Adenoviruses and acute respiratory infections in children living in an equatorial area of Brazil.

Human adenoviruses (HAdVs) are important respiratory pathogens, found in 2-27% of acute respiratory infection (ARI) cases. Few studies have analysed the diversity of species and types of HAdVs associated with ARI in Brazil. The purpose of this study was to determine the circulation patterns of the different HAdV species and respective types associated with ARI in children in the city of Fortaleza, northeastern Brazil. HAdVs were screened by an indirect immunofluorescence assay, and subsequently identified as species and types by PCR and sequencing of the hexon gene (HVR1-HVR6). Between 2001 and 2013, a total of 290 HAdV strains were detected, 190 of which were identified as belonging to the HAdV-B, -C and -E species. Seven types of HAdVs circulated in the studied population during the analysed period, with HAdV-3 being predominant.

Electropositive filter membrane as an alternative for the elimination of PCR inhibitors from sewage and water samples.

PCR is the best method for the detection of enteric viruses present at low concentrations in environmental samples. However, some organic and inorganic compounds present in these samples can interfere in the reaction. Many of these substances are cytotoxic, too. The ZP60S filter membranes used in addition to fluorpentane treatment are quite efficient for virus concentration and simultaneous elimination of cytotoxicity from environmental samples. In this study, both procedures were used to promote the elimination of reverse transcriptase PCR (RT-PCR) inhibitors from sewage and sewage-polluted creek water. Samples were subjected separately to each of the following procedures: filtration through electropositive filter membranes (ZP60S), organic extraction with Vertrel XF, and filtration through ZP60S followed by organic extraction. Afterwards, aliquots were experimentally inoculated with rotavirus SA-11 RNA and subjected to RT-seminested PCR for amplification of the VP7 gene. Results showed that the ZP60S membranes efficiently eliminated the RT-PCR inhibitors from water samples. The sample processing method was also applied to 31 in natural sewage and creek water samples for detection of naturally occurring rotavirus. A duplex seminested PCR was used for the quick detection of couples of the four rotavirus genotypes (G1 to G4). Eight samples (25.8%) were positive, and rotavirus sequences were not detected in 23 (74.2%). Results were confirmed by direct immunoperoxidase method. In summary, the use of electropositive filter membrane is appropriate for the elimination of substances that can interfere with RT-PCR, obviating additional sample purification methods.

Factors that can interfere with virus concentration from wastewater when using zeta plus 60S filter membranes

Zeta plus filter membranes (ZP60S) have been shown to be efficient for rotavirus concentration from wastewater and for the reduction of cytotoxicity for cell cultures. Recently a variability in both properties was observed. In view of the low costs and the high virus recovery rates obtained in the past, we re-evaluated the application of ZP60S filter membranes for virus concentration from environmental samples. Some factors that could interfere with the concentration strategy using ZP60S were also considered and assessed including the type of water to be filtered and the possible release of toxic substances from the membrane matrix during filtration.

Factors that can interfere with virus concentration from wastewater when using Zeta Plus 60S filter membranes.

Zeta plus filter membranes (ZP60S) have been shown to be efficient for rotavirus concentration from wastewater and for the reduction of cytotoxicity for cell cultures. Recently a variability in both properties was observed. In view of the low costs and the high virus recovery rates obtained in the past, we re-evaluated the application of ZP60S filter membranes for virus concentration from environmental samples. Some factors that could interfere with the concentration strategy using ZP60S were also considered and assessed including the type of water to be filtered and the possible release of toxic substances from the membrane matrix during filtration.

Genetic characterization of adenovirus strains isolated from patients with acute conjunctivitis in the city of São Paulo, Brazil.

Genome analysis was carried out on adenovirus strains isolated from patients with acute follicular conjunctivitis in the city of São Paulo, Brazil. Eighteen conjunctival scrapings, collected between December 1993 and March 1994, were analyzed by two methods: a combination of polymerase chain reaction with restriction fragment length polymorphism and viral DNA restriction analysis, carried out using 10 restriction endonucleases: BamHI, BglI, BglII, HindIII, KpnI, SacI, SalI, SmaI, XbaI, and XhoI. Among 11 adenovirus detected by cell culture isolation, nine were Ad8, and two were Ad7. By restriction analysis the Ad8 isolates were typed as two new variants-Ad8/D11 (seven of nine samples) and Ad8/D12 (two of nine samples). Ad7 isolates were identified as a subtype of the widespread genome type Ad7b and the virulent type Ad7h, a predominant genome type circulating in Argentina, Chile, and Uruguay but absent in Brazil until 1991.

Detection of rotavirus in sewage and creek water: efficiency of the concentration method

Simian rotavirus SA-11, experimentally seede, was recovered from raw domestic sewage by a two-step concentration procedure, using filtration through a positively charged microporous filter (Zeta Plus 60 S) followed by ultracentrifugation, effecting an 8000-fold concentration. By this method, a mean recovery of 81 per centñ7.5 of the SA-11 virus was achieved.

Detection of rotavirus in sewage and creek water: efficiency of the concentration method.

Simian rotavirus SA-11 experimentally seeded, was recovered from raw domestic sewage by a two-step concentration procedure, using filtration through a positively charged microporous filter (Zeta Plus 60 S) followed by ultracentrifugation, effecting an 8,000-fold concentration. By this method, a mean recovery of 81% +/- 7.5 of the SA-11 virus, was achieved.

Humoral immunity patterns based on antibody reactivity to rotavirus antigens in Brazilian children under 5 years of age.

The age distribution of antibody to simian rotavirus (SA-11) was studied in serum specimens obtained from 399 children aged to 5 years and living in the city of Recife (PE), located in the north eastern region of Brazil. Sera were examined for group-specific rotavirus antibody using a blocking enzyme immunoassay (bELISA) and a hemagglutination inhibition antibody (HIA) test, and for anti-VP2, anti-VP4, anti-VP6, and anti-VP7 antibodies using an immunoblotting assay (IBA). Antibody prevalence was similar in all bELISA and HIA assays, showing a steep rise in the 6-to 17-month-old age groups. The results indicate early acquisition of antibody to rotavirus. The majority of children aged 2 to 4 years had bELISA (50% to 60%) and HIA (70% to 81%) antibodies. There was an association in prevalence data obtained by HIA and bELISA with immunoblotting (IBA), revealing four serologic profiles. Children with profiles I and II (60%) respectively had HAI and ELISA antibody or HAI antibody alone and all had immunoprotective antibodies to VP4 and/or VP7. These children were regarded as "immune," resembling convalescent patients with a rotavirus infection. Children with profile III (4%) had no HIA antibody and only non-protective anti-VP6 and/or VP7 antibody, and were considered to be "partially immune." Children with profile IV (36%) had no detectable antibody and were classified as "nonimmune." These children should be considered to be susceptible to rotavirus infection, with the risk of developing clinically severe diarrhea.

Adenovirus genome types isolated from stools of children with gastroenteritis in São Paulo, Brazil.

In a prospective one-year study of acute gastroenteritis in hospitalized children less than 2 years of age, in São Paulo (Brazil), adenoviruses were detected by specific enzyme immunoassay (El-ARA) in 7 of 67 (10%) ill children and in 9 of 79 (11.4%) controls. They were the sole recognizable agent of diarrhea in 6 ill children. In another child these viruses were detected in a dual infection with astrovirus. Enteric adenoviruses (Ad40/41) were the most common serotypes detected in children with diarrhea (3/7) and Ad7 the serotype most detected in the controls (5/9), associated with lower respiratory tract infection. Thirteen adenovirus strains, isolated in HEp2 or HEK-293 cells, were characterized by seroneutralization and restriction enzyme analysis. The established adenoviruses were typed as AV-7-D5 (five associated to lower respiratory tract infection and one to diarrhea), AV-1-D10 (one diarrhea case), AV-31-D2 (two controls with respiratory infection), and two isolates as AV-12-D7, a new genome type. One subgenus D isolate, serotype 28, with restriction patterns different from those of the prototype, remained untyped. Only one enteric adenovirus could be typed. The restriction patterns of this isolated were similar to those of the prototype AV-41-D1. The genome type of the other three enteric adenoviruses could not be determined.

Serotypes and electropherotypes of human rotavirus detected in the city of São Luís (MA), Brazil.

A total of 22 (65%) of 34 representative rotavirus-positive specimens from infants with acute gastroenteritis were electropherotyped (RNA genome segments) and serotyped using an enzyme immunoassay with monoclonal antibodies (ELISA with MAbs). Serotype 3 was predominant during the 10-month study period (41%), followed by serotype 1 (27%) and serotype 4 (9%). Serotype 2 was not found. Rotavirus strains were grouped into 3 major electropherotypes designated V, W and Z, each corresponding to a single serotype, i.e., serotypes 1, 3 and 4, respectively. Three strains that could not be typed by the serologic technique showed the W electrophoretic profile. The relative migration of the gene segments 7-9 was the main feature distinguishing the predominant serotype 3 from the other serotypes. The migration of segments 2 and 5 was also important for differentiating serotype 4 strains. The present study strengthens the view that electropherotyping, when used in conjunction with serotyping, can help characterize atypical and unusual strains, as well as rotaviruses that cannot be typed by the serologic technique.

Serotypes and electropherotypes of human rotavirus detected in the city of São Luís (MA), Brazil

A total of 22 (65) of 34 representative rotavirus-positive specimens from infants with acute gastroenteritis were electropherotyped (RNA genome segments) and serotyped using an enzyme immunoassay with monoclonal antibodies (ELISA with MAbs). Serotype 3 was predominant during the 10-month study period (41), followed by serotype 1 (27) and serotype 4 (9). Serotype 2 was not found. Rotavirus strains were grouped into 3 major electropherotypes designated V, W and Z, each corresponding to a single serotype, i.e., serotypes 1, 3 and 4, respectively. Three strains that could not be typed by the serologic technique showed the W electrophoretic profile. The relative migration of the gene segments 7-9 was the main feature distinguishing the predominant serotype 3 from the other serotypes. The migration of segments 2 and 5 was also important for differentiating serotype 4 strains. The present study strengthens the view that electropherotyping, when used in conjunction with serotyping, can help characterize atypical and unusual strains, as well as rotaviruses that cannot be typed by the serologic technique.

Viral, bacterial and parasitic pathogens associated with severe diarrhoea in the city of São Paulo, Brazil.

Viral, bacterial and parasitic pathogens associated with diarrhoea were studied in infants and young children admitted to the paediatric clinic of the University Hospital of São Paulo during the period of 13 months. A recognised pathogenic organism was detected in 78% of the diarrhoeic patients, 6% of whom had a mixed infection with two agents. Enteropathogenic E. coli (EPEC) was the most common enteropathogen detected (22%), followed by rotavirus (19%) and adenovirus (10%). Altogether 6% had diarrhoea associated with Salmonella or Shigella and 3% showed diarrhoeal illness associated with astrovirus. Infants less than 6 months of age were most commonly infected with enterobacteria (35%), mainly enteropathogenic E. coli (30%), whereas children 6 months to 2 years presented more often with viruses (38%), mainly rotaviruses (25%). Enteropathogens were found during all seasons of the year and rotaviruses showed a seasonal variation.