Os parasitas pertencentes ao gênero Leishmania têm distribuição ubíqua. Este táxon inclui Leishmania infantum chagasi, agente etiológico da leishmaniose visceral nas Américas, uma zoonose negligenciada cujas metodologias diagnósticas acumulam uma série de limitações, requerendo a validação e padronização de metodologias diagnósticas satisfatórias. Vários fatores estão relacionados à patogênese causada por este protozoário, entre eles a catepsina L-like, uma cisteíno protease envolvida em processos regulatórios metabólicos e infecciosos. Portanto, este trabalho teve como objetivo avaliar a eficácia do gene de catepsina L-like isoforma CPA como alvo de diagnóstico molecular e como marcador filogenético que permita a compreensão das variações intraespecíficas e elucidem a história evolutiva de L. infantum chagasi no Brasil. Foram utilizados 44 isolados de L. infantum chagasi de diferentes estados brasileiros. Os fragmentos do gene de catepsina L-like foram amplificados, purificados, sequenciados, alinhados manualmente e analisados por métodos filogenéticos de máxima parcimônia e inferência bayesiana. As sequências geradas foram usadas para pesquisar e sintetizar iniciadores a serem usados em reações específicas para o parasita alvo. O gene de catepsina L-like não mostrou variabilidade intraespecífica entre os isolados analisados, sugerindo um evento recente de introdução do mesmo nas Américas. O par de iniciadores propostos amplificou o DNA alvo de isolados de L. infantum chagasi, sendo efetivo na amplificação de DNA em concentrações de até 10-11g / µl. O marcador proposto não apresentou reações cruzadas com outros hemoparasitas de importância clínica. Quando utilizado para o diagnóstico em um painel de amostras clínicas de cães, obteve-se uma frequência de positividade de 49,03% (102/208), contrastando com o valor de 14,42% (30/208) obtido com o marcador para o gene do espaçador ribossomal interno ITS. Quando testado em amostras de flebotomíneos se obteve um valor de 6,25% e em amostras de pacientes humanos o valor foi de 14,28%. Os marcadores também foram eficazes em amplificar DNA extraído de amostras de urina, de sangue fixado em papel filtro e mesmo em amostras de swab de lesões conjuntivas. Este conjunto de parâmetros permite inferir que o CatLeish- PCR é sensível e específico para o diagnóstico de L. infantum chagasi podendo ser aplicado tanto em pesquisas clínicas quanto em inquéritos epidemiológicos de vigilância. / The parasites belonging to the Leishmania genus have a wide distribution. This taxon includes Leishmania infantum chagasi, the etiologic agent of Visceral Leishmaniasis in the Americas, a neglected zoonosis that requires the validation and standardization of satisfactory diagnostic methodologies. Several factors are related to the pathogenesis caused by this protozoan, as Catepsin L-like, a cysteine protease involved in regulatory and infectious processes. Given this information this work aimed to evaluate the effectiveness of Cathepsin L-like isoform CPA as a target for molecular diagnosis and as a phylogenetic marker that allows understanding the intraspecific variations and the evolutionary history of L. infantum chagasi in Brazil. We used 44 isolates of L. infantum chagasi from different Brazilian states. The cathepsin L-like gene fragments were amplified, purified, sequenced, manually aligned and analyzed by maximum parsimony and Bayesian inference methods. The sequences generated were researched to construction of oligonucleotide primers to be used in reactions specific to the target parasite. The Cathepsin L-like gene did not show intraspecific variability among the isolates analyzed, suggesting a recent event of introduction of the same in the Americas. The pair of proposed primers amplified the target DNA of L. infantum chagasi isolates, being effective in DNA amplification at concentrations of up to 10-11g/µl. The proposed marker did not present cross-reactions with other hemoparasites of clinical importance. When used for the diagnosis in a panel of clinical samples of dogs obtained a positive frequency of 49.03% (102/208), against the 14.42% (30/208) to ribosomal ITS marker. Samples of sandflies obtained a value of 6.25% and in humans the value was 14.28%. The markers were also effective in blood samples fixed on filter paper and even in samples from conjunctival lesion swabs. This set of parameters allows to infer that CatLeish-PCR is a sensitive and specific marker for the diagnosis of L. infantum chagasi in clinical and epidemiological surveys.

