Cytokine and nitric oxide production by mouse macrophages infected with brazilian flaviviruses

The Flaviviridae family, Flavivirus genus includes viruses that are transmitted to vertebrates by infected mosquitoes or ticks. The genus Flavivirus includes a variety of viruses that cause diseases such as acute febrile illness, encephalitis, and hemorrhagic fever. Flaviviruses primarily infect blood monocytes and tissue macrophages, which have been shown to be permissive, supporting viral replication and serving as virus reservoirs. On the other hand, these cells may have an important antiviral activity related to modulation by cytokine production and by the capacity of these cells to synthesize reactive free radicals such as nitric oxide (NO) which can have a microbicidal effect. The present study was performed in order to determine the production of cytokines interleukin-1beta (IL-1β), tumor necrosis factor -alpha (TNF-α), transforming growth factor- beta (TGF-β) and interferon -alpha (IFN-α) and NO by macrophages infected with one of four Brazilian flaviviruses, Bussuquara virus (BUSV), Yellow Fever virus (YFV), Rocio virus (ROCV) and Encephalitis Saint Louis virus (SLEV), and to verify the possible antiviral effect of NO during macrophage infection with ROCV. Moreover, we asked if the different viruses were able to regulate bacterial lipopolysaccharide (LPS) induced cytokine production. Our results showed that YFV and SLEV reduced the production of IL-1β and TGF-β by LPS-stimulated macrophages, while ROCV only diminished LPS-stimulated TGF-β synthesis. On the other hand, BUSV more likely favored an enhancement of the LPS-induced production of IL-1β by macrophages. Additionally, while most of the viruses stimulated the production of IFN-α, none of them altered the production of TNF-α by murine macrophages. Interestingly, all viruses induced synthesis of NO that was not correlated with antiviral activity for ROCV.

A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes

Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. Besides genotype, quantitative analysis of HBV infection is extensively used for monitoring disease progression and treatment. Affordable viral load monitoring is desirable in resource-limited settings and it has been already shown to be useful in developing countries for other viruses such as Hepatitis C virus (HCV) and HIV. In this paper, we describe the validation of a real-time PCR assay for HBV DNA quantification with TaqMan chemistry and MGB probes. Primers and probes were designed using an alignment of sequences from all HBV genotypes in order to equally amplify all of them. The assay is internally controlled and was standardized with an international HBV panel. Its efficacy was evaluated comparing the results with two other methods: Versant HBV DNA Assay 3.0 (bDNA, Siemens, NY, USA) and another real-time PCR from a reference laboratory. Intra-assay and inter-assay reproducibilities were determined and the mean of CV values obtained were 0.12 and 0.09, respectively. The assay was validated with a broad dynamic range and is efficient for amplifying all HBV genotypes, providing a good option to quantify HBV DNA as a routine procedure, with a cheap and reliable protocol.

Pathogenicity of different rabies virus isolates and protection test in vaccinated mice

This study was aimed to evaluate and compare the pathogenicity of rabies virus isolated from bats and dogs, and to verify the efficacy of a commercial rabies vaccine against these isolates. For evaluation of pathogenicity, mice were inoculated by the intramuscular route (IM) with 500MICLD50/0.03mL of the viruses. The cross-protection test was performed by vaccinating groups of mice by the subcutaneous route and challenged through the intracerebral (IC) route. Isolates were fully pathogenic when inoculated by the IC route. When inoculated intramuscularly, the pathogenicity observed showed different death rates: 60.0% for the Desmodus rotundus isolate; 50.0% for dog and Nyctinomops laticaudatus isolates; 40.0% for Artibeus lituratus isolate; 9.5% Molossus molossus isolate; and 5.2% for the Eptesicus furinalis isolate. Mice receiving two doses of the vaccine and challenged by the IC route with the isolates were fully protected. Mice receiving only one dose of vaccine were partially protected against the dog isolate. The isolates from bats were pathogenic by the IC route in mice. However, when inoculated through the intramuscular route, the same isolates were found with different degrees of pathogenicity. The results of this work suggest that a commercial vaccine protects mice from infection with bat rabies virus isolates, in addition to a canine rabies virus isolate.

