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El presente estudio tuvo como objetivo determinar la seroprevalencia de los agentes neumotrópicos como los virus de la Diarrea Viral Bovina (VDVB), Respiratorio Sincitial (VRS) y Parainfluenza 3 (VPI3) en rumiantes de la comunidad de Chahuaytiri, provincia de Calca, Cusco, a través de la detección de anticuerpos en el suero sanguíneo de alpacas (n igual 21), bovinos (n igual 66) y ovinos (n igual 152) mediante la prueba de neutralización viral (NV). El 15.8±16.4% (3/21), 4.8 ± 9.1% (1/21) y el 23.8±18.2% (5/21) de las alpacas presentaron anticuerpos neutralizantes contra los virus: DVB, RS y PI3. El 90.9±6.9% (60/66), 87.88±7.9% (58/66) y el 81.8 ± 9.3% (54/66) de los bovinos y el 28.29±7.1% (43/152), 49.34 ±7.9% (75/152) y 50.0 ±7.9% (76/152) de los ovinos presentaron anticuerpos contra la DVB, RS y PI3, respectivamente. Estos resultados confirman la presencia de infecciones virales en rumiantes bajo sistema de crianza mixto de la comunidad campesina del Cusco. / --- The seroprevalence of Bovine Viral Diarrhoea Virus (BVDV), Parainfluenza Virus Type 3 (PI3V) and Respiratory Syncytial Virus (RSV) in serum samples of alpacas (n igual 21), bovine (n igual 66) and sheep (n igual 152) of a rural community of Cusco, Peru. It was carried out by virus-neutralization test. The 15.8±16.4% (3/21), 4.8 ±9.1% (1/21) and 23.8±18.2% (5/21) of alpacas had neutralizing antibodies against BVD, RS and PI3 virus. The 90.9±6.9% (60/66), 87.88±7.9% (58/66) and 81.8 ±9.3% (54/66) of bovine and the 28.29±7.1% (43/152), 49.34 ±7.9% (75/152) and 50.0 ±7.9% (76/152) of sheep had antibodies to BVDV, RSV and PI3V respectively. These results confirm the presence of viral infections in ruminants of mixed breeding system of a rural community.

El dengue es una enfermedad febril aguda viral causada por la infección con uno de los cuatro serotipos del virus del dengue (DENV-1, 2, 3 y 4) que se trasmite al hombre a través del mosquito del género Aedes aegypti. En el presente estudio se identificaron los genotipos y linajes de los cuatro serotipos del virus dengue a partir de los aislamientos obtenidos en muestras procedentes de diferentes regiones geográficas de Perú durante los años 1998 - 2012. El diseño del estudio fue descriptivo - retrospectivo de las características genéticas, 109 cepas que pertenecen a uno de los cuatro serotipos del virus dengue fueron procesadas por la RT-Nested PCR para amplificar la región del gen E/NS1 y se secuenció el genoma completo de una cepa de DENV-2. Las secuencias fueron alineadas con el programa CLUSTAL X y analizadas con el programa MEGA 5.0 con el algoritmo de distancia Neighbor Joining para E/NS1 y el método de Maximum Likelihood para el genoma completo. Se identificó el serotipo DENV-1, genotipo V, con tres linajes; respecto al serotipo DENV-2, se estableció dos genotipos: el genotipo América con dos linajes y genotipo América/Asia con cinco linajes. El serotipo DENV-3, presentó el genotipo III con cinco linajes y finalmente se halló el serotipo DENV-4 con dos linajes. Se concluye que los cuatro serotipos de los virus dengue aislados en diferentes áreas geográficas de Perú durante los años 1998 - 2012 presentaron variabilidad genética a nivel de genotipos y linajes, siendo el DENV-3 más divergente con cinco linajes y el DENV-4 menos divergente con dos linajes, respecto a los otros serotipos estudiados. Palabras clave: virus dengue, serotipo s virus dengue, genotipo viral, linaje viral, genoma viral / Dengue is an acute febrile viral disease caused by infection with one of the four dengue virus serotypes (DENV-1, 2, 3 and 4) that is transmitted to humans by the mosquito Aedes aegypti. In the present study identified genotypes and line ages of the four dengue virus serotypes from isolates from different geographic regions of Peru from 1998 - 2012. Design of the study was descriptive – retrospective of the genetic features, 109 strains belonging to one of the four dengue virus serotypes were processed by RT-Nested PCR to amplify the gene region E/NS1 and sequenced the full genome of a strain of DENV-2. Sequences were aligned with the program CLUSTAL X and analyzed with the software MEGA 5.0 with the Neighbor-Joining distance algorithm for E/NS1 and Maximum Likelihood method for full genome. Was identified serotype DENV-1, genotype V, with three lineages; respect to serotype DENV-2, were described two genotypes: genotype America with two lineages and genotype Americas/Asia with five lineages. DENV-3 genotype III presented the five lineages and ultimately was found DENV-4 with two lineages. We conclude that the four serotypes of dengue viruses isolated in different geographical areas of Peru during 1998 - 2012 showed genetic variation at the level of genotypes and lineages, DENV-3 remains the most divergent with five lineages and least divergent DENV-4 with two lineages, compared to the other serotypes examined. Keywords: dengue virus, dengue virus serotype, viral genotype, viral lineage, viral genome.

