Made available in DSpace on 2015-04-22T22:06:44Z (GMT). No. of bitstreams: 1 ALEX_GONCALVES.pdf: 1200334 bytes, checksum: d88f52a9c61abd36247626ef3528ba98 (MD5) Previous issue date: 2011-02-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The EEEV and WEEV are Arboviruses of the family Togaviridae, genus Alphavirus, which measure 60-70 nm in diameter and is composed of transmembrane glycoproteins E1 and E2, the capsid protein or the nucleocapsid, which is derived from the lipid bilayer of the host, and a single molecule single-stranded RNA and positive polarity. They are transmitted by mosquitoes, primarily Culex, Aedes, whose natural reservoir of birds and humans as accidental hosts, horses and other small mammals. Infected individuals are asymptomatic or develop flu-like illness that may progress to severe neurological disease with a prevalence in humans in North America and horses across America. The fatality rate for EEE in humans is 30% and 40% for WEE is 10%. The study was observational, cross-sectional analytical study carried out in individuals of both sex, aged over 18, resident of the lake Tucuruí power plant and from the RDS and Alcobaça Pucurui-Ararão. The collection of blood samples and filling in the questionnaire were performed at two different times, in September/2008 and March/2009. All samples were analyzed by the Evandro Chagas Institute where they were tested with hemagglutination inhibition for detection of antibodies against 19 types of Arboviruses. Data analysis was descriptive and analytical, and used the chi-square or Fisher exact test to verify the statistical significance of associations between variables of the study with an alpha level of 0,05. Altogether 365 samples were analyzed, which showed 1.1% positive for EEE and 1.6% for WEE. Most positive cases had poor sanitary conditions (100% for EEA and 83.3% for WEE) using cesspits (75% to 100% for EEE and WEE) and water from rivers and / or lakes ( 100% to 83.3% for EEE and WEE). The positive cases for EEE and WEE were related to other Arboviruses, where 100% of EEE and WEE were positive for HI-Flavivirus, 25% for EEE virus Maguari, HI-Mayaro and HI-Oropouche and 33.3 % of WEE to HI-Mayaro and 16.7% for the virus Guaroa. The study confirmed the presence of EEE virus and WEE in the region and some socio-environmental factors such as use of cesspits and poor sanitation conditions, conducive to public exposure to vectors and raised the possibility of cross protection with other alphaviruses. / O EEEV e o WEEV são Arbovírus da família Togaviridae, gênero Alphavirus, que medem 60 a 70 nm de diâmetro e é composto por glicoproteínas transmembrana E1 e E2, o capsídio ou proteína do nucleocapsídio, que deriva da bicamada lipídica do hospedeiro, e uma única molécula de RNA de fita única e de polaridade positiva. São transmitidos por mosquitos, principalmente Culex e Aedes, tendo como reservatório natural os pássaros e como hospedeiros acidentais os humanos, equinos e outros pequenos mamíferos. Os indivíduos infectados são assintomáticos ou desenvolvem síndrome gripal que podem evoluir para doença neurológica graves com predominância em humanos na América do Norte e em equinos em toda América. A taxa de letalidade em humanos para EEE é de 30% a 40% e para WEE é de 10%. O estudo foi observacional do tipo transversal analítico, realizado em indivíduos de ambos os sexos, maiores de 18 anos, residentes do lago UHE de Tucuruí e proveniente das RDS de Alcobaça e Pucuruí-Ararão. A coleta das amostras de sangue e o preenchimento do questionário foram realizados em dois momentos distintos, em setembro/2008 e março/2009. Todas as amostras foram analisadas pelo Instituto Evandro Chagas onde foram submetidas ao teste de Inibição da Hemaglutinação para a pesquisa de anticorpos contra 19 tipos de Arbovírus. A análise dos dados foi descritiva e analítica, sendo empregado o teste qui-quadrado e/ou exato de Fisher a fim de verificar a significância das associações estatísticas entre as variáveis do estudo com um nível alfa de 0,05. Ao todo foram analisadas 365 amostras, que mostraram positividade de 1,1% para EEE e de 1,6% para WEE. A maioria dos casos positivos tinha condições de saneamento ruim (100% para EEE e 83,3% para WEE), com uso fossa negra (75% para EEE e 100% para WEE) e abastecimento de água de rios e/ou lagos (100% para EEE e 83,3% para WEE). Os casos positivos para EEE e WEE tiveram relação com outros Arbovírus, sendo que 100% de EEE e WEE eram positivos para HI-Flavivírus, 25% de EEE para o vírus Maguari, HI-Mayaro e HI-Oropouche e 33,3% de WEE para HI-Mayaro e 16,7% para o vírus Guaroa. O estudo confirmou a presença dos vírus da EEE e WEE na região e que alguns fatores sócio-ambientais, como uso de fossa negra e condições de saneamento ruins, favorecem a exposição da população aos vetores e levantou a hipótese de proteção cruzada com outros Alphavírus.