Identification of respiratory virus in infants with congenital heart disease by comparison of different methods

Respiratory virus infections are the main cause of infant hospitalization and are potentially severe in children with congenital heart disease (CHD). Rapid and sensitive diagnosis is very important to early introduction of antiviral treatment and implementation of precautions to control transmission, reducing the risk of nosocomial infections. In the present study we compare different techniques in the diagnosis of respiratory viruses in CHD infants. Thirty-nine samples of nasopharyngeal aspirate were obtained from CHD infants with symptoms of respiratory infection. The Multiplex PCR (Seeplex® RV 12 ACE Detection) driven to the detection of 12 respiratory viruses was compared with the direct immunofluorescence assay (DFA) and PCR, both targeting seven respiratory viruses. The positivity found by DFA, Multiplex and PCR was 33.3%, 51.3% and 48.7%, respectively. Kappa index comparing DFA and Multiplex, DFA and PCR and PCR and Multiplex PCR was 0.542, 0.483 and 0.539, respectively. The concordance between techniques was considered moderate. Both Multiplex PCR (p = 0.001) and PCR (p = 0.002) detected significantly more respiratory virus than DFA. As the performance of the tests may vary, the combination of two or more techniques may increase diagnostic sensitivity favoring the diagnosis of co-infections, early introduction of antiviral therapy and implementation of appropriate measures.

Acute respiratory viral infections in children in Rio de Janeiro and Teresópolis, Brazil

The frequency of viral pathogens causing respiratory infections in children in the cities of Rio de Janeiro and Teresópolis was investigated. Nasal swabs from children with acute respiratory illnesses were collected between March 2006 and October 2007. Specimens were tested for viral detection by conventional (RT)-PCR and/or real time PCR. Of the 205 nasal swabs tested, 64 (31.2%) were positive for at least one of the viral pathogens. Single infections were detected in 56 samples, 50 of those were caused by RNA viruses: 33 samples tested positive for rhinovirus, five for influenza A, five for metapneumovirus, four for coronavirus and, three for respiratory syncytial virus. For the DNA viruses, five samples were positive for bocavirus and one for adenovirus. Co-infections with these viruses were detected in eight samples. Our data demonstrate a high frequency of viral respiratory infections, emphasizing the need for a more accurate diagnosis particularly for the emerging respiratory viruses. The fact that the emerging respiratory viruses were present in 9.2% of the tested samples suggests that these viruses could be important respiratory pathogens in the country.

Strategies of the African swine fever virus to manipulate innate immunity

Dissertation presented to obtain the Ph.D degree in Biology

Genetic Control of Mosquitoes: population suppression strategies

Over the last two decades, morbidity and mortality from malaria and dengue fever among other pathogens are an increasing Public Health problem. The increase in the geographic distribution of vectors is accompanied by the emergence of viruses and diseases in new areas. There are insufficient specific therapeutic drugs available and there are no reliable vaccines for malaria or dengue, although some progress has been achieved, there is still a long way between its development and actual field use. Most mosquito control measures have failed to achieve their goals, mostly because of the mosquito's great reproductive capacity and genomic flexibility. Chemical control is increasingly restricted due to potential human toxicity, mortality in no target organisms, insecticide resistance, and other environmental impacts. Other strategies for mosquito control are desperately needed. The Sterile Insect Technique (SIT) is a species-specific and environmentally benign method for insect population suppression, it is based on mass rearing, radiation mediated sterilization, and release of a large number of male insects. Releasing of Insects carrying a dominant lethal gene (RIDL) offers a solution to many of the drawbacks of traditional SIT that have limited its application in mosquitoes while maintaining its environmentally friendly and species-specific utility. The self-limiting nature of sterile mosquitoes tends to make the issues related to field use of these somewhat less challenging than for self-spreading systems characteristic of population replacement strategies. They also are closer to field use, so might be appropriate to consider first. The prospect of genetic control methods against mosquito vectored human diseases is rapidly becoming a reality, many decisions will need to be made on a national, regional and international level regarding the biosafety, social, cultural and ethical aspects of the use and deployment of these vector control methods.