Memoria para optar al Título Profesional de Médico Veterinario / El Virus Diarrea Viral Bovina (VDVB) es el agente causal del complejo Diarrea Viral Bovina / Enfermedad de las Mucosas (DVB/EM), que afecta productiva y reproductivamente al bovino, provocando grandes pérdidas económicas en la industria bovina mundial. El virus se presenta en todo el mundo, con prevalencias que van del 50 al 90%, diseminándose vía horizontal y vertical. En las infecciones prenatales se puede observar muerte fetal, momificación, aborto, alteraciones teratogénicas o generación de animales persistentemente infectados (PI); y en las infecciones posnatales, generalmente cursa en forma subclínica, pero también hay problemas reproductivos, inmunosupresión y cuadros más severos (incluso letales) como la diarrea viral bovina, enfermedad de las mucosas y síndrome hemorrágico. El VDVB presenta una gran variabilidad genómica, antigénica y biológica. El VDVB se clasifica en dos genogrupos o genotipos: el genotipo VDVB1 (con actualmente 15 subgrupos) y el genotipo VDVB2. Antigénicamente, aunque presenta sólo un serotipo tiene una gran variabilidad antigénica. Biológicamente presenta dos biotipos: biotipo citopático (cp) y no citopático (ncp) y diversos estudios evidencian la presencia de virus de distinta virulencia. En esta memoria de título se determinó la variabilidad genética de aislados chilenos del VDVB por filogenia molecular de la región E2 del genoma viral. Para ello, se analizaron genéticamente virus obtenidos desde animales con signología sospechosa de infección por VDVB, como así también de animales aparentemente normales, que en su conjunto provenían de la Región Metropolitana, VIII y X regiones. También se analizaron algunos virus aislados entre los años 1993-2001, que se mantenían guardados a -20°C. De dieciocho virus analizados, catorce pertenecen al genotipo VDVB-1 y cuatro al genotipo VDVB-2. En los virus del genotipo VDVB-1, 6 pertenecen al subgrupo 1a, 7 al subgrupo 1b, y 1 al subgrupo 1e. En los virus del genotipo VDVB-2, todos corresponden al período 1993-2001, no siendo aislado el VDVB-2 en las muestras posteriores al 2001. Esto concluye que el perfil genético de los aislados virales chilenos del VDVB analizados en esta memoria presenta una alta variabilidad, distinto a lo reportado en el estudio anterior, pero muy similar al perfil genético reportado en Argentina. / Proyecto FONDECYT 1060581