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes disease in humans and equines eliciting both an agricultural and public health threat. In humans, the disease typically presents as a febrile illness with common signs of fever and malaise. Four to fourteen percent of Venezuelan equine encephalitis (VEE) cases are associated with severe neurological complications due to encephalitis caused by VEEV's propensity to infect the brain. Public health concerns are exacerbated by VEEV's aerosolization capabilities, low infectious dose and affordability to mass produce. These qualities drove interest in the pathogen as a bioweapon by the US and the former Soviet Union during the cold war. As a precautionary response to VEEV's notoriety as a biothreat, the National Institute of Allergies and Infectious Diseases has classified VEEV as a category B priority pathogen, and the Human Health Services and United States Department of Agriculture list live virulent strains of VEEV as a select agent and require the pathogen to be manipulated in highly regulated biosafety level 3 (BSL3) facilities. There are currently no FDA approved vaccines or antivirals to target VEEV or other closely related alphaviruses associated with clinical disease in humans. The research performed in this dissertation aimed to elucidate new antiviral targets and treatments to help bridge gaps in current understanding of alphaviruses. The current market lacks available antibodies for E1 specific isolation. In response, a recombinant VEEV TC-83 was produced with a V5 tag at the C-terminal of the E1 sequence to enable VEEV E1 detection. Sequencing was used to verify V5 insertion in the plasmid and immunoprecipitation was used to verify V5 insertion within the E1 glycoprotein. Replication kinetics experiments verified the virus replicated similarly to the parental VEEV TC-83 strain, while passaging experiments verified the tag was highly stable for up to 10 passages. This research produced a cost-effective and highly efficient means to probe and isolate the E1 glycoprotein without modifying the viability of the virus. Knowledge of host protein interactions with VEEV E1 glycoprotein has been limited, with most E1 research focusing on its fusion capabilities. Utilizing 293-T cells infected with E1-V5 TC-83, co-immunoprecipitation was performed to isolate E1 and associated interactors. A total of 486 host and 5 viral protein interactors of E1 were discovered after normalization to the negative control. The top peptide spectrum matches (PSMs) revealed a number of chaperone proteins and ubiquitin proteins as top interactors of VEEV E1. These results effectively revealed a number of previously unknown alphavirus interactions that can be targeted by antivirals and explored further for implications in viral replication. LC-MS/MS results showed that protein disulfide isomerase family A member 6 (PDIA6) interacted with E1. High PSMs, presence in all 3 replicates, similar cellular localization to E1 and known associations between other viruses and protein disulfide isomerase (PDI) family members made this protein an optimum target for further analysis. Co-immunoprecipitation and co-localization experiments were used to validate the LC-MS/MS results. Involvement of PDIs in VEEV replication were explored utilizing two known PDI inhibitors, LOC14 and Nitazoxanide. LOC14, a non-FDA approved broad-spectrum PDI inhibitor, showed broad-spectrum alphavirus antiviral potential, decreasing titers of VEEV TC-83, VEEV Trinidad Donkey strain, eastern equine encephalitis virus (EEEV), chikungunya virus (CHIKV) and Sindbis (SINV) virus in a dose dependent manner. Nitazoxanide, an FDA approved drug known to inhibit PDIA3, was shown to have minimal toxicity and effectively reduced VEEV TC-83 and EEEV titers at concentrations with 100% cell viability. Time of addition assays, E1 expression time course studies, and early event assays showed PDI inhibition with these drugs effects early viral production events. RNA quantification, confocal microscopy and biotin switch assay experiments show that the drugs also prevented proper folding of the E1 glycoprotein and decreased expression of E1 on the peripheral membrane. With no current treatments for alphaviruses, these data provide an effective broad-spectrum target that affects viral replication at multiple stages in-vitro. Nitazoxanide also presents as a promising, non-toxic drug that could be repurposed to combat a number of clinically relevant alphaviruses. Valosin containing protein (VCP) was also shown to interact with the E1 glycoprotein. Exploration of VCP's interaction with alphavirus E1 has never been explored, yet it was previously shown to be involved in alphavirus replication. Co-localization and co-immunoprecipitation experiments were performed validating the interaction between VCP and E1. siRNA knockdown of VCP in 293-T cells and U87-MG cells showed a significant reduction in VEEV TC-83 titers. The allosteric VCP inhibitor, NMS-873, also reduced VEEV TC-83 titers, but was shown to be less effective against CHIKV, SINV and EEEV, suggesting the NMS-873 mechanism is more selective for VEEV. Mechanism experiments showed that reduction of VCP with NMS-873 inhibits early events of VEEV replication. These