O conhecimento sobre hantavirus patogênicos no Brasil até o momento indica que roedores silvestres são os reservatórios naturais. Estes roedores podem eliminar grandes quantidades do vírus através das fezes, urina e saliva (Khaiboullina et al., 2005). A infecção humana pode ocorrer por contato com roedores ou inalação dos aerossóis que contêm o vírus. A síndrome cardiopulmonar por hantavirus (HCPS) tem sido relatada em humanos em muitas regiões do Brasil. Tendo em vista a quantidade de casos de doenças emergentes e re-emergentes nas últimas décadas elegemos algumas regiões para um estudo detalhado sobre a distribuição desses reservatórios virais e consequentemente a caracterização da variante circulante. Com objetivo de adquirir um diagnóstico rápido e sensível, desenvolvemos primers específicos que pela técnica de Real- time RT- PCR é capaz de detectar o genoma viral em apenas uma hora em amostras de pulmões, rins e urina de roedores. Ao todo, nosso trabalho analisou 153 amostras sendo: 126 de roedores silvestres e domésticos, (de cinco diferentes regiões do Estado de São Paulo onde: 10 amostras colhidas no município de Jacupiranga, 61 oriundas de Teodoro Sampaio, 7 de Salesópolis, 48 provenientes de Biritiba Mirim, 16 de Nazaré Paulista) e mais 27 soros de pacientes de diferentes localidades de Minas Gerais. Neste estudo foi possível detectar dez amostras positivas em Biritiba Mirim, quatro em Nazaré Paulista e em vinte e dois soros de pacientes com hantavirose. O monitoramento contínuo através de testes moleculares em animais silvestres é imprescindível para um melhor entendimento da circulação dos hantavirus nessas regiões. Acreditamos que este teste possa auxiliar na vigilância e também no diagnóstico dos hantavirus no Brasil. / Current knowledge of the pathogenic hantavirus indicates that wild rodents are its primary natural reservoir. These rodents can eliminate large amounts of the virus through feces, urine and saliva. Human infection can occur through contact with rodents or inhalation of aerosols containing of the virus. Hantavirus cardiopulmonary syndrome (HCPS) has been reported in humans in many regions of Brazil. In view the number of cases of emerging diseases and re- emerging in recent decades, some regions to were select a detailed study on the distribution of these viral reservoirs, and consequently viral characterization. We developed specific primers to detect the presence of viral genomes using Real- time RT- PCR (Reverse- Transcriptase Polymerase Chain Raction). Altogether, our study analyzed 153 samples: 126 of wild rodents and domestic, of five different regions of São Paulo. 10 samples collected in Jacupiranga municipality, 61 in Teodoro Sampaio, 7 Salesópolis, 48 samples in Biritiba Mirim, 16 of Nazaré Paulista and 27 samples of the serum from patients from different sites of Minas Gerais State. Their samples were tested for hantavirus using the described SYBR Green-based real-time RT-PCR protocol and the specific primers. The presence of hantavirus RNA was detected in 10 rodents from Biritiba Mirim, 4 in Nazaré Paulista and 22 in serum of patients with hantaviruse. The surveillance by molecular tests in wild animals is essential to understood of hantavirus circulation in these regions. This SYBR Green real-time RTPCR method for detection of hantavirus may be useful for surveying hantaviruses in Brazil.