A Malária autóctone de Mata Atlântica é caracterizada por baixa incidência de casos com pouca ou nenhuma manifestação clínica e baixa parasitemia, tendo como principal agente etiológico o Plasmodium vivax (ou plasmódios muito semelhantes a este). O principal mosquito envolvido na transmissão é o Anopheles (Kerteszia) cruzii, cujas formas imaturas se desenvolvem na água acumulada nas imbricações das folhas de bromélias, vegetal muito abundante neste bioma. As formas adultas de An. cruzii tendem a viver e se alimentar com maior frequência na copa das árvores, comportamento conhecido como acrodendrofilia, no entanto, em diversas situações esta espécie tem sido observada em elevada densidade no estrato inferior da floresta. Evidências sugerem que a malária possa ser transmitida de forma zoonótica nestas áreas, uma vez que primatas das famílias Atelidae e Cebidae são encontrados portando plasmódios muito próximos geneticamente aos que infectam humanos e An. cruzii pode se alimentar do sangue de ambos os hospedeiros. O presente estudo teve como objetivo investigar a influência da composição e configuração da paisagem sobre as variações na abundância e no comportamento acrodendrófilo de An. cruzii e como estas variações atuam sobre a dinâmica de transmissão de malária na Mata Atlântica, considerando um cenário de transmissão zoonótica. Coletas entomológicas mensais foram conduzidas no período de março de 2015 a abril de 2017 em duas unidades de conservação situadas no município de São Paulo, ambas com histórico de malária autóctone. Em cada área foram selecionados pontos de coleta com diferentes graus de interferência antrópica e os mosquitos foram coletados utilizando armadilhas do tipo CDC e Shannon. Métricas de composição e configuração da paisagem foram mensuradas para cada ponto de estudo e modelos estatísticos foram utilizados para avaliar a relação entre estas métricas e variações na abundância e acrodendrofilia de An. cruzii. Foi proposto um modelo matemático de transmissão zoonótica que permitiu observar, por meio de simulações, como a dinâmica de transmissão da malária é influenciada por variações na abundância e acrodendrofilia do vetor. Os resultados obtidos sugerem que a perda de vegetação natural e aumento das áreas de borda levam a uma redução na abundância de An. cruzii mas favorecem uma maior atividade desta espécie próximo ao solo. As análises baseadas no modelo teórico corroboram com observações anteriores, apontando que as variações na acrodendrofilia do vetor podem ter um papel importante na dinâmica de transmissão de plasmódios entre símios e humanos na Mata Atlântica. Em condições nas quais An. cruzii tende a se alimentar quase exclusivamente na copa das árvores ou quase exclusivamente próximo ao solo a transmissão zoonótica parece não se sustentar, ainda que a abundância do vetor seja elevada. De outra forma, condições nas quais An. cruzii se desloque com maior frequência entre os estratos copa e solo tendem a favorecer a transmissão zoonótica, sendo que quanto maior o deslocamento menor é a abundância relativa do vetor necessária para que um símio infectado gere mais do que um caso novo na população de humanos suscetível ou vice-versa. / The autochthonous Malaria of the Atlantic Forest is characterized by a low incidence of cases with little or no clinical manifestation and low charge of parasites in the blood stream, being Plasmodium vivax (or plasmodiums very similar to it) the main etiologic agent. The main mosquito involved in transmission is the Anopheles (Kerteszia) cruzii, whose immature forms develop in the water held in bromeliads, which are very abundant plants in this biome. The adult forms of An. cruzii prefer to live and feed on the treetops, a behavior known as acrodendrophily, however, in several situations this species has been observed in high density near the ground of the forest. Some evidences suggest that malaria can be transmitted zoonotically in these areas, since monkeys of Atelidae and Cebidae families are found carrying plasmodiums very close to those that infect humans and An. cruzii can feed on the blood of both hosts. This study aimed to investigate the influence of the landscape composition and configuration on the variations in the abundance and acrodendrophic behavior of An. cruzii and how