results elucidate VCP's association with E1 and show that VCP can be targeted to decrease VEEV viral replication. / Doctor of Philosophy / Venezuelan equine encephalitis virus (VEEV) causes disease in humans, as well as horses, donkeys and other closely related animals. In humans, the virus causes a flu-like disease and sometimes swelling of the brain. This can be associated with symptoms such as light sensitivity, confusion and sometimes coma. Prior to the Cold War, VEEV was researched by the US and previous Soviet Union's militaries in hopes to deploy the virus as a bioweapon. Current treaties prevent active production of such weapons, yet allows for defensive research to continue in preparation for a worst-case scenario. Currently no FDA approved medications or vaccines exist to combat the virus further exacerbating concerns. In order to protect laboratorians and prevent unintentional or intentional introduction of the virus into the community, the virus is only manipulated in highly secure facilities with barriers that separate the virus from personnel and the outside environment. A component of the virus called E1, allows for the virus to be released from a structure, called an endosome, that transports the virus into the cell. Currently, E1 is mostly known for this function, yet our research found that E1 interacts with 486 protein components of the host cell, suggesting a more elaborate role of E1 than previously understood. This list of interactors provides numerous new targets for potential medications to combat VEEV and other closely related viruses. Discovered E1 interactors, protein disulfide isomerase family A member 6 (PDIA6) and valosin containing protein (VCP), were validated through extensive experimentation and their function in viral replication was further explored. Protein disulfide isomerases (PDI), such as PDIA6, play an important role in folding proteins, which are cellular components made of organic building blocks called amino acids. PDIs do so by creating organic pillars, called disulfide bonds, between two cysteine amino acid residues. These disulfide bonds contribute to the 3D shape of the proteins they fold which are essential for the protein's function. E1 of VEEV has a total of eight disulfide bonds within its structure, highlighting that disulfide bonds are likely essential for the protein's structure, and therefore, function. We verified that E1 could not properly fold without PDI function by using two compounds that prevented PDI from forming or breaking disulfide bonds, specifically LOC14 and FDA approved drug nitazoxanide. Cells treated with one of either compound before and after infection with VEEV, were found to produce E1 protein with significantly less disulfide bonds therefore producing less viable virus. Further experiments also showed that the compounds also affected early stages in the virus production cycle. These two mechanisms explain the significant reduction in production of VEEV and related viruses when PDI is inhibited. These results provide a new VEEV drug target, PDIs, as well as two compounds that can potentially be used to combat VEEV and other related viruses that have no current treatment options. Another host interactor, VCP, functions throughout the cell and is known for unfolding of numerous substrates, including proteins. It is involved in numerous cellular functions thus making this interactor a promising target for drug treatment. Cells with reduced VCP function were shown to produce less progeny VEEV. Cells treated with NMS-873, a compound that reduces VCP function was also shown to reduce VEEV production. NMS-863 inhibition of VCP was shown to effect early events in VEEV replication. These results further emphasize the E1 interactors discovered are invaluable novel targets for VEEV drug treatment.

Eastern Equine Encephalitis virus is a vector-borne virus amplified by the Culiseta melanura mosquito in an enzootic avian cycle, causing high morbidity and mortality to horses and humans when contracted as incidental hosts. The virus is distributed across most of the eastern United States, Canada, and Gulf coast, and has been expanding in geographic range and season of activity over time. Spatial-temporal trends in Cs. melanura abundance were correlated with available meteorological (temperature and precipitation) and remotely sensed environmental data for the period of 1997-2012 in Connecticut. The effects of inter-annual changes in precipitation, temperature, and groundwater levels on Cs. melanura abundances using time-series linear regression and cross-correlation analyses were inconclusive. Habitat modeling using logistic regression and landscape-based predictive variables demonstrated strong efficiency (46.2%) and acceptable sensitivity and specificity (65.6 and 78.6%, respectively) using NDVI difference and distance from palustrine areas as predictive factors. Remotely sensed data can improve the understanding of vector abundance patterns, helping to forecast future outbreaks and regional expansions by guiding surveillance efforts.

