Infections with dengue viruses (DENV) constitute both a global problem as well as locally in Florida. DENV comprise four distinct serotypes of single-stranded RNA viruses and belong to the family Flaviviridae. DENV are among the most medically important arboviruses in the world and cases may currently exceed 400 million per annum. Additionally, dengue established its first recorded endemic transmission cycle in the state of Florida in over a half century, first within the Florida Keys during 2009-10 followed by an unrelated outbreak in Martin County in 2013. The clinical profile of DENV infections ranges from a mild febrile illness to severe illness including hemorrhaging and/or shock, occasionally leading to death. Asymptomatic and mild cases also play a role in maintaining transmission cycles. The early diagnosis and management of patients at the clinical level have both proven to be major obstacles in the control of DENV and are important at both the individual and community levels. Individually, the proper management of patients that will progress to severe illness demands that they are identified in order to receive supportive treatment and mitigate associated morbidity and mortality. At the community level, early diagnosis may reduce transmission by limiting the possibility of vector contact with viremic individuals. Early diagnosis is dependent on the detection of viral markers, while a number of host factors may inform prognosis. The microsphere-based immunoassay (MIA) is capable of detecting up to 100 different targets in a single sample and therefore would be useful as a single assay for determining both. This study attempted to develop a diagnostic and prognostic MIA using the DENV NS1 glycoprotein and 5 host markers as targets. For the purposes of DENV NS1 detection in MIA, a set of monoclonal antibodies (mAbs) were subjected to immunoprecipitation and/or Western blot analysis in order to determine immunoreactivity. Two mAbs, 3A5.4 and 3D1.4, were chosen for use in MIA as a capture antibody and a detection antibody, respectively, and the results compared to a commercially available DENV NS1 ELISA. The 5 markers chosen for MIA trials included GM-CSF, IFN-γ, IP-10, IL-10, and MCP-1 and were selected from a panel of 27 markers screened initially in two in vitro models of DENV infection in addition to serum samples. The two cell lines investigated were HPMEC ST1.6R and u937 as both are thought to play important roles in models of DENV pathogenesis. The results of the DENV NS1 detection MIA were initially promising but were ultimately unsuccessful. When measuring host markers in the MIA, results pointed towards certain profiles that may be of future use. IL-10 was found to be elevated in a statistically significant manner in DENV qRT-PCR+ samples (p=0.035) when compared to negative sera. MCP-1 elevation was found to be of borderline significance (p=0.058). Other potential markers based on the results reported here include IP-10, IL-6, IL-8, VEGF, and RANTES. The ultimate goal of measuring host markers is to gain the ability to differentiate patients that will progress to severe illness from those that will recover. In conclusion, despite the failure of the MIA to detect DENV NS1 in human sera, our results in determining host markers and developments leading to successful DENV NS1 detection ELISAs elsewhere lead us to believe that this approach remains promising. Major drawbacks of this study included the lack of samples from patients that experienced severe DENV illness as a comparative group in addition to small sample sizes. Future goals should include determining the reasons for the failure of the MIA in detecting DENV NS1, selecting a panel of appropriate markers to differentiate non-severe from severe cases of DENV prior to progression, and optimizing the inclusion of these markers to an appropriate number.

No description available.

African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

<p>Abstract</p><p>Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.</p>

Abstract Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.

African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication. SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production. The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.