O Vírus da influenza, apesar de todas as epidemias e pandemias referirem-se a infecções em seres humanos, não está restrita a espécie humana e é capaz de causar debilidade ou mortalidade em várias outras espécies, incluindo cavalos, suínos, mamíferos marinhos e aves, entre outros. Estudos ecológicos das viroses de influenza conduziram a hipótese que todas as que acometem mamíferos derivam de reservatórios destes vírus em aves. Mesmo com programas de monitoramento contínuo de aves silvestres em alguns países do mundo que possuem casos originados pelos vírus aviário H5N1, pouco foi feito na Antártica e por isso, o presente trabalho foi realizado nas estações de verão antártico de 2006, 2007 e 2008 em duas localidades no território Antártico, a Península Keller, localizada na Ilha Rei George e na ilha Elefante 61°08S, 55°07W, a primeira onde está situada a Estação Antártica Comandante Ferraz-EACF e a segunda onde está localizada uma base de apoio a estudos avançados. Para este estudo foi realizada a coleta de 283 amostras de quatro diferentes espécies de pinguins: Pygoscelis adeliae; P. papua; P. antarctica; Aptenodytes patagonicus. Para o diagnóstico das amostras colhidas, foi aplicada a detecção direta dos produtos amplificados pelo método de RT-PCR em gel de agarose confirmados pelo método de Real-Time PCR (Applied Biosystems) e pelo RT-PCR-GeneScan no laboratório de Virologia Clínica e Molecular, do Departamento de Microbiologia, da Universidade de São Paulo. Os resultados obtidos em nosso estudo foram 8 amostras positivas em pinguins para o vírus Influenza A. As amostras positivas por RT-PCR foram encaminhadas para o laboratório de Influenza do Department of Infectious Diseases, St. Jude Children\'s Research Hospital, Memphis, TN, USA, para isolamento em ovos embrionados, não havendo crescimento de vírus da influenza A. Quatro destas amostras positivas puderam ser sequenciadas e comparadas com sequências de Influenza A depositadas no Genbank apresentando uma identidade de 96,8 % a 100 % entre elas e o controle tendo este último uma identidade de 100% com as do banco de dados, confirmando a presença do vírus nestas aves. / Epidemics and pandemics of influenza usually refer to infections in human beings. The influenza virus is not, however, restricted to humans and can cause infirmity and death in other species including horses, swine, marine mammals, birds, and others. Ecological studies of viral infections have led to the hypothesis that the influenza viruses that attack mammals have their origin in the accumulation of these viruses in birds (avian flu). In some countries with influenza cases caused by the avian H5N1 virus, there was monitoring of wild birds but little had been done in Antarctica. The present work was therefore carried out during the Antarctic summer seasons of 2006, 2007, and 2008 in two Antarctic locations: The Commander Ferraz Antarctic Station, on the Keller Peninsula of King George Island, and at the Base of Advanced Studies located on Elephant Island (61°08S, 55°07W). Two hundred eighty-three (283) samples from four different penguin species Pygoscelis adeliae, Pygoscelis papua, Pygoscelis antarctica; and Aptenodytes patagonicus were collected for this study. Diagnoses of the samples were performed not only by application of direct detection and amplification according to the RT-PCR method in agar-gel, but also by Real-Time PCR (Applied Biosystems), and by RT-PCR gene scan at the Laboratory of Clinical and Molecular Virology of the Department of Microbiology of the University of Sao Paulo. Eight of the penguin samples tested positive for the Influenza-A virus. The positive samples, as determined by RT-PCR, were sent to the Influenza Laboratory of the Department of Infectious Diseases of the St. Jude Research Hospital in Memphis, Tennessee, USA, to be isolated in egg embryos where no further growth of the Influenza-A virus took place. Four of these positive samples could be sequenced and compared with those of Influenza-A on deposit at the Gene Bank and ranged from 96.85 to 100% when compared with the control samples (100% positive), thus confirming the presence of the virus in the tested birds.

