O gênero Ranavirus, especialmente a espécie Frog virus 3 (FV3), é considerado uma ameaça crescente às populações de anfíbios em diversas partes do mundo, desencadeando surtos que frequentemente resultam em mortalidade em massa e perdas econômicas substanciais. Neste sentido, propusemos o isolamento de uma estirpe patogênica de FV3-like, associada a surto com alta mortalidade de anfíbios adultos (Lithobates catesbeianus) em uma ranicultura do Estado de São Paulo, Brasil, bem como a caracterização molecular e fenotípica do vírus isolado. No mais, objetivamos verificar a possível indução de morte celular por apoptose por essa estirpe. O isolamento viral foi realizado a partir de fragmentos de órgãos de animais que vieram à óbito, os quais foram inoculados em células BF-2 (Lepomis macrochirus). A técnica de reação em cadeia da polimerase (PCR) foi conduzida com primers específicos e dirigidos para duas regiões altamente conservadas do genoma dos ranavírus: MCP e 53R. O sequenciamento parcial do gene que codifica a proteína principal do capsídeo (MCP) foi realizado, seguido de alinhamento e análise filogenética com outros ranavírus. Outras técnicas diagnósticas, incluindo microscopia eletrônica de transmissão, imunofluorescência indireta e Western blot foram utilizadas na caracterização e confirmação do isolamento. Dois marcadores apoptóticos foram utilizados para investigar a possível ativação da apoptose em células BF-2 infectadas e amostradas de 4 à 16 horas, incluindo a ativação de caspases efetoras e a fragmentação do DNA celular pelo ensaio de TUNEL. Obtivemos o isolamento de uma estirpe de FV3-like com efeitos citopáticos típicos para ranavírus. A PCR confirmou a presença de DNA viral nas culturas de células BF-2 infectadas, com resultados positivos para os oligonucleotídeos direcionados para MCP e 53R. A análise da sequência nucleotídica obtida para MCP revelou alta homologia (99%) com Frog virus 3, espécie-tipo do gênero Ranavirus (família Iridoviridae) e, na reconstrução filogenética, a cepa isolada mostrou ser intimamente relacionada com outros ranavírus detectados no Brasil. As micrografias eletrônicas mostraram partículas icosaédricas em células BF-2 infectadas, com nucleocapsídeo medindo cerca de 150 ηm, semelhante aos ranavírus. No ensaio de imunofluorescência indireta, células BF-2 infectadas com o isolado FV3-like, apresentaram marcação de imunofluorescência positiva para MCP, enquanto que MCP foi demonstrada por um ensaio de Western blot, onde observamos um polipeptídeo com massa molecular estimada em 50 kDa. Por fim, verificamos que o isolado FV3-like é capaz de induzir apoptose em células BF-2, uma vez que foram detectadas caspases efetoras em todos os tempos experimentais, sugerindo ser um mecanismo caspase-dependente. A fragmentação do DNA celular, claramente observada em todos os tempos experimentais, confirmou a indução de apoptose pela estirpe brasileira de FV3-like. Os resultados aqui obtidos tornam-se a base para diversos estudos futuros, podendo ainda contribuir como subsídio para a melhor compreensão dos surtos provocados por estes vírus no país. / The genus Ranavirus, especially the Frog virus 3 (FV3) species, is considered a growing threat to amphibian populations in various parts of the world, triggering outbreaks that often result in mass mortality and substantial economic losses. In this sense, it was proposed the isolation of a pathogenic FV3-like strain associated with an outbreak with high mortality of adult amphibians (Lithobates catesbeianus) in a frog farm in the State of São Paulo, Brazil, as well as the molecular and phenotypic characterization of the isolated virus. In addition, we aimed to verify the possible induction of the mechanism of cell death by apoptosis by this strain. Virus isolation was performed from organ fragments of animals that died, which were inoculated into BF-2 cells (Lepomis macrochirus). The polymerase chain reaction (PCR) technique was conducted with specific primers and directed to two highly conserved genome regions of the ranavirus: MCP and 53R. Partial sequencing of the gene encoding the major capsid protein (MCP) was performed, followed by alignment and phylogenetic analysis with other ranaviruses. Other diagnostic techniques, including transmission electron microscopy, indirect immunofluorescence and Western blot were used in the characterization and confirmation of the isolation. Two apoptotic markers were used to investigate the possible activation of apoptosis in infected BF-2 cells and sampled from 4 to 16 hours, including the activation of effector caspases and the fragmentation of cellular DNA by the TUNEL assay. We obtained the isolation of an FV3-like strain with typical cytopathic effects for ranavirus. PCR confirmed the presence of viral DNA in cultures of infected BF-2 cells, with positive results for MCP and 53R oligonucleotides. Analysis of the nucleotide sequence obtained for MCP revealed high homology (99%) with Frog virus 3, type species member of the