Western equine encephalitis virus (WEEV) is an arboviral pathogen naturally found in North America. The primary disease phenotype associated with WEEV infection in susceptible hosts is a relatively long prodromal period followed by viral encephalitis. By contrast, in the current work, experimental inoculation of WEEV into the peritoneum of Syrian golden hamsters produced rapid death within approximately 96 h. It was determined that direct virus killing of lymphoid cells leads to death in WEEV-infected Syrian golden hamsters, and that inflammatory cytokines have the potential to enhance virus-induced lymphoid cell destruction. It was further concluded that WEEV retains its ability to cause encephalitis in Syrian golden hamsters, if hamsters survive the early stages of virus infection or if virus is introduced directly into the CNS. Death in WEEV-infected hamsters is associated with lymphonecrotic lesions in the absence of pathological lesions in the central nervous system (CNS). Few clinical parameters were altered by WEEV infection, with the exception of circulating lymphocyte numbers. Circulating lymphocyte numbers decreased dramatically during WEEV infection, and lymphopenia was identified as a consistent indicator of eventual death. Virus infection also increased serum concentrations of the cytokines interferon and tumor necrosis factor-alpha (TNF-alpha). Hamster peritoneal macrophages exposed to WEEV expressed TNF-alpha in a dose-responsive manner. Macrophage expression of TNF-alpha could be significantly inhibited by treatment of cells with anti-inflammatory agents flunixin meglumine (FM) or dexamethasone (Dex). Anti-inflammatory treatment also protected macrophages from cytotoxicity associated with exposure to WEEV. Treatment of WEEV-infected hamsters with either FM or Dex significantly improved survival compared to placebo-treated controls. WEEV induced cytotoxicity in hamster splenocytes exposed to WEEV in a virus dose-responsive manner. Supernatant from WEEV-exposed macrophages significantly enhanced WEEV killing of splenocytes. Hamsters that survived the early stages of WEEV infection occasionally developed signs of neurological disease and died approximately 6 to 9 d after virus inoculation. These animals had histopathological lesions in the CNS consistent with alphavirus-induced encephalitis. Inoculation of WEEV directly into the CNS caused apparent encephalitic disease. Death following CNS inoculation of WEEV was rapid and concurrent with histopathological lesions in the CNS similar to lesions seen in encephalitic hamsters following peripheral inoculation.

Within the state of Florida, there are three arboviruses of public health importance that can cause neuroinvasive disease in humans: West Nile Virus, Saint Louis Encephalitis Virus, and Eastern Equine Encephalitis Virus. Mosquitoes (Diptera: Culicidae) within the genus Culex are known and suspected vectors of these diseases. The vectors of these diseases can be present in urban wetland habitats that allow for exposure to residential communities. Vector ecology must be investigated in order to understand the dynamics of disease transmission. In Hillsborough County, Florida the spatial and temporal distribution of these vectors are not well established. An ecological study was conducted in the University of South Florida's Ecopreserve using trapping methodologies to sample the adult and gravid females as well as collect the egg population. Collections were made at three spatial points for the duration of July through December 2013 and compared to meteorological variables. Culex erraticus, a proposed bridge vector of Eastern Equine Encephalitis, was the most abundant adult species and gravid female captured. Culex nigripalpus, primary Floridian vector of Saint Louis Encephalitis and bridge vector of West Nile Virus, was the second most abundant adult species caught as well as the majority of eggs collected. Based on the results collected, the presence of Culex erraticus and Culex nigripalpus was confirmed. The majority of Culex erraticus adults were collected in September and October and Culex nigripalpus adults were the highest in July and August. The results of the gravid and egg collection generated crucial insight regarding methodology for studying vector ecology within this urban wetland habitat. However, modeling at spatial points based on meteorological variables yielded inconsistent results that illicit further investigation regarding these arboviral vectors of disease.

