Detection of Herpesvirus, Enterovirus, and Arbovirus infection in patients with suspected central nervous system viral infection in the Western Brazilian Amazon.

Acute infections of the central nervous system (CNS) can be caused by various pathogens. In this study, the presence of herpesviruses (HHV), enteroviruses (EVs), and arboviruses were investigated in CSF samples from 165 patients with suspected CNS viral infection through polymerase chain reaction (PCR) and reverse transcriptase PCR. The genomes of one or more viral agents were detected in 29.7% (49/165) of the CSF samples. EVs were predominant (16/49; 32.6%) followed by Epstein-Barr virus (EBV) (22.4%), Varicella-Zoster virus (VZV) (20.4%), Cytomegalovirus (CMV) (18.4%), herpes simplex virus (HSV-1) (4.1%), (HSV-2) (4.1%), and the arboviruses (14.3%). Four of the arboviruses were of dengue virus (DENV) and three of oropouche virus (OROV). The detection of different viruses in the CNS of patients with meningitis or encephalitis highlight the importance of maintaining an active laboratory monitoring diagnostics with rapid methodology of high sensitivity in areas of viral hyperendemicity that may assist in clinical decisions and in the choice of antiviral therapy.

Analysis of the nucleocapsid gene brings new insights to the classification of Sigmodontinae-borne hantaviruses.

Hantaviruses, members of the family Bunyaviridae, are the causative agents of hantavirus cardiopulmonary syndrome in South America. Hantaviruses are currently classified into species based on the guidelines provided by the International Committee on Taxonomy of Viruses. However, a new taxonomic system was proposed recently to classify Sigmodontinae-borne hantaviruses, which are divided currently into three phylogenetic clades corresponding to Andes, Laguna Negra, and Rio Mamore. Analyzing complete nucleocapsid gene sequences of all Sigmodontinae-borne hantaviruses, we propose the addition of a new clade and a fourth group to the already established Andes clade, allowing a better classification of the Sigmodontinae-borne hantaviruses.

Pathophysiology of chikungunya virus infection associated with fatal outcomes.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes acute, subacute, and chronic human arthritogenic diseases and, in rare instances, can lead to neurological complications and death. Here, we combined epidemiological, virological, histopathological, cytokine, molecular dynamics, metabolomic, proteomic, and genomic analyses to investigate viral and host factors that contribute to chikungunya-associated (CHIK) death. Our results indicate that CHIK deaths are associated with multi-organ infection, central nervous system damage, and elevated serum levels of pro-inflammatory cytokines and chemokines compared with survivors. The histopathologic, metabolite, and proteomic signatures of CHIK deaths reveal hemodynamic disorders and dysregulated immune responses. The CHIKV East-Central-South-African lineage infecting our study population causes both fatal and survival cases. Additionally, CHIKV infection impairs the integrity of the blood-brain barrier, as evidenced by an increase in permeability and altered tight junction protein expression. Overall, our findings improve the understanding of CHIK pathophysiology and the causes of fatal infections.

[Dengue: sero-epidemiological survey and virological surveillance in Campinas, São Paulo, Brazil]. / Dengue: inquérito populacional para pesquisa de anticorpos e vigilância virológica no Município de Campinas, São Paulo, Brasil.

The epidemiology of dengue in the municipality of Campinas, São Paulo, Brazil, was studied in 1998 using a randomized sero-epidemiological survey. Epidemiological surveillance data from 1996-2003 were also analyzed, with an emphasis on virological surveillance. 1,260 individuals participated in the survey and had blood samples drawn by finger stick on filter paper. Blood samples were tested by EIA-ICC, an enzyme immunoassay using infected cells as antigen. Dengue antibody prevalence (14.79%) was lower than in other surveys in other States of Brazil, but higher than in two other serological surveys in São Paulo State. Dengue antibody prevalence was far higher than the reported case incidence during the 1996, 1997, and 1998 epidemics. Antibody prevalence and reported case incidence in different health districts were disproportional. The article concludes by recommending further research on the significance of transmission rates during epidemics and more intensive virological surveillance, especially in years with few reported cases.

Serologic Evidence of Mammarenaviruses among Wild Rodents in Brazil.

We screened blood samples from 560 wild rodents collected in southeastern Brazil for antibodies to a recombinant nucleoprotein (rN) of Junín virus. Six rodents were antibody positive (1.1%), demonstrating evidence of infection with mammarenaviruses in several species of Brazilian rodents.

Linear amplification followed by single primer polymerase chain reaction to amplify unknown DNA fragments: complete nucleotide sequence of Oropouche virus M RNA segment.