genus Ranavirus (family Iridoviridae) and, in phylogenetic reconstruction, the isolated strain showed to be closely related to other ranaviruses detected in Brazil. Electron micrographs showed icosahedral particles in infected BF-2 cells, with a nucleocapsid measuring about 150 ηm, similar to ranaviruses. In the indirect immunofluorescence assay, BF-2 cells infected with the FV3-like isolate showed positive immunofluorescence labeling for MCP, whereas MCP was demonstrated by a Western blot assay, where we observed a polypeptide with a molecular mass estimated at 50 kDa. Finally, we verified that the FV3-like isolate is able to induce apoptosis in BF-2 cells, since effector caspases were detected at all experimental times, suggesting to be a caspase-dependent mechanism. The fragmentation of cellular DNA, clearly observed at all experimental times, confirmed the induction of apoptosis by the Brazilian FV3-like strain. The results obtained here become the basis for several future studies, and may contribute as a subsidy to better understand the outbreaks caused by these viruses in the country.

A aquicultura é apontada como um mercado estratégico para o desenvolvimento sustentável, produção de alimentos e ampliação de fronteiras inexploradas no Brasil. No entanto, como outros sistemas de produção animal, este setor enfrenta problemas com doenças resultantes de sua intensificação, como os aspectos sanitários da produção e a falta de estrutura para o diagnóstico das principais enfermidades infecciosas. Durante os últimos 20 anos, os vírus da família Iridoviridae, em especial membros do gênero Ranavirus, têm sido responsáveis por epizootias de grande impacto ecológico e econômico, envolvendo um grande número de espécies de peixes, anfíbios e répteis de importância na aquicultura de várias partes do mundo. No entanto, as informações sobre a ocorrência de infecções de peixes e anfíbios causadas por ranavírus no Brasil são limitadas. O presente projeto compreendeu o isolamento em cultivo celular de estirpe de Frog Virus 3-símile, no Estado de São Paulo, confirmada por sequenciamento nucleotídico do gene MCP e subsequente RFLP, assim como caracterização fenotípica e de cinética de replicação do isolado. Amostras de fígado, baço e rins de rãs-touro gigante provenientes de ranário comercial, positivas ao diagnóstico molecular para Ranavirus, foram utilizadas para isolamento viral em cultivo celular. Efeito citopático foi detectado na segunda passagem em células BF-2 e posterior confirmação do isolamento foi feita por PCR e RFLP. A caracterização fenotípica viral foi feita com microscopia eletrônica de transmissão, a qual permitiu a confirmação de morfologia semelhante às partículas de Ranavirus, além da realização de curva de replicação, indicando maiores títulos virais no quarto dia após inoculação. Também foi feito teste de susceptibilidade a solventes, que confirmou a presença de partículas envelopadas. Os resultados obtidos nesse estudo poderão contribuir para a futura compreensão da biologia dos iridovírus circulantes como agentes etiológicos de ranaviroses em anfíbios no Estado de São Paulo. / Aquaculture is appointed as a strategic market for sustainable development, food production and expansion of unexplored frontiers in Brazil. However, just like other animal production systems, this area faces problems due its intensification, like the production sanitation aspects and the lack of structure for the diagnosis of major infectious diseases. During the last 20 years, viruses from Iridoviridae family, especially Ranavirus genera, have been responsible for major economic and ecological impactant epizootic diseases in a great number of fish, amphibian and reptile species in many parts of the world. However, information on the occurrence of infections in fishes and amphibians caused by Ranavirus in Brazil are limited. In this context, this project aimed to isolate a Frog Virus 3-like strain detected in São Paulo state in cell culture, which was later confirmed by nucleotide sequencing of MCP gene and further RLFP technique and also phenotipical characterization and replication kinetics of the obtained strain. Liver, spleen and kidney samples from american bullfrogs from commercial frog ponds, positive by molecular diagnostic for Ranavirus, were used for viral isolation in cell culture. Citopathic effect was detected during the second passage in cells and later confirmed by PCR and RFLP. The viral characterization was carried out with transmission electronic microscopy, which confirmed similar morphology of viral particles to those of Ranavirus, besides the construction of a growth curve which indicated larger titres on the fourth day post inoculation. A test for solvent sensitivity was also performed and confirmed the presence of enveloped particles. The results obtained in this study will contribute to the understanding of the biology of the circulating iridovirus in São Paulo state as an etiological agent of Ranavirus infection in amphibians.