these variations affect the dynamics of malaria transmission in the Atlantic Forest, considering a zoonotic transmission scenario. Monthly entomological collections were conducted from March 2015 to April 2017 in two conservation units located in the municipality of São Paulo, Brazil, where human and simian malaria occurs. In each area, collection points with different degrees of anthropic intervention were selected and mosquitoes were collected using CDC and Shannon traps. Metrics of landscape composition and configuration were measured for each study point and generalized linear models were used to evaluate the relationship between these metrics and variations in abundance and acrodendrophily of An. cruzii. It was proposed a mathematical model of zoonotic transmission that allowed to observe, through simulations, how the dynamics of malaria transmission is influenced by variations in the abundance and acrodendrophily of the vector. The results suggest that loss of natural vegetation and increase in edge effect lead to a reduction in the abundance of An. cruzii but favor a higher activity of this species near the ground level. The analyzes based on the theoretical model corroborate previous observations, pointing out that the variations in the acrodendrophily of the vector may play an important role in the dynamics of plasmodium transmission between monkeys and humans in the Atlantic Forest. In situations in which An. cruzii seeks to feed almost exclusively on the treetops or in situations where it feeds almost exclusively near the ground, the zoonotic transmission does not seem to occur, even in situations of high abundance of the vector. Otherwise, conditions in which An. cruzii moves more frequently between canopy and ground level tend to favor zoonotic transmission, and the more vertical movement occurs, the lower is the needed relative abundance of the vector for an infected monkey to generate more than a new case in the susceptible human population or vice versa.

Three main research products are reported in this dissertation. This research focused on estimation of the seroprevalence rates in agricultural workers with exposure to pigs and poultry, and determination of risk factors for infection. Chapter 2, "Are swine workers in the United States at increased risk of infection with zoonotic influenza virus?", reports controlled, cross-sectional seroprevalence studies among farmers, meat processing workers, veterinarians, and control subjects. Using a hemagglutination inhibition assay against six influenza A virus isolates, all 3 exposed study groups demonstrated markedly elevated titers against the H1N1 and H1N2 swine influenza virus isolates, compared with control subjects. Chapter 3, "Infection due to 3 avian influenza subtypes in United States veterinarians", describes a controlled, cross-sectional seroprevalence study that examined veterinarians in the United States for evidence of previous avian influenza virus infection. Using a microneutralization assay against 9 influenza A virus strains, veterinarians exposed to birds demonstrated statistically significant elevated titers against the H5, H6, and H7 avian influenza virus isolates compared with control subjects. In chapter 4, "Cases of swine influenza in humans: a review of the literature", all known human cases of swine influenza are compiled and analyzed. Fifty cases of apparent zoonotic swine influenza virus infection, including 37 civilians and 13 military personnel, were identified, with a case-fatality rate of 14% (7 of 50 persons). Most civilian subjects (61%) reported exposure to swine. These studies provide strong evidence that transmission of zoonotic influenza likely occurs much more frequently than previously thought, and that individuals with occupational exposure to pigs and birds are at elevated risk for acquiring zoonotic influenza infections. Agricultural workers should be included in pandemic influenza planning, should receive information and training on how to use personal protective equipment, and should be offered human influenza vaccine to reduce the risk of creating viral reassortants. In the event of a pandemic, workers should be considered for antiviral medications and pandemic strain vaccines.

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.