Low genetic diversity in Wolbachia-Infected Culex quinquefasciatus (Diptera: Culicidae) from Brazil and Argentina

Culex quinquefasciatus is a vector of human pathogens, including filarial nematodes and several viruses. Although its epidemiological relevance is known to vary across geographical regions, an understanding of its population genetic structure is still incipient. In light of this, we evaluated the genetic diversity of Cx. quinquefasciatus and Cx. pipiens x Cx. quinquefasciatus hybrids collected from nine localities in Brazil and one site in Argentina. We used mitochondrial genes cox1 and nd4, along with the coxA and wsp genes of the maternally-inherited Wolbachia endosymbiont. The nd4 fragment was invariant between samples, whilst cox1 exhibited four haplotypes that separated two types of Cx. quinquefasciatus, one clustered in southern Brazil. Low sequence diversity was generally observed, being discussed. Both Brazilian and Argentinian mosquitoes were infected with a single Wolbachia strain. As reported in previous studies with these populations, cox1 and nd4 diversity is not congruent with the population structure revealed by nuclear markers or alar morphology. Future Cx. quinquefasciatus research should, if possible, evaluate mtDNA diversity in light of other markers.

MOLECULAR CHARACTERIZATION OF INFLUENZA B VIRUS OUTBREAK ON A CRUISE SHIP IN BRAZIL 2012

In February 2012, an outbreak of respiratory illness occurred on the cruise ship MSC Armonia in Brazil. A 31-year-old female crew member was hospitalized with respiratory failure and subsequently died. To study the etiology of the respiratory illness, tissue taken at necropsy from the deceased woman and respiratory specimens from thirteen passengers and crew members with respiratory symptoms were analyzed. Influenza real-time RT-PCR assays were performed, and the full-length hemagglutinin (HA) gene of influenza-positive samples was sequenced. Influenza B virus was detected in samples from seven of the individuals, suggesting that it was the cause of this respiratory illness outbreak. The sequence analysis of the HA gene indicated that the virus was closely related to the B/Brisbane/60/2008-like virus, Victoria lineage, a virus contained in the 2011-12 influenza vaccine for the Southern Hemisphere. Since the recommended composition of the influenza vaccine for use during the 2013 season changed, an intensive surveillance of viruses circulating worldwide is crucial. Molecular analysis is an important tool to characterize the pathogen responsible for an outbreak such as this. In addition, laboratory disease surveillance contributes to the control measures for vaccine-preventable influenza.

ENTEROPATHOGENS DETECTED IN A DAYCARE CENTER, SOUTHEASTERN BRAZIL: BACTERIA, VIRUS, AND PARASITE RESEARCH

Introduction: The objective of this study was to determine the prevalence and etiological profile of enteropathogens in children from a daycare center. Methods: From October 2010 to February 2011 stool samples from 100 children enrolled in a government daycare center in the municipality of São José do Rio Preto, in the state of São Paulo, were collected and analyzed. Results: A total of 246 bacteria were isolated in 99% of the fecal samples; 129 were in the diarrheal group and 117 in the non-diarrheal group. Seventy-three strains of Escherichia coli were isolated, 19 of Enterobacter, one of Alcaligenes and one of Proteus. There were 14 cases of mixed colonization with Enterobacter and E. coli. Norovirus and Astrovirus were detected in children with clinical signs suggestive of diarrhea. These viruses were detected exclusively among children residing in urban areas. All fecal samples were negative for the presence of the rotavirus species A and C. The presence of Giardia lamblia, Entamoeba coli, Endolimax nana and hookworm was observed. A significant association was found between food consumption outside home and daycare center and the presence of intestinal parasites. Conclusions: For children of this daycare center, intestinal infection due to pathogens does not seem to have contributed to the occurrence of diarrhea or other intestinal symptoms. The observed differences may be due to the wide diversity of geographical, social and economic characteristics and the climate of Brazil, all of which have been reported as critical factors in the modulation of the frequency of different enteropathogens.