M.Med. Faculty of Health Sciences, University of the Witwatersrand, 2008 / Objectives: Characterization of HIV-1 from slow progressors (SP) is important to facilitate vaccine and antiviral drug development. In order to identify virus attenuations that may contribute to slower rates of disease progression, the full viral genomes from primary isolates of six slow progressing HIV-positive children were sequenced. Methods: Primary virus biological phenotypes were determined by growth in CCR5- and CXCR4-expressing U87.CD4 cell lines. Proviral DNA was isolated from co-cultured PBMCs, and the near full-length genomes and LTR regions were PCR amplified, sequenced and analysed. Predicted amino acid (aa) sequences for all the HIV-1 proteins were extensively analyzed. Results: All primary HIV-1 isolates utilized CCR5, and were determined to be HIV-1 subtype C by phylogenetic analysis. Predicted aa sequence analysis revealed open reading frames for all HIV-1 genes which encoded for proteins of the expected length, with several exceptions. For example, isolate LT5 had a 2 aa insertion in the Vpr mitochondriotoxic domain. Isolate LT21 contained an additional 5aa in the C-terminus of tat exon 2, while the integrase enzyme in isolate LT39 had an additional 3aa at the Cterminus. Rev from isolates LT45 and LT46 did not have the characteristic subtype C 16aa truncation, and in addition, had a further 3aa. In addition, altered functional domains was noted in several isolates, such as the cAMP-dependent kinase PKA phosphorylation site in Nef (LT5), a Vpr mutation involved in decreasing pro-apoptotic activity (LT42), and the Nef ExxxLL motif involved in the interaction with AP-1 and AP-2 (LT46). Conclusions: The slower HIV disease progression in these six children may be attributed to altered protein functions. For example, LT46 Nef is unable to bind AP-1 and AP-2 and therefore inactive on CD4 endocytosis. The biological relevance of these findings requires further investigation.

O Vírus Respiratório Sincicial Humano (HRSV) é um dos patógenos mais importantes do trato respiratório. Analisamos as interações das proteínas virais nucleoproteína (N), fosfoproteína (P) e matriz (M) em células HEK-293T. N interage com as proteínas celulares Hsp70, PRMT5 e WDR77; P com Hsp70 e Tropomiosina; e M com Nucleofosmina e Tropomiosina. Cada gene celular foi co-expresso em bactérias com um gene viral possibilitando a co-precipitação das proteínas. Analisamos a interação entre Hsp70 e N ou P, confirmando sua ocorrência em bactérias. Com um conjunto de proteínas mutantes, definimos que as interações são através dos amino terminais de N e P, ou do carboxi terminal de P, e do domínio amino terminal de Hsp70. Superexpressão de Hsp70 por transfecção provocou efeito de estímulo sobre a replicação de HRSV. Imunizações em camundongos com vacinas de DNA para N e P mostraram a indução de resposta celular e humoral. Ensaios de desafio resultaram em redução da carga viral após imunização com N, indicando potencial para sua aplicação em formulação vacinal. / Human respiratory syncytial virus (HRSV) is one of the most important pathogens of the respiratory tract. We analyzed the interactions of viral nucleoprotein (N), phosphoprotein (P) and matrix (M) in HEK-293T. N interacts with the cellular proteins Hsp70, PRMT5 and WDR77; P interacts with Hsp70 and Tropomyosin; and M with Nucleophosmin and Tropomyosin. Each cellular gene was co-expressed with a viral gene in bacteria allowing co-precipitation of proteins. Hsp70 co-expression with N or P proteins confirmed that these interactions also occur in bacteria. Using a set of mutants we found that the N and P amino terminus, P carboxy terminus, and Hsp70 amino terminal domain participate in the interactions. The overexpression of Hsp70 by transfection resulted in stimulation of HRSV replication. Mice immunization with N and P showed that DNA vaccines were capable of inducing humoral and cellular response. In challenge assays it was possible to detect significant virus titer reduction in animals immunized with N, indicating its potential for a vaccine formulation.

A rainbow trout (Slamo gairdneri Richardson) population of IPN virus carriers was studied over a one-year period using both homogenisatian and co-cultivation for virus isolation. The percentage of virus-yielding fish was high between March and June but then declined. This was diametrically opposite to the trend in the serum antibody levels indicating that the marked humoral immune response resulted in a very significant reduction in the virus titres. The highest isolation rate was obtained from the kidneys after co-cultivation (from seventeen of the twenty-three virus-positive fish) underlining the very high sensitivity of this recently developed method for virus detection. Twelve of the twenty-three virus-positive fish yielded virus from the pyloric caeca after homogenisation. Virus was occasionally isolated from the faeces indicating that this may well be a possible avenue for horizontal transmission of the virus. No virus was ever detected in gonadal tissue. The virulence of the rainbow trout virus was enhanced in various ways and used to infect tilapia Oreochromis spilurus Gunther of different ages through a variety of routes. Fry infected by direct immersion, orally and by force feeding showed little or no signs of infection. Intraperitoneally and intramuscularly injected fingerling and adult fish developed marked haemorrhaging, severe loss of skin mucus and up to 50% mortalities were recorded. Gross pathology included enlarged and liquifying liver, gastroenteritis and mild brain haemorrhaging. Histopathologically there was extensive cellular vacuolation and degeneration as well as marked leucocytic infiltration in the liver, intestine and swimbladder. Eosinophilic granule cell infiltration of the intestinal wall was also very prominent. Virus was recovered from several organs and determination of virus titres revealed that active viral replication had occurred in the tilapia tissues, a finding further supported by electron microscopical evidence. The fish showed a clear humoral antibody response.