The flavivirus West Nile virus (WNV) encodes a single polyprotein that is processed into three structural and seven nonstructural proteins. Various WNV bicistronic replicons that direct cap-dependent translation of an N-terminal viral capsid or capsid/Renilla luciferase fusion protein as well as IRES-dependent translation of the nonstructural proteins were constructed. An original replicon consisting of the WNV 5' NCR, the 5' 198 nts of the capsid coding sequence, which included the 5' cyclization sequence (Cyc), and an EMCV IRES followed by the WNV nonstructural genes and 3' NCR was generated. Real time qRT-PCR analysis of intracellular levels of this replicon RNA showed a 4 fold increase by 96 hr after transfection of BHK cells. Increasing the distance between the 5' Cyc and IRES by insertion of a 5' IRES flanking sequence alone or together with a Renilla luciferase reporter did not increase RNA replication. Addition of only a reporter decreased RNA replication. The insertion of an extended capsid coding sequence also did not enhance RNA replication, but did enhance both cap- and IRES-dependent translation of replicon RNA, as indicated by immunofluorescence and Western blot analysis. These results suggest the presence of a translation enhancer in the 3' portion of the capsid coding region. Simian hemorrhagic fever virus (SHFV) is a member of the family Arteriviridae, order Nidovirales. SHFV is unique among Nidoviruses in having three instead of two papain-like cysteine protease (PCP) motifs designated alpha, beta, and gamma, within the N-terminal region of its ORF1a. Mutations of putative PCP cleavage sites showed that the most efficient cleavage was by PCP beta at its downstream cleavage site. A large deletion located between the two catalytic residues of PCP alpha was hypothesized to render this protease inactive. However, processing was observed at the cleavage site following PCP alpha. Mutational analyses confirmed that PCP alpha is an inactive protease, and that the cleavage sites downstream of PCP alpha are cleaved by PCP gamma. When the catalytic residues of PCP gamma were mutated, PCP beta was also able to back cleave at these sites. This "back" cleavage is a previously unreported activity for an arterivirus PCP.

Puumala hantavirus (PUUV) causes hemorrhagic fever with renal syndrome in Europe. After inhalation of virus shed by bank voles, the virus systemically targets the vascular endothelium leading to vascular dysfunction and leakage. Many patients with PUUV infection experience cardiopulmonary manifestations but the underlying mechanisms have not been determined. The aims of the studies presented were to describe cardiopulmonary manifestations, investigate pathogenetic mechanisms including presence of virus in the lungs and the local immune response in PUUV infection. The results showed cardiopulmonary involvement of varying severity in almost all studied patients. High-resolution computed tomography frequently revealed vascular leakage into the lungs or pleural cavities. Pulmonary function tests generally showed reduced gas diffusing capacity, evidenced in patients as dyspnea, poor oxygenation and frequent need of oxygen treatment. Among patients who were not fully recovered at 3 months follow-up, remaining decreased gas diffusing capacity was highly common. Echocardiography revealed mainly right heart dysfunction which was related to manifestations within the lungs, in terms of increased estimated pulmonary vascular resistance, mild to moderate pulmonary hypertension, and reduced right ventricular systolic function in patients with more pronounced lung involvement, as indicated by need of oxygen treatment. Analyses on bronchoalveolar lavage (BAL) and bronchial biopsies revealed a highly activated cytotoxic T cell (CTL) response in the lungs. The CTL response was not balanced by the expansion of regulatory T cells and high numbers of CTLs were associated with more severe disease. PUUV RNA was detected in almost all patients’ BAL samples and the viral load was inversely correlated to the number of CTLs. Three patients presenting with severe and fatal cardiopulmonary distress were also described. Autopsies revealed PUUV protein in vascular endothelium in all investigated organs, including the heart and lungs, along with a massive CTL response mainly in the lungs. In conclusion, cardiopulmonary involvement of varying severity was present in almost all patients with PUUV infection. Cytotoxic immune responses could contribute to disease development but also help in clearing the infection. Long lasting fatigue after hantavirus infection may be explained by remaining manifestations within the lungs.