The lack of vaccines for emerging and re-emerging diseases highlights technical gaps and indicates a need for innovative approaches to produce new vaccines. Vaccines may be improved by knowledge of host responses to vaccination, disease pathogenesis, and the effect of age and genetics on vaccine outcome. This study's purpose was to quantitatively assess the molecular epidemiology of Francisella tularensis (Ft) and Venezuelan Equine Encephalitis Virus (VEEV). Study results support the Epidemiology Nexus model which holds that association of changes in gene expression to vaccination facilitate understanding the mechanisms of immune development and link public health and disease epidemiology. My research questions assessed the relationship between gene expression following vaccination, the relationship between age and vaccine response, and the association between Human Leukocyte Antigen (HLA) allele and vaccine response. The study was a novel secondary analysis of human data subjected to ANOVA to measure association between treatment and outcome, correlation to measure association of age with vaccine outcome, and Mann-Whitney U tests to measure association of HLA allele with vaccine outcome. Both Ft and VEEV vaccination elicited significant changes in gene expression. A highly positive relationship between age and vaccine outcome was shown for VEEV. The results may affect positive social change by contributing to a growing compendium of evidence of vaccine efficacy mechanisms that may function to assure the public of vaccine safety, combat vaccine hesitancy, and promote vaccine acceptance, as well as contribute mechanistic knowledge to reduce developmental costs of novel vaccines.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-05-15T16:43:54Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_EncefalomieliteEquinaIlha.PDF: 1366808 bytes, checksum: ba2d7733f0ff4fb35353b9c21dc7a614 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-05-19T12:26:25Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_EncefalomieliteEquinaIlha.PDF: 1366808 bytes, checksum: ba2d7733f0ff4fb35353b9c21dc7a614 (MD5) / Made available in DSpace on 2017-05-19T12:26:25Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_EncefalomieliteEquinaIlha.PDF: 1366808 bytes, checksum: ba2d7733f0ff4fb35353b9c21dc7a614 (MD5) Previous issue date: 2012-02-29 / Nove casos de encefalomielite equina foram estudados na Ilha de Marajó, estado do Pará, Brasil. Os animais apresentavam dificuldade em se manter em estação, andar em círculo, acentuada depressão, pálpebras cerradas, paralisia da língua, tremores musculares, bruxismo, anorexia e desidratação. Alguns apresentavam diminuição dos reflexos auricular, palpebral, de ameaça, diminuição do tônus da língua e taquicardia. Posição de auto-auscultação foi observada com frequência. Os animais muitas vezes eram encontrados apoiados em troncos e cercas para se manterem em estação. À necropsia verificou-se hemorragia das leptomeninges e medula, alguns animais apresentaram ainda aderencia das leptomeninges. Na histopatologia verificou-se encefalite difusa afetando principalmente a substância cinzenta, com meningite e coroidite. Foi observada presença de manguitos perivasculares constituídos por células inflamatórias mononucleadas. Em dois animais identificou-se o Eastern equine encephalitis virus por semi nested transcrição reversa reação de polimerase em cadeia (Semi-Nested RT-PCR). / Nine cases of equine encephalomyelitis were studied in the Marajó Island, State of Pará, Brazil. The animals had difficulty in maintaining a station, walk in a circle, marked depression, eyelids closed, tongue paralysis, muscle tremors, bruxism, anorexia and dehydration. Some had their ear and eyelid reflexes diminished, decreased tongue tone and tachycardia. Position of self-hearing was observed frequently. The animals were often found leaning on tree trunks and fences to keep themselves on station. At necropsy, they showed hemorrhage of the meninges and spinal cord, and some animals also showed adhesion of the meninges. Histologically there was diffuse encephalitis affecting mainly the gray matter, with meningitis and choroiditis. It was observed the presence of perivascular cuffs consisting of mononuclear inflammatory cells. In two animals it was possible to identificate the Eastern equine encephalitis virus by semi-nested reverse transcription polymerase chain reaction (semi-nested RT-PCR).

Várias zoonoses virais acometem equídeos causando quadros neurológicos, entre as quais a raiva e as encefalites equinas do Leste (EEE) e Oeste (WEE). O diagnóstico clínico geralmente não é conclusivo, o que torna imprescindível o diagnóstico laboratorial. Dados do Laboratório de Diagnóstico de Raiva do Instituto Pasteur de São Paulo, entre os anos 2000 e 2010, mostram que aproximadamente 75% das amostras enviadas foram negativas para raiva, ressaltando a relevância da realização de um diagnóstico diferencial para as encefalites equinas causadas por alfavírus. Os objetivos do estudo foram testar a adequação do uso de multiplex hemi-nested RT-PCR para o diagnóstico de raiva, EEE e WEE em amostras de sistema nervoso central de equídeos e realizar uma análise de custo das reações de cada técnica. Foram utilizados os primers 21G, 304 e 504 dirigidos ao