Orientador: Cáris Maroni Nunes / Coorientador: Luzia Helena Queiroz / Banca: Adolorata Aparecida Bianco Carvalho / Banca: Rafael Silva Cipriano / Resumo: A raiva é uma zoonose globalmente distribuída que apresenta alta letalidade, constituindo grande problema em saúde pública. A persistência do vírus da raiva nas populações é mantida devido sua capacidade de se replicar em diversas espécies de mamíferos, incluindo os humanos. O controle da raiva urbana tem se baseado na vacinação de cães e gatos, reservatórios importantes no ciclo urbano, bem como no atendimento de indivíduos após exposição ao vírus da raiva. Adicionalmente, a profilaxia pré-exposição (PrPE) tem sido aplicada em indivíduos que podem ser expostos à infecção, por atividade ocupacional. Objetivando proteger os estudantes ingressantes do Curso de Medicina Veterinária, grupo de risco em potencial para a raiva, a Faculdade de Medicina Veterinária (FMVA) da Unesp, Câmpus Araçatuba, promove anualmente a vacinação em esquema PrPE, seguida de avaliação sorológica, a fim de habituá-los a anualmente verificarem a resposta de anticorpos. Assim, a presente pesquisa teve como objetivo avaliar, por meio de levantamento de dados de PrPE dos arquivos do Serviço de Vigilância Epidemiológica da Secretaria de Saúde da Prefeitura Municipal de Araçatuba, SP, Brasil a adesão à PrPE, a proteção conferida e a dinâmica dos anticorpos (IgG) neutralizantes contra o vírus da raiva, nos estudantes da FMVA durante o período de 2000 a 2017. Foram avaliados 2.404 registros, 86,4% dos quais apresentaram título protetor contra o vírus da raiva (≥0,5UI/mL). A PrPE teve boa adesão pelos estudantes... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Rabies is a globally distributed zoonosis that shows high lethality and is also a major public health problem. Persistence of rabies virus in populations is maintained due to its ability to replicate in several mammals species, including humans. Rabies control has been based on dogs and cats' vaccination, the most important reservoirs in the urban cycle, as well as on the treatment of individuals after exposure to the rabies virus. In addition, pre-exposure prophylaxis (PEPr) has been applied in individuals who might be exposed to infection due to their occupational activities. In order to protect the incoming students of the veterinary medicine course, a group of potential risk for rabies, the School of Veterinary Medicine (FMVA) of Unesp, Araçatuba, SP, Brazil, promotes annual vaccination in a PEPr scheme, followed by a serological evaluation, in order to habituate them to annually check their antibody response. Thus, by analyzing the PEPr data from the archives of the Epidemiological Surveillance Service of the Health Department of Araçatuba, SP, Brazil, during the period from 2000 to 2017, the present study aimed at evaluating the adhesion of the students to the rabies PEPr, the protection conferred by it and the dynamics of the neutralizing IgG antibodies against rabies virus. A total of 2,404 records were evaluated, 86.4% of which presented protective titers against rabies (≥0.5 IU/mL). PEPr had good adhesion by the students, has proved to be effective for generating an... (Complete abstract click electronic access below) / Mestre

La faune sauvage a été depuis longtemps incriminée dans la survenue de zoonoses et joue le rôle de réservoir d'agents pathogènes (virus Nipah, Hendra, Ebola, Hantaan etc.) pour l'homme. Les îles tropicales et subtropicales du Sud-Ouest de l'Océan Indien (SOOI) constituent l'une des 34 régions reconnues comme « hotspot » de biodiversité au niveau mondial. Elles sont caractérisées par un très fort endémisme de la faune sauvage surtout sur l'Ile de Madagascar. Le caractère multi-insulaire de la région du SOOI, la diversité de ses biotopes et ses disparités biogéographiques et humaines offrent un champ d'investigation unique pour explorer « in natura » la dynamique évolutive des agents infectieux et les relations hôtes-virus. Nos travaux de recherche ont porté sur deux modèles de virus à ARN de polarité négative, les paramyxovirus et les lyssavirus. Le premier modèle viral nous a permis d'aborder les questions relatives à la dynamique de transmission virale au sein de communauté d'hôtes, plus particulièrement, les chauves-souris et les petits mammifères terrestres de Madagascar et d'identifier les facteurs agissant sur cette dynamique de transmission et de diversification virale, en particulier les facteurs bio-écologiques associés à leurs hôtes. Le second modèle viral, les lyssavirus, nous a permis de décrire sur l'ensemble des îles du SOOI échantillonés, la circulation virale dans ce système