A aquicultura é apontada como um mercado estratégico para o desenvolvimento sustentável, produção de alimentos e ampliação de fronteiras inexploradas no Brasil. No entanto, como outros sistemas de produção animal, este setor enfrenta problemas com doenças resultantes de sua intensificação, como os aspectos sanitários da produção e a falta de estrutura para o diagnóstico das principais enfermidades infecciosas. Durante os últimos 20 anos, os vírus da família Iridoviridae, em especial membros do gênero Ranavirus, têm sido responsáveis por epizootias de grande impacto ecológico e econômico, envolvendo um grande número de espécies de peixes, anfíbios e répteis de importância na aquicultura de várias partes do mundo. No entanto, as informações sobre a ocorrência de infecções de peixes e anfíbios causadas por ranavírus no Brasil são limitadas. O presente projeto compreendeu o isolamento em cultivo celular de estirpe de Frog Virus 3-símile, no Estado de São Paulo, confirmada por sequenciamento nucleotídico do gene MCP e subsequente RFLP, assim como caracterização fenotípica e de cinética de replicação do isolado. Amostras de fígado, baço e rins de rãs-touro gigante provenientes de ranário comercial, positivas ao diagnóstico molecular para Ranavirus, foram utilizadas para isolamento viral em cultivo celular. Efeito citopático foi detectado na segunda passagem em células BF-2 e posterior confirmação do isolamento foi feita por PCR e RFLP. A caracterização fenotípica viral foi feita com microscopia eletrônica de transmissão, a qual permitiu a confirmação de morfologia semelhante às partículas de Ranavirus, além da realização de curva de replicação, indicando maiores títulos virais no quarto dia após inoculação. Também foi feito teste de susceptibilidade a solventes, que confirmou a presença de partículas envelopadas. Os resultados obtidos nesse estudo poderão contribuir para a futura compreensão da biologia dos iridovírus circulantes como agentes etiológicos de ranaviroses em anfíbios no Estado de São Paulo. / Aquaculture is appointed as a strategic market for sustainable development, food production and expansion of unexplored frontiers in Brazil. However, just like other animal production systems, this area faces problems due its intensification, like the production sanitation aspects and the lack of structure for the diagnosis of major infectious diseases. During the last 20 years, viruses from Iridoviridae family, especially Ranavirus genera, have been responsible for major economic and ecological impactant epizootic diseases in a great number of fish, amphibian and reptile species in many parts of the world. However, information on the occurrence of infections in fishes and amphibians caused by Ranavirus in Brazil are limited. In this context, this project aimed to isolate a Frog Virus 3-like strain detected in São Paulo state in cell culture, which was later confirmed by nucleotide sequencing of MCP gene and further RLFP technique and also phenotipical characterization and replication kinetics of the obtained strain. Liver, spleen and kidney samples from american bullfrogs from commercial frog ponds, positive by molecular diagnostic for Ranavirus, were used for viral isolation in cell culture. Citopathic effect was detected during the second passage in cells and later confirmed by PCR and RFLP. The viral characterization was carried out with transmission electronic microscopy, which confirmed similar morphology of viral particles to those of Ranavirus, besides the construction of a growth curve which indicated larger titres on the fourth day post inoculation. A test for solvent sensitivity was also performed and confirmed the presence of enveloped particles. The results obtained in this study will contribute to the understanding of the biology of the circulating iridovirus in São Paulo state as an etiological agent of Ranavirus infection in amphibians.