A dengue à transmitida por mosquitos hematÃfagos do gÃnero Aedes das espÃcies aegypti e albopictus. O vÃrus dengue (DENV) pertence à famÃlia Flaviviridae do gÃnero FlavivÃrus e possui quatro sorotipos que foram designados como: DENV-1, DENV-2, DENV-3 e DENV-4. A doenÃa pode manifestar-se como uma enfermidade infecciosa aguda, caracterizada por um amplo espectro clÃnico que varia desde formas de infecÃÃo assintomÃtica ou febre indiferenciada atà as formas graves, com hemorragia e/ou choque. Este estudo apresentou como objetivo descrever os aspectos epidemiolÃgicos, clÃnicos e laboratoriais de pacientes com suspeita de dengue atendidos no Hospital SÃo Josà de DoenÃas Infecciosas (HSJ) e no Hospital Nossa Senhora da ConceiÃÃo (HDNSC) no perÃodo de fevereiro a dezembro de 2010. Dessa forma, foram recrutados 93 pacientes, sendo que, 86 preencheram os critÃrios de inclusÃo. Os pacientes foram recrutados por busca ativa nas emergÃncias e nas enfermarias dos referidos hospitais e submetidos a um protocolo de acompanhamento por meio de uma ficha de avaliaÃÃo clÃnica inicial (1 ao 5Âdia de doenÃa) e de uma ficha de avaliaÃÃo subsequente (6 ao 7 dia de doenÃa). Foram realizadas pelo menos duas mensuraÃÃes de hematÃcrito, plaquetas, alÃm de exames bioquÃmicos e exames especÃficos para dengue. Os sinais e os sintomas mais prevalentes nos pacientes com suspeita de dengue foram: febre, cefaleia e mialgia. Vinte e cinco pacientes (29%) apresentavam manifestaÃÃes hemorrÃgicas espontÃneas, sendo que, as hemorragias cutÃneas (petÃquias e equimoses) foram as mais encontradas (15%). Quando avaliada a populaÃÃo feminina em idade reprodutiva, 6% apresentaram metrorragia. A prova do laÃo foi realizada em 80 pacientes, sendo positiva em 20 pacientes (25%). Dos 86 pacientes, 48 (55,8%) foram positivos para dengue por pelo menos uma das tÃcnicas: imunocromatografia NS1 (16%), RT-PCR (19%), ELISA IgM (44%), imunocromatografia IgM (42%) e ELISA NS1 (27%). O vÃrus dengue foi detectado em 16 pacientes, sendo, DENV-1 em 1 paciente (6,2%), DENV-2 em 14 pacientes (87,5%) e DENV-3 em 1 paciente (6,2%). Vinte e seis pacientes (54,1%) preencheram os critÃrios do MinistÃrio da SaÃde (MS) de Dengue ClÃssica (DC), 10 (20,8%) de Febre HemorrÃgica de Dengue (FHD) e 12 (25%) de Dengue com ComplicaÃÃo (DCC). A relaÃÃo entre sexo feminino e masculino foi de aproximadamente 1,1/1, com predomÃnio maior de adultos jovens. Quanto ao critÃrio de gravidade do MS, 60% dos casos suspeitos de dengue foram classificados como grau II e nenhum caso como grau IV. Dos critÃrios de extravasamento plasmÃtico preconizado pelo MS, a hipoalbuminemia esteve presente em 5 pacientes (10,4%). A queda do hematÃcrito acima de 20% apÃs hidrataÃÃo foi observada em apenas 4 pacientes (8,3%). No perÃodo do estudo, foram diagnosticados dois pacientes com dengue e leptospirose, sendo que um paciente complicou com pÃrpura trombocitopÃnica idiopÃtica, esses pacientes tiveram evoluÃÃo benigna. Portanto salientamos a necessidade de um diagnÃstico precoce, antes do desenvolvimento das manifestaÃÃes graves, de polÃticas de erradicaÃÃo do Aedes e da estruturaÃÃo de um serviÃo de referÃncia. / Dengue is transmitted by blood-sucking mosquitoes of the species of the genus Aedes aegypti and albopictus. Dengue virus (DENV) belongs to the genus Flavivirus of the Flaviviridae family and has four serotypes that were designated as: DENV-1, DENV-2, DENV-3 and DENV-4. The disease can manifest as an acute infectious disease characterized by a wide clinical spectrum ranging from asymptomatic forms of infection or undifferentiated fever to severe forms, with bleeding and / or shock. This study had as objective to describe the epidemiological, clinical and laboratory features of patients with suspected dengue fever treated at the St Joseph Hospital of Infectious Diseases (HSJ) and the Hospital Nossa Senhora da ConceiÃÃo (HDNSC) in the period from February to December 2010. Thus, we recruited 93 patients, 86 met the inclusion criteria. Patients were recruited by an active search in emergencies and in the ward of these hospitals and underwent a follow-up protocol through an initial clinical evaluation form (from day 1 to day 5 of illness) and an evaluation form following (from the 6 to 7 day of illness). Were performed at least two measurements of hematocrit, platelets, and biochemical tests and specific tests for dengue. The signs and symptoms more prevalent in patients with suspected dengue were fever, headache and myalgia. Twenty-five patients (29%) had spontaneous bleeding manifestations and the cutaneous bleeding (petechiae and ecchymosis) were the most frequent (15%). When evaluating the female population of reproductive age, 6% had metrorrhagia. The tourniquet test was performed in 80 patients and was positive in 20 patients. Of 86 patients, 48 (55,8%) were positive for dengue at least one of the techniques: immunochromatography NS1 (16%), RT-PCR (19%), IgM ELISA (44%), IgM immunochromatography (42%) and NS1 ELISA (27%). The dengue virus was detected in 16 patient and, DENV-1 in 1 patient (6.2%), DENV-2 in 14 patients (87.5%) and DENV-3 in 1 patient (6.2%). Twenty- six patients (54,1%) met the criteria of the Ministry of Health (MOH) Classic Dengue (DC), 10 (20,8%) of Dengue Hemorrhagic Fever (DHF) and 12 (25%) of Dengue with complication (DCC). The relationship bet ween females and males were approximately 1.1/1, with higher prevalence in young adults The criterion for severity of MOH, 60% of suspected dengue cases were classified as grade II and none as grade IV. Plasma extravasation of criteria recommended by MOH, hypoalbuminemia was present in 5 patients (10,4%). The drop in hematocrit higher than 20% after hydration was observed in only 4 patients (8,3%). During the study period, two patients were diagnosed with dengue and leptospirosis, and one patient complicated with idiopathic thrombocytopenic purpura, these patients had a benign outcome. Therefore we stress the need for early diagnosis, before the development of severe manifestations, of policies to eradicate Aedes and structure a reference service.