gene N do vírus da raiva, e os primers cM3W, M2W, nEEE e nWEE dirigidos ao gene NSP1 dos vírus da EEE e WEE. Procedeu-se a um estudo preliminar dos primers e de seu uso em uma hemi-nested RT-PCR, avaliando a temperatura ótima de anelamento, a sensibilidade e especificidade analíticas e a reprodutibilidade da técnica em amostras de campo positivas para raiva e para EEE. A partir do protocolo estabelecido na reação de hemi-nested RT-PCR, realizaram-se variações de concentração de reagentes no protocolo para a reação de multiplex hemi-nested RT-PCR. Após o estabelecimento do protocolo para esta reação, os mesmos testes para verificação da sensibilidade e especificidade analíticas e da reprodutibilidade foram realizados, comparando-se os resultados com os obtidos pela hemi-nested RT-PCR. No teste de limiar de detecção, a sensibilidade analítica foi semelhante para as duas técnicas, obtendo-se 10&#45;1,7 para os três vírus padrão CVS, EEEV e WEEV. No teste de limiar de detecção utilizando uma amostra com os três vírus verificou-se uma alta especificidade dos primers, sendo que na reação de multiplex hemi-nested RT-PCR foi possível detectar simultaneamente os três vírus padrão. Não houve diferença nas proporções de amostras detectadas como positivas para raiva obtidas pelas duas técnicas, analisando-se pelo teste exato de Fisher (P=1,0000). No entando, para amostras de campo positivas para EEE, a proporção de amostras detectadas como positivas pela hemi-nested RT-PCR foi maior do que a proporção obtida pela multiplex hemi-nested RT-PCR (P<0,0001). Apesar de não ter sido possível o uso de amostras de campo positivas para WEE nesse estudo, os resultados sugerem que seria possível a detecção pela multiplex hemi-nested RT-PCR. Estes dados sugerem que a técnica de multiplex hemi-nested RT-PCR poderia ser aplicada para detecção de raiva e WEE, mas com limitações para a detecção de EEE. Pela análise de custo dos reagentes, o valor de uma reação de multiplex hemi-nested RT-PCR é semelhante ao de uma hemi-nested RT-PCR, podendo representar uma economia de pelo menos 49,17%. / Several viral zoonoses affect the equids causing neurological diseases, including rabies and Eastern and Western equine encephalitides (EEE and WEE). Clinical diagnosis is often not conclusive, in a way that laboratory diagnosis is essential. Data from the Laboratory of Rabies Diagnosis at the Pasteur Institute of São Paulo, between 2000 and 2010, demonstrate that approximately 75% of submitted equid samples were negative for rabies, emphasizing the importance of achieving a differential diagnosis for equine encephalitis caused by alphaviruses. The aims of this study were to test the suitability of using multiplex hemi-nested RT-PCR for the diagnosis of rabies, EEE and WEE in equids central nervous system samples and to perform a cost analysis of the reactions of each technique. We used the primers 21G, 304 and 504 directed to the N gene of rabies virus, and the primers cM3W, M2W, nEEE and nWEE directed the NSP1 gene of WEE and EEE viruses. A preliminary study of the primers was carried out, as well as their use in a hemi-nested RT-PCR, evaluating the optimal annealing temperature, the analytical sensitivity and specificity and the reproducibility of the technique in positive field samples for rabies and EEE. From the protocol established for the hemi-nested RT-PCR, variations in reagents concentrations for the multiplex hemi-nested RT-PCR protocol were perfomed. After establishing the protocol for this reaction, the same tests to verify the analytical sensitivity and specificity and reproducibility were performed and the results compared to those obtained by hemi-nested RT-PCR. In the detection threshold test, the analytical sensitivity was similar for both techniques, resulting in 10&#45;1.7 for the three virus standard CVS, and EEEV WEEV. In the detection threshold test using a sample with the three viruses, a high specificity of the primers was verified and the multiplex hemi-nested RT-PCR was able to detect the three viruses simultaneously. There was no difference in the proportions of samples detected as positive for rabies obtained by both techniques, according to the Fisher exact test (P = 1.0000). However, for EEE positive field samples, the proportion of samples detected as positive by the hemi-nested RT-PCR was higher than the proportion obtained by multiplex hemi-nested RT-PCR (P <0.0001). Although it was not possible to use WEE positive field samples in this study, the results suggest that its detection would be possible by multiplex hemi-nested RT-PCR. Thus, data suggest that the multiplex hemi-nested RT-PCR technique could be applied to detect rabies and WEE, but with limitations for the EEE detection. For the analysis of reagent costs, the cost of one multiplex hemi-nested RT-PCR is similar to one hemi-nested RT-PCR, and may represent a saving of 49,17% at least.