Ranavirus infection has caused severe disease and mass mortality in UK common frogs for more than twenty years resulting in serious declines in some populations. The pathogen has been studied since 1992. These studies generated two valuable resources exploited in this thesis: an archive of tissues and virus isolates and a database of reports from citizen scientists on ranavirus-consistent mortality. The previous studies yielded modest evidence suggesting that introductions from North America initiated ranavirus emergence in the UK, though little else was known about the pattern of introduction or spread. This thesis conducts a more detailed investigation, extending existing knowledge of ranavirus diversity and spread through molecular epidemiology and phylogenetics, an in vivo infection experiment, and in silico models. Non-lethal sampling protocols for ranavirus screening were assessed in a controlled setting and shown to be as effective as traditional protocols. The database of citizen science reports was utilised in spatio-temporal models of the spread of ranavirus disease, finding that ranavirus infection is spreading by transmission between ponds but that new outbreaks are also correlated with both human population density and regional temperatures. The first whole genome sequence from a UK ranavirus is presented. Analysis of the genome shows that it is an isolate of the ranavirus type species, FV3, on the basis of its near identical genome arrangement and a ‘supergene’ phylogenetic analysis. An unexpected finding was evidence for recent lateral transfer of host DNA into the FV3 genome. A candidate gene survey of European ranaviruses revealed considerable diversity that may explain the variation in virulence and host range in Spain. Two proposed new species of Ranavirus are described there - one highly virulent, the other seemingly asymptomatic – and the previously described CMTV is shown to be a likely cause of catastrophic decline across multiple hosts. A lack of monophyly among Spanish ranaviruses and the spatial pattern of incidence suggest recent introduction(s). Together, the evidence presented in this thesis underlines the key role that humans have played in the spread of this group of virulent wildlife pathogens in two European countries.

Anthropogenic impacts to the environment are unavoidable currently; however, my research investigates a potential mitigation method for amphibians dealing with poor health outcomes caused by detrimental anthropogenic changes to their wetlands. Environmental stressors such as antibiotics leeching from manure of domesticated farm animals into local wetlands can cause a dysbiosis of the gastrointestinal bacterial flora within tadpoles. Dysbiosis of gastrointestinal bacteria during early tadpole development is associated with a decrease in development rate, decrease in body mass accumulation, and other poor health outcomes. I investigated if increasing the indigestible fiber (prebiotic) content in wood frog tadpole’s alfalfa based diet could return tadpoles with stripped microbiomes (dysbiotic gastrointestinal bacterial community composition) to the same phenotype of healthy control tadpoles. I also did a pilot study to see if diet could help in increasing survival post infection with Ranavirus, and from both studies, I created NGSS aligned curriculum and activities. I found that a 10% corn starch enriched alfalfa diet significantly increased the body mass accumulation and development rate of stripped tadpoles. I found there was an association with metabolism and gut dysbiosis. Unfortunately, the connection in regards to corticosterone release was unclear. There was an association with diet and survival, but it needs to be repeated with a larger sample size.