The whole nucleotide sequence of Oropouche virus medium (M) RNA, Orthobunyavirus genus, Bunyaviridae family, was obtained using a new genomic amplification method. This method is based on the use of a single and specific primer of high melting temperature in a linear amplification (LA), followed by a single primer polymerase chain reaction (LASP-PCR). The LASP-PCR was used to walk along the Oropouche M RNA completing the sequence in seven successive walks. The amplicons obtained in each walking step ranged from 300 to 1100 bp; however, amplicons of up to 3970 bp was obtained when the extension time of the LASP-PCR was increased from 120 to 270 s. This method was tested successfully for Escherichia coli and cytomegalovirus obtaining amplicons of up to 2130 and 6500 bp, respectively, indicating that it can be applied to amplify unknown DNA sequences adjacent to a short stretch of known sequence of more complex genomes.

Multiplex nested PCR for Brazilian Alphavirus diagnosis.

A reverse-transcriptase PCR (RT-PCR) and a multiplex nested PCR were developed for the rapid detection and identification of 14 Brazilian alphaviruses. Using Alphavirus genus-specific primers in a RT-PCR, we obtained amplified products of 434 bp. Species-specific primers were selected and simultaneously tested in a multiplex nested PCR. The nested PCR increased the test sensitivity 1000-fold and was capable of identifying Brazilian Alphavirus showing the expected bands with diagnostic sizes for Venezuelan (400 bp), Eastern (124 bp), and Western (208 bp) equine encephalitis, Aura (86 bp), and Mayaro (270 bp) viruses. This strategy for diagnosis is fast, sensitive, specific and it can be used as a reliable alternative for routine Brazilian Alphavirus diagnosis.

Mechanisms of shock in hantavirus pulmonary syndrome.

PURPOSE OF REVIEW: Despite abundant literature on hantavirus, few reports have focused on the shock in hantavirus pulmonary syndrome. This review approaches recent advances that allow us to better understand the pathogenesis of hantavirus pulmonary syndrome shock. RECENT FINDINGS: Hantavirus pulmonary syndrome has been studied in a hamster model that mimics human shock and respiratory failure. In-vitro experiments show that pathogenic hantaviruses are able to inhibit antiviral responses, and that cytotoxicity of hantavirus-specific T cells enhances the permeability of infected endothelial cells. The idea that the primary cardiac lesion of shock is mostly functional has been shaken by the report of a typical myocarditis in hearts from human hantavirus pulmonary syndrome fatal cases. The involvement of regulatory T cells on hantavirus persistence in its rodent reservoir suggests that these cells could protect from severe hantavirus pulmonary syndrome and shock. SUMMARY: Hantavirus pulmonary syndrome shock is probably related to an exacerbated immune response of CD8+ T cells producing cytotoxicity on infected endothelial cells, presence of myocarditis and myocardial depression induced by nitric oxide. The virulence elements in G1 glycoprotein could also contribute to shock. Active suppression of immune T regulatory cells is probably involved in hantavirus pulmonary syndrome pathogenesis. These are all new aspects of hantavirus pulmonary syndrome pathogenesis that stimulate further studies to elucidate mechanisms of shock and to develop effective treatment strategies.

Diagnosis of Oropouche virus infection by RT-nested-PCR.

Using the RT-PCR with primers that anneal to the 5' and the 3' extremities of the genome segments of bunyaviruses and internal primers that anneal to the S segment of Simbu serogroup viruses in a nested PCR it was possible to amplify the Oropouche virus (ORO) genome from the sera of three patients. These results show that this RT-nested-PCR is a useful tool for rapid diagnosis of Oropouche fever infections.

PCR with the aqueous humor, blood leukocytes and vitreous of patients affected by cytomegalovirus retinitis and immune recovery uveitis.

PURPOSE: To detect the cytomegalovirus (CMV) genome by PCR in the aqueous humor, blood leukocytes and vitreous of patients affected by retinitis and immune recovery uveitis (IRU). METHODS: A PCR for CMV genome detection was carried out with the aqueous humor, vitreous and blood leukocytes of 54 patients with retinitis, including 25 HIV-infected patients presenting CMV retinitis in different stages (active lesion 6 cases, healed lesion 14 cases and IRU 5 cases), and 29 non-HIV-infected patients (retinitis unrelated to CMV) as negative controls. RESULTS: The CMV genome was detected in the vitreous, aqueous humor and blood leukocytes of 3 out of 6 HIV-infected patients, presenting active lesions in the retina. No CMV genome was detected in the vitreous, aqueous humor and blood leukocytes of the 5 HIV-infected patients presenting IRU. CONCLUSIONS: CMV genome detection by PCR in aqueous humor could be used as a specific and highly predictive technique for confirmation of this infection in the retina. The absence of CMV, based on the results of PCR done in clinical samples of the 5 IRU cases, does not confirm the hypothesis of a viral replication in the vitreous body and aqueous humor of these patients.

