Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health concern in many developing countries. It mainly infects young adults and has a mortality of up to 25% in pregnant women. Although hepatitis E is only sporadic in industrialized countries including the United States, a relative high seroprevalence rate has been reported in healthy individuals. Evidence suggests that there exist animal reservoirs for HEV and HEV transmission is zoonotic. Animal strains of HEV, swine HEV and avian HEV have been identified from a pig and a chicken, respectively, in the United States. Studies showed that swine HEV and avian HEV are genetically and antigenically related to human HEV, and that pigs and chickens are useful animal models to study HEV replication, pathogenesis and cross-species infection. The objectives of this dissertation were to genetically characterize both avian HEV and swine HEV, to determine their serological and molecular epidemiology in the United States, to assess the ability of avian HEV cross-species infection in non-human primates, to determine the full-length genomic sequence and genome organization, and to construct an infectious cDNA clone of avian HEV. The prevalence of swine HEV infections in US swine herds and the heterogeneity of swine HEV isolates from different geographic regions of the United States were determined. We found that 35% pigs and 54% swine herds were positive for swine HEV RNA. Partial capsid gene region of twenty-seven US swine HEV isolates was sequenced and was showed to share 88%-100% nucleotide sequence identity to each other and 89-98% identity with the prototype US swine HEV, but only <79% identity with Taiwanese swine HEV isolates and most known human strains of HEV worldwide. All US swine HEV isolates belong to the same genotype 3 with the prototype US swine HEV and the two US strains of human HEV. Similarly, the prevalence of avian HEV infections in US chicken flocks and the heterogeneity of avian HEV isolates were also determined. Helicase gene region of eleven field isolates of avian HEV from chickens with hepatitis-splenomegaly (HS) syndrome was sequenced and was found to share 78-100% nucleotide sequence identities with each other, 79-88% identities with the prototype avian HEV, 76-80% identities with Australian chicken big liver and spleen disease virus (BLSV), and 56-61% identities with other known strains of mammalian HEV. A relative high prevalence of anti-avian HEV antibodies was found in apparently healthy chicken flocks in 5 states. Like swine HEV, the seropositivity of avian HEV in adult chickens was higher than that in young chickens. To genetically characterize the avian HEV genome, we determined the full-length genomic sequence of avian HEV, which is 6,654 bp in length excluding the poly (A) tail, and 600 bp shorter than that of mammalian HEVs. Avian HEV has similar genomic organization with human and swine HEVs, but shared only about 50% nucleotide sequence identity with mammalian HEVs in the complete genome. Significant genetic variations such as deletions and insertions, particularly in the ORF1 of avian HEV, were observed, but motifs in the putative functional domains of the ORF1 were relatively conserved between avian HEV and mammalian HEVs. Phylogenetic analyses based on the full-length genomic sequence revealed that avian HEV represents a branch distinct from human and swine HEVs. Since swine HEV infects non-human primates and possibly humans, the ability of avian HEV cross-species infection in non-human primates was also assessed. However, unlike swine HEV, avian HEV failed to infect two rhesus monkeys under experimental conditions. With the availability of the complete genome sequence of avian HEV, we constructed three full-length cDNA clones of avian HEV and tested their infectivity by in vitro transfection of the LMH chicken liver cells and by in vivo intrahepatic inoculation of specific-pathogen-free (SPF) chickens. The results showed that all 3 cDNA clones of avian HEV were infectious both in vitro and in vivo, as the capped RNA transcripts from each of the clones were replication-competent in transfected LMH cells and developed active infection in inoculated SPF chickens. In summary, avian HEV and swine HEV infections are enzootic in chicken flocks and in swine herds in the United States, respectively. Like human HEV, swine HEV and avian HEV isolates from different geographic regions are also genetically heterogenic. Complete genomic sequence analyses showed that avian HEV is related to, but distinct from, human and swine HEVs. Unlike swine HEV, avian HEV is probably not transmissible to non-human primates. Infectious cDNA clones of avian HEV have been successfully constructed. The availability of the infectious clones for a chicken strain of HEV now affords us an opportunity to study the mechanisms of HEV replication, pathogenesis and cross-species infection. / Ph. D.