Algumas doenças associadas à piscicultura de tilápias trazem enormes prejuízos aos criadores, resultando em expressivos coeficientes de morbidade e mortalidade entre animais de todas as idades. Nesse sentido, exigências para o aprimoramento do diagnóstico laboratorial de doenças de natureza infecto-contagiosa tornaram-se constantes, face às perdas vinculadas. Os Ranavirus são vírus que infectam vertebrados ectotérmicos causando necrose generalizada, hemorragias focais e apoptose celular nos animais infectados. O presente estudo objetivou a realização de infecções experimentais em larvas e alevinos de tilápia do Nilo (Oreochromis niloticus), através de 3 diferentes modelos de infecção, utilizando estirpe de Ranavirus Frog vírus 3-like; recentemente detectada e isolada no Brasil, para avaliação das patologias associadas, incluindo o sequenciamento e análise do genoma da cepa viral. Vários sinais clínicos macroscópicos e microscópicos foram observados, hemólise, hemácias com anisocitose e policromasia, alterações relacionadas a média dos volumes das hemácias e hemoglobina, índices hepato-somáticos e espleno-somáticos com interações estatisticamente significantes, linfócitos reativos, alterações nos níveis das enzimas alanina aminotransferase e corpúsculos de inclusão basofílicos em diferentes tecidos dos animais experimentados com Ranavirus FV3-like foram observados dentro do período de 60 dias pós-infecção. Além disso, animais experimentalmente infectados foram positivos ao Ranavirus através de qPCR. O genoma de cepa brasileira de Ranavirus FV3-like apresentou 105 kilobases, contendo 54,98% de guanina + citosina, sendo 94 potenciais ORFs anotadas. A reconstrução filogenética, baseada em sequencias nucleotídicas, agrupou a amostra brasileira de Ranavirus no clado dos Frog vírus 3-like e as maiores identidades do genoma se deram com cepas norte-americanas de FV3-like. Doze potenciais eventos de recombinação, estatisticamente significantes (p < 0,05), foram identificados entre a cepa brasileira e amostras de referência de Ranavirus FV3-like. Nesse sentido, o presente trabalho contribui para o melhor entendimento da infecção causada por estirpe brasileira de Ranavirus FV3-like em tilápias do Nilo (Oreochromis niloticus). Além disso, adiciona e reforça a potencial recombinação entre diferentes estirpes de Ranavirus, colaborando para uma melhor compreensão das características de agentes virais associados a graves surtos em vertebrados ectotérmicos no país. / Some diseases associated with tilapia fish farming cause enormous losses to breeders, resulting in significant morbidity and mortality rates among animals of all ages. In this sense, requirements for the improvement of the laboratory diagnosis of diseases of infectious-contagious nature, became constant, in view of the related losses. Ranaviruses are viruses that infect ectothermal vertebrates causing widespread necrosis, focal haemorrhages and cell apoptosis in infected animals. The present study aimed to perform experimental infections in Nile tilapia (Oreochromis niloticus) fry and larvae through 3 different infection models, using Ranavirus Frog virus 3-like strain recently detected and isolated in Brazil, to evaluate the associated pathologies, including sequencing and genome analysis of the viral strain. Several macroscopic and microscopic clinical signs were observed, hemolysis, red blood cells presenting anisocytosis and polychromasia, alterations related to the mean red blood cell volumes and hemoglobin present in the red blood cells, hepato-somatic and spleno-somatic indexes showing statistically significant interactions, reactive lymphocytes, alterations in the levels of the enzymes alanine aminotransferase and basophilic inclusion corpuscles in different tissues of animals tested with Ranavirus FV3-like were observed within the 60-day post-infection period. In addition, experimentally infected animals were positive to Ranavirus through qPCR. The genome of the Ranavirus FV3-like Brazilian strain presented 105 kilobases, containing 54.98% of guanine + cytosine, with 94 potential ORFs annotated. The phylogenetic reconstruction, based on nucleotide sequences, grouped the Brazilian sample of Ranavirus in the clade of Frog virus 3-like, the greater identities of the genome were with North American strains of FV3-like. Twelve potential recombination events, statistically significant (p <0.05), were identified between the Brazilian strain and reference samples of Ranavirus FV3-like. The present work contributes to a better understanding of the infection caused by the Brazilian strain of Ranavirus FV3-like in Nile tilapia (Oreochromis niloticus). In addition, it adds and reinforces the potential recombination between different strains of Ranavirus, contributing to a better understanding of the characteristics of viral agents associated with severe outbreaks in ectothermic vertebrates.

