Diversidade genética de papilomavírus bovino no Brasil

Os papilomavírus (PV) são vírus epiteliotrópicos que infectam um grande número de vertebrados, incluindo os mamíferos, aves, répteis e peixes. Eles são vírus não envelopados, compostos de um capsídeo estruturado pelas proteínas L1 e L2, que abriga uma molécula de DNA dupla fita e circular. Os PV são vírus oncogênicos que causam lesões benignas e malignas na epiderme e mucosas de seus hospedeiros. Em bovinos, o papilomavírus bovino (BPV) causa papilomas extensivos em animais suscetíveis. Apesar das infecções por BPV apresentarem alta morbidade e baixa mortalidade, elas causam prejuízos econômicos aos produtores, que frequentemente descartam animais precocemente devido à extensão e o local das lesões. Atualmente, 23 tipos de BPV foram classificados e caracterizados, contrastando com o alto número de papilomavírus humanos (HPV) sequenciados e caracterizados (204). Desta forma, acredita-se que a diversidade genética dos BPV seja provavelmente similar à diversidade genética dos HPV existentes. Este trabalho objetivou investigar a diversidade genética dos BPV existentes na região norte e sul do Brasil, assim como realizar o sequenciamento completo de novos tipos virais e tipos já descritos e, desta forma, contribuir para futuros estudos epidemiológicos, filogenéticos e de caracterização genética de BPV. Ainda, esta tese objetivou prover uma atualização da atual epidemiologia, classificação e características genéticas dos PV de ruminantes, com o foco principal nos BPV. Metodologias convencionais (baseadas em amplificação de ácidos nucléicos virais por PCR utilizando oligonucletotídeos específicos para PV) e de última geração (amplificação randômica de genomas circulares e sequenciamento de alta eficiência) foram empregadas, proporcionando a identificação dos vírus circulantes em rebanhos da região Norte e da região Sul do país. Nove novos tipos de BPV foram sequenciados e caracterizados neste estudo (BPV16, 17, 18, 19, 20, 21 e os prováveis BPV24, 25 e 26), evidenciando a grande diversidade genética de BPV presente na região amazônica quando comparada com amostras do sul do país. Além disso, o sequenciamento de genomas completos de outros tipos virais já descritos também foi realizado. Esses resultados proporcionaram uma visão geral dos tipos virais presentes em rebanhos de dois extremos do Brasil, assim como possibilitou a identificação de prováveis novos tipos e a caracterização de novos tipos de BPV. Esses resultados são importantes para entender a evolução e a distribuição dos papilomavírus, bem como para o desenvolvimento de estudos vacinais, que envolvem papilomavírus humano e bovino. Adicionalmente, podemos verificar que pesquisas envolvendo ruminantes selvagens são esporádicas, no entanto seriam importantes para o melhor entendimento da biologia e das relações intra e interespecíficas dos PV.Papillomaviruses (PV) are epitheliotropic viruses that infect a large number of vertebrates, including mammals, birds, reptiles and fish. They are non-enveloped viruses composed of a capsid that is structured by the L1 and L2 proteins, which harbous a circular double-stranded DNA molecule. PVs are oncogenic viruses that cause benign and malignant lesions in the epidermis and mucosa of their hosts. In cattle, bovine papillomavirus (BPV) causes extensive papillomas in susceptible animals. Although BPV infections present high morbidity and low mortality, they cause economic losses to producers, who often discard animals prematurely because of the extent and location of the lesions. Currently, 23 types of BPVs have been classified and characterized, contrasting with the high number of human papillomaviruses (HPVs) sequenced and characterized (204). Therefore, it is believed that the genetic diversity of BPV is probably similar to the genetic diversity of existing HPV. This work aimed to investigate the genetic diversity of BPV in the Northern and Southern regions of Brazil, as well as complete sequencing of new viral types and types already described, and thus contribute to future epidemiological, phylogenetic and genetic characterization studies of BPV. Furthermore, this thesis aimed to provide an update of the current epidemiology, classification and genetic characteristics of ruminant PV, with the main focus on BPV. Conventional methodologies (based on amplification of viral nucleic acids by PCR using specific oligonucleotides for PV) and last generation methodologies (random amplification of circular genomes and high efficiency sequencing) were employed, providing the identification of circular viruses in herds of the North and South regions of the country. Nine new types of BPV were sequenced and characterized in this study (BPV16, 17, 18, 19, 20, 21 and the putatives BPV24, 25 and 26), evidencing the great genetic diversity of BPV present in the Amazon region when compared with samples from the South region. In addition, sequencing of complete genomes from other viral types already described has also been performed. These results provided an overview of the viral types present in Brazilian herds from two distant regions, as well as the identification of potential new types and the characterization of new types of BPV. These results are important to understand the papillomavirus evolution and distribution as well as to development of vaccinal studies, which involve human and bovine papillomaviruses. Additionally, we could verify that researches involving wild ruminants are sporadic, but it would be important for the better understanding of the biology as well as the intra and interspecific of PV relationship

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

Occurrence of Ascaridia galli and Heterakis gallinarum in Guinea Fowl (Numida meleagris) in the State of Rondônia, Brazil