Várias zoonoses virais acometem equídeos causando quadros neurológicos, entre as quais a raiva e as encefalites equinas do Leste (EEE) e Oeste (WEE). O diagnóstico clínico geralmente não é conclusivo, o que torna imprescindível o diagnóstico laboratorial. Dados do Laboratório de Diagnóstico de Raiva do Instituto Pasteur de São Paulo, entre os anos 2000 e 2010, mostram que aproximadamente 75% das amostras enviadas foram negativas para raiva, ressaltando a relevância da realização de um diagnóstico diferencial para as encefalites equinas causadas por alfavírus. Os objetivos do estudo foram testar a adequação do uso de multiplex hemi-nested RT-PCR para o diagnóstico de raiva, EEE e WEE em amostras de sistema nervoso central de equídeos e realizar uma análise de custo das reações de cada técnica. Foram utilizados os primers 21G, 304 e 504 dirigidos ao gene N do vírus da raiva, e os primers cM3W, M2W, nEEE e nWEE dirigidos ao gene NSP1 dos vírus da EEE e WEE. Procedeu-se a um estudo preliminar dos primers e de seu uso em uma hemi-nested RT-PCR, avaliando a temperatura ótima de anelamento, a sensibilidade e especificidade analíticas e a reprodutibilidade da técnica em amostras de campo positivas para raiva e para EEE. A partir do protocolo estabelecido na reação de hemi-nested RT-PCR, realizaram-se variações de concentração de reagentes no protocolo para a reação de multiplex hemi-nested RT-PCR. Após o estabelecimento do protocolo para esta reação, os mesmos testes para verificação da sensibilidade e especificidade analíticas e da reprodutibilidade foram realizados, comparando-se os resultados com os obtidos pela hemi-nested RT-PCR. No teste de limiar de detecção, a sensibilidade analítica foi semelhante para as duas técnicas, obtendo-se 10&#45;1,7 para os três vírus padrão CVS, EEEV e WEEV. No teste de limiar de detecção utilizando uma amostra com os três vírus verificou-se uma alta especificidade dos primers, sendo que na reação de multiplex hemi-nested RT-PCR foi possível detectar simultaneamente os três vírus padrão. Não houve diferença nas proporções de amostras detectadas como positivas para raiva obtidas pelas duas técnicas, analisando-se pelo teste exato de Fisher (P=1,0000). No entando, para amostras de campo positivas para EEE, a proporção de amostras detectadas como positivas pela hemi-nested RT-PCR foi maior do que a proporção obtida pela multiplex hemi-nested RT-PCR (P<0,0001). Apesar de não ter sido possível o uso de amostras de campo positivas para WEE nesse estudo, os resultados sugerem que seria possível a detecção pela multiplex hemi-nested RT-PCR. Estes dados sugerem que a técnica de multiplex hemi-nested RT-PCR poderia ser aplicada para detecção de raiva e WEE, mas com limitações para a detecção de EEE. Pela análise de custo dos reagentes, o valor de uma reação de multiplex hemi-nested RT-PCR é semelhante ao de uma hemi-nested RT-PCR, podendo representar uma economia de pelo menos 49,17%. / Several viral zoonoses affect the equids causing neurological diseases, including rabies and Eastern and Western equine encephalitides (EEE and WEE). Clinical diagnosis is often not conclusive, in a way that laboratory diagnosis is essential. Data from the Laboratory of Rabies Diagnosis at the Pasteur Institute of São Paulo, between 2000 and 2010, demonstrate that approximately 75% of submitted equid samples were negative for rabies, emphasizing the importance of achieving a differential diagnosis for equine encephalitis caused by alphaviruses. The aims of this study were to test the suitability of using multiplex hemi-nested RT-PCR for the diagnosis of rabies, EEE and WEE in equids central nervous system samples and to perform a cost analysis of the reactions of each technique. We used the primers 21G, 304 and 504 directed to the N gene of rabies virus, and the primers cM3W, M2W, nEEE and nWEE directed the NSP1 gene of WEE and EEE viruses. A preliminary study of the primers was carried out, as well as their use in a hemi-nested RT-PCR, evaluating the optimal annealing temperature, the analytical sensitivity and specificity and the reproducibility of the technique in positive field samples for rabies and EEE. From the protocol established for the hemi-nested RT-PCR, variations in reagents concentrations for the multiplex hemi-nested RT-PCR protocol were perfomed. After establishing the protocol for this reaction, the same tests to verify the analytical sensitivity and specificity and reproducibility were performed and the results compared to those obtained by hemi-nested RT-PCR. In the detection threshold test, the analytical sensitivity was similar for both techniques, resulting in 10&#45;1.7 for the three virus standard CVS, and EEEV WEEV. In the detection threshold test using a sample with the three viruses, a high specificity of the primers was verified and the multiplex hemi-nested RT-PCR was able to detect the three viruses simultaneously. There was no difference in the proportions of samples detected as positive for rabies obtained by both techniques, according to the Fisher exact test (P = 1.0000). However, for EEE positive field samples, the proportion of samples detected as positive by the hemi-nested RT-PCR was higher than the proportion obtained by multiplex hemi-nested RT-PCR (P <0.0001). Although it was not possible to use WEE positive field samples in this study, the results suggest that its detection would be possible by multiplex hemi-nested RT-PCR. Thus, data suggest that the multiplex hemi-nested RT-PCR technique could be applied to detect rabies and WEE, but with limitations for the EEE detection. For the analysis of reagent costs, the cost of one multiplex hemi-nested RT-PCR is similar to one hemi-nested RT-PCR, and may represent a saving of 49,17% at least.