O objetivo do trabalho foi realizar investigação sobre ocorrência de ranavirose, infecção causada por vírus do gênero Ranavirus, família Iridoviridiae, em anfíbios anuros silvestres nos municípios de Porto Ferreira e Pirassununga, cidades da região centro-leste do estado de São Paulo, Brasil, utilizando diagnóstico molecular. Ao todo, 40 anuros adultos silvestres da espécie Leptodactylus fuscus foram capturados nos municípios citados, sendo coletados três órgãos alvos (fígado, baço e rins) de replicação viral, com registro de eventuais alterações macroscópicas nesses órgãos, e dos quais extraiu-se DNA. Na sequência, realizou a reação em cadeia pela polimerase (PCR), utilizando-se primers específicos para o gene MCP (major capsid protein) de Frog Virus 3 (FV3), ranavírus circulante no Brasil, com isolado de FV3 pertencente ao biobanco do Laboratório de Higiene Zootécnica da FZEA-USP empregado como controle positivo e água livre de nuclease como controle negativo. Como resultados, em relação às alterações macroscópicas, somente um espécime apresentou hepatoesplenomegalia e hipopigmentação do coração. No entanto, ao diagnóstico molecular, nenhum dos 40 animais amostrados foi positivo à ranavirose. Tal resultado pode estar associado tanto a fatores intrínsecos (espécie, idade, resposta imune) como extrínsecos ao hospedeiro (alterações ambientais, patogenicidade viral). Em paralelo, em rãs de criação comercial, surtos por FV3 já foram detectados em vários ranários do país; contudo, essa é a primeira vez que uma prospecção para ranavirose é realizada especificamente com representantes da espécie Leptodactylus fuscus, em municípios da região centro-leste do estado de São Paulo. Assim, são necessárias mais pesquisas com espécies silvestres de rãs, particularmente no entorno de ranários com histórico de animais positivos para FV3, para melhor conhecimento da epidemiologia e dinâmica de transmissão da ranavirose entre rãs silvestres. / The objective of this study was to investigate the occurrence of ranavirus infection caused by virus belonging to genus Ranavirus, Iridoviridiae family, in wild anuran amphibians from Porto Ferreira and Pirassununga municipalities, located in the central-eastern region of the state of São Paulo, Brazil, using molecular diagnostics. In all, 40 wild adult anurans of the species Leptodactylus fuscus were captured in the mentioned municipalities, and three target organs (liver, spleen and kidneys) of the viral replication were collected, being possible macroscopic changes in these organs recorded, and from which DNA was extracted. In the sequence, the polymerase chain reaction (PCR) was carried out using primers specific for the MCP (major capsid protein) gene of Frog Virus 3 (FV3), a circulating ranavirus in Brazil, with a FV3 isolate belonging to the biobank of the Laboratory of Zootechnical Hygiene of FZEA-USP employed as positive control and nuclease-free water as negative control. As a result, in relation to macroscopic alterations, only one specimen showed hepatosplenomegaly and heart hypopigmentation. However, for molecular diagnostics, none of the 40 animals sampled were positive for ranavirus. This result may be associated to both intrinsic (species, age, immune response) and extrinsic (environmental changes, viral pathogenicity) factors to the host. In parallel, FV3 outbreaks have already been detected in several frog farms in the country. However, this is the first time that a prospection for ranavirus infection is performed specifically with specimens of the Leptodactylus fuscus from municipalities in the central-eastern region of the state of São Paulo. Thus, more studies on wild-type frogs are needed, particularly in the settings of frog farms with positive animal history for FV3, to better understand the epidemiology and dynamics of ranavirus transmission among wild frogs.

Algumas doenças associadas à piscicultura de tilápias trazem enormes prejuízos aos criadores, resultando em expressivos coeficientes de morbidade e mortalidade entre animais de todas as idades. Nesse sentido, exigências para o aprimoramento do diagnóstico laboratorial de doenças de natureza infecto-contagiosa tornaram-se constantes, face às perdas vinculadas. Os Ranavirus são vírus que infectam vertebrados ectotérmicos causando necrose generalizada, hemorragias focais e apoptose celular nos animais infectados. O presente estudo objetivou a realização de infecções experimentais em larvas e alevinos de tilápia do Nilo (Oreochromis niloticus), através de 3 diferentes modelos de infecção, utilizando estirpe de Ranavirus Frog vírus 3-like; recentemente detectada e isolada no Brasil, para avaliação das patologias associadas, incluindo o sequenciamento e análise do genoma da cepa viral. Vários sinais clínicos macroscópicos e microscópicos foram observados, hemólise, hemácias com anisocitose e policromasia, alterações relacionadas a média dos volumes das hemácias e hemoglobina, índices hepato-somáticos e espleno-somáticos com interações estatisticamente