Genomic Signatures and Antiviral Drug Susceptibility Profile of A(H1N1)pdm09

Background: Genetic changes in influenza surface and internal genes can alter viral fitness and virulence. Mutation trend analysis and antiviral drug susceptibility profiling of A(H1N1)pdm09 viruses is essential for risk assessment of emergent strains and disease management. Objective: To profile genomic signatures and antiviral drug resistance of A(H1N1)pdm09 viruses and to discuss the potential role of mutated residues in human host adaptation and virulence. Study design: A(H1N1)pdm09 viruses circulating in Portugal during pandemic and post-pandemic periods and 2009/2010 season. Viruses were isolated in MDCK-SIAT1 cell culture and subjected to mutation analysis of surface and internal proteins, and to antiviral drug susceptibility profiling. Results: The A(H1N1)pdm09 strains circulating during the epidemic period in Portugal were resistant to amantadine. The majority of the strains were found to be susceptible to oseltamivir and zanamivir, with five outliers to neuraminidase inhibitors (NAIs) identified. Specific mutation patterns were detected within the functional domains of internal proteins PB2, PB1, PA, NP, NS1, M1 and NS2/NEP, which were common to all isolates and also some cluster-specific. Discussion: Modification of viral genome transcription, replication and apoptosis kinetics, changes in antigenicity and antiviral drug susceptibility are known determinants of virulence. We report several point mutations with putative roles in viral fitness and virulence, and discuss their potential to result in more virulent phenotypes. Monitoring of specific mutations and genetic patterns in influenza viral genes is essential for risk assessing emergent strains, disease epidemiology and public health implications.

SAINT LOUIS ENCEPHALITIS VIRUS IN MATO GROSSO, CENTRAL-WESTERN BRAZIL

The dengue virus (DENV), which is frequently involved in large epidemics, and the yellow fever virus (YFV), which is responsible for sporadic sylvatic outbreaks, are considered the most important flaviviruses circulating in Brazil. Because of that, laboratorial diagnosis of acute undifferentiated febrile illness during epidemic periods is frequently directed towards these viruses, which may eventually hinder the detection of other circulating flaviviruses, including the Saint Louis encephalitis virus (SLEV), which is widely dispersed across the Americas. The aim of this study was to conduct a molecular investigation of 11 flaviviruses using 604 serum samples obtained from patients during a large dengue fever outbreak in the state of Mato Grosso (MT) between 2011 and 2012. Simultaneously, 3,433 female Culex spp. collected with Nasci aspirators in the city of Cuiabá, MT, in 2013, and allocated to 409 pools containing 1-10 mosquitoes, were also tested by multiplex semi-nested reverse transcription PCR for the same flaviviruses. SLEV was detected in three patients co-infected with DENV-4 from the cities of Cuiabá and Várzea Grande. One of them was a triple co-infection with DENV-1. None of them mentioned recent travel or access to sylvatic/rural regions, indicating that transmission might have occurred within the metropolitan area. Regarding mosquito samples, one pool containing one Culex quinquefasciatus female was positive for SLEV, with a minimum infection rate (MIR) of 0.29 per 1000 specimens of this species. Phylogenetic analysis indicates both human and mosquito SLEV cluster, with isolates from genotype V-A obtained from animals in the Amazon region, in the state of Pará. This is the first report of SLEV molecular identification in MT.

Evolution of the Human Immunodeficiency Virus Type 2 Envelope in the First Years of Infection is Associated with the Dynamics of the Neutralizing Antibody Response

Background: Differently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4+ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth. Results: CD4+ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a β-hairpin structure were related with rate of escape from the neutralizing antibodies. Conclusion: Our data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the neutralizing antibody response providing further support for a model in which Nabs play an important role in HIV-2 pathogenesis.