Globally, it is estimated that 240 million people are infected with chronic viral hepatitis B and in excess of 185 million people with chronic hepatitis C. The burden of disease from hepatitis is concentrated in developing countries where transmission of HBV occurs predominantly from mother to child (vertical transmission) and transmission of HCV through unsafe medical procedures and the transfusion of unscreened blood products. Global patterns of migration favour the movement of individuals from countries with medium or high risk prevalence of chronic viral hepatitis to countries with traditionally low prevalence among their indigenous populations, including the United Kingdom (UK). In excess of 3.2% of the global population are international migrants, posing important implications for healthcare systems in host nations. It is predicted that up to 7 million first and second generation immigrants, originating from high prevalence countries for viral hepatitis now reside permanently in the UK. However, as a result of deficiencies in screening initiatives, the prevalence and associated burden of these diseases in these high-risk populations residing in the UK is yet to be determined. In order to establish the feasibility of inviting first and second generation immigrant populations to participate in viral hepatitis testing in primary care, as well to determine the prevalence and demography of viral hepatitis in four areas of the UK, a randomised controlled cross sectional cluster trial was conducted. In HepFree clinical computer systems in general practice surgeries were interrogated to identify the target population that was then approached using a variety of different invitations to determine the most appropriate method for engaging this population. The outcomes of viral hepatitis testing from practices in one area of the UK are described in this thesis. Despite multiple challenges encountered both in engaging practices and individuals in trial participation, results of this investigation suggest that if it is found to be cost effective, then viral hepatitis screening is feasible and the burden of disease in the UK is concentrated in first generation immigrants.

Ebolavirus (EBOV) is a negative sense, single stranded RNA virus that causes Ebola hemorrhagic fever. This disease causes substantial morbidity and mortality in humans, with death occurring in 50-90% of cases. Despite years of intensive research, much of the molecular mechanism underlying the entry of EBOV remains unknown. We performed a bioinformatics screen to identify novel entry cofactors by correlating mRNA expression in a panel of human cancer cell lines with permissivity to the EBOV entry glycoprotein. This assay identified several known EBOV entry cofactors such as actin and the tyrosine kinase Axl. In addition, several genes involved in macropinocytosis and endosomal maturation were also correlated with EBOV permissivity. Subsequent evaluation of plasma membrane proteins correlated by this screen showed T-cell immunoglobulin and mucin domain-1 (TIM-1) mRNA expression correlated extremely well with EBOV pseudovirion transduction. Depletion of TIM-1 from highly-permissive cells inhibits EBOV pseudovirion transduction. Conversely, expression of TIM-1 in poorly-permissive cells significantly and specifically enhances EBOV pseudovirion transduction and infection. TIM-1 binds to EBOV GP and this binding is important in the initial interaction between the virus and the host cell. ARD5, a TIM-1 mAb, significantly inhibits EBOV GP-mediated entry into several cell lines and primary human airway epithelia in a dose and time-dependent manner. Therefore, TIM-1 is the first receptor identified for EBOV. Additionally, AMP-activated protein kinase (AMPK) mRNA correlated strongly with EBOV pseudovirion transduction. Compound C, a specific AMPK inhibitor, inhibited EBOV pseudovirion transduction and infection in a time and dose-dependent manner into several cell lines and primary human monocyte derived macrophages. Mouse embryonic fibroblasts (MEFs) lacking functional AMPK were significantly less permissive to EBOV GP-mediated infection that WT MEFs. Visualization of virus entry into these cells revealed that EBOV causes actin polymerization independently of AMPK, but AMPK-/- cells do not form lamellipodia in the presence of EBOV and, consequently, cannot internalize virus into cells by macropinocytosis.