Background: Parasitic diseases are among the problems that most affect birds. The Ascaridia spp. is one of the genera which is responsible for the main infections, and the Ascaridia. galli is the most frequent species found in birds. The Ascaridia spp. egg is oval and present a smooth shell which could be mistaken with the Heterakis gallinarum eggs, once they are the most frequent parasites in birds. Therefore, this case report describes the occurrence of Ascaridia galli and Heterakis gallinarum in a guinea fowl (Numida meleagris) feces and gut.Case: In January 2017, rain season in northern Brazil, a visit was performed to a free range avian breeding property in the municipality of São Miguel do Guaporé in the state of Rondônia, Brazilian Amazonian Region. The property breeds two chicken species: Gallus gallus domesticus and Numida meleagris as well as one quail species. The problem was related to the guinea fowl breeding which was composed by 50 adults and 45 young birds about two or three weeks old. All 45 young guinea fowl were presenting various clinical signs, which included: yellowish catarrhal diarrhea, isolation, loss of appetite, body weight, ruffled feathers, drooping wings and sternal prominence. All animals that presented the clinical signs as ruffled feathers, drooping wings and sternal prominence died on an average of 24 h and the mortality was of 65.9% (29/45). Subsequently, a three-week old bird corpse was necropsied, and it was observed that its intestines presented a large amount of endoparasites besides presenting thickening. As there were a lot of helminths, they were collected along with the intestinal content for examination. The parasitological examinations of the feces were performed by Willys Molay's technique. As a result of the fluctuation technique, were observed and identified large quantities of Heterakis gallinarum and Ascaridia galli eggs. The adult worms of H. gallinarum and A. galli were identified using dichotomous key.Discussion: Ascaridia galli and Heterakis gallinarum infection were reported in guinea fowl in a free range chicken breeding property in the municipality of São Miguel do Guaporé in the state of Rondônia, Brazilian Amazonian Region. Ascaridia galli and Heterakis gallinarum eggs have already been identified in free-range chickens (Gallus gallus domesticus) by flotation techniques, as Willys Mollay and Faust and they are the main species of helminths housed by chickens. However, these parasites are not commonly reported in guinea fowl worldwide, especially in the Numida meleagris species. Regarding necropsy, intestinal wall thickening is generally a common finding which has been reported in chickens, however it is not commonly reported in guinea fowl. Additionally, the presence of clinical signs of nematode parasitosis frequently occurs in birds with high parasitic load. In this report, we believe that the guinea fowls births on the rain season was one of the factors to the high parasites incidence found, since both nematodes species are frequently identified in the rain seasons. Additionally, the management adopted by the breeder, using concomitantly the same nursery for guinea fowl and chicken, added up to inadequate sanitary management, seems to have directly influenced the contamination and recontamination of the different brood litter of guinea fowls. It is concluded that guinea fowl (Numida meleagris) located in the state of Rondônia were affected by high amounts of Ascaridia galli and Heterakis gallinarum and caused high mortality in the young specimens and consequently causing economic losses to the proprietary

Wild capybaras as reservoir of shiga toxin-producing Escherichia coli in urban Amazonian Region

Capybaras are rodent widely distributed in South America, which inhabit lakeside areas including ecological parks and urban sites. Due to anthropological interaction, monitoring zoonotic pathogens in wildlife is essential for One Health. We investigated faecal samples from capybaras living in an urban area in Rio Branco (Acre, Brazil) for the presence diarrhoeagenic E. coli. Virulence factors from shiga toxin-producing E. coli (STEC), enterohaemorrhagic E. coli (EHEC), and enteropathogenic E. coli (EPEC) were screened by PCR. We detected at least one virulence factor in 81% of the animals, being classified as STEC and EHEC pathotypes. The presence of zoonotic E. coli in capybaras is a warning due to the highly frequent anthropological interactions with wild animals in this area. Our findings highlight the importance of investigating wild animals as carriers of zoonotic E. coli, requiring further investigations into wildlife surveillance and epidemiological monitoring

Complete genome sequence of Deltapapillomavirus 4 (bovine papillomavirus 2) from a bovine papillomavirus lesion in Amazon Region, Brazil

The complete genome sequence of bovine papillomavirus 2 (BPV2) from Brazilian Amazon Region was determined using multiple-primed rolling circle amplification followed by Illumina sequencing. The genome is 7,947 bp long, with 45.9% GC content. It encodes seven early (E1, E2,E4, E5, E6,E7, and E8) and two late (L1 and L2) genes. The complete genome of a BPV2 can help in future studies since this BPV type is highly reported worldwide although the lack of complete genome sequences available

Complete genome sequence of deltapapillomavirus 4 (bovine papillomavirus 2) from a bovine papillomavirus lesion in Amazon region, Brazil

The complete genome sequence of bovine papillomavirus 2 (BPV2) from Brazilian Amazon Region was determined using multiple-primed rolling circle amplification followed by Illumina sequencing. The genome is 7,947 bp long, with 45.9% GC content. It encodes seven early (E1, E2, E4, E5, E6, E7, and E8) and two late (L1 and L2) genes. The complete genome of a BPV2 can help in future studies since this BPV type is highly reported worldwide although the lack of complete genome sequences available