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-05-19T13:08:10Z No. of bitstreams: 1 Maria Fernanda Melo Monteiro.pdf: 639501 bytes, checksum: 8ac4c3c948754bb276d69058aaf30f8b (MD5) / Made available in DSpace on 2016-05-19T13:08:10Z (GMT). No. of bitstreams: 1 Maria Fernanda Melo Monteiro.pdf: 639501 bytes, checksum: 8ac4c3c948754bb276d69058aaf30f8b (MD5) Previous issue date: 2016-02-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Rickettsial diseases are zoonotic infections caused by gram-negative bacteria of the family Rickettsiaceae which are transmitted by several ectoparasites such as ticks belonging to the genus Amblymomma, however, the species Rhipicephalus sanguineus sensu lato (s.l.) and Ctenocephalides felis felis have been reported as potential vectors.The aim of this study was to detect Rickettsia spp. DNA in R. sanguineus s.l. and C. felis felis through molecular examination (Polymerase Chain Reaction PCR). A total of 728 ectoparasites were collected of 155 dogs from the states of Pernambuco (municipalities of Recife and Bezerros) and Alagoas (municipalities of Viçosa and Arapiraca) were used in this study. All specimens were morphologically identified (R. sanguineus s.l. and C. felis felis), separated in pools (n = 3) and analyzed molecularly through PCR. Out of the 136 pools of R. sanguineus s.l. analyzed, 20.58% (28/136) and 80 pools of C. felis felis analyzed 31.20% (39/80) it was observed amplification fragment of 401 bp compatible with Rickettsia spp. In relation to the positivity of ectoparasites in the municipalities studied, it was observed that R. sanguineus s.l. was positive in the Metropolitan Area of Recife (20.49%) and Bezerros (22.22%), however in the state of Alagoas was not observed the positivity for Viçosa municipality, but, Arapiraca got 25% positive. With regard to C. felis felis it was observed that in Pernambuco 54.54% of samples were positive being derived from Metropolitan Area of Recife, while Bezerros municipality was not observed amplification of the fragment compatible with Rickettsia spp. In Alagoas, it was observed 12.50% of positivity for Arapiraca and 50.00% for Viçosa. This study reports, for the first time, the detection of Rickettsia spp. in R. sanguineus s.l. parasitizing dogs from the state of Pernambuco and R. sanguineus s.l. and C. felis felis parasitizing dogs from the state of Alagoas. Although there are no reports of Brazilian Spotted Fever (BSF) in the areas studied, the presence of infected ticks and fleas suggests the circulation of the pathogen between vertebrate hosts and vectors. / As riquetsioses são doenças zoonóticas causadas por bactérias gram-negativas da família Rickettsiaceae sendo transmitidas por diversos ectoparasitos, destacando-se os carrapatos do gênero Amblymomma, entretanto, as espécies Rhipicephalus sanguineus sensu lato (s.l.) e Ctenocephalides felis felis vêm sendo relatadas como vetores em potencial. Desta forma, o objetivo deste trabalho foi diagnosticar a infecção por Rickettsia spp. em R. sanguineus s.l. e C. felis felis através da Reação em Cadeia da Polimerase (PCR). Para tanto foram coletados 728 ectoparasitos de 155 cães de dois municípios do estado de Pernambuco (Recife e Bezerros) e de dois municípios do estado de Alagoas (Viçosa e Arapiraca). Todos os espécimes foram identificados morfologicamente (R. sanguineus s.l. e C. felis felis), sendo separados em pools (n = 3) e analisados através da PCR. Dos 136 pools de R. sanguineus s.l. analisados 20,58% (28/136) e dos 80 pools de C. felis felis analisadas 31,20% (39/80) foi observado amplificação de fragmento de 401 pb compatíveis com Rickettsia spp. Em relação à positividade dos ectoparasitos nos municípios estudados, observou-se que R. sanguineus s.l. foi positivo na Região Metropolitana do Recife (20,49%) e em Bezerros (22,22%), entretanto, no estado de Alagoas não foi observada positividade para o município de Viçosa, porém, Arapiraca obteve 25% de positividade. Em relação à C. felis felis observou-se que em Pernambuco 54,54% das amostras foram positivas sendo elas procedentes da Região Metropolitana do Recife, enquanto que no município de Bezerros não foi observada amplificação do fragmento compatível com Rickettsia spp. Já em Alagoas observou-se positividade de 12,50% para Arapiraca e 50,00% para Viçosa. Este estudo reporta pela primeira vez a detecção de Rickettsia spp. em R. sanguineus s.l. parasitando cães provenientes do estado de Pernambuco e R. sanguineus s.l. e C. felis felis parasitando cães provenientes do estado de Alagoas. Apesar de não existirem relatos de Febre Maculosa Brasileira (FMB) nas regiões estudadas, a presença de carrapatos e pulgas infectados sugere a circulação do patógeno entre hospedeiros vertebrados e vetores.