsid gene (<85%) indicate a possible new classification between the two genotypes MAstV5 species. We conclude that the MAstV5 occurs in dogs in Brazil and circulating strains have a high genetic diversity

Genotyping of canine distemper virus strains circulating in Brazil from 2008 to 2012

AbstractCanine distemper virus (CDV) is a major pathogen of dogs and represents a serious threat to both unvaccinated and vaccinated animals. This study surveyed dogs with or without clinical signs related to canine distemper from different regions of Brazil from 2008 to 2012. A total of 155 out of 386 animals were found to be CDV positive by RT-PCR; 37 (23.8%) dogs were asymptomatic at the time of sampling, and 90 (58%) displayed clinical signs suggestive of distemper. Nineteen (12.2%) dogs had a record of complete vaccination, 15 (9.6%) had an incomplete vaccination protocol, and 76 (49%) had no vaccination record. Based on the sequence analysis of the complete hemagglutinin gene of 13 samples, 12 of the strains were characterized as Genotype South America-I/Europe. Considering criteria of at least 95% nucleotide identity to define a genotype and 98% to define a subgenotype, South America-I/Europe sequences segregated into eight different phylogenetically well-defined clusters that circulated or co-circulated in distinct geographical areas. Together, these findings highlight the relevance of CDV infection in Brazilian dogs, demonstrate the predominance of one genotype in Brazil and support the need to intensify the current control measures

Detection and genetic characterization of Mamastrovirus 5 from brazilian dogs

Mamastrovirus 5 (MAstV5), belonging to the Astroviridae (AstV) family, previously knownas canine astrovirus or astrovirus-like particles, has been reported in several countries tobe associated with viral enteric disease in dogs since the 1980s. Astroviruses have beendetected in fecal samples from a wide variety of mammals and birds that are associ-ated with gastroenteritis and extra enteric manifestations. In the present study, RT-PCRwas used to investigate the presence of MAstV5 in 269 dog fecal samples. MAstV5 wasdetected in 26% (71/269) of the samples. Interestingly, all MAstV5-positive samples derivedfrom dogs displaying clinical signs suggestive of gastroenteritis, other enteric viruses weresimultaneously detected (canine parvovirus, canine distemper virus, canine coronavirus,canine adenovirus and canine rotavirus). Based on genomic sequence analysis of MAstV5a novel classification of the species into four genotypes, MAstV5a-MAstV5d, is proposed.Phylogenetic analyses based on the ORF2 amino acid sequences, samples described hereingrouped into the putative genotype ‘a’ closed related with Chinese samples. Other studiesare required to attempt the clinical and antigenic implications of these astrovirus genotypesin dogs

Detection and genetic characterization of Mamastrovirus 5 from brazilian dogs

Mamastrovirus 5 (MAstV5), belonging to the Astroviridae (AstV) family, previously knownas canine astrovirus or astrovirus-like particles, has been reported in several countries tobe associated with viral enteric disease in dogs since the 1980s. Astroviruses have beendetected in fecal samples from a wide variety of mammals and birds that are associ-ated with gastroenteritis and extra enteric manifestations. In the present study, RT-PCRwas used to investigate the presence of MAstV5 in 269 dog fecal samples. MAstV5 wasdetected in 26% (71/269) of the samples. Interestingly, all MAstV5-positive samples derivedfrom dogs displaying clinical signs suggestive of gastroenteritis, other enteric viruses weresimultaneously detected (canine parvovirus, canine distemper virus, canine coronavirus,canine adenovirus and canine rotavirus). Based on genomic sequence analysis of MAstV5a novel classification of the species into four genotypes, MAstV5a-MAstV5d, is proposed.Phylogenetic analyses based on the ORF2 amino acid sequences, samples described hereingrouped into the putative genotype ‘a’ closed related with Chinese samples. Other studiesare required to attempt the clinical and antigenic implications of these astrovirus genotypesin dogs

Identification of enteric viruses circulating in a dog population with low vaccine coverage

Although the use of vaccines has controlled enteric diseases in dogs in many developedcountries, vaccine coverage is still under optimal situation in Brazil. There is a large popula-tion of nonimmunized dogs and few studies about the identification of the viruses associatedwith diarrhea. To address this situation, stool samples from 325 dogs were analyzed bypolymerase chain reaction for the detection of common enteric viruses such as Canine ade-novirus (CAdV), Canine coronavirus (CCoV), Canine distemper virus (CDV), Canine rotavirus (CRV)and Carnivorous protoparvovirus 1 (canine parvovirus 2; CPV-2). At least one of these specieswas detected in 56.6% (184/325) of the samples. The viruses detected most frequently ineither diarrheic or nondiarrheic dog feces were CPV-2 (54.3% of the positive samples), CDV(45.1%) and CCoV (30.4%), followed by CRV (8.2%) and CAdV (4.9%). Only one agent wasdetected in the majority of the positive samples (63%), but co-infections were present in 37%of the positive samples and mainly included CDV and CPV-2. The data presented herein canimprove the clinical knowledge in regions with low vaccine coverage and highlight the needto improve the methods used to control these infectious diseases in domestic dogs

Canine papillomavirus type 16 associated to squamous cell carcinoma in a dog : virological and pathological findings

Papillomaviruses (PVs) are circular double-stranded DNA virus belonging to Papillomaviridae family. During the infection cycle, PVs translate proteins that can influence cell growth and differentiation, leading to epidermal hyperplasia and papillomas (warts) or malignant neoplasms. Canis familiaris papillomaviruses (CPVs) have been associated with different lesions, such as oral and cutaneous papillomatosis, pigmented plaques, and squamous cell carcinomas (SCCs). Here, we report a clinical case of a mixed bred female dog with pigmented plaques induced by CPV16 (Chipapillomavirus 2) that progressed to in situ and invasive SCCs. Gross and histological findings were characterized, and the lesions were mainly observed in ventral abdominal region and medial face of the limbs. In situ hybridization (ISH) revealed strong nuclear hybridization signals in the neoplastic epithelial cells, as well as in the keratinocytes and koilocytes of the pigmented viral plaques. The full genome of the CPV16 recovered directly from the lesions was characterized, and the phylogenetic relationships were determined. The identification of oncoprotein genes (E5, E6, and E7) by high throughput sequencing (HTS) and their expected domains are suggestive of the malignant transformation by CPV16