Dengue: inquérito populacional para pesquisa de anticorpos e vigilância virológica no Município de Campinas, São Paulo, Brasil / Dengue: sero-epidemiological survey and virological surveillance in Campinas, Sao Paulo, Brazil

Estudou-se a epidemiologia do dengue no Município de Campinas, São Paulo, Brasil, por meio de um inquérito populacional aleatório realizado em 1998, visando à detecção dos níveis de anticorpos para dengue, e de dados de vigilância epidemiológica do período de 1996 a 2003, com ênfase na vigilância virológica. Foram coletadas 1.260 amostras, por meio de punção digital, utilizando-se papel de filtro, sendo as amostras testadas pelo teste imunoenzimático em culturas celulares infectadas (EIA-ICC). Observou-se que a prevalência de soro reagentes (14,79 por cento) é mais baixa que as encontradas em outros inquéritos realizados no país e superior às encontradas em dois inquéritos realizados em cidades do Estado de São Paulo. Detectou-se uma prevalência de soro reagentes muito superior à incidência de casos notificados e confirmados laboratorialmente durante as epidemias de 1996, 1997 e 1998. Não se encontrou proporcionalidade entre a prevalência de anticorpos para dengue e a incidência de casos durante a epidemia nos diferentes Distritos de Saúde da cidade. Sugerem-se um estudo aprofundado do significado dos indicadores de transmissão utilizados em epidemias e uma vigilância virológica mais intensa, principalmente em anos com níveis de transmissão baixos.

The epidemiology of dengue in the municipality of Campinas, São Paulo, Brazil, was studied in 1998 using a randomized sero-epidemiological survey. Epidemiological surveillance data from 1996-2003 were also analyzed, with an emphasis on virological surveillance. 1,260 individuals participated in the survey and had blood samples drawn by finger stick on filter paper. Blood samples were tested by EIA-ICC, an enzyme immunoassay using infected cells as antigen. Dengue antibody prevalence (14.79 percent) was lower than in other surveys in other States of Brazil, but higher than in two other serological surveys in São Paulo State. Dengue antibody prevalence was far higher than the reported case incidence during the 1996, 1997, and 1998 epidemics. Antibody prevalence and reported case incidence in different health districts were disproportional. The article concludes by recommending further research on the significance of transmission rates during epidemics and more intensive virological surveillance, especially in years with few reported cases.

A simple reverse transcription-polymerase chain reaction for dengue type 2 virus identification

We show here a simplified reverse transcription-polymerase reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reation vessel was carried out following a digestion of virus with 1 per cent Nondet P-40. The 50 µl assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37ºC for RT followed by a variable amount of cycles of two-step PCR amplification (92ºC for 60 sec, 53ºC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7 per cent agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 10².8 TCID 50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique.

Infective endocarditis (IE) first diagnosed at autopsy: analysis of 31 cases in Ribeiräo Preto, Brazil

Thirty one infective endocarditis (IE) fatal cases whose diagnosis was first obtained at autopsy were studied. The clinical data of these patients (Group 1) showed significant differences compared to other 141 IE cases (Group 2). The average age of 53 years in Group 1 patients was 18 years higher than that of Group 2. The Group 1 patients had a low frequency of IE predisposing heart disease. Both patient groups presented fever (about 87 percent), but a significant low frequency of cardiac murmur (25.8 percent) was observed in Group 1 patients and echocardiography tests were performed in only 16.1 percent, suggesting that IE diagnosis was not suspected. Likewise, although most Group 1 patients appeared with severe acute illness, they did not present the classic IE clinical presentation. Blood cultures were performed in only 64.5 percent of the Group 1 patients. However, bacteria were isolated in 70 percent of these blood cultures and Staphylococcus aureus was isolated in 71.4 percent. The bacteria attacked mitral and aortic valves. Complications such as embolizations and cardiac failure occurred in almost half of the cases and they also presented with infections of the lungs, urinary tract, and central nervous system. Medical procedures were performed in practically all fatal cases whose diagnosis was first obtained at autopsy. Sepsis occurred in about half of the patients and it was followed by shock in more than 25 percent. This form of IE must be suspected in mature and in old febrile hospitalized patients having infection predisposing diseases, embolization, and suffering medical procedures

Jatobal virus antigenic characterization by ELISA and neutralization test using EIA as indicator, on tissue culture

A caracterizaçäo antigênica do vírus Jatobal (BeAn 423380) foi efetuada utilizando uma técnica de ELISA para deteccäo de anticorpos que utiliza culturas celulares infectadas como antígeno e um micro teste de neutralizaçäo para vírus que utiliza o método imunoenzimático como auxiliar para a leitura dos resultados (NT-EIA). O vírus Jatobal foi caracterizado como um Bunyaviridae, gênero Bunyavirus, pertencente ao sorogrupo Simbu. A técnica de ELISA, utilizando culturas celulares infectadas como antígeno, trata-se de método sensível e confiável na identificaçäo de agentes virais, possuindo muitas vantagens sobre ELISA convencionais e outros testes: elimina a preparaçäo laboriosa de antígenos para o revestimento em fase sólida; permite que se teste de forma mais rápida e fácil que por CF, HAI e neutralizaçäo por reduçäo de plaques um grande número de antisoros de vírus. ELISA e NT-EIA podem ser utilizados para a classificaçäo de vírus que näo produzem efeito citopático e podem ser aplicáveis na identificaçäo de vírus que crescem em células oriundas de mosquitos

Hantavirus pulmonary syndrome (HPS) in Guariba, SP, Brazil: report of 2 cases

Casos de hantavirose foram notificados em diferentes regioes do Estado de Sao Paulo (SP), Brasil, durante o primeiro semestre de 1998. Dois casos fatais de sindrome pulmonar ocorreram em maio de 1998 na Cidade de Guariba, localizada na Regiao Nordeste de SP. Ambos os pacientes trabalhavam no mesmo local, estocando milho em um paiol infestado de roedores. Este pacientes, apos 2 ou 3 dias de doenca febril aguda inespecifica, desenvolveram uma grave pneumonia intersticial, que espalhou-se difusamente por ambos os pulmoes causando insuficiencia respiratoria e obito. A autopsia, ambos os casos apresentavam edema pulmonar intersticial com infiltrado de celulas mononucleares (imunoblastos) sugestivo de etiologia viral...

Condutas terapêuticas e de suporte no paciente com dengue hemorrágico / Therapeutics behavior and supportin patiente with hemorrhagic fever of dengue

Aborda o fluxo de atendimento do paciente com febre hemorrágica do dengue, quanto à relevancia da organizaçäo da rede de serviços de saúde.

Vírus brasileiros da família Bunyaviridae / Brazilian viruses in the family Bunyaviridae

Os vírus brasileiros da família Bunyaviridae säo vírus RNA, isolados principalmente na Regiäo Amazônica, pertencentes aos gêneros Bunyavirus, Hantavirus e Phlebovirus. A grande maioria destes vírus säo transmitidos por mosquitos e flebótomos, exceto os Hantavirus que têm transmissäo relacionada à inalaçäo de aerossóis dos excretas de roedores. A estrutura, a composiçäo e o mecanismo de replicaçäo dos Bunyaviridae säo brevemente revistos neste trabalho. Também, säo analisados aspectos epidemiológicos e clínicos das doenças causadas pelos seguintes Bunyaviridae brasileiros: Oropouche, Apeú, Caraparu, Marituba, Guaroa, Tacaiuma, Guamá, Maguari, Candiru e Hantavirus.

Patogenia das infecçöes pelos vírus do dengue / Pathogenesis of dengue virus infections

Neste trabalho de revisäo, analisam-se aspectos das infecçöes primárias e secundárias pelos vírus do dengue, ressaltando a resposta imune benéfica, que impede a reinfecçäo e a resposta imune que facilita a entrada dos vírus do dengue em macrófagos e que é parte importante no mecanismo fisiopatológico do dengue hemorrágico. Esta forma de dengue é conseqüência de uma anômala resposta imune, envolvendo leucócitos, citocinas e imunocomplexos, causando aumento da permeabilidade por má funçäo vascular endotelial, sem destruiçäo do endotélio, com extravasamento de líquidos para o interstício, causando queda da pressäo arterial e manifestaçöes hemorrágicas, associadas a trombocitopenia.

Vacinas contra o dengue / Dengue vaccines

O controle do dengue é feito, nos dias atuais, em todo o mundo, seguindo normas de combate aos mosquitos vetores preconizadas por sanitaristas do começo do século. O controle e a erradicaçäo do Aedes aegypti, vetor do dengue, é bastante difícil e necessita de grandes investimentos com funcionários, máquinas, venenos e campanhas educacionais permanentes. A alternativa ideal para o controle do dengue seria através do uso de vacinas. Neste trabalho de revisäo, analisam-se pesquisas para o desenvolvimento de vacinas contra dengue, incluindo: as do vírus vivo atenuado; as de engenharia genética (vacinas recombinantes) tendo abordagens relativas à expressäo de proteínas de dengue em células eucarióticas, aos vírus recombinantes, aos vírus mutantes ou quiméricos, e às vacinas com vetores vivos. Ainda, abordam-se as vacinas de DNA. Observa-se que as vacinas de vírus vivo e atenuado säo aquelas que têm as melhores perspectivas para serem utilizadas de forma generalizada, no controle do dengue, dentro de alguns anos.