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Objective To investigate the prevalence of AAV and HPV DNA and their types in cervical secretion from pregnant and non-pregnant women. Study design The samples were obtained from 40 pregnant and 62 non-pregnant women who were attended at the outpatient clinic of the Federal University Hospital of Espirito Santo, Southeastern Brazil. AAV and HPV were investigated by PCR and typed by PCR and/or RFLP. Results The occurrence of AAV in all samples was 25.5% (26/102): 81% (21/26) and 19% (5/26) for AAV2/3 and AAV5 species, respectively. AAV were observed in 35% (14/40) and 19% (12/62) of pregnant and non-pregnant women, respectively. HPV occurred in 22% of all samples; 25% (10/40) in pregnant and 20% (12/60) in non-pregnant women. HPV types were determined for 72.7% of the strains, most of which classified as high-risk. AAV–HPV co-infection was observed in 15.4% (4/26), mostly in pregnant women. Conclusions There was a greater prevalence of AAV and HPV in pregnant than in non-pregnant women, which suggests that the gestational state may play a role in reactivating the viruses.

This study was conducted to analyse the course and the outcome of the liver disease in the co-infected animals in order to evaluate a possible synergic effect of human parvovirus B19 (B19V) and hepatitis A virus (HAV) co-infection. Nine adult cynomolgus monkeys were inoculated with serum obtained from a fatal case of B19V infection and/or a faecal suspension of acute HAV. The presence of specific antibodies to HAV and B19V, liver enzyme levels, viraemia, haematological changes, and necroinflammatory liver lesions were used for monitoring the infections. Seroconversion was confirmed in all infected groups. A similar pattern of B19V infection to human disease was observed, which was characterised by high and persistent viraemia in association with reticulocytopenia and mild to moderate anaemia during the period of investigation (59 days). Additionally, the intranuclear inclusion bodies were observed in pro-erythroblast cell from an infected cynomolgus and B19V Ag in hepatocytes. The erythroid hypoplasia and decrease in lymphocyte counts were more evident in the co-infected group. The present results demonstrated, for the first time, the susceptibility of cynomolgus to B19V infection, but it did not show a worsening of liver histopathology in the co-infected group.

Human cytomegalovirus (HCMV) displays genetic variability in several regions, supposed to be related with strain-specific tissue tropism and immunopathogenesis. Based on sequence variation in the UL55 gene that encodes gB glycoprotein, HCMV strains can be assigned to one of four genotypes. Previous studies have addressed gB genotyping mostly by investigating strains derived from immunosuppressed patients, sometimes without previous knowledge about genotype distribution in a geographic area. The present study verified the distribution of HCMV gB genotypes of strains obtained from immunocompetent women at Vitória City, Espírito Santo State, Southeastern, Brazil. The HCMV genome was extracted from their cervical secretion, fetal and maternal placenta tissues (chorionic villous and decidua) from abortion cases and from white blood cells (WBCs). HCMV genotyping was performed by restriction fragment length polymorphism analyses of amplified product from the high variability site of the UL55 gene. All four genotypes were observed in both cervical secretion and placenta, whereas in WBCs a single gB1 genotype was detected. HCMV gB1 and gB2 genotypes were detected, respectively, in nine and in six of the 23 studied samples, while gB3 and gB4 were each found in four separate samples of the total. The differences in genotype frequency were not considered statistically significant. No mixed genotype infection was observed. The results indicated that the four gB HCMV genotypes had no particular tropism for placenta tissues and that all genotypes circulated within immunocompetent women at the time and in the region of study. O citomegalovírus humano (HCMV) apresenta variabilidade em diversas regiões do genoma, supostamente relacionada ao tropismo tecidual e imunopatogênese viral. Baseando-se na variação de seqüência do gene UL55 que codifica a glicoproteína gB, o HCMV pode ser classificado em um dos quatro genótipos. Estudos prévios têm investigado a associação destes genótipos a partir de cepas obtidas de pacientes imunossuprimidos. O presente estudo determinou os genótipos gB de cepas de HCMV obtidas de mulheres imunocompetentes em Vitória, Espírito Santo, Sudeste do Brasil. O genoma do HCMV foi extraído de secreção cervical, tecidos placentários fetais e maternos (vilosidade coriônica e decídua) obtidos de casos de aborto e de leucócitos do sangue periférico. A genotipagem foi realizada através da análise de polimorfismo de fragmentos de restrição do produto amplificado da região de alta variabilidade do gene UL55. Todos os quatro genótipos foram detectados na secreção cervical e na placenta, enquanto que somente o genótipo gB1 foi detectado em leucócitos. Genótipos gB1 e gB2 foram detectados em nove e seis das 23 cepas estudadas, respectivamente, enquanto gB3 e gB4 foram encontrados cada um em quatro casos. A diferença na freqüência de genótipos não foi estatisticamente significante. Infecção mista não foi detectada. Estes resultados indicam que os quarto genótipos de HCMV apresentam tropismo para os tecidos placentários e que todos eles circularam nas mulheres imunocompetentes no período e região geográfica do estudo.

Com o objetivo de medir a prevalência de anticorpos IgG contra o parvovírus B19 em gestantes com até 24 semanas de idade gestacional e detectar a ocorrência de casos de hidropisia fetal não-imune atribuídos a esse vírus, coletamos 249 amostras de soro em uma maternidade de referência na cidade do Rio de Janeiro, entre junho de 2003 e março de 2005. As gestantes foram acompanhadas até o termo da gestação, sendo detectados 17 casos de hidropisia fetal. Quatro casos foram atribuídos ao parvovírus B19 e dois destes ocorreram em gestantes residentes na zona oeste da cidade, em fevereiro de 2005. Resultados positivos para anticorpos IgG antiparvovírus B19 foram encontrados em 172 (71,6%) gestantes (IC 95% 65,5-77,7%), sendo esta prevalência de anticorpos comparável à encontrada em outras cidades brasileiras. A única variável associada com aquisição prévia de anticorpos IgG foi número de gestações anteriores maior que um(p= 0,02, IC 95% 0,36-0,94).

Biotin-labeled probe was used in an in situ hybridisation assay to localize virus infection in formalin-fixed, paraffin embedded tissues taken from eleven abortion cases. Probes for human cytomegalovirus (HCMV), human Parvovirus B19 (B19) and human adenovirus type 2 (HAd2), were labeled with biotin-11-dUTP by nick-translation reaction. Streptavidin-alkaline-phosphatase (SAP) was used to detect biotin, followed by 4-nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) solution. Positive reaction was observed in nucleus of glandular ephitelium cells of decidua either in positive or in negative control at first and second gestational trimester. The reaction was not inhibited with blocking solution for alkaline phosphatase endogenous activity and it persisted even with probes omission. The use of adequate negative control permitted to reveal the presence of nuclear biotin in glandular epithelium of decidua, responsible for false positivity in detection systems involving streptavidin biotin system (StrepABC). The stained cells resembled to cytophatic effect due to herpesvirus, which could induce further misinterpretation. The results obtained in this study strongly recommend that DNA detection by in situ hybridisation reaction in gestational endometrium should be done without using StrepABC system.Sondas marcadas com biotina foram utilizadas neste trabalho para detecção de infecção viral por hibridização in situ em tecidos fixados com formalina e embebidos em parafina de 11 casos obtidos de abortamento. Sondas para citomegalovírus humano (HCMV), parvovírus B19 humano (B19) e adenovírus humano tipo 2 (HAd2), foram marcadas com biotina-11-dUTP através da reação de nick-translation. Estreptavidina conjugada com fosfatase alcalina (SAP) seguida por solução de 4-nitro-azul de tetrazolio/5-bromo-4-cloro-3-indolil fosfato (NBT/BCIP) foram utilizadas para detecção da biotina após a reação de hibridização. Reação positiva foi observada no núcleo de células do epitélio glandular da decídua tanto no controle positivo quanto no negativo em tecidos de primeiro e segundo trimestre gestacional. Esta reação não foi inibida com solução bloqueadora da atividade endógena de fosfatase alcalina e persistiu mesmo com a omissão das sondas. O uso de controles negativos permitiu revelar atividade endógena de biotina nuclear em epitélio glandular da decídua, responsável por reações falso positivas em sistemas de detecção estreptavidina-biotina (StrepABC). Os resultados obtidos neste estudo fortemente recomendam que a detecção de ADN por hibridização in situ em endométrio gestacional seja feita com outro sistema de detecção que não o StrepABC.

Forty isolates of adenovirus type 7 were analized by restriction enzyme digestion with BamHI, SmaI, EcoRI and HindIII. These isolates were obtained from acute respiratory disease patients during the years 1980 to 1991. Only two genomic types were found: Ad7b and Ad7e, with Ad7b (87.5%) being more frequent than Ad7e (12.5%). The genomic type Ad7e appeared in the years 1980, 1981 and 1983. Ad7b appeared in 1982 and it was the only genomic type found from 1984 to 1991. Both genomic types were responsible for lower (LRTI) and upper (URTI) respiratory tract infection, but the proportion LRTI/URTI is higher for Ad7b (25/6) than for Ad7e (1/4). Quarenta amostras de Adenovírus tipo 7 foram analizadas por digestão com as enzimas de restrição BamHI, SmaI, EcoRI e HindIII. Estas amostras foram coletadas de pacientes apresentando doença respiratória aguda durante os anos de 1980 a 1991. Somente dois tipos genômicos foram encontrados: Ad7b e Ad7e, sendo que Ad7b (87,5%) foi mais freqüente que Ad7e (12,5%). O tipo genômico Ad7e apareceu nos anos 1980, 1981 e 1983. Ad7b apareceu durante 1982 e seguiu sendo o único tipo genômico encontrado de 1984 a 1991. Ambos os tipos genômicos foram responsáveis por infecções do trato respiratório superior (ITRS) e inferior (ITRI), porém a proporção ITRI/ITRS é mais alta para Ad7b (25/6) que para Ad7e(1/4).

Pregnancy failure is a common event and often of unknown cause. Some viruses are thought to cause abortions including the adeno-associated viruses (AAV), viruses which are regarded as being without any definitive association to any human disease. This study investigated AAV infection in 81 human abortions, both spontaneous and intentional that occurred up to the 23rd week of gestation. Nucleic acid of AAV-2, 3, and 5 types from 118 decidual and chorionic tissues, collected from the patients in this study, was amplified by nested-PCR. In situ hybridization (ISH) was developed with a digoxigenin-labeled AAV probe in paraffin embedded tissues from the AAV positive cases. AAV was observed in 28.4% (23/81) of the cases, of which, 78.3% (18/23) were in the decidua and 21.7% (5/23) in the extravillous trophoblast, the chorionic plate, or chorionic villi fragments. AAV-2, the only type detected, occurred in 32.3% (22/68) and in 7.7% (1/13) of the spontaneous and intentional abortions, respectively. ISH revealed AAV in the decidua, chorionic tissue or chorionic plate and extravillous trophoblast. The detection of only AAV-2 type indicates that it is the most frequent in the population studied and/or shows tissue tropism. The presence of AAV in decidual or trophoblastic cells in cases of abortion, as observed by ISH, implies that the virus could jeopardize the pregnancy. The significant predominance in spontaneous cases suggests possibly a causal association between AAV and abortion. J. Med. Virol. 82:1689–1693, 2010. © 2010 Wiley-Liss, Inc.

With the aim of measuring the prevalence of anti-parvovirus B19 IgG antibodies during pregnancy up to 24 weeks of gestation and detecting cases of nonimmune hydrops fetalis, 249 sera from pregnant women attending a reference hospital in Rio de Janeiro city, from June 2003 to November 2004 were collected. They were followed-up until the end of pregnancy, with 17 cases of fetal hydrops detected. Four cases were caused by parvovirus B19 and two of them occurred in pregnant women living in the western zone of the city, during February 2005. Anti-parvovirus B19 IgG antibodies were found in 172 (71.6%) pregnant women (CI 95% 65.5%-77.7%); this antibody prevalence is similar to results found for others Brazilian cities. The only variable associated with previous acquisition of IgG antibodies to parvovirus B19 was number of pregnancies greater than one (p= 0.02, CI 95% 0.36-0.94).

Biotin-labeled probe was used in an in situ hybridisation assay to localize virus infection in formalin-fixed, paraffin embedded tissues taken from eleven abortion cases. Probes for human cytomegalovirus (HCMV), human Parvovirus B19 (B19) and human adenovirus type 2 (HAd2), were labeled with biotin-11-dUTP by nick-translation reaction. Streptavidin-alkaline-phosphatase (SAP) was used to detect biotin, followed by 4-nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) solution. Positive reaction was observed in nucleus of glandular ephitelium cells of decidua either in positive or in negative control at first and second gestational trimester. The reaction was not inhibited with blocking solution for alkaline phosphatase endogenous activity and it persisted even with probes omission. The use of adequate negative control permitted to reveal the presence of nuclear biotin in glandular epithelium of decidua, responsible for false positivity in detection systems involving streptavidin biotin system (StrepABC). The stained cells resembled to cytophatic effect due to herpesvirus, which could induce further misinterpretation. The results obtained in this study strongly recommend that DNA detection by in situ hybridisation reaction in gestational endometrium should be done without using StrepABC system. Sondas marcadas com biotina foram utilizadas neste trabalho para detecção de infecção viral por hibridização in situ em tecidos fixados com formalina e embebidos em parafina de 11 casos obtidos de abortamento. Sondas para citomegalovírus humano (HCMV), parvovírus B19 humano (B19) e adenovírus humano tipo 2 (HAd2), foram marcadas com biotina-11-dUTP através da reação de nick-translation. Estreptavidina conjugada com fosfatase alcalina (SAP) seguida por solução de 4-nitro-azul de tetrazolio/5-bromo-4-cloro-3-indolil fosfato (NBT/BCIP) foram utilizadas para detecção da biotina após a reação de hibridização. Reação positiva foi observada no núcleo de células do epitélio glandular da decídua tanto no controle positivo quanto no negativo em tecidos de primeiro e segundo trimestre gestacional. Esta reação não foi inibida com solução bloqueadora da atividade endógena de fosfatase alcalina e persistiu mesmo com a omissão das sondas. O uso de controles negativos permitiu revelar atividade endógena de biotina nuclear em epitélio glandular da decídua, responsável por reações falso positivas em sistemas de detecção estreptavidina-biotina (StrepABC). Os resultados obtidos neste estudo fortemente recomendam que a detecção de ADN por hibridização in situ em endométrio gestacional seja feita com outro sistema de detecção que não o StrepABC.

Erythrovirus B19 infection is usually benign but may have serious consequences in patients with hemolytic anemia (transient aplastic crisis), immunodeficiency (in whom persistent infection can lead to chronic bone marrow failure with anemia), or who are in the first or second trimester of gestation (spontaneous abortion, hydrops fetalis, and fetal death). Being non-enveloped, B19 resists most inactivation methods and can be transmitted by transfusion. B19 is difficult to cultivate and native virus is usually obtained from viremic blood. As specific antibodies may be absent, and there is no reliable immunological method for antigen detection, hybridization or polymerase chain reaction are needed for detecting viremia. A rapid method, gel hemagglutination (Diamed ID-Parvovirus B19 Antigen Test), can disclose highly viremic donations, whose elimination lessens the viral burden in pooled blood products and may even render them non-infectious. In order to obtain native antigen and to determine the frequency of viremic donors, we applied this test to blood donors in a period of high viral activity in our community. Positive or indeterminate results were re-tested by dot-blot hybridization. We tested 472 donors in 1998 and 831 ones in 1999. One viremic donor was found in 1999. We suggest that in periods of high community viral activity the gel hemagglutination test may be useful in avoiding highly viremic blood being added to plasma pools or directly transfused to patients under risk.

Enteroviruses were investigated in respiratory secretions collected from patients with acute respiratory infections (ARI) over a seven year period (1985-1991), as part of a longitudinal study of ARI aetiology. All the viruses that are most commonly associated with ARI were found in this study. Among the virus isolates, enteroviruses were only less frequent than respiratory syncytial viruses, adenoviruses and influenzaviruses. Forty five enterovirus samples were isolated from patients with either upper respiratory tract infections (URTI) or lower respiratory tract infections (LRTI). From these enterovirus isolates, thirty one samples were identified as poliovirus (n=18) and non polio enterovirus (n=13) by serum neutralization. Poliovirus were identified as type 1 and 2 and all of them were vaccinal strains. From thirteen non polio enterovirus, twelve were identified as echovirus serotypes 1, 2, 7, 11, 19 and 31. The remainder was identified as coxsackievirus B4.Os enterovírus foram investigados em secreções respiratórias coletadas de pacientes com infecção respiratória aguda (IRA), durante um período de sete anos (1985-1991), dentro de um estudo longitudinal da etiologia das IRAs. Neste estudo foram encontrados todos os vírus que são mais comumente associados com IRA. Entre os vírus isolados, os enterovírus foram apenas menos frequentes que vírus respiratório sincicial, adenovírus e influenzavírus. Quarenta e cinco amostras de enterovirus foram isoladas de pacientes com infecção do trato respiratório superior ou inferior. Entre estes enterovírus isolados, trinta e uma amostras foram identificadas como poliovírus (n=18) e enterovírus não polio (n=13) pelo teste de soroneutralização. As amostras de poliovirus isoladas foram classificadas como amostras vacinais do tipo 1 e 2. Dos treze enterovírus não polio, doze foram identificados como echovírus sorotipos 1, 2, 7, 11, 19 e 31 e apenas um foi identificado como coxsackievírus B4.