O Epstein-Barr vírus (EBV) é a única espécie humana pertencente ao gênero Lymphocryptovirus. A transmissão ocorre através da saliva contaminada e geralmente ainda na infância. Nosso estudo analisou 165 amostras clínicas de pacientes, portadores de HIV, em tratamento com antiretrovirais, atendidos no Sistema Hospitalar do Sistema Penitenciário do Estado de São Paulo. Nosso enfoque foi pesquisar o EBV nas células mononucleares do sangue periférico, através das técnicas de PCR, Nested-PCR e seqüenciamento de nucleotídeos. Os resultados obtidos, indicaram que 11,51% (19) das amostras analisadas, apresentaram-se positivas para o EBV. Essas 19 amostras, foram seqüenciadas com primers específicos para a região da EBNA-1 (Epstein Barr Nuclear Antigen 1). As amostras foram alinhadas com o auxílio do DNASTAR. Ao alinharmos as amostras, encontramos uma troca de base (de G para A) em 7 amostras e essa troca não alterou a conformação da proteína EBNA-1. Na análise filogenética de nossas sequências com as depositadas no GenBank, foi possível observar dois grupos, que representam tipo 1 e o tipo 2 do EBV. 100% das amostras estudadas por nós foram identificadas como pertencentes ao grupo que caracteriza o tipo 2. Sendo assim, as 7 amostras que apresentaram a troca sugerem a origem um novo subtipo. / The Epstein-Barr Virus (EBV) is the only species to the genus Lymphocriptovirus that infects humans. One of the possible route for its transmission thought by contamined saliva and usually occurs in the childhood. This study analysed 165 clinical samples from HIV infected patients, treated by HARRT, attended in the Hospitalar System in the Penitentiary System from Sao Paulo State. The aim of this study was to search EBV in peripheral blood mononuclear cells by PCR, Nested-PCR and sequencing analysis. The results showed 11,51% of the analysed samples, positive for EBV. This samples, was sequenced with specifics primers from the EBNA-1 (Epstein Barr Nuclear Antigen 1) region. The samples were aligned by DNASTAR program. The aligned sequences showed the base conversion G to A in seven samples. This conversion caused no alteration in the EBNA-1 protein conformation. In the phylogenetic analysis the studied sequences with the sequences from GenBank was possible to observe two groups represented with type 1 and type 2 from EBV. 100% the samples studied was identified with the group characterized by the type 2 to EBV. So the seven samples showed the conversion, suggesting the origin of the one new subtype.

Tomato spotted wilt virus (TSWV), a Tospovirus, is one of the ten most economically destructive plant viruses worldwide, causing losses exceeding one billion U.S. dollars annually on several crops. In South Africa (SA), TSWV has become an important virus in many economically important crops. The main objective of this research project was to isolate, identify and characterise TSWV isolates occurring in SA. A review of current literature assembled background information on TSWV molecular biology, epidemiology, transmission, detection and control. A TSWV isolate infecting pepper (Capsicum sp.) occurring in KZN was isolated and partially characterised. The virus was positively identified as TSWV using the enzyme-linked immunosorbent assay (ELISA) and the presence of typical necrotic TSWV symptoms on Nicotinia rustica L. Symptomatic leaves were harvested and the virus was partially purified using standard procedures. Under the transmission electron microscope (TEM), typical quasi-spherical and dumbbell-shaped particles of 80-100nm in diameter were observed in negatively stained preparations of both crude and purified virus samples. In negatively stained ultra-thin virus infected leaf sections, an abundance of mature viral particles (100nm) housed in the cisternae of the endoplasmic reticulum (ER) were observed among typical viroplasm inclusions (30nm) and hollow tubules (200-300nm). A viral protein migrating as a 29kDa band, which corresponds to the TSWV nucleocapsid (N) protein, was observed after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Total plant RNA, isolated from N. rustica displaying typical symptoms was subjected to reverse-transcription polymerase chain reaction (RT-PCR) using .primers specific to the nucleocapsid (N) gene. An expected 760bp product was amplified. The results obtained in this study confirm the presence of TSWV in infected pepper plants from KZN. The genetic diversity of TSWV isolates occurring in SA was examined. The nucleocapsid (N) gene sequences of six SA TSWV isolates originating from Gauteng, KwaZulu-Natal, North West, Limpopo and Mpumulanga provinces were determined and used in a phylogenetic tree comparison with TSWV isolates occurring in different geographical locations in the world. Nucleotide sequence comparisons of the N gene revealed high levels of similarity between