Improving Influenza Surveillance in Portuguese Preschool Children by Parents' Report

Influenza surveillance is usually based on nationally organized sentinel networks of physicians and on hospital reports. This study aimed to test a different report system, based on parents' phone contact to the research team and in home collection of samples by a dedicated team. The identification of influenza and other respiratory viruses in children who attended a Hospital Emergency Department was also recorded. Real-time PCR and reverse transcription PCR were performed for influenza A and B, parainfluenza 1-4, adenovirus, human metapneumovirus, respiratory syncytial virus A and B, rhinovirus, enterovirus, group 1 coronaviruses, group 2 coronaviruses, and human bocavirus. One hundred children were included, 64 from the day care centers and 36 from the Hospital. Overall, 79 samples were positive for at least one respiratory virus. Influenza A (H3) was the virus most frequently detected: 25 cases, 20 of these in children under 5 years of age (ten from day care centers and ten who went to the hospital) which was higher than those reported by the National Influenza Surveillance Programme for this age. CONCLUSION: The results obtained in this study suggest that a surveillance system based on parents' reports could complement the implanted system of the National Influenza Surveillance Programme.

Pandemia influenza A/H1N1 2009 em Portugal : estratégias de intervenção e saúde pública

RESUMO - Introdução: A necessidade de gestão da ameaça de uma pandemia obriga à gestão da incerteza absoluta. O desconhecimento científico quanto a uma série de factores tais como, as características dos vírus causadores de infecções, a efectividade das medidas de prevenção e de tratamento, contribuiu para a dificuldade de actuação a vários níveis. Face à evolução da situação epidemiológica mundial no campo da gripe, Portugal reviu e adaptou o seu plano de contingência para a gripe tendo sido homologado, em Janeiro de 2006, um novo plano – Plano de Contingência Nacional do Sector da Saúde para a Pandemia. Objectivos: O presente estudo apresenta como principais objectivos (1) a avaliação das estratégias de intervenção e respectiva implementação das medidas de saúde pública não farmacológicas, durante a pandemia da gripe de 2009; (2) a identificação dos pontos críticos na gestão das medidas de saúde pública não farmacológicas, para preparação e resposta a futuras pandemias da gripe e, por último, (3) a definição de um modelo de acompanhamento, de monitorização e de avaliação das medidas de saúde pública não farmacológicas, que permita identificar os pontos importantes a ter em conta na implementação de boas práticas de saúde pública, nos diferentes níveis de prestação de cuidados de saúde, tendo como limitação o estado da arte dos sistemas de informação e avaliação, nacionais e internacionais, existentes. Material e Métodos: Para a avaliação das estratégias de intervenção e respectiva implementação das medidas de saúde pública não farmacológicas bem como, para a identificação dos pontos críticos na gestão das respectivas medidas foram consideradas duas perspectivas: a avaliação dos resultados através do Relatório da Pandemia da Gripe e a avaliação dos resultados através das avaliações da OMS e da Euro OMS. É apresentado um modelo de acompanhamento, de monitorização e de avaliação das medidas de saúde pública não farmacológicas, a implementar na classe médica que esteve na “linha da frente”, durante a pandemia H1N1 2009 em Portugal, nos cuidados primários, hospitalares e intensivos. Resultados: Deverá existir uma representação dos profissionais de saúde para suporte das decisões de forma a assegurar uma representação alargada não só na preparação mas também, na implementação e na aplicação do Plano de Contingência. O processo de planemanto deverá ter acesso público com possibilidade de participação activa do cidadão na sua implementação. O Plano deve ter a capacidade para se adaptar à situação epidemiológica e, no terreno os exercícios de simulações durante o processo de planeamento são importantes. As orientações técnicas devem ser dinâmicas, práticas, específicas e úteis e, dever-se investir numa cultura de confiança (“right people communicating to right people”). As alterações ao plano devem partir da coordenação, comando e controlo que, nos vários níveis de decisão, devem definir claramente quem toma as decisões. Conclusões: As estratégias para as intervenções ao nível da saúde pública, continuam, no mundo hodierno a desempenhar um papel fundamental e crucial na contenção das pandemias. O modelo apresentado neste estudo procura uma abordagem objectiva para destacar não só, os elementos essenciais de actividades bem-sucedidas, mas também as áreas em que a experiência pandémica sugere que o futuro planeamento deve dar maior ênfase.