Les arboviroses représentent un fardeau sanitaire considérable et croissant à l’échelle mondiale. La complexité des facteurs biotiques et abiotiques qui interviennent dans la transmission de ces arbovirus pose un défi de taille aux scientifiques qui tentent de comprendre, de modéliser ou d’anticiper leur transmission ainsi qu’aux intervenants de santé publique qui ont la responsabilité de surveiller, d’évaluer et gérer le risque que posent les arbovirus pour la santé des populations. Cette étude visait à estimer et caractériser le risque d’exposition à plusieurs arbovirus suspectés d’être actifs et émergents au Québec mais dont la distribution avait peu ou n’avait pas été étudiée au Québec : le virus du Nil occidental (VNO), le virus de l’encéphalite équine de l’est (VEEE) et deux virus du sérogroupe de la Californie (VSGC), le virus de Jamestown Canyon (VJC) et le virus du lièvre d’Amérique (VLA). Basée notamment sur l’hypothèse selon laquelle les animaux d’espèces différentes qui partagent un environnement commun partagent également un risque environnemental commun, cette étude visait également à évaluer si les populations d’animaux de compagnie pouvaient aider à estimer et caractériser le risque d’infection arbovirale chez l’humain. L’échantillonnage sérologique de populations humaines, canines et équines du sud-ouest du Québec a permis d’évaluer et de comparer la séroprévalence aux arbovirus étudiés chez chacune de ces trois espèces. Les estimations de séroprévalence ont révélé un niveau d’activité arbovirale significative pour chacun des arbovirus. Des différences ont été remarquées quant au pourcentage de sujets séropositifs chez chacune des espèces. Les facteurs environnementaux ayant une influence sur le risque d’infection par le VEEE ont été modélisés à partir de données sérologiques et cliniques chez les chevaux. Les milieux humides boisés ont été identifiés comme les principaux environnements à risque pour le VEEE au Québec alors que les zones agricoles ont été identifiées comme des environnements protecteurs. Les facteurs environnementaux ayant un impact sur le risque d’infection par le VNO ont été modélisés à partir des données sérologiques chez le chien et des données cliniques agrégées chez l’humain. Cette modélisation a suggéré un risque singulièrement plus élevé en zone agricole chez le chien et un risque plus faible en zone forestière chez l’humain, des facteurs rarement identifiés dans la littérature Nord-Américaine. Les facteurs environnementaux et individuels ayant un impact sur le risque d’infection par les VSGC chez l’humain et le chien ont par la suite été modélisés à partir des données sérologiques chez ces deux espèces. D’après nos modèles, le risque d’infection par ces virus serait supérieur en zone forestière et le degré d’exposition aux piqures de moustiques serait un facteur déterminant du risque d’infection chez les deux espèces. Cette étude a permis de bonifier de façon substantielle le portrait de l’activité arbovirale au Québec. Elle a permis de caractériser la distribution du risque et a fourni des données probantes pouvant soutenir la recherche ainsi que la planification des interventions en santé publique. La méthodologie utilisée dans le cadre de cette étude supporte la pertinence de l’approche « One Health » pour l’étude des maladies vectorielles émergentes. / Arboviral infections represent a considerable and growing health burden globally. The complexity of biotic and abiotic factors involved in the transmission of these arboviruses pose a challenge to scientists trying to understand, model or anticipate arboviral transmission as well as to public health authorities who have the responsibility to monitor, assess and manage the public health risk posed by arboviruses. This study aimed at estimating and characterizing the risk of exposure to several arboviruses suspected of being active and emerging in Québec but whose distribution had not been studied thoroughly in Québec: West Nile virus (WNV), eastern equine encephalitis virus (EEEV) and two viruses of the California serogroup (CSG), Jamestown Canyon virus (JCV) and Snowshoe hare virus (SHV). Based on the assumption that animals of different species sharing a common environment also share similar environmental risk, this study also aimed to assess whether some populations of domestic animals could help to estimate and characterize the risk of arboviral infection in humans. Serological sampling of human, canine and equine populations from southwestern Québec was used to evaluate and compare the seroprevalence to the selected arboviruses in each of these three species. Seroprevalence estimates showed a significant level of arboviral activity for all arboviruses. Differences were noted in the percentage of seropositive individuals in each species. Environmental factors that influence the risk of infection by EEEV were modeled based on serological and clinical data in horses. Wooded wetlands were identified as the main risk environments for EEEV in Québec while agricultural areas were identified as protective environments. Environmental factors affecting the risk of WNV infection were modeled based on serological data in dogs and aggregated clinical data in humans. These models suggested a higher risk in agricultural areas in dogs and a lower risk in forest areas in humans, two factors rarely identified in the North American literature. Environmental and individual factors affecting the risk of infection by CSGV in humans and dogs have subsequently been modeled based on serological data in these two species. According to our models, the risk of infection with these viruses would be higher in forested areas and the degree of exposure to mosquito bites would be a risk factor for infection in both species. This study substantially enhanced the comprehension of arboviral activity in Québec. It allowed for characterizing the distribution of risk and provided evidence that may support research and planning of public health interventions. The methodology used in this study supports the relevance of the "One Health" approach for the study of emerging vector-borne diseases.