Rodents have been reported to play a significant role as reservoirs of over 22 rodent-associated Bartonella species. In this study, we contrast prevalence and diversity of Bartonella infections in 377 small mammals, representative of three terrestrial rodent genera, namely Aethomys, Gerbilliscus and Rhabdomys and one subterranean mole-rat species (Bathyergus suillus). The latter was sampled in close proximity to an informal human settlement, whereas the afore-mentioned murid rodent genera were sampled across a range of landscapes inclusive of natural, agricultural, urban, peri-urban and rural settings, from three provinces (Free State, Gauteng and Western Cape) in South Africa. Molecular estimates of Bartonella infection rates were determined through multi-gene screening of DNA extracted from clinical samples, primarily heart and spleen. PCR assays targeting the citrate synthase (gltA) and NADH dehydrogenase gamma subunit (nuoG) and/or beta subunit of bacterial RNA polymerase (rpoB) genes were used to ensure enhanced molecular estimates of Bartonella prevalence. Aethomys had the highest infection rate (86.7%), whereas Rhabdomys had the lowest (15%). Nucleotide sequencing and phylogenetic analyses revealed that the different primers sets used for Bartonella screening have different affinities to the different strains present in rodents from South Africa. Furthermore, the presence of Bartonella co-infections, confirmed through the presence of multiple peaks at 15% of the nucleotide sequences sites, ranged from 33.8% (in Aethomys) to 42.9% (in Gerbilliscus species) for the gltA gene region. For Aethomys ineptus, of the discrete Bartonella lineages recovered, one was closely related to zoonotic B. elizabethae. The latter species, which is associated with Rattus hosts worldwide and has been linked to cases of human endocarditis, suggests spillover from invasive to indigenous rodents. This is supported by previous studies indicating that indigenous Micaelamys namaquensis, a highly adaptable species, which like Aethomys is capable of utilizing natural and modified landscapes also hosts B. elizabethae-related lineages. Of potential public health importance, Bathyergus suillus were shown to be infected with a zoonotic Bartonella species, B. rochalimae. Our results further indicate that the level of anthropogenic transformation is significantly correlated with Bartonella prevalence, with Rhabdomys sampled from rural settings in the Western Cape Province having infection rates of 36% versus 0% in a nearby urban setting. This study also uncovered high levels of strain diversity in members of the Gerbilliscus cryptic species complex, sampled from an agricultural setting. The overall Bartonella PCR-positivity rate was 67.5 % and the gltA gene phylogeny confirmed the presence of six discrete Bartonella gerbil-specific lineages (I-VI). Lineages I and II clustered with Bartonella strains identified previously in G. leucogaster sampled from Sandveld nature reserves in the Free State Province South Africa, whereas lineages III-VI comprised of lineages that were restricted to either G. leucogaster or to G. brantsii, indicative of host-specificity. From the findings of this study, it is clear that the public health importance of the Bartonella species present in indigenous rodents warrants further investigation as at least two species, with known zoonotic potential (B. elizabethae and B. rochalimae) were shown to be present in rodents sampled in close proximity to human settlements. / Thesis (PhD (Zoology))--University of Pretoria, 2019. / This research was made possible through the financial support from the (AZD-IRT), CDC Co-Ag 5 NU2GGH001874-02-00 and through the NRF incentive and SARChI PI funding awarded to ADSB. / Zoology and Entomology / PhD (Zoology) / Unrestricted

Tularemia is a vector-borne disease of global concern with diverse regional foci. Arkansas is an endemic state with differences in case distribution and land suitability supporting host and vector sustainment. The aim of this study was to conduct a geospatial and spatiotemporal assessment of factors associated with case distribution and timeliness and completeness of public reporting. Guided with direction from spatial epidemiology and nidality, referring to the association of ecology, climate, and proximity of disease, analysis included secondary data collected from the Arkansas Department of Health between 1995 and 2018. Using Poisson-based software, 2 clusters were found: a high-risk cluster encompassing 23% of the total population within 24 counties spanning an 8-year period (RR = 4.98, p < 0.05), and a low risk cluster that included 25% of the population within 28 counties during a 12-year period (RR 0.14, p < 0.05). Analysis of ecological data revealed associations between annual precipitation within the high-risk cluster and total number of cases (AUC = 0.716 and AUC = 0.726, respectively) with trends toward higher incidence rates in suitable land cover and moderate to high elevation using maximum entropy software. Analysis of timeliness and completeness revealed gaps for clinical form and transmission mode determination (p < 0.05), while increases in probable cases followed decreases in confirmed cases revealing gaps in laboratory diagnostics. Positive social change necessitates multidisciplinary collaboration between climatologists, clinicians, and epidemiologists to reach high-risk populations and promote educational awareness. The potential for social change includes predictive modeling optimizing funding while representing underserved populations.