the SA isolates and TSWV isolates from Asia and Europe. SA isolates showed a high degree of sequence similarity (99-100%) which was reflected in their distinct clustering pattern. The resistance of tomato (Lycopersicon escuJentum Mill.) plants with natural and transgenic resistance against mechanical inoculation with TSWV isolates occurring in SA was evaluated. The Stevens cultivar which has natural resistance conferred by the Sw-5 gene and the transgenic 13-1 line, which expresses the nucleocapsid (N) protein gene of the TSWV-BL isolate, was used as test cultivars. Plants were assessed for TSWV resistance using a disease severity rating scale and measurements of virion accumulation levels (A405nm). There were no significant differences among the reactions produced by the six TSWV isolates on the test plants. Although both plants were susceptible to the SA TSWV isolates by exhibiting similarly high viral accumulation levels, the transgenic tomato line showed milder disease severity compared to the natural resistant cultivar. Results suggest that transgenic resistance is a more effective approach in the control of TSWV in SA. The information generated in this study will be useful in formulating effective control measures using genetic engineering approaches for this economically important virus. Such approaches will be used as a tool to make strategic decisions in an integrated control programme for ISWV. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

African horse sickness (AHS) is a viral disease with high mortality rates, vectored by the Culicoides midge and affecting members of the Equidae family. AHS is endemic to South Africa, and, as a result, affects export and international competitiveness in equine trade, and impacts significantly on the South African racehorse and performance horse industries. AHS also has devastating consequences for rural and subsistence equine ownership. The protocol developed in this dissertation has the potential to serotype and confirm the AHS virus within a few hours at significantly less cost than current methods. It will ease the financial and time constraints of studying an outbreak in real time and has the potential to solve many of the unknown factors surrounding AHS, particularly and most importantly, the role that each serotype plays in outbreaks and the form of the disease contracted by horses. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Human cytomegalovirus (HCMV) is a widespread human herpesvirus pathogen and prototypical member of the β-herpesvirus subfamily. Like all herpesviruses, the virus establishes a lifelong latent infection following host exposure, which has the potential to reactivate periodically and contribute to recurrent disease processes. In individuals with weak or compromised immune systems, such reactivation can lead to profound pathology. Understanding how latent infections are maintained is important for uncovering how HCMV causes disease. The study of viral genes that are expressed during latent infection grants insight into how latency is regulated and how it could be therapeutically targeted. To that end, this project has sought to evaluate the functional significance of one such viral gene termed LUNA in the context of latency. In models of experimental latent infection based on primary myeloid cells, levels of viral gene transcription were found to be significantly reduced following infection with LUNA deletion mutant viruses, consistent with corresponding observable changes in post-translational histone modifications over the viral promoters of latency-associated genes. Additionally, using luciferase reporter systems, latency-associated viral gene promoters became activated in response to the expression of wild-type LUNA. Together, these findings argue for a role of LUNA in regulating viral gene expression during latent HCMV infection. One possible mechanism by which LUNA may fulfil its role is by targeting cellular ND10 structures, known intrinsic inhibitors of herpesvirus gene expression, for disruption. In support of this, latently infected cells were found to be devoid of ND10, a phenotype that was recapitulated by the direct expression of wild-type LUNA. Furthermore, mutation studies confirmed the identification of a novel deSUMOylase activity encoded by LUNA that was responsible for mediating ND10 disruption. Use of a catalytically inactive LUNA mutant in transcriptional analyses of latent infection also generated similar results as with the LUNA deletion viruses. Overall, these data support the hypothesis that LUNA serves as an important regulator of viral gene expression during latency, which is likely linked to its ability to target ND10 structures for disruption, thus raising the possibility that inhibition of deSUMOylation may serve as a novel therapeutic strategy to target latent HCMV infection.

