El presente trabajo de tesis tuvo como objetivo titular anticuerpos neutralizantes contra una cepa endémica del virus Chikungunya que circula en la costa norte peruana. Para desarrollar esta investigación, se empleó la prueba de neutralización por reducción en placas (PRNT), la cual se realizó en el Instituto Nacional de Salud (INS)– Laboratorio de Aislamiento y Cultivo Celular perteneciente al área de Metaxénicas Virales. La justificación de la investigación se basa en la necesidad de comprobar que los anticuerpos detectados por la prueba de ELISA son neutralizantes y logran inhibir la dispersión del CHIKV en el cuerpo humano. Además, la prueba es necesaria para evaluar drogas antivirales y futuras vacunas que lleguen al Perú. El diseño metodológico utilizado fue analítico y experimental. La cepa y las muestras fueron proporcionadas por el INS y provinieron del departamento de Tumbes. Las muestras positivas fueron previamente diagnosticadas con ELISA utilizando IgM e IgG y las muestras negativas fueron de individuos sanos sin contacto previo con arbovirus. La prueba de PRNT se realizó en 24 horas usando la línea celular VERO CCL-81 en monocapa. La valoración de la prueba se realizó al 50% de neutralización. Se obtuvo como resultado títulos de anticuerpos en el rango de 1/8 y 1/16. Ésta variación responde a una relación inversa con el inicio de los días de síntomas. Por lo tanto, se concluye que la población de la costa norte del Perú sí está desarrollando anticuerpos neutralizantes para contrarrestar el virus Chikungunya endémico en la región.The present thesis aims to titrate neutralizing antibodies against an endemic strain of the Chikungunya virus that circulates in the northern coast of Peru. In order to develop this research, the plaque reduction neutralization test (PRNT) has been used, which has been carried out at the National Institutes of Health (INS) - Isolation and Cell Culture Laboratory belonging to the Viral Metaxenics area. The justification of the investigation is based on the need to verify that the antibodies detected by the ELISA test are neutralizing and manage to inhibit the dispersion of CHIKV in the human body. In addition, the test is necessary to evaluate antiviral drugs and future vaccines that arrive in Peru. The methodological design used was analytical and experimental. The strain and samples were provided by the INS. The strain is endemic to the department of Tumbes. The positive samples were previously diagnosed with ELISA using IgM and IgG and the negative samples were from individuals that had not had contact with any arbovirus. The PRNT test was performed in 24 hours using the VERO CCL-81 cell line in monolayer. The titration of the test was performed at 50% neutralization. Antibody titres were obtained in the range of 1/8 and 1/16. This variation responds to an inverse relationship with the onset of symptom days. Therefore, it is concluded that the population of the northern coast of Peru is developing neutralizing antibodies to counteract the endemic Chikungunya virus in the region.

West Nile virus (WNV) and St. Louis Encephalitis (SLEV) are arthropod-borne viruses belonging to the genus Flavivirus and are classified as significant human pathogens of global epidemiological importance. Since its introduction into the United States in 1999, WNV has spread throughout most of the country and has caused major epidemics of neuroinvasive disease (Hayes and Gubler, 2005). SLEV is endemic to the United States and is maintained in an enzootic transmission cycle in Florida.The Florida Sentinel Chicken Arboviral Surveillance Network was established in 1978 following a widespread rural epidemic of SLEV in central Florida to monitor the activity of arboviruses (Day and Stark, 1996). This program ultimately impacts vector control strategies and may warrant medical alerts to warn the population. Current serological detection methods for sentinel chickens include hemagglutination inhibition antibody test (HAI), IgM antibody capture enzyme-linkedimmunosorbent assay (MAC-ELISA), and Plaque Reduction Neutralization Test (PRNT).These serological assays may take over three weeks to generate a final result. A more rapid and equally sensitive test to replace these current serological methods would be of benefit. Microsphere-based immunoassays (MIAs) are a more rapid serological option for laboratory diagnosis of many diseases (Kellar et al, 2001). The objective of this study was to develop and validate a protocol for a MIA to detect antibodies to WNV and SLEV in sentinel chicken sera. A total of 385 sentinel chicken sera from 2005 were assayed using the MIA for WNV and 424 sera from multiple years were assayed for SLEV. The capability of the MIA to multiplex allowed for simultaneous detection of antibodies to WNV and SLEV in sentinel chicken sera. The MIA was found to be more sensitive and specific than both the HAI and MAC-ELISA for the detection of antibodies to WNV, and just as sensitive and specific as the MAC-ELISA for the detection of antibodies to SLEV in sentinel chicken sera. These results indicate that there is a potential of the MIA to decrease turn-around time and allow for earlier detection and improvement to the current surveillance system.