The great variation displayed by nucleic acid molecules in human cells, and the continuous discovery of their impact on life, consequently require continuous refinements of molecular analysis techniques. Padlock probes and rolling circle amplification offer single nucleotide discrimination in situ, a high signal-to-noise ratio and localized detection within cells and tissues. In this thesis, in situ detection of nucleic acids with padlock probes and rolling circle amplification was applied for detection of DNA in the single cell gel electrophoresis assay to detect nuclear and mitochondrial DNA. This assay is used to measure DNA damage and repair.  The behaviour of mitochondrial DNA in the single cell gel electrophoresis assay has earlier been controversial, but it was shown herein that mitochondrial DNA diffuses away early in the assay. In contrast, Alu repeats remain associated with the nuclear matrix throughout the procedure. A new twelve gel approach was also developed with increased throughput of the single cell gel electrophoresis assay. DNA repair of three genes OGG1, XPD and HPRT and of Alu repeats after H2O2 induced damage was further monitored. All three genes and Alu repeats were repaired faster than total DNA. Finally, padlock probes and rolling circle amplification were applied for detection of the single stranded RNA virus Crimean Congo hemorrhagic fever virus. The virus was detected by first reverse transcribing RNA into cDNA.. The virus RNA together with its complementary RNA and the nucleocapsid protein were detected in cultured cells. The work presented here enables studies of gene specific damage and repair as well as viral infections in situ. Detection by ligation offers high specificity and makes it possible to discriminate even between closely related molecules. Therefore, these techniques will be useful for a wide range of applications within research and diagnostics.