multi-insulaire diversifié, au sein des chauves-souris dont la plupart des espèces sont endémiques à cette région. Dans l'ensemble, nos investigations ont permis de mettre en évidence des échanges viraux (« host-switch ») importants entre chauves-souris, petits mammifères terrestres endémiques de Madagascar et les rongeurs introduits, le rôle de ces mammifères en tant que réservoir viral majeur et souligner le rôle disséminateur de Rattus rattus. Par ailleurs, nous avons pu identifier ce phénomène de « host-switch » comme étant le mécanisme macro-évolutif prépondérant et l'importance des facteurs biotiques et abiotiques à l'origine de la dynamique de transmission et de la diversification virale observée chez les paramyxovirus de chauves-souris de Madagascar. / Since many decades, the wild fauna has been incriminated as an important reservoir of many zoonotic pathogens (Nipah, Hendra, Ebola, Hantaan viruses etc.) at risk for humans. Tropical and subtropical islands of the South West Indian Ocean (SWIO) are part of the 34 areas of the world recognized as "hotspot" of biodiversity. They are characterized by a strong wildlife endemism especially on Madagascar. The multi-island structure of the SWIO region, the diversity of its biotopes and its biogeographical human disparities, offer a unique opportunity to investigate "in natura" the evolutionary dynamics of infectious agents and the host-virus relationships. Our research has focused on two models of negative RNA viruses, paramyxoviruses and lyssaviruses. The first virus model allowed us to address issues related to the dynamics of viral transmission within a host community, in particular, bats and small terrestrial mammals of Madagascar and to identify the driving factors, especially bio-ecological factors associated with their hosts, affecting the dynamic of transmission and of viral diversification. The second model allowed us to describe on the islands of the SWIO, the intense circulation of bats lyssaviruses in this multi-island system which bats are endemic to this region. Overall, our investigations highlighted (i) intense viral exchanges ("host-switch") between bats, endemic terrestrial small mammals and introduced rodents from Madagascar, (ii) the role of these mammals as major viral reservoir and (iii) the key role played by Rattus rattus as viral spreader. Furthermore, we identified both the phenomenon of "host-switch" as the major macro-evolutionary mechanism among bat paramyxoviruses from Madagascar and the importance of biotic and abiotic factors in shaping the transmission dynamics and viral diversification.

Following rabies virus (RABV) exposure, a combination of thorough wound washing, multiple-dose vaccine administration and the local infiltration of rabies immune globulin (RIG) are essential components of modern post-exposure prophylaxis (PEP). Although modern cell-culture-based rabies vaccines are increasingly used in many countries, RIG is much less available. The prohibitive cost of polyclonal serum RIG products has prompted a search for alternatives and design of anti-RABV monoclonal antibodies (MAbs) that can be manufactured on a large scale with a consistent potency and lower production costs. Robust in vitro neutralization activity has been demonstrated for the CL184 MAb cocktail, a 1:1 protein mixture of two human anti-RABV MAbs (CR57/CR4098), against a large panel of RABV isolates. In this study, we used a hamster model to evaluate the efficacy of experimental PEP against a lethal challenge. Various doses of CL184 and commercial rabies vaccine were assessed for the ability to protect against lethal infection with representatives of four distinct bat RABV lineages of public health relevance: silver-haired bat (Ln RABV); western canyon bat (Ph RABV); big brown bat (Ef-w1 RABV) and Mexican free-tailed bat RABV (Tb RABV). 42–100% of animals survived bat RABV infection when CL184 (in combination with the vaccine) was administered. A dose-response relationship was observed with decreasing doses of CL184 resulting in increasing mortality. Importantly, CL184 was highly effective in neutralizing and clearing Ph RABV in vivo, even though CR4098 does not neutralize this virus in vitro. By comparison, 19–95% survivorship was observed if human RIG (20 IU/kg) and vaccine were used following challenge with different bat viruses. Based on our results, CL184 represents an efficacious alternative for RIG. Both large-scale and lower cost production could ensure better availability and affordability of this critical life-saving biologic in rabies enzootic countries and as such, significantly contribute to the reduction of human rabies deaths globally.

Chapter one introduces the concept of disease emergence and factors associated with emergence. The role of wildlife as reservoirs of emerging diseases and specifically the history of bats as reservoirs of zoonotic diseases is previewed. Finally, the aims and structure of the thesis are outlined. In Chapter two, the literature relating to the emergence of Hendra virus, Nipah virus, and Australian bat lyssavirus, the biology of flying foxes, methodologies for investigating wildlife reservoirs of disease, and the modelling of disease in wildlife populations is reviewed. Chapter three describes the search for the origin of Hendra virus and investigations of the ecology of the virus. In a preliminary survey of wildlife, feral and pest species, 6/21 Pteropus alecto and 5/6 P. conspicillatus had neutralizing antibodies to Hendra virus. A subsequent survey found 548/1172 convenience-sampled flying foxes were seropositive. Analysis using logistic regression identified species, age, sample method, sample location and sample year, and the interaction terms age*species and age* sample method as significantly associated with HeV serostatus. Analysis of a subset of the data also identified a significant or near-significant association between time of year of sampling and HeV serostatus. In a retrospective survey, 16/68 flying fox sera collected between 1982 and 1984 were seropositive. Targeted surveillance of non-flying fox wildlife species found no evidence of Hendra virus. The findings indicate that flying foxes are a likely reservoir host of Hendra virus, and that the relationship between host and virus is mature. The transmission and maintenance of Hendra virus in a captive flying fox population is investigated in Chapter four. In study 1, neutralizing antibodies to HeV were found in 9/55 P. poliocephalus and 4/13 P. alecto. Titres ranged from 1:5 to 1:160, with a median of 1:10. In study 2, blood and throat and urogenital swabs from 17 flying foxes from study 1 were collected weekly for 14 weeks. Virus was isolated from the blood of a single aged non-pregnant female on one occasion. In study 3, a convenience sample of 19 seropositive and 35 seronegative flying foxes was serologically monitored monthly for all or part of a two-year period. Three individuals (all pups born during the study) seroconverted, and three individuals that were seropositive on entry became seronegative. Two of the latter were pups born during the study period. Dam serostatus and pup serostatus at second bleed were strongly associated when data from both years were combined (p<0.001; RR=9, 95%CI 1.42 to 57.12). The serial titres of 19 flying foxes monitored for 12 months or longer showed a rising and falling pattern (10), a static pattern (1) or a falling pattern (8). The findings suggest latency and vertical transmission are features of HeV infection in flying foxes. Chapter five describes Australian bat lyssavirus surveillance in flying foxes, insectivorous bats and archived museum bat specimens. In a survey of 1477 flying foxes, 69/1477 were antigen-positive (all opportunistic specimens) and 12/280 were antibody-positive. Species (p<0.001), age (p=0.02), sample method (p<0.001) and sample location (p<0.001) were significantly associated with fluorescent antibody status. There was also a significant association between rapid focus fluorescent inhibition test status and species (p=0.01), sample method (p=0.002) and sample location (p=0.002). There was a near-significant association (p=0.067) between time of year of sampling and fluorescent antibody status. When the analysis was repeated on P. scapulatus alone, the association stronger (p=0.054). A total of 1234 insectivorous bats were surveyed, with 5/1162 antigen–positive (all opportunistic specimens) and 10/390 antibody-positive. A total of 137 archived bats from 10 species were tested for evidence of Australian bat lyssavirus infection by immunohistochemistry (66) or rapid focus fluorescent inhibition test (71). None was positive by either test but 2 (both S. flaviventris) showed round basophilic structures consistent with Negri bodies on histological examination. The findings indicate that Australian bat lyssavirus infection is endemic in Australian bats, that submitted sick and injured bats (opportunistic specimens) pose an increased public health risk, and that Australian bat lyssavirus infection may have been present in Australian bats 15 years prior to its first description. In Chapter six, deterministic state-transition models are developed to examine the dynamics of HeV infection in a hypothetical flying fox population. Model 1 outputs demonstrated that the rate of transmission and the rate of recovery are the key parameters determining the rate of spread of infection, and that population size is positively associated with outbreak size and duration. The Model 2 outputs indicated that that long-term maintenance of infection is inconsistent with lifelong immunity following infection and recovery. Chapter seven discusses alternative hypotheses on the emergence and maintenance of Hendra virus and Australian bat lyssavirus in Australia. The preferred hypothesis is that both Hendra virus and Australian bat lyssavirus are primarily maintained in P. scapulatus populations, and that change in the population dynamics of this species due to ecological changes has precipitated emergence. Future research recommendations include further observational, experimental and/or modeling studies to establish or clarify the route of HeV excretion and the mode of transmission in flying foxes, the roles of vertical transmission and latency in the transmission and maintenance of Hendra virus in flying foxes, and the dynamics of Hendra virus infection in flying foxes.

Chapter one introduces the concept of disease emergence and factors associated with emergence. The role of wildlife as reservoirs of emerging diseases and specifically the history of bats as reservoirs of zoonotic diseases is previewed. Finally, the aims and structure of the thesis are outlined. In Chapter two, the literature relating to the emergence of Hendra virus, Nipah virus, and Australian bat lyssavirus, the biology of flying foxes, methodologies for investigating wildlife reservoirs of disease, and the modelling of disease in wildlife populations is reviewed. Chapter three describes the search for the origin of Hendra virus and investigations of the ecology of the virus. In a preliminary survey of wildlife, feral and pest species, 6/21 Pteropus alecto and 5/6 P. conspicillatus had neutralizing antibodies to Hendra virus. A subsequent survey found 548/1172 convenience-sampled flying foxes were seropositive. Analysis using logistic regression identified species, age, sample method, sample location and sample year, and the interaction terms age*species and age* sample method as significantly associated with HeV serostatus. Analysis of a subset of the data also identified a significant or near-significant association between time of year of sampling and HeV serostatus. In a retrospective survey, 16/68 flying fox sera collected between 1982 and 1984 were seropositive. Targeted surveillance of non-flying fox wildlife species found no evidence of Hendra virus. The findings indicate that flying foxes are a likely reservoir host of Hendra virus, and that the relationship between host and virus is mature. The transmission and maintenance of Hendra virus in a captive flying fox population is investigated in Chapter four. In study 1, neutralizing antibodies to HeV were found in 9/55 P. poliocephalus and 4/13 P. alecto. Titres ranged from 1:5 to 1:160, with a median of 1:10. In study 2, blood and throat and urogenital swabs from 17 flying foxes from study 1 were collected weekly for 14 weeks. Virus was isolated from the blood of a single aged non-pregnant female on one occasion. In study 3, a convenience sample of 19 seropositive and 35 seronegative flying foxes was serologically monitored monthly for all or part of a two-year period. Three individuals (all pups born during the study) seroconverted, and three individuals that were seropositive on entry became seronegative. Two of the latter were pups born during the study period. Dam serostatus and pup serostatus at second bleed were strongly associated when data from both years were combined (p<0.001; RR=9, 95%CI 1.42 to 57.12). The serial titres of 19 flying foxes monitored for 12 months or longer showed a rising and falling pattern (10), a static pattern (1) or a falling pattern (8). The findings suggest latency and vertical transmission are features of HeV infection in flying foxes. Chapter five describes Australian bat lyssavirus surveillance in flying foxes, insectivorous bats and archived museum bat specimens. In a survey of 1477 flying foxes, 69/1477 were antigen-positive (all opportunistic specimens) and 12/280 were antibody-positive. Species (p<0.001), age (p=0.02), sample method (p<0.001) and sample location (p<0.001) were significantly associated with fluorescent antibody status. There was also a significant association between rapid focus fluorescent inhibition test status and species (p=0.01), sample method (p=0.002) and sample location (p=0.002). There was a near-significant association (p=0.067) between time of year of sampling and fluorescent antibody status. When the analysis was repeated on P. scapulatus alone, the association stronger (p=0.054). A total of 1234 insectivorous bats were surveyed, with 5/1162 antigen–positive (all opportunistic specimens) and 10/390 antibody-positive. A total of 137 archived bats from 10 species were tested for evidence of Australian bat lyssavirus infection by immunohistochemistry (66) or rapid focus fluorescent inhibition test (71). None was positive by either test but 2 (both S. flaviventris) showed round basophilic structures consistent with Negri bodies on histological examination. The findings indicate that Australian bat lyssavirus infection is endemic in Australian bats, that submitted sick and injured bats (opportunistic specimens) pose an increased public health risk, and that Australian bat lyssavirus infection may have been present in Australian bats 15 years prior to its first description. In Chapter six, deterministic state-transition models are developed to examine the dynamics of HeV infection in a hypothetical flying fox population. Model 1 outputs demonstrated that the rate of transmission and the rate of recovery are the key parameters determining the rate of spread of infection, and that population size is positively associated with outbreak size and duration. The Model 2 outputs indicated that that long-term maintenance of infection is inconsistent with lifelong immunity following infection and recovery. Chapter seven discusses alternative hypotheses on the emergence and maintenance of Hendra virus and Australian bat lyssavirus in Australia. The preferred hypothesis is that both Hendra virus and Australian bat lyssavirus are primarily maintained in P. scapulatus populations, and that change in the population dynamics of this species due to ecological changes has precipitated emergence. Future research recommendations include further observational, experimental and/or modeling studies to establish or clarify the route of HeV excretion and the mode of transmission in flying foxes, the roles of vertical transmission and latency in the transmission and maintenance of Hendra virus in flying foxes, and the dynamics of Hendra virus infection in flying foxes.