significantes, linfócitos reativos, alterações nos níveis das enzimas alanina aminotransferase e corpúsculos de inclusão basofílicos em diferentes tecidos dos animais experimentados com Ranavirus FV3-like foram observados dentro do período de 60 dias pós-infecção. Além disso, animais experimentalmente infectados foram positivos ao Ranavirus através de qPCR. O genoma de cepa brasileira de Ranavirus FV3-like apresentou 105 kilobases, contendo 54,98% de guanina + citosina, sendo 94 potenciais ORFs anotadas. A reconstrução filogenética, baseada em sequencias nucleotídicas, agrupou a amostra brasileira de Ranavirus no clado dos Frog vírus 3-like e as maiores identidades do genoma se deram com cepas norte-americanas de FV3-like. Doze potenciais eventos de recombinação, estatisticamente significantes (p < 0,05), foram identificados entre a cepa brasileira e amostras de referência de Ranavirus FV3-like. Nesse sentido, o presente trabalho contribui para o melhor entendimento da infecção causada por estirpe brasileira de Ranavirus FV3-like em tilápias do Nilo (Oreochromis niloticus). Além disso, adiciona e reforça a potencial recombinação entre diferentes estirpes de Ranavirus, colaborando para uma melhor compreensão das características de agentes virais associados a graves surtos em vertebrados ectotérmicos no país. / Some diseases associated with tilapia fish farming cause enormous losses to breeders, resulting in significant morbidity and mortality rates among animals of all ages. In this sense, requirements for the improvement of the laboratory diagnosis of diseases of infectious-contagious nature, became constant, in view of the related losses. Ranaviruses are viruses that infect ectothermal vertebrates causing widespread necrosis, focal haemorrhages and cell apoptosis in infected animals. The present study aimed to perform experimental infections in Nile tilapia (Oreochromis niloticus) fry and larvae through 3 different infection models, using Ranavirus Frog virus 3-like strain recently detected and isolated in Brazil, to evaluate the associated pathologies, including sequencing and genome analysis of the viral strain. Several macroscopic and microscopic clinical signs were observed, hemolysis, red blood cells presenting anisocytosis and polychromasia, alterations related to the mean red blood cell volumes and hemoglobin present in the red blood cells, hepato-somatic and spleno-somatic indexes showing statistically significant interactions, reactive lymphocytes, alterations in the levels of the enzymes alanine aminotransferase and basophilic inclusion corpuscles in different tissues of animals tested with Ranavirus FV3-like were observed within the 60-day post-infection period. In addition, experimentally infected animals were positive to Ranavirus through qPCR. The genome of the Ranavirus FV3-like Brazilian strain presented 105 kilobases, containing 54.98% of guanine + cytosine, with 94 potential ORFs annotated. The phylogenetic reconstruction, based on nucleotide sequences, grouped the Brazilian sample of Ranavirus in the clade of Frog virus 3-like, the greater identities of the genome were with North American strains of FV3-like. Twelve potential recombination events, statistically significant (p <0.05), were identified between the Brazilian strain and reference samples of Ranavirus FV3-like. The present work contributes to a better understanding of the infection caused by the Brazilian strain of Ranavirus FV3-like in Nile tilapia (Oreochromis niloticus). In addition, it adds and reinforces the potential recombination between different strains of Ranavirus, contributing to a better understanding of the characteristics of viral agents associated with severe outbreaks in ectothermic vertebrates.

O objetivo do trabalho foi realizar investigação sobre ocorrência de ranavirose, infecção causada por vírus do gênero Ranavirus, família Iridoviridiae, em anfíbios anuros silvestres nos municípios de Porto Ferreira e Pirassununga, cidades da região centro-leste do estado de São Paulo, Brasil, utilizando diagnóstico molecular. Ao todo, 40 anuros adultos silvestres da espécie Leptodactylus fuscus foram capturados nos municípios citados, sendo coletados três órgãos alvos (fígado, baço e rins) de replicação viral, com registro de eventuais alterações macroscópicas nesses órgãos, e dos quais extraiu-se DNA. Na sequência, realizou a reação em cadeia pela polimerase (PCR), utilizando-se primers específicos para o gene MCP (major capsid protein) de Frog Virus 3 (FV3), ranavírus circulante no Brasil, com isolado de FV3 pertencente ao biobanco do Laboratório de Higiene Zootécnica da FZEA-USP empregado como controle positivo e água livre de nuclease como controle negativo. Como resultados, em relação às alterações macroscópicas, somente um espécime apresentou hepatoesplenomegalia e hipopigmentação do coração. No entanto, ao diagnóstico molecular, nenhum dos 40 animais amostrados foi positivo à ranavirose. Tal resultado pode estar associado tanto a fatores intrínsecos (espécie, idade, resposta