Potato virus Y (PVY) is an economically important virus worldwide. In South Africa, PVY has been shown to be a major limiting factor in the production of important solanaceous crops, including potato (Solanum tuberosum L.), pepper (Capsicum annuum L.), tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana spp). The variability that PVY displays, wherever the virus occurs, merits the study of the isolates occurring in KwaZulu-Natal (KZN) in the Republic of South Africa (RSA). This characterization will provide a clear understanding of strains/isolates from local vegetables and how they relate to the other PVY strains already identified, as well as information that can be used to manage the diseases they cause. Hence, the aim of this project was to study the biological and genetic properties of PVY isolates infecting potato, tomato and pepper in KZN. Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies and reverse transcription polymerase chain reaction (RT-PCR) using primers specific to all PVY strains were used to detect the virus in plant material showing PVY-like symptoms collected from various locations in KZN. A total of 39 isolates (18 isolates infecting tomato, 12 infecting potato and 9 infecting pepper) were further differentiated into strains by means of ELISA using strain specific antibodies and RT-PCR using primers specific to the different strains of PVY identified around the world. All PVY isolates infecting tomato and pepper tested positive for the ordinary PVYO strain with both ELISA and RT-PCR. PVY isolates infecting potato were more diverse and comprised the PVYN, PVYNTN and PVYNWilga strains, with mixed infections noted in some cases. The biological properties were studied by mechanically inoculating Chenopodium quinoa, Nicotiana tabacum cv Xanthi, N. tabacum cv Samsun, N. glutinosa, and N. rustica with leaf extracts from plants infected with the different PVY strains detected in this study. All inoculated C. quinoa plants did not show symptoms. All tobacco plants showing symptoms were tested for the presence of PVY by means of ELISA using monoclonal antibodies targeting all strains and electron microscopy using the leaf dip technique. Not all the inoculated tobacco tested positive with ELISA. The symptoms observed were therefore divided into PVY-related and PVY non- related. PVY-related symptoms included vein clearing, mosaic chlorosis, stunting, and vein necrosis. PVY non-related symptoms included wrinkles and leaf distortions. Potyvirus-like particles of about 700 nm were observed under the transmission electron microscope (TEM) from plants showing PVY-related symptoms while rod shaped viral particles of sizes varying between 70 and 400 nm were observed from plants showing non-PVY related symptoms. A portion of the virus genome (1067 bp) covering part of the coat protein gene and the 3’ non-translated region (NTR) of three PVYO isolates infecting tomato, one PVYO isolate infecting pepper and one PVYNWilga isolate infecting potato were amplified, cloned and sequenced. The 5’ NTR, P1, HC-Pro and part of P3 regions (2559 bp) of a PVYN isolate infecting potato were also amplified, cloned and sequenced. Sequence data was compared with selected PVY sequences from different geographical locations around the world. These were available on the NCBI website and subsequently used for phylogenic analyses. The sequenced genomic regions of the PVYN isolate were found to be 99% similar to the New Zealand PVYN isolate (GenBank accession number: AM268435), the Swiss PVYN isolate CH605 (X97895) and the American PVYN isolate Mont (AY884983). Moreover, the deduced amino acid sequence comparison of the genomic regions of the PVYN isolate revealed the presence of five distinct amino acids residues. The three amino acid residues (D205, K400, and E419), which determine the vein necrosis phenotype in tobacco, were also identified. The coat protein and 3’ NTR sequences of all KZN PVYO isolates infecting pepper and tomato were closely similar to each other than to KZN PVYNWilga isolate infecting potato. The phylogenic analysis clustered the KZN PVYN isolate with the European sublineage N, PVYNWilga isolate infecting potato with the American PVYO isolate Oz (EF026074) in the O lineage and all PVYO isolates infecting tomato and pepper in a new sublineage within the O lineage. Taken together, these results point to the presence of PVY in solanaceous vegetables cultivated in KZN and they lay the foundation for the formulation of effective control measure against PVY diseases in KZN. / Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2009.

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VV) is the prototypical member of the orthopoxvirus genus of the Poxviridae family, and is currently being evaluated as a vector for vaccine development and cancer cell-targeting therapy. Despite the importance of studying poxvirus effects on the human immune system, reports of the direct interactions between poxviruses and primary human leukocytes (PHLs) are limited. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias towards binding and infecting monocytes among PHLs. VV binding strongly co-localized with lipid rafts on the surface of these cell types, even when lipid rafts were relocated to the cell uropods upon cell polarization. In humans, monocytic and professional antigen-presenting cells (APCs) have so far only been reported to exhibit abortive infections with VV. We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, were permissive to VV replication. The majority of virions produced in MDMs were extracellular enveloped virions (EEV). Visualization of infected MDMs revealed the formation of VV factories, actin tails, virion-associated branching structures and cell linkages, indicating that infected MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. Classical activation of MDMs by LPS plus IFN-γ stimulation caused no effect on VV replication, whereas alternative activation of MDMs by IL-10 or LPS plus IL-1β treatment significantly decreased VV production. The IL-10-mediated suppression of VV replication was largely due to STAT3 activation, as a STAT3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data indicate that PHL subsets express and share VV protein receptors enriched in lipid rafts. We also demonstrate that primary human macrophages are permissive to VV replication. After infection, MDMs produced EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread.