The presence of pathogenic viruses in coastal environments is an important tool in evaluating water quality and health risks. Millions of viruses are excreted in fecal matter and bacterial indicators do not correlate with the presence of pathogenic viruses. Enteroviruses have been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter could be used to indicate fecal pollution. The purpose of this research is to develop a baseline understanding of the diversity of viruses found in raw sewage and to assess their presence in the marine environment. PCR was used to detect adenoviruses, herpesviruses, hepatitis B viruses, morbilliviruses, noroviruses, papillomaviruses, pepper mild mottle viruses, picobirnaviruses, reoviruses, rotaviruses, and sapporoviruses in raw sewage collected from throughout the United States and from five marine environments ranging in their proximity to dense human populations. Adenoviruses, noroviruses, pepper mild mottle viruses, and picobirnaviruses were detected in raw sewage but absent in the marine environment, making these viruses potential indicators of fecal pollution in marine environments. These viruses were also found in many of the final effluent samples. Pepper mild mottle viruses may be useful for source tracking fecal contamination since it was consistently found in human sewage and is not expected in the feces of other animals due to its dietary origin. Furthermore, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage and the marine environment will enable educated decisions to be made regarding the use of viruses in water quality assessments.

Orientador: Maria Silvia Viccari Gatti / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T08:58:49Z (GMT). No. of bitstreams: 1 Fregolente_MariaClaraDuarte_D.pdf: 7203529 bytes, checksum: acf60e6e770f96882a60213754fcb52d (MD5) Previous issue date: 2010 / Resumo: Picobirnavírus (PBV) pertencem à família Picobirnaviridae, gênero Picobirnavirus e têm como espécie tipo Human picobirnavirus e Rabbit picobirnavirus. Estes pequenos vírus não envelopados, de dois segmentos genômicos de RNA dupla fita, são encontrados em amostras de fezes diarreicas ou não de diferentes hospedeiros mamíferos, incluindo o homem, aves e répteis. Os mecanismos da infecção por PBV e sua associação a gastroenterites ainda não estão esclarecidos, mas são colocados como agentes emergentes e oportunistas e seu potencial zoonótico foi sugerido. As técnicas utilizadas para a identificação desses vírus são: eletroforese em gel de poliacrilamida (EGPA) e RT-PCR. A primeira permite diferenciar os PBV pelas diferenças de migração dos seus segmentos genômicos. Já na RT-PCR são sete os pares de iniciadores descritos, incluindo aqueles que permitem sua diferenciação em genogrupo I ou II. Este projeto objetivou caracterizar genética e filogeneticamente PBV de diferentes hospedeiros naturais e as estratégias utilizadas foram o sequenciamento total e parcial dos segmentos genômicos de PBV e a definição de uma região conservada para o desenho de iniciadores capazes de diagnosticar todos os PBV por RT-PCR. Foram analisadas para a presença de PBV amostras de fezes de suínos, coelhos, ratos, cães, cobras, ratos silvestres, capivaras, cavalos e bovinos. Utilizando a EGPA, PBV foram identificados em todos os hospedeiros estudados e pela RT-PCR identificou-se genogrupo I de PBV em quase todos, com exceção de capivaras e bovinos. O genogrupo II não foi identificado. A circulação do genogrupo I em diferentes hospedeiros sugere que não existe especificidade genogrupo-espécie de hospedeiro. O sequenciamento parcial do segmento menor dos PBV identificados em cães, cobra e ratos mostrou uma relativa homologia principalmente com sequências de PBV identificados em humanos. A coexistência de duas ou mais populações de PBV em um mesmo hospedeiro foi identificada em cavalos, suínos, rato, rato silvestre e coelho a partir do sequenciamento parcial do segmento menor após clonagem, sugerindo um possível mecanismo de reassortment, o que pode levar a salto entre espécies. Esses resultados suportam o potencial emergente e zoonótico dos PBV. A heterogeneidade nas sequências de nucleotídeos verificada por esse sequenciamento sugere a presença de quasiespécies de PBV nesses hospedeiros. A menor variação observada nas sequências de nucleotídeos de PBV identificados em animais não confinados pode ser justificada pela tendência ao menor contato entre esses animais do que entre os de cativeiro, fazendo com que a transmissão viral também seja menor. Foi proposta uma padronização para a nomenclatura dos PBV, baseada em seu hospedeiro, país e ano de identificação. O atual sistema de classificação para os PBV não é apropriado, devido à identificação de PBV não pertencentes a nenhum dos genogrupos já descritos e à presença de heterogeneidade nas sequências de PBV do genogrupo I. Infelizmente, não foi possível sequenciar o genoma completo dos PBV estudados, não sendo identificada nenhuma sequência conservada que permitisse o desenho de iniciadores capazes de unificar o diagnóstico dos PBV. Estudos tentativos estão em andamento para que, a partir do sequenciamento completo e análise do genoma de diferentes PBV, seja possível definir as porcentagens de identidade mínimas para sua classificação em genogrupos e/ou genotipos / Abstract: Picobirnaviruses (PBV) belong to the Picobirnaviridae family, genus Picobirnavirus, and Human picobirnavirus and Rabbit picobirnavirus are the type species. These small non-enveloped viruses, with two genetic segments of double-stranded RNA, can be found in diarrheic or nondiarrheic fecal samples from different hosts like mammals, including humans, birds and reptiles. PBV infection and its association with gastroenteritis are still unknown, but they are considered opportunistic and emergent pathogens, and their zoonotic potential has also been suggested. Techniques for PBV identification include: polyacrylamide gel electrophoresis (PAGE) and RTPCR. The first one allows characterization of PBV according to the migration pattern of their genomic segments. In the RT-PCR, seven primers' pairs have been designed, including one that allows classification of PBV into genogroups I or II. The aim of this project was the genetic and phylogenetic characterization of PBV identified in fecal samples from different natural hosts by complete and partial sequencing of PBV genomic segments and set up of a conserved region for designing primers able to detect all PBV by RT-PCR. Fecal samples from pigs, rabbits, rats, dogs, snakes, wild rats, capybaras, horses and cattle were analyzed for PBV occurrence. PBV were identified in all studied hosts by PAGE and genogroup I was identified in the majority of them by RT-PCR, except in capybaras and cattle. Genogroup II was not identified. Genogroup I circulation in different hosts suggests that there is no genogroup-host species' specificity. Partial sequencing of small PBV's genomic segment identified in fecal samples from dogs, snake and rats showed homology mainly to human PBV sequences. Coexistence of two or more PBV population in the same host could be determined in fecal samples from horses, pigs, rat, wild rat and rabbit by partial sequencing of small PBV's genomic segment after cloning, suggesting that reassortment may occur in nature, allowing host species jump. These results support the emergent and zoonotic potential of PBV. The heterogeneity found in PBV's nucleotide sequences after cloning suggests the existence of PBV quasispecies in these hosts. The little variation in nucleotide sequences of PBV identified in hosts living in an open environment could be justified by a tendency of less contact among these animals, allowing less viral spread. The classification system used nowadays is cannot be considered appropriated as it doesn't consider the heterogeneity found in PBV's genogroup I sequences. Also, PBV that don't belong to any of the described genogroups, remain with no classification. Therefore, a new standard nomenclature for PBV, based on its host, country and year of identification was proposed. Unfortunately, it was not possible to sequence the complete genome of PBV found in this study. Also, no conserved sequence could be identified for primers' design, which would be capable of standardized PBV detection. Additional studies are ongoing to try to define nucleotide sequences identity percentages for genogroups and/or genotypes classification / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular