Klaus E. Stewien

Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a “flip-flop” phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Clinical patterns and seasonal trends in respiratory syncytial virus hospitalizations in São Paulo, Brazil

The respiratory viruses are recognized as the most frequent lower respiratory tract pathogens for infants and young children in developed countries but less is known for developing populations. The authors conducted a prospective study to evaluate the occurrence, clinical patterns, and seasonal trends of viral infections among hospitalized children with lower respiratory tract disease (Group A). The presence of respiratory viruses in childrens nasopharyngeal was assessed at admission in a pediatric ward. Cell cultures and immunofluorescence assays were used for viral identification. Complementary tests included blood and pleural cultures conducted for bacterial investigation. Clinical data and radiological exams were recorded at admission and throughout the hospitalization period. To better evaluate the results, a non- respiratory group of patients (Group B) was also constituted for comparison. Starting in February 1995, during a period of 18 months, 414 children were included- 239 in Group A and 175 in Group B. In Group A, 111 children (46.4%) had 114 viruses detected while only 5 children (2.9%) presented viruses in Group B. Respiratory Syncytial Virus was detected in 100 children from Group A (41.8%), Adenovirus in 11 (4.6%), Influenza A virus in 2 (0.8%), and Parainfluenza virus in one child (0.4%). In Group A, aerobic bacteria were found in 14 cases (5.8%). Respiratory Syncytial Virus was associated to other viruses and/or bacteria in six cases. There were two seasonal trends for Respiratory Syncytial Virus cases, which peaked in May and June. All children affected by the virus were younger than 3 years of age, mostly less than one year old. Episodic diffuse bronchial commitment and/or focal alveolar condensation were the clinical patterns more often associated to Respiratory Syncytial Virus cases. All children from Group A survived. In conclusion, it was observed that Respiratory Syncytial Virus was the most frequent pathogen found in hospitalized children admitted for severe respiratory diseases. Affected children were predominantly infants and boys presenting bronchiolitis and focal pneumonias. Similarly to what occurs in other subtropical regions, the virus outbreaks peak in the fall and their occurrence extends to the winter, which parallels an increase in hospital admissions due to respiratory diseases.

Epidemiology and genetic variability of human metapneumovirus during a 4-year-long study in Southeastern Brazil.

Epidemiological and molecular characteristics of human metapneumovirus (hMPV) were compared with human respiratory syncytial virus (hRSV) in infants and young children admitted for acute lower respiratory tract infections in a prospective study during four consecutive years in subtropical Brazil. GeneScan polymerase chain assays (GeneScan RT‐PCR) were used to detect hMPV and hRSV in nasopharyngeal aspirates of 1,670 children during January 2003 to December 2006. hMPV and hRSV were detected, respectively, in 191 (11.4%) and in 702 (42%) of the children admitted with acute lower respiratory tract infections at the Sao Paulo University Hospital. Sequencing data of the hMPV F gene revealed that two groups of the virus, each divided into two subgroups, co‐circulated during three consecutive years. It was also shown that a clear dominance of genotype B1 occurred during the years 2004 and 2005, followed by genotype A2 during 2006. J. Med. Virol. 81:915–921, 2009.

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.

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. J. Med. Virol. 61:143–149, 2000.

The influence of maternally derived antibody on the efficacy of further attenuated measles vaccine.

SummaryThe natural decline of passively acquired maternal antibody titers and the influence of these antibodies on the efficacy of live, further attenuated measles vaccine were studied in 7 to 12 month old infants. Hemagglutination-inhibition (HI) antibody titers were determined in the pre- and postvaccination sera. HI antibodies at low titers were detected in 20 (46%) of the 43 infants investigated, in five of six infants at seven months and two of nine infants at 12 months of age. The serological conversion rate following vaccination of the infants exhibiting transplacental maternal antibody was as low as 38%, in contrast with 100% sero-conversion of the infants without measurable antibodies of maternal origin. HI antibody titers were low in the infants seven to nine months of age, reflecting poor antibody responses to the administered vaccine. For the 12 month old infants the geometrical mean antibody titer was 1 : 52. It is concluded that transplacental maternal antibody, even at low concentration, inhibits induction of HI antibody in the vaccinees and therefore measles vaccination must be initiated after 12 months of age in order to achieve successful immunization of the children. Infants who receive the vaccine before their first birthday have to be revaccinated at or after 15 months of age.ZusammenfassungDie Abnahme passiv erworbener mütterlicher Antikörpertiter und der Einfluß dieser Antikörper auf die