Giardia intestinalis is a single-celled intestinal parasite infecting humans and animals. The species is divided into eight genetic groups, assemblages, with different host specificity. Stool samples from 99 dogs, 61 cats and 22 chinchillas were examined for the presence of Giardia using microscopy and PCR diagnostics. The found populations were assigned to assemblages using a multi-locus genotyping scheme, with the goal of mapping the occurrence of zoonotic assemblages A and B and evaluating the risk of transmission of Giardia from pets to humans. The Giardia prevalence in examined dogs was 36,4%. The majority of dog infections was caused by dog-specific assemblages D and C. Individual cases of infection with assemblage F, or a mix of assemblages A+D, A+F, B+D, C+D and D+F were also detected. The prevalence in cats was 14.8%, and the dog assemblages C and D prevailed as well. In individual cases, cats were infected with assemblages A or F, which is specific for cats. The highest prevalence, 85.7%, was detected in chinchillas. The majority of chinchilla infections was caused by the zoonotic assemblage B (88.9%). The found sequences were compared to those obtained from animals with clinical giardiasis, but no identical matches were found between these two pools. The nature of mixed infections was studied by...

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Recent research has shown that climate change had and will likely continue to have impacts on biological processes, including the propagation of infectious and zoonotic diseases. Assessments of local level impacts at the human-wildlife interface are imperative for stakeholders and policy makers, and empirical review of such research is undoubtedly necessary to understand the current state of the field, gaps of knowledge, and to identify future lines of research. In that vein, this thesis focuses on the impacts of climate change on disease at the human-wildlife interface. Specifically, my thesis works to quantify the recent temporal and spatial distribution of empirical research linking climate change with changes in the burden of infectious diseases (Chapter 2). This retrospective scoping of the last five years of empirical research identified if, and to what extent, there are biases in the diseases, species, or geographic areas studied within this scientific field. My study revealed both geographic and topical biases within the scope of recent literature, with an overwhelming emphasis on vector-borne diseases in temperate areas. There was also unequal representation in publication demographics of authors and institutions with most research originating from well developed countries. As a proof-of-concept case study, my thesis provides an empirical assessment of the plausible climatic drivers of a wildlife-disease transmission risk in an understudied region (Chapter 3), which could function to fill some of the identified research gaps in Chapter 2. Therein, my work assessed the impacts of climate variation from the last century on the environmental suitability of the rabies host Desmodus rotundus (common vampire bat) in Latin America. Findings revealed that average and standard deviation of temperature were the most important drivers of D. rotundus geographic distribution according to species' records between 1901 and 2019. Nevertheless, high uncertainty was detected regarding the predictability of D. rotundus environmental suitability across the United States-Mexico border and in the Andes Mountains of Chile. The overall modeling efforts did, however, reveal a northward distributional shift of the rabies reservoir as a likely response to climate change. Together, studies contained in this thesis provide empirical, retrospective evidence that demonstrates the effects of climate change on the increased risk of disease transmission at the human-wildlife interface. / Master of Science / Climate change is understood as the change in global or regional climate patterns, including variations of temperature and humidity factors beyond normal ranges, mostly attributed to increased levels of atmospheric carbon dioxide. Climate change is expected to influence many biological systems and presents an imminent threat to almost all organisms and geographic areas across the globe. Previous studies suggest that climate change will increase the burden of infectious diseases, including those originating from wildlife. This thesis aims to assess the availability of empirical evidence supporting the idea of a link between climate change and infectious diseases of wildlife origin. Chapter 2 examines recent scientific literature assessing climate change and infectious disease, and identifies biases in the diseases, species, and geographic areas commonly studied. This study found that literature generally focused on diseases transmitted by arthropods (e.g., insects, arachnids, or crustaceans) in temperate areas. There was little focus on diseases transmitted directly (e.g., via bites) or in non-temperate areas (e.g., tropics). Chapter 3 attempts to address issues detected in Chapter 2 by studying a directly-transmitted infectious disease in the tropics. More specifically, Chapter 3 assessed the impacts of climate variation from the last century on the distribution of the common vampire bat (Desmodus rotundus), which is a known rabies host in Latin America. Chapter 3 revealed that temperature variables were the largest drivers of common vampire bat distribution. Nevertheless, high uncertainty was detected regarding the vampire bat's ability to invade new areas such as the continental United States-Mexico border or the lowlands to the Andes Mountains in Chile. Together, studies contained in this thesis provide support for current and future research on the study of climate change as an amplifier for the risk of infectious diseases.