Por representar a mais importante arbovirose em nível mundial, as infecções causadas pelos vírus da dengue são de grande importância em nosso país, apresentando uma ampla variedade de sintomas clínicos que vão desde infecção assintomática até formas mais graves da doença. O título de anticorpos neutralizantes produzidos frente à infecção por dengue parece ser determinante na forma de apresentação da doença no paciente. Atualmente, a forma com que o teste de neutralização é realizado demanda tempo para sua realização. O objetivo deste trabalho foi padronizar um ensaio de neutralização viral por RT-PCR em tempo real em cepas virais dos quatro sorotipos dengue para, posteriormente, ser empregada na detecção rápida e em grande escala de anticorpos em soro de pacientes e de candidatos vacinais. Para isso, foram construídas curvas padrão, para cada sorotipo viral, por meio da transcrição in vitro do RNA viral. O ensaio de neutralização padronizado nesse estudo reduziu o número de dias de detecção da neutralização em 48 horas quando comparada com a técnica de neutralização tradicional (PRNT), além de utilizar técnicas moleculares sensíveis e específicas para detecção como a RT-PCR em tempo real que garantem maior aplicabilidade do teste. / Because Dengue virus is the most important arboviral disease worldwide, infections caused by this pathogen are of great importance in Brazil, producing a wide variety of clinical symptoms ranging from asymptomatic infection to more serious forms of the disease. The title of neutralizing antibodies produced against the dengue infection appears to be determinant in the outcome of the disease. Currently, neutralization tests that have been performed take time for its execution. The aim of this study was to standardize a viral neutralization assay by real-time RT-PCR of viral strains of the four Dengue serotypes to subsequently be used for rapid detection and large-scale using antibodies of patients and for vaccine candidates. For this matter, standard curves were constructed for each Dengue serotype, by in vitro transcription of viral RNA. The neutralization assay in this study reduced the period of neutralization to 48 hours, compared to traditional neutralization test (PRNT), and it uses a more sensitive and specific molecular technique for detection of neutralizing antibodies, such as real time RT-PCR, to ensure greater applicability of the test

Por representar a mais importante arbovirose em nível mundial, as infecções causadas pelos vírus da dengue são de grande importância em nosso país, apresentando uma ampla variedade de sintomas clínicos que vão desde infecção assintomática até formas mais graves da doença. O título de anticorpos neutralizantes produzidos frente à infecção por dengue parece ser determinante na forma de apresentação da doença no paciente. Atualmente, a forma com que o teste de neutralização é realizado demanda tempo para sua realização. O objetivo deste trabalho foi padronizar um ensaio de neutralização viral por RT-PCR em tempo real em cepas virais dos quatro sorotipos dengue para, posteriormente, ser empregada na detecção rápida e em grande escala de anticorpos em soro de pacientes e de candidatos vacinais. Para isso, foram construídas curvas padrão, para cada sorotipo viral, por meio da transcrição in vitro do RNA viral. O ensaio de neutralização padronizado nesse estudo reduziu o número de dias de detecção da neutralização em 48 horas quando comparada com a técnica de neutralização tradicional (PRNT), além de utilizar técnicas moleculares sensíveis e específicas para detecção como a RT-PCR em tempo real que garantem maior aplicabilidade do teste. / Because Dengue virus is the most important arboviral disease worldwide, infections caused by this pathogen are of great importance in Brazil, producing a wide variety of clinical symptoms ranging from asymptomatic infection to more serious forms of the disease. The title of neutralizing antibodies produced against the dengue infection appears to be determinant in the outcome of the disease. Currently, neutralization tests that have been performed take time for its execution. The aim of this study was to standardize a viral neutralization assay by real-time RT-PCR of viral strains of the four Dengue serotypes to subsequently be used for rapid detection and large-scale using antibodies of patients and for vaccine candidates. For this matter, standard curves were constructed for each Dengue serotype, by in vitro transcription of viral RNA. The neutralization assay in this study reduced the period of neutralization to 48 hours, compared to traditional neutralization test (PRNT), and it uses a more sensitive and specific molecular technique for detection of neutralizing antibodies, such as real time RT-PCR, to ensure greater applicability of the test

Florida's mild climate supports year round enzootic transmission of arthropod-borne (arbo) viruses, such as St. Louis Encephalitis virus (SLEV), West Nile virus (WNV), and Eastern Equine Encephalomyelitis virus (EEEV). First isolated in 1960 from two Florida blue jays, Highlands J virus (HJV) is endemic to the state and vectored by the same mosquitoes as EEEV (Henderson et al, 1962). EEEV and HJV are both alphaviruses, but HJV is not pathogenic to humans, occasionally causes encephalitis in horses, and is a recognized pathogen in some bird species (turkeys, emus, etc) (Cilnis et al, 1996). The Florida Sentinel Chicken Arboviral Surveillance Program, established in 1978, utilizes sentinel chickens to detect arboviral activity throughout the state. Current serologic antibody detection methods include the hemagglutination inhibition (HAI), IgM antibody capture enzyme-linked immunosorbent (MAC-ELISA), and serum neutralization plaque reduction (PRNT) assays (Blackmore et al, 2003). In 2003, sentinel chickens detected significantly greater alphavirus activity than seen in the previous 15 years (Stark & Kazanis, 2003). This increase raised concerns that bridging into the human population had become a serious threat as well as an important issue for veterinary health. The objective of this study was to determine if cross-reactions with Highlands J virus were impacting the serologic diagnostic tests routinely performed for identification of EEEV. For 2003, the HAI test detected 476 alphavirus positive sentinels. We tested 316 of these chickens in the PRNT, which identified 176 EEEV positive sentinels and 75 HJV positive sentinels. Our results indicate that Highlands J virus is extensively cross-reactive in the HAI test and that the MAC-ELISA is more specific for the detection of antibodies solely to EEEV. We demonstrated that EEEV antibody titers in the HAI test were positively correlated to antibody titers in the PRNT assay. Analysis of alphaviral activity by county indicates widespread transmission of HJV across the northern and panhandle regions of the state; however EEEV activity was greater than HJV activity in all but four counties. Consequently, distinguishing between the two agents can reduce the expenditure of resources on unnecessary vector control and medical alerts to protect the public health from Highlands J virus.

Made available in DSpace on 2017-07-25T19:29:58Z (GMT). No. of bitstreams: 1 Maik Rodrighero.pdf: 828244 bytes, checksum: b17d81b60eaad6eff20ad64666064e48 (MD5) Previous issue date: 2012-12-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / To control soil acidity in no-till systems, lime is broadcast on the surface without incorporation. In order to test the hypothesis that the source and particle size of the corrective influence the soil reaction and crop response to lime on the surface in no-till system, two experiments were conducted in Tibagi (PR), one in clayey Oxisol and another in sandy Litholic Neosol. A randomized complete block design was used in a factorial 2 × 2 × 4 with three replications, in each experiment. The treatments consisted of four rates of lime on the surface, estimated to raise the base saturation of the topsoil (0-20 cm), at 50, 70 and 90%; two sources of lime were used, calcitic and dolomitic,and two ranges of lime material effective calcium carbonate equivalent (ECCE), range B (ECCE 60-75%) and range D (ECCE > 90%). In both experiments, the lime was applied on the soil surface in August 2010. During the spring-summer season in 2010- 11 and 2011-12, corn and soybean were grown on the clayey soil, and soybean and corn on the sandy soil, respectively. After 12 months of liming, soil chemical analyses were performed in samples taken at the 0-5, 5-10, 10-20, 20-40, and 40-60 cm depths. Leaf samples of corn and soybean were taken during crop flowering, in the second year of cultivation, for foliar diagnosis. Grain yields of corn and soybean, in both experiments,were evaluated in two years, after the physiological maturity, and grain moisture was corrected to 130 g kg-1. Surface application of lime in both soils, after 12 months, promoted amelioration of soil acidity mainly in the 0-5 cm layer and, to a lesser extent in the 5-10 cm, regardless of the source and range of lime ECCE. Calcitic lime showed a stronger reaction than dolomitic lime in the soil surface layers. The extraction with cation exchange resin overestimated the exchangeable Ca and Mg content in relation to the 0.1 mol L-1 KCl solution when coarser grain size lime was applied on the surface. Surface liming increased the Ca-leaf content, especially with the use of calcitic lime, and Mg-leaf content mainly with the use of dolomitic limestone, and reduced Mn and Zn contents in the leaves, regardless of the source of lime, in the corn and soybean crops. Grain yields of corn and soybean in clayey Oxisol and corn in sandy Litholic Neosol were increased with the lime rates, but were not affected by sources and ECCE ranges of material lime. Surface application of lime in soils under no-till was proved of fundamental importance to maximize crop grain yield, regardless of the lime source being calcitic or dolomitic, or the ECCE range, B or D of the material lime. / No sistema plantio direto, a correção da acidez é feita por meio da aplicação de calcário na superfície sem incorporação. Com a hipótese de que a fonte e a granulometria dos corretivos interferem na reação do solo e na resposta das culturas à calagem superficial em plantio direto, foram realizados dois experimentos em Tibagi (PR), sendo um em Latossolo Vermelho argiloso e outro em Neossolo Litólico arenoso, no período de 2010 a 2012. O delineamento empregado, em cada experimento, foi o de blocos completos ao acaso, em esquema fatorial 2 × 2 × 4, com três repetições. Os tratamentos foram constituídos de quatro doses de calcário na superfície, estimadas para elevar a saturação por bases do solo (0–20 cm), a 50, 70 e 90%; duas fontes de calcário, calcítico e dolomítico; e duas faixas de poder relativo de neutralização total (PRNT), faixa B (PRNT de 60 a 75%) e faixa D (PRNT > 90%). Nos dois experimentos, o calcário foi aplicado a lanço sobre a superfície do solo em agosto de 2010. Realizaram-se dois cultivos em 2010–11 e 2011–12, com milho e soja no solo argiloso, e soja e milho no solo arenoso. Após 12 meses da aplicação, análises químicas de solo foram realizadas em amostras coletadas nas camadas de 0–5, 5–10, 10–20, 20–40 e 40–60 cm de profundidade. Amostras de folhas de milho e soja foram coletadas por ocasião do florescimento das culturas, no segundo ano de cultivo, para fins de diagnose foliar. A produtividade de grãos de milho e soja, nos dois experimentos, foi avaliada, nos dois anos, após a maturação fisiológica, corrigindo-se a umidade dos grãos para 130 g kg-1 de água. A aplicação superficial de calcário nos dois solos, após 12 meses, promoveu correção da acidez principalmente na camada de 0-5 cm e, em menor grau, na de 5-10 cm, independentemente da fonte e da faixa de PRNT dos corretivos. O calcário calcítico apresentou maior reação do que o calcário dolomítico nas camadas superficiais do solo. A extração com a resina de troca catiônica superestimou os teores de Ca e Mg trocáveis em relação à solução de KCl 1 mol L-1 quando houve aplicação superficial de calcário com granulometria mais grossa. A calagem superficial aumentou as concentrações de Ca-foliar, principalmente com a utilização de calcário calcítico, e de Mg-foliar,especialmente com o uso de calcário dolomítico, e reduziu os teores foliares de Mn e Zn, independentemente da fonte de calcário, nas culturas de milho e soja. As produtividades de milho e soja, no Latossolo argiloso, e de milho, no Neossolo arenoso,foram aumentadas com as doses de calcário, mas não foram influenciadas pelas fontes e faixas de PRNT dos corretivos. A calagem superficial em solos sob plantio direto se mostrou de fundamental importância para maximizar a produtividade de grãos das culturas, independentemente da fonte de calcário, calcítico ou dolomítico, e da faixa, B ou D, de PRNT dos corretivos.