Vanessa Salete de Paula

Age-Specific Prevalence and Transmission of TT Virus

TT virus (TTV) is an unenveloped, single‐stranded DNA virus that was discovered recently in the sera of Japanese patients with posttransfusion hepatitis of unknown etiology. A high prevalence of TTV infection in blood donors of several countries, including Brazil, has been demonstrated. To study the variation in TTV prevalence between different age groups, sera from 223 individuals without liver disease, aged 0–80 years, were tested by the polymerase chain reaction for the presence of TTV DNA. All subjects were inhabitants of the city of Rio de Janeiro, Brazil. The prevalence increased continuously with age (P < .001), from 17% among children under the age of 11 years, to 57% in people older than 50 years. To assess vertical transmission, sera from 105 unselected, consecutive parturient women attending a public maternity hospital were paired with cord bloods and examined for the presence of TTV DNA. Thirty‐seven (35%) mothers were found to be TTV infected. Seven cord bloods were also positive, suggesting the possible transplacental transmission of the virus. Furthermore, a direct correlation between TTV viremia and presence of antibodies to the enterically transmissible hepatitis A virus (HAV) was observed in this group of women, with a relative risk of TTV infection of 5.09 (95% confidence interval 0.76–34.03) for women with anti‐HAV, compared with women without. This finding suggested that the fecal‐oral route might be an important route of TTV transmission. J. Med. Virol. 59:318–322, 1999.

Hepatitis A virus: Host interactions, molecular epidemiology and evolution

Infection with hepatitis A virus (HAV) is the commonest viral cause of liver disease and presents an important public health problem worldwide. Several unique HAV properties and molecular mechanisms of its interaction with host were recently discovered and should aid in clarifying the pathogenesis of hepatitis A. Genetic characterization of HAV strains have resulted in the identification of different genotypes and subtypes, which exhibit a characteristic worldwide distribution. Shifts in HAV endemicity occurring in different parts of the world, introduction of genetically diverse strains from geographically distant regions, genotype displacement observed in some countries and population expansion detected in the last decades of the 20th century using phylogenetic analysis are important factors contributing to the complex dynamics of HAV infections worldwide. Strong selection pressures, some of which, like usage of deoptimized codons, are unique to HAV, limit genetic variability of the virus. Analysis of subgenomic regions has been proven useful for outbreak investigations. However, sharing short sequences among epidemiologically unrelated strains indicates that specific identification of HAV strains for molecular surveillance can be achieved only using whole-genome sequences. Here, we present up-to-date information on the HAV molecular epidemiology and evolution, and highlight the most relevant features of the HAV-host interactions.

Serological and molecular evidence of hepatitis E virus in swine in Brazil

Active hepatitis E virus (HEV) infections in two Brazilian swine herds were investigated. In study 1, 26 piglets born to five anti-HEV positive sows were monitored from birth to post-partum week 22. Serum samples were screened for the detection of anti-HEV antibodies and a nested RT-PCR used to examine the HEV genome. Passive transfer of immunity was confirmed. At week 22, 23/26 (88.4%) of the piglets had seroconverted. Genome amplification was achieved in a feces pool from one holding pen and in one serum sample, both from 13-week-old animals. Histology was suggestive of a potential HEV infection. In the second study, 47 piglets born to six anti-HEV-positive sows were monitored after weaning. Seroconversion was determined in eight animals at 6-8 weeks of age. HEV RNA was detected in two pools from a holding pen for 12-16-week-old animals. Brazilian isolates were classified as genotype 3. This is the first molecular evidence of HEV infection in Brazilian pig herds.

Detection of hepatitis A, B, and C virus-specific antibodies using oral fluid for epidemiological studies

In this report, we examine the adaptability of commercially available serological kits to detect antibodies markers for viral hepatitis in oral fluid samples. We also assessed the prevalence of hepatitis A, B, and C virus-specific antibodies, and related risk factors for these infectious diseases through sensitivity of the tests in saliva samples to evaluate if oralfluid can be an alternative tool to substitute serum in diagnosis of acute viral hepatitis and in epidemiological studies. One hundred and ten paired serum and saliva specimens from suspect patients of having acute hepatitis were collected to detect antibodies to hepatitis A (total and IgM), hepatitis B (anti-HBs, total anti-HBc and IgM anti-HBc), and hepatitis C (anti-HCV) using commercially available enzyme-linked immunossorbent assay (EIA). In relation to serum samples, oral fluid assay sensitivity and specificity were as follows: 87 and 100% for total anti-HAV, 79 and 100% for anti-HAVIgM, 6 and 95% for anti-HBs, 13 and 100%for total anti-HBc, 100 and 100% for anti-HBc IgM, and 75 and 100% for anti-HCV The consistency observed between antibodies tests in saliva and expected risk factors for hepatitis A and C suggests that the saliva method could replace serum in epidemiological studies for hepatitis A and C.

Hepatitis E virus in swine and effluent samples from slaughterhouses in Brazil.

Hepatitis E is an infectious disease which virus (HEV) is highly disseminated in swine herd populations. Sporadic acute human hepatitis E cases have been associated to genotype 3 and 4 strains of HEV also reported in swine populations of endemic and non-endemic areas. With the aim to evaluate the incidence of animals with current infection of HEV, 115 bile samples were collected from three slaughterhouses under inspection by Animal Sanitary Protection Agency of Rio de Janeiro, Brazil. In parallel, effluent samples were collected from six sewage pipe exit sites of two slaughterhouses. HEV RNA was detected in 11 out of 115 (9.6%) bile samples collected and three waste samples from one slaughterhouse. Viral loads observed for bile samples varied from 10(1)-10(5) genome copies/mL and for effluent samples mean load was 10(2) genome copies/mL. Sequencing and phylogenetic analysis classified samples within genotype 3 subtype 3b closely related to the sample obtained from the first reported autochthonous human case and samples from swine of commercial herds in Brazil. Our data demonstrates that although most animals achieve slaughter age (around 20 weeks old) already immune to HEV, a significant number of animals are with current infection at commercial age. Further studies should be addressed to consider risk analysis and possible evaluation of inspection regulations considering food safety measures regarding hepatitis E zoonotic aspect in Brazil.

Investigação de acidentes biológicos entre profissionais de saúde

Resumen 1Aluna do Curso de Pós-Graduação em Análises Clínicas, Faculdade Redentor, Itaperuna, RJ. Bióloga, Laboratório de Referência Regional de Campos dos Goytacazes, Hospital Geral de Guarus, Campos dos Goytacazes, RJ. Brasil. E-mail: jazevedo_silva@hotmail.com, 2 Pesquisadora em Saúde Pública, Doutora em Ciências. Laboratório de Desenvolvimento Tecnológico em Virologia, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ. Brasil. E-mail: vdepaula@ioc.fiocruz.br, 3 Professor Adjunto, Doutor em Ciências. Setor de Hematologia da Clínica Médica “B” do Hospital Universitário Gaffrée e Guinle, Escola de Medicina e Cirurgia, UNIRIO, Rio de Janeiro. Brasil. E-mail:adilsonjoal@ig.com.br, 4 Tecnologista em Saúde Pública, Doutora em Ciências. Laboratório de Hepatites Virais, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ. Brasil. E-mail: lvillar@ioc.fiocruz.br PESQUISA RESEARCH INVESTIGACIÓN The aims of this study were to identify the major professional category exposed to biological risk and the principal type of accident occurred among health care workers in Campos dos Goytacazes, RJ. Based on the analysis of 183 records filled out by the workers who suffered biohazardous accidents between January 2005 and September 2005, we found the nursing auxiliaries and technicians as the professional category more exposed to biohazardous accidents (54.1%), followed by undergraduate medical and dental students (10.4%). The occurence of acidents with piercing-cutting materials was related to frequent handling of these instruments, and to the behavior of workers who maintain practices providing risks of needlestick injuries, such as inappropriate disposal of piercing-cutting materials.Os objetivos deste trabalho foram identificar a principal categoria profissional exposta a risco biologico e os principais tipos de acidentes ocorridos entre trabalhadores da area de saude, em Campos dos Goytacazes, RJ. A partir da analise das fichas de notificacao de acidentes biologicos dos 183 profissionais acidentados entre janeiro de 2005 e setembro de 2005, observamos que a categoria profissional mais exposta foi a dos auxiliares/tecnicos de enfermagem (54,1%), seguida pela dos academicos de medicina e odontologia (10,4%). A ocorrencia de acidentes com materiais perfurocortantes foi relacionada a manipulacao frequente desses objetos e ao comportamento dos profissionais que utilizam praticas que oferecem riscos de acidentes com agulhas, tais como o descarte inadequado de objetos perfurocortantes.

Hepatitis A Outbreak in a Public School in Rio de Janeiro, Brazil

From June 1 to July 1 1999, an outbreak involving 25 cases of hepatitis A occurred in a public school in Rio de Janeiro, Brazil. Since these cases were notified to the State Health Department, the National Reference Center for Hepatitis Viruses (CNRHV) was required to investigate the extent of hepatitis A virus (HAV) dissemination. Blood samples from all students were tested for IgM and total anti-HAV antibodies using a commercial enzyme-linked immunoassay (ELISA). At the same time, a questionnaire was completed in order to identify possible risk factors for HAV infection. The environmental investigation showed that there was no fecal contamination of the water supply. The epidemiological investigation demonstrated that almost 50% of this population was susceptible to HAV infection and probably person-to-person transmission was the principal mode of virus dissemination. In this situation, a massive vaccination campaign could control the HAV infection.

Mixed infection of a child care provider with hepatitis A virus isolates from subgenotypes IA and IB revealed by heteroduplex mobility assay.

Phylogenetic analysis based on a 168 base segment encompassing the putative VP1/2A junction of the hepatitis A virus (HAV) genome has enabled the classification of HAV isolates into seven genotypes (I-VII). Genotype I, which includes the vast majority of the human HAV isolates, has been divided further into subgenotypes IA and IB. An heteroduplex mobility assay was designed with amplification products from the VP1/2A junction region, and used as a genotyping method able to discriminate HAV isolates belonging to IA, IB and non-I genotypes. The method was used to successfully genotype 48 samples (16 IA and 32 IB). However, one HAV RNA positive serum sample (AUX-23), collected from a 15 year old female employed at a child care center located in Rio de Janeiro, Brazil, showed an unusual pattern. PCR products from sample AUX-23 gave rise to heteroduplex bands when mixed with IA products as well as with IB products, suggesting the presence of HAV isolates from both subgenotypes in the serum. PCR products from sample AUX-23 were then cloned and 20 clones were analyzed by heteroduplex mobility assay. Eleven were subgenotype IA and 9 were IB. Three clones of each subgenotype were then sequenced to confirm the results. These data constitute the first report of mixed infection of a single individual with different HAV isolates.

Experimental hepatitis A virus (HAV) infection in cynomolgus monkeys (Macaca fascicularis): evidence of active extrahepatic site of HAV replication

This work studied the replication sites of hepatitis A virus (HAV) in cynomolgus monkeys (Macaca fascicularis) after intravenous inoculation. The cynomolgus monkeys were inoculated with the Brazilian hepatitis A virus strain (HAF‐203). Monkeys were euthanized on days 15, 30, 45 and 60 postinoculation (pi). Liver samples, submandibular salivary gland, mesenteric lymph node and tonsils were removed for virological and pathological evaluation. Immunofluorescence analyses on liver and salivary gland sections using confocal laser scanning microscopy revealed the presence of HAV antigen (HAV Ag). The presence of HAV genome was monitored by real‐time PCR. The HAV RNA was detected at 7 days postinoculation (dpi), concomitantly in serum, saliva and faeces. The highest HAV viral load was observed in faeces at 15 dpi (105 copies/ml), followed by serum viral load of 104 copies/ml at 20 dpi and saliva viral load of 103 copies/ml at 7 dpi. The animals showed first histological and biochemical signs of hepatitis at 15 dpi. The HAV antigen (Ag) was present from day 7 until day 60 pi in the liver and salivary glands. The HAV replicative intermediate was also detected in the liver (4.5 × 104 copies/mg), salivary glands (1.9 × 103 copies/mg), tonsils (4.2 × 101 copies/mg) and lymph nodes (3.4 × 101 copies/mg). Our data demonstrated that the salivary gland as an extrahepatic site of early HAV replication could create a potential risk of saliva transmitted infection. In addition, the cynomolgus monkey was confirmed as a suitable model to study the pathogenesis of HAV human infection.

Applied biotechnology for production of immunoglobulin Y specific to hepatitis A virus

A new protocol for producing polyclonal antibody against hepatitis A virus (HAV) is described. Twenty hens were immunized three times with a commercial HAV vaccine and HAV from a cell culture with three types of adjuvants: CpG oligodeoxynucleotides (CpG-ODN), incomplete Freunds adjuvant and an alum adjuvant. In each of the last two booster inoculations, blood from the birds was collected and tested for HAV antibodies. Egg yolk was separated from egg white and immunoglobulin Y (IgY) antibody was then purified by polyethylene glycol 6000. The mean yield of total protein in yolk was 22.62 mg/mL. Specific activity of the antibody was tested using commercial ELISA, Western blotting, and in vitro neutralization assay demonstrating that anti-HAV IgY bound specifically. After the first immunization, birds immunized with HAV from cell culture plus incomplete Freunds adjuvant with/without CpG-ODN showed highest levels of anti-HAV IgY in serum (p<0.05). Viral combination with CpG-ODN resulted in early response of anti-HAV serum in hens, reflecting the amount of IgY transferred to the egg yolk (p<0.05). The results suggest that egg yolk may be a large scale source of specific antibodies against hepatitis A virus. Further applications of this method have yet to be tested.