O sequenciamento de alto desempenho, pela redução dos custos nos últimos anos, vem sendo cada vez mais utilizado para prospectar e identificar vírus. Estes métodos são extremamente mais sensíveis que outros métodos moleculares, e capazes de sequenciar genomas virais sem conhecimento prévio, clonagem ou isolamento. Neste estudo, utilizamos o sequenciamento de alto desempenho para conhecer, e caracterizar genomas completos de arbovírus isolados nas Américas, incluindo a prospecção de vírus em amostras de pequenos mamíferos do estado de São Paulo, Brasil. Assim, sequenciamos e caracterizamos 44 Bunyavirales, 35 no gênero Orthobunyavirus, família Peribunyaviridae, oito no gênero Phlebovirus, família Phenuiviridae, e um orthonairovírus, família Nairoviridae. Entre os Bunyavirales identificamos uma provável nova estratégia de codificação da proteína não estrutural do segmento pequeno, e ainda identificados sete vírus que são reassortants naturais. Caracterizamos o genoma completo do vesiculovírus Piry, determinando sua relação filogenética com arbovírus pertencentes ao gênero Vesiculovirus, família Rhabdoviridae. Prospectamos novos vírus, os quais incluímos em três famílias, Parvoviridae, Anelloviridae e Hepeviridae. Na família Parvoviridae, identificamos 20 chapparvovírus endógenos e exógenos, oriundos de grande diversidade de hospedeiros vertebrados e invertebrados, e que representam uma nova subfamília, a Chapparvovirinae. Também, descrevemos onze novas espécies de Anelloviridae em roedores silvestres e marsupiais, fornecendo importantes informações sobre a diversidade, a taxonomia, e ainda ampliamos a gama de hospedeiros de anellovírus conhecidos. Por fim, identificamos e caracterizamos uma nova espécie de Orthohepevirus de roedores Sigmodontinae, nomeada \"Orthohepevirus E\". Acreditamos que estamos a fornecer relevantes informações sobre genômica, epidemiologia molecular, evolução e taxonomia de 45 arbovírus americanos, bem como sobre 13 novas espécies virais encontradas em pequenos mamíferos. Tais informações deverão dar subsídios para múltiplos futuros estudos visando compreender a importância destes novos vírus e a desenvolver métodos diagnósticos. / In last years, high-throughput sequencing (HTS) has been cost-effective and increasingly used for prospection and identification of viruses. These methods are extremely more sensitive than other molecular methods and are capable of sequencing viral genomes without prior knowledge, cloning or isolation. In this study, we used HTS approach to identify and characterize complete genomes of arbovirus isolated in the Americas, as well as viral prospection in samples of small mammals from São Paulo State, Brazil. Thus, we sequenced and characterized 44 viruses from Bunyavirales order, including 35 in Orthobunyavirus genus, family Peribunyaviridae, eight in Phlebovirus genus, family Phenuiviridae, and one in Orthonairovirus genus, family Nairoviridae. Among the Bunyavirales we identified a novel putative strategy for encoding the non-structural protein of the small segment, as well as we identified seven viruses that are natural reassortants. Also, we characterized the complete genome of the Piry vesiculovirus, determining its phylogenetic relationship with arboviruses belonging to the Vesiculovirus genus, family Rhabdoviridae. On the other hand, we have prospected novel viruses, which included in three families, Parvoviridae, Anelloviridae, and Hepeviridae. In the Parvoviridae family, we identified 20 endogenous and exogenous chapparvoviruses from a broad diversity of vertebrate and invertebrate hosts, representing a new subfamily, the Chapparvovirinae. Also, we have described eleven new species of Anelloviridae in wild rodents and marsupials, providing important information on diversity, taxonomy and even broadening the range of known anelloviruses hosts. Finally, we identified and characterized a novel species of orthohepevirus in Sigmodontinae rodent, named \"Orthohepevirus E\". We believe that we are providing relevant relevant on genomics, molecular epidemiology, evolution and taxonomy of 45 American arboviruses, as well as on 13 new viral species found in small mammals. Thus, these informations should provide support for multiple future studies to understand the importance of these new viruses, as well as to develop diagnostic methods.