imune) como extrínsecos ao hospedeiro (alterações ambientais, patogenicidade viral). Em paralelo, em rãs de criação comercial, surtos por FV3 já foram detectados em vários ranários do país; contudo, essa é a primeira vez que uma prospecção para ranavirose é realizada especificamente com representantes da espécie Leptodactylus fuscus, em municípios da região centro-leste do estado de São Paulo. Assim, são necessárias mais pesquisas com espécies silvestres de rãs, particularmente no entorno de ranários com histórico de animais positivos para FV3, para melhor conhecimento da epidemiologia e dinâmica de transmissão da ranavirose entre rãs silvestres. / The objective of this study was to investigate the occurrence of ranavirus infection caused by virus belonging to genus Ranavirus, Iridoviridiae family, in wild anuran amphibians from Porto Ferreira and Pirassununga municipalities, located in the central-eastern region of the state of São Paulo, Brazil, using molecular diagnostics. In all, 40 wild adult anurans of the species Leptodactylus fuscus were captured in the mentioned municipalities, and three target organs (liver, spleen and kidneys) of the viral replication were collected, being possible macroscopic changes in these organs recorded, and from which DNA was extracted. In the sequence, the polymerase chain reaction (PCR) was carried out using primers specific for the MCP (major capsid protein) gene of Frog Virus 3 (FV3), a circulating ranavirus in Brazil, with a FV3 isolate belonging to the biobank of the Laboratory of Zootechnical Hygiene of FZEA-USP employed as positive control and nuclease-free water as negative control. As a result, in relation to macroscopic alterations, only one specimen showed hepatosplenomegaly and heart hypopigmentation. However, for molecular diagnostics, none of the 40 animals sampled were positive for ranavirus. This result may be associated to both intrinsic (species, age, immune response) and extrinsic (environmental changes, viral pathogenicity) factors to the host. In parallel, FV3 outbreaks have already been detected in several frog farms in the country. However, this is the first time that a prospection for ranavirus infection is performed specifically with specimens of the Leptodactylus fuscus from municipalities in the central-eastern region of the state of São Paulo. Thus, more studies on wild-type frogs are needed, particularly in the settings of frog farms with positive animal history for FV3, to better understand the epidemiology and dynamics of ranavirus transmission among wild frogs.

Ranaviruses are large dsDNA viruses that are considered emerging pathogens, and they are known to cause mortality events in amphibian and fish populations. This research utilizes experimental and genomic data to elucidate the mechanisms driving the evolution and spread of ranaviruses, with a focus on host switching within the genus. In Chapter 1, we utilize virus challenge assays to examine potential transfer of ranaviruses between cultured juvenile largemouth bass (M. salmoides) and bullfrog tadpoles (Rana catesbeiana). Additionally, a commonly used antiparasitic treatment containing malachite green and formalin (MGF) was utilized to suppress the immune system of largemouth bass to assess the susceptibility of immunocompromised fish to ranaviruses. The results indicate that tadpoles are not susceptible to Largemouth Bass Virus (LMBV), but that bass are susceptible to ranaviruses isolated from amphibians. Furthermore, immunocompromised fish were more susceptible to both LMBV and FV3 infections than immunocompetent fish. In Chapter 2, we used eight sequenced ranavirus genomes and two selection-detection methods (site-based and branch-based) to identify genes that exhibited signatures of positive selection, potentially due to the selective pressures at play during host switching. We found evidence of positive selection acting on four genes via the site-based method, three of which are newly-acquired genes unique to ranavirus genomes. Our results suggest that the group of newly acquired genes in the ranavirus genome may have undergone recent adaptive changes that have facilitated interspecies and interclass host switching. In Chapter 3, we annotated and analyzed the nearly complete genomic sequence of LMBV to determine its taxonomic classification. The available genomic content and phylogenetic evidence suggests that LMBV is more closely related to amphibian-like ranaviruses (ALRVs) than grouper ranaviruses, and this is further supported by greater genomic collinearity between LMBV and ALRVs. This data suggests that the classification of LMBV as a ranavirus is warranted. The results presented here will help to clarify the taxonomic relationships of ranaviruses, and will also be useful in developing management strategies to limit interspecific and intraspecific viral spread. The information garnered from this research will have far-reaching implications in studies of amphibian conservation, disease evolution, and virology. / text