Rubella virus genotype 1G and echovirus 9 as etiologic agents of exanthematous diseases in Brazil: insights from phylogenetic analysis.

The aim of the present study was to identify the rubella virus (RV) and enterovirus (EV) genotypes detected during the Epidemiological Surveillance on Exanthematic Febrile Diseases (VIGIFEX) study and to perform phylogenetic analysis. Ten RV- and four EV-positive oropharyngeal samples isolated from cell culture were subjected to RT-PCR and sequencing. Genotype 1G and echovirus 9 (E-9) was identified in RV- and EV-positive samples, respectively. The RV 1G genotype has been persisting in Brazil since 2000-2001. No evidence of E-9 being involved in exanthematic illness in Brazil has been reported previously. Differential laboratory diagnosis is essential for management of rash and fever disease.

Molecular epidemiology of rubella viruses involved in congenital rubella infections in São Paulo, Brazil, between 1996 and 2009.

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as congenital rubella syndrome (CRS). This retrospective study was conducted between 1996 and 2009 with surveillance specimens collected from patients suspected of congenital rubella infection (CRI) and CRS. The clinical samples (nine amminiotic fluid, eight urine, eight blood, one conception product, and one placenta) were sent for viral isolation and genotyping. Twenty-seven sequences were analysed and four genotypes (1a, 1B, 1G, and 2B) were identified in São Paulo that were involved in congenital infection. To our knowledge, this study is the first report that describes genetic diversity of the circulating rubella strains involved in CRI.

Single-dose varicella vaccine effectiveness in Brazil: A case-control study.

BACKGROUND: Varicella vaccine was introduced into the Brazilian Immunization Program in October 2013, as a single-dose schedule administered at 15 months of age. Its effectiveness had not yet been assessed in the country. METHODS: A matched case-control study was carried out in São Paulo and Goiânia (Southeast and Midwest regions, respectively), Brazil. Suspected cases, were identified through a prospective surveillance established in the study sites. All cases had specimens from skin lesion collected for molecular laboratory testing. Cases were confirmed by either clinical or PCR of skin lesions and classified as mild, moderate, and severe disease. Two neighborhood controls were selected for each case. Cases and controls were aged 15-32 months and interviewed at home. Evidence of prior vaccination was obtained from vaccination cards. Univariate and multivariate logistic regression models were used, and odds ratio and its respective 95% confidence intervals were estimated. Vaccine effectiveness was estimated by comparing de odds of having received varicella vaccine among cases and controls. RESULTS: A total of 168 cases and 301 controls were enrolled. Moderate and severe illness, was found in 33.3% and 9.9% of the cases. Effectiveness of a single dose varicella vaccine was 86% (95%CI 72-92%) against disease of any severity and 93% (95%CI 82-97%) against moderate and severe disease. Out of 168 cases, 81.8% had positive PCR results for wild-type strains, and 22.0% were breakthrough varicella cases. Breakthrough cases were milder compared to non-breakthrough cases (p < .001). CONCLUSIONS: Effectiveness of single dose varicella vaccine in Brazil is comparable to that in other countries where breakthrough varicella cases have also been found to occur. The goal of the varicella vaccination program, along with disease burden and affordability should be taken into consideration when considering the adoption of a second dose of varicella vaccine into national immunization programs.

Genotype 1 of human parvovirus B19 in clinical cases.

INTRODUCTION:: Virus surveillance strategies and genetic characterization of human parvovirus B19 (B19V) are important tools for regional and global control of viral outbreak. In São Paulo, Brazil, we performed a study of B19V by monitoring the spread of this virus, which is an infectious agent and could be mistakenly reported as a rash and other types of infection. METHOD:: Serum samples were subjected to enzyme immunoassay, real time polymerase chain reaction, and sequencing. RESULTS:: From the 462 patients with suspected cases of exanthematic infections, the results of the 164 serum samples were positive for B19V immunoglobulin M. Among these cases, there were 38 patients with erythema infections and B19-associated with other infections such as encephalitis, hydrops fetalis, chronic anemia, hematological malignancies. These samples were sequenced and identified as genotype 1. CONCLUSION:: This study showed patients with infections caused by B19V and sequencing genotype 1. Continuous monitoring is necessary to detect all known genotypes, and the emergence of new genotypes of these viruses for case management in public health control activities.

Rubella in pregnancy: intrauterine transmission and perinatal outcome during a Brazilian epidemic.

Sixty pregnant women with clinical signs of rubella and specific rubella antibodies were studied between January 1999 and December 2002 in order to determine the intrauterine rubella transmission rate and the presence of the virus in amniotic fluid and fetal tissues by nested PCR. Thirty-three patients presented rubella before 12 weeks of gestation and 27 after 12 weeks. Gestational age at the time of acute rubella was determined on the basis of the date of last menstruation and the first trimester ultrasound scan. Thirteen patients with clinical features of rubella before 12 weeks of gestation were submitted to amniocentesis. Three products of conception were analyzed. The presence of the rubella virus was determined by nested PCR. IgM and IgG antibodies were analyzed in neonatal samples at birth and at 3 months of age using a capture immunoassay. Newborn follow-up was based on the presence of congenital rubella syndrome-compatible defects, anti-rubella antibodies, echocardiographic alterations, brainstem evoked response audiometry, and ophthalmological pathology. Five miscarriages and four fetal deaths were observed in the group of patients presenting clinical features before 12 weeks of gestation. IgM antibodies were detected in seven neonates at birth and at 3 months of age. Deafness was observed in three cases and pigmentary retinopathy in one case. Fourteen of the 16 samples (13 amniotic fluid and 3 fetal tissue samples) submitted to virological analysis tested positive. Four fetal deaths, five miscarriages (one with negative virology) and seven newborns with anti-rubella IgM at birth and/or at 3 months age were observed in the group with rubella before 12 weeks of gestation. There were three cases in which virological analysis of the amniotic fluid samples was positive (infected) while the newborn showed no signs of congenital rubella syndrome and anti-rubella IgM were absent. When maternal rubella occurred after 12 weeks of gestation, no fetal or neonatal rubella signs were observed. Eradication of congenital rubella syndrome is possible since vaccination campaigns continue and all services related to the health care of children, adolescents and women have become aware of the significance of the problem and are collaborating. All pregnant women in Brazil should be screened for the rubella antibody and the susceptible group should be vaccinated after giving birth.

Antiviral Action of Hydromethanolic Extract of Geopropolis from Scaptotrigona postica against Antiherpes Simplex Virus (HSV-1).

The studies on chemical composition and biological activity of propolis had focused mainly on species Apis mellifera L. (Hymenoptera: Apidae). There are few studies about the uncommon propolis collected by stingless bees of the Meliponini tribe known as geopropolis. The geopropolis from Scaptotrigona postica was collected in the region of Barra do Corda, Maranhão state, Brazil. The chemical analysis of hydromethanolic extract of this geopropolis (HMG) was carried out through HPLC-DAD-ESI-MS/MS and the main constituents found were pyrrolizidine alkaloids and C-glycosyl flavones. The presence of alkaloids in extracts of propolis is detected for the first time in this sample. The antiviral activity of HMG was evaluated through viral DNA quantification experiments and electron microscopy experiments. Quantification of viral DNA from herpes virus showed reduction of about 98% in all conditions and concentration tested of the HMG extract. The results obtained were corroborated by transmission electron microscopy, in which the images did not show particle or viral replication complex. The antiviral activity of C-glycosyl flavones was reported for a variety of viruses, being observed at different points in the viral replication. This work is the first report about the antiviral activity of geopropolis from Scaptotrigona postica, in vitro, against antiherpes simplex virus (HSV).

Discovery of a new antiviral protein isolated Lonomia obliqua analysed by bioinformatics and real-time approaches.

This study presents a new recombinant protein that acts as a powerful antiviral (rAVLO-recombinant Antiviral protein of Lonomia obliqua). It was able to reduce the replication by 10(6) fold for herpes virus and by 10(4) fold for rubella virus. RT-PCR of viral RNA rAVLO treated infected cells also showed similar rate of inhibition in replication. The analysis of this protein by bioinformatics suggests that this protein is globular, secreted with a signal peptide and has the ability to bind to MHC class I. It was found that there are several protein binding sites with various HLA and a prevalence of α-helices in the N-terminal region (overall classified as a α/ß protein type). BLAST similarity sequence search for corresponding cDNA did not reveal a similar sequence in Genbank, suggesting that it is from a novel protein family. In this study we have observed that this recombinant protein and hemolymph has a potent antiviral action. This protein was produced in a baculovirus/Sf-9 system. Therefore, these analyses suggest that this novel polypeptide is a candidate as a broad spectrum antiviral.

Human vaccinia-like virus outbreaks in São Paulo and Goiás States, Brazil: virus detection, isolation and identification.

Since October 2001, the Adolfo Lutz Institute has been receiving vesicular fluids and scab specimens of patients from Paraíba Valley region in the São Paulo and Minas Gerais States and from São Patricio Valley, in the Goiás State. Epidemiological data suggested that the outbreaks were caused by Cowpox virus or Vaccinia virus. Most of the patients are dairy milkers that had vesiculo-pustular lesions on the hands, arms, forearms, and some of them, on the face. Virus particles with orthopoxvirus morphology were detected by direct electron microscopy (DEM) in samples of 49 (66.21%) patients of a total of 74 analyzed. Viruses were isolated in Vero cell culture and on chorioallantoic membrane (CAM) of embryonated chicken eggs. Among 21 samples submitted to PCR using primers for hemagglutinin (HA) gene, 19 were positive. Restriction digestion with TaqI resulted in four characteristic Vaccinia virus fragments. HA nucleotide sequences showed 99.9% similarity with Cantagalo virus, described as a strain of Vaccinia virus. The only difference observed was the substitution of one nucleotide in the position 616 leading to change in one amino acid of the protein in the position 206. The phylogenetic analysis showed that the isolates clustered together with Cantagalo virus, other Vaccinia strains and Rabbitpox virus.

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.

Two novel circo-like viruses detected in human feces: complete genome sequencing and electron microscopy analysis.

The application of viral metagenomic techniques and a series of PCRs in a human fecal sample enabled the detection of two novel circular unisense DNA viral genomes with 92% nucleotide similarity. The viruses were tentatively named circo-like virus-Brazil (CLV-BR) strains hs1 and hs2 and have genome lengths of 2526 and 2533 nucleotides, respectively. Four major open reading frames (ORFs) were identified in each of the genomes, and differences between the two genomes were primarily observed in ORF 2. Only ORF 3 showed significant amino acid similarities to a putative rolling circle replication initiator protein (Rep), although with low identity (36%). Our phylogenetic analysis, based on the Rep protein, demonstrated that the CLV-BRs do not cluster with members of the Circoviridae, Nanoviridae or Geminiviridae families and are more closely related to circo-like genomes previously identified in reclaimed water and feces of a wild rodent and of a bat. The CLV-BRs are members of a putative new family of circular Rep-encoding ssDNA viruses. Electron microscopy revealed icosahedral (~23 nm) structures, likely reflecting the novel viruses, and rod-shaped viral particles (~65-460 × 21 × 10 nm in length, diameter, and axial canal, respectively). Circo-like viruses have been detected in stool samples from humans and other mammals (bats, rodents, chimpanzees and bovines), cerebrospinal fluid and sera from humans, as well as samples from many other sources, e.g., insects, meat and the environment. Further studies are needed to classify all novel circular DNA viruses and elucidate their hosts, pathogenicity and evolutionary history.

Fulminant encephalitis associated with a vaccine strain of rubella virus.

Involvement of the central nervous system is common in measles, but rare in rubella. However, rubella virus (RV) can cause a variety of central nervous system syndromes, including meningitis, encephalitis, Guillain-Barré syndrome and sub acute sclerosing panencephalitis. We report the occurrence of one fatal case of the encephalitis associated with measles-rubella (MR) vaccine during an immunization campaign in São Paulo, Brazil. A 31 year-old-man, previously in good health, was admitted at emergency room, with confusion, agitation, inability to stand and hold his head up. Ten days prior to admission, he was vaccinated with combined MR vaccine (Serum Institute of India) and three days later he developed 'flu-like' illness with fever, myalgia and headache. Results of clinical and laboratory exams were consistent with a pattern of viral encephalitis. During hospitalization, his condition deteriorated rapidly with tetraplegia and progression to coma. On the 3rd day of hospitalization he died. Histopathology confirmed encephalitis and immunohistochemistry was positive for RV on brain tissue. RV was also detected by qPCR and virus isolation in cerebrospinal fluid, brain and other clinical samples. The sequence obtained from the isolated virus was identical to that of the RA 27/3 vaccine strain.

Genotype 1 of human parvovirus B19 in clinical cases / Genótipo 1 do parvovírus humano em casos clínicos

Summary Introduction: Virus surveillance strategies and genetic characterization of human parvovirus B19 (B19V) are important tools for regional and global control of viral outbreak. In São Paulo, Brazil, we performed a study of B19V by monitoring the spread of this virus, which is an infectious agent and could be mistakenly reported as a rash and other types of infection. Method: Serum samples were subjected to enzyme immunoassay, real time polymerase chain reaction, and sequencing. Results: From the 462 patients with suspected cases of exanthematic infections, the results of the 164 serum samples were positive for B19V immunoglobulin M. Among these cases, there were 38 patients with erythema infections and B19-associated with other infections such as encephalitis, hydrops fetalis, chronic anemia, hematological malignancies. These samples were sequenced and identified as genotype 1. Conclusion: This study showed patients with infections caused by B19V and sequencing genotype 1. Continuous monitoring is necessary to detect all known genotypes, and the emergence of new genotypes of these viruses for case management in public health control activities.

Resumo Introdução: Estratégias de vigilância para o parvovírus humano B19 e caracterização genética são ferramentas importantes para o controle regional e global do surto viral. Em São Paulo, Brasil, foi realizado um estudo de parvovírus B19, monitorando a disseminação desse vírus, que é um agente infeccioso e poderia ser erroneamente relatado como uma erupção cutânea e outros tipos de infecções. Método: As amostras de soro foram submetidas ao ensaio imunoenzimático, PCR quantitativo em tempo real e sequenciamento. Resultados: Dos 462 pacientes com casos suspeitos de infecções exantemáticas, os resultados das 164 amostras de soro foram positivos para parvovírus B19 imunoglobulina M. Entre eles, 38 pacientes com eritema infeccioso apresentaram B19 associado com outras infecções, como encefalite, hidropisia fetal, anemia crônica, doenças hematológicas malignas. Essas amostras foram sequenciadas e identificadas como genótipo 1. Conclusão: Os pacientes foram infectados com parvovírus B19 e apresentaram genótipo 1. Monitoração contínua é necessária para detectar todos os genótipos conhecidos e o surgimento de novos genótipos para o controle de casos em saúde pública.

Prenatal diagnosis of congenital rubella infection in São Paulo / Diagnóstico pré-natal de infecção congênita por rubéola, em São Paulo, Brasil

Objective: rubella during the early stages of pregnancy can lead to severe birth defects known as congenital rubella syndrome (CRS). Samples collected from pregnant women with symptoms and suspected of congenital rubella infection between 1996 and 2008 were analyzed. Methods: a total of 23 amniotic fluid samples, 16 fetal blood samples, 1 product of conception and 1 placenta were analyzed by serology and RT-PCR. Results: all patients presented positive serology for IgG / IgM antibodies to rubella virus. Among neonates, 16 were IgG-positive, 9 were IgM-positive and 4 were negative for both antibodies. Of the 25 samples analyzed in this study, 24 were positive by RT-PCR. Changes in ultrasound were found in 15 (60%) of 25 fetuses infected with rubella virus. Fetal death and miscarriage were reported in 10 (40%) of the 25 cases analyzed. The rubella virus was amplified by PCR in all fetuses with abnormal ultrasound compatible with rubella. Fetal death and abortion were reported in 10 of 25 cases analyzed. Conclusion: this study, based on primary maternal rubella infection definitely confirms the good sensitivity and specificity of RT-PCR using amniotic fluid and ultrasound. The results showed that molecular assays are important tools in the early diagnosis of rubella and congenital rubella syndrome. .

Objetivo: a rubéola, durante os primeiros estágios da gravidez, pode levar a graves defeitos congênitos, conhecidos como síndrome da rubéola congênita (SRC). Amostras de gestantes com sintomas e suspeitas da rubéola congênita foram coletadas entre 1996 e 2008. Métodos: um total de 23 amostras de fluido amniótico, 16 amostras de sangue fetal, um produto da concepção e uma placenta foram analisados por sorologia e PCR. Resultados: todas as gestantes apresentaram sorologia positiva para IgG/IgM para o vírus da rubéola. Entre os recém-nascidos, 14 apresentaram anticorpos IgG positivos e 11 foram os anticorpos IgM positivos. Das 25 amostras analisadas neste estudo, 24 eram positivas por RT-PCR. Alterações na ultrassonografia foram encontradas em 15 (60%) dos 25 fetos infectados com o vírus da rubéola. Morte fetal e aborto espontâneo foram reportados em 10 (40%) dos 25 casos analisados. O vírus da rubéola foi amplificado por PCR em todos os fetos que apresentaram alterações na ultrassonografia, compatíveis com a rubéola. Morte fetal e aborto foram relatados em 10 dos 25 casos analisados. Conclusão: os resultados mostraram que os ensaios moleculares são ferramentas importantes para o diagnóstico precoce da rubéola e da síndrome da rubéola congênita. .

Molecular charatterization of influenza b virus outbreak on a cruise ship in Brazil 2012 / Caracterização molecular de surto de vírus influenza B em cruzeiro marítimo no Brasil 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.

Em fevereiro de 2012, durante a temporada de verão no Brasil, um surto de doença respiratória ocorreu no navio de cruzeiro MSC Armonia. Mulher de 31 anos, membro da tripulação, foi internada com insuficiência respiratória e morreu. Com o objetivo de estudar a etiologia da doença foram investigadas necrópsia de tecido do caso fatal e secreções respiratórias de 13 passageiros e membros da tripulação com sintomas respiratórios. O teste de influenza por RT-PCR em tempo real foi realizado e o gene completo da hemaglutinina (HA) das amostras positivas foi sequenciado. O vírus influenza B foi detectado em sete indivíduos, sugerindo-o como a causa do surto de doença respiratória a bordo do navio. A análise da sequência do gene da HA indicou que os vírus estão fortemente relacionados com o vírus B/Brisbane/60/2008, linhagem Victoria, componente da vacina de influenza para 2011-2012 no hemisfério sul. Uma vez que a composição da vacina foi alterada para uso na temporada de 2012-2013, é essencial a vigilância ativa dos vírus circulantes em todo o mundo. A análise molecular é uma ferramenta importante para caracterização do patógeno responsável pelo surto. Além disso, a vigilância de doenças baseada em dados laboratoriais contribui para as medidas de controle da influenza, uma doença imunoprevinível.

Molecular characterization of human parvovirus B19 associated with neuromyelitis / Caracterização molecular do parvovirus humano B19 associado com neuromielite

INTRODUCTION: Human parvovirus B19 (B19V) is associated with a wide range of clinical symptoms. An acute infection can lead to anemia, erythema infectiosum, or hydrops fetalis, as well as arthritis and other clinical manifestations. Among other diseases associated with B19V, several demyelinating disorders may occur as a result of B19V infection. Phylogenetic analysis of partial erythrovirus sequences resulted in three possible B19V genotype groups. Our study involved analysis of samples from a patient with neuromyelitis that enabled the molecular characterization of human parvovirus B19. OBJECTIVE: To study B19V infection associated with a neurological manifestation and to perform phylogenetic analysis.METHODS: Serum and cerebrospinal fluid samples were tested for B19V infection. The serological assay for B19V was a commercial IgG and IgM enzyme immunoassay kit. Nucleic acid detection was performed using a PCR assay. The phylogenetic analyses were performed using PAUP and other software programs. RESULTS: Our study reports that the topology of viral isolate sequences from a patient with neuromyelitis fell within a clade consisting of B19V genotype 1.CONCLUSION: This result indicates that this virus genotype group has been circulating in Sao Paulo, Brazil. This report represents a rare case in which B19V might be the responsible agent for central nervous system infection.

INTRODUÇÃO: O parvovírus B19 humano (B19V) é associado a vários sintomas clínicos. Uma infecção aguda pode levar à ocorrência de anemia, eritema infeccioso ou hidropsia fetal, bem como artrite e outras manifestações clínicas. Infecções por B19V podem também ocasionar várias doenças desmielinizantes. A análise filogenética das sequências parciais de eritrovírus resultou em três possíveis grupos de genótipos de B19V. Este estudo consistiu na análise de amostras de uma paciente com neuromielite, o que possibilitou a caracterização molecular do parvovírus B19 humano. OBJETIVO: Estudar a infecção por B19V associada a uma manifestação neurológica e realizar sua análise filogenética. MÉTODOS: Amostras de soro e de líquido cefalorraquidiano foram testadas para a observação de infecção por B19V. O teste sorológico utilizado para a pesquisa de B19V foi um kit comercial de ensaio imunoenzimático para IgG e IgM. A detecção de ácido nucleico foi realizada utilizando-se um teste de PCR. As análises filogenéticas foram realizadas utilizando PAUP e outros programas. RESULTADOS: Este artigo registra que a topologia de sequências de isolados virais de uma paciente com neuromielite foram inseridas em um clado constituído de grupos dentro do genótipo 1 de B19V. CONCLUSÃO: Esses achados indicam que este genótipo do vírus tem se mantido circulante em São Paulo, Brasil. Este relato representa um caso raro no qual o B19V pode ter sido o agente responsável pela infecção no sistema nervoso central.

Growth of rubella virus into SIRC (rabbit cornea cell line) using various culture media

The present study evaluated the efficacy of various culture media for performing the isolation and growth of the rubella virus inoculated into SIRC cells. Rubella virus RA-27/3 strain and RVi/São Paulo/BRA99 wild type strain (Gen Bank number DQ458965) were inoculated into SIRC cell line and cultivated in 199, DMEM, MEM and RPMI media. The inoculated cells when examined on phase contrast microscopy showed the characteristic rounded and multipolar cells. The CPE was observed at the first 48 hours cultivation in the respective tested media. The curve of the infectivity increase was higher in the cultures maintained in DMEM and RPMI media. Hence, the SIRC cellular lineage cultivated in DMEM or RPMI media is an excellent substratum for performing the rubella virus isolation. These findings are relevant since the SIRC has been one of the few cell lines described in the literature which presents a cytopathic effect, and on that account it can be useful for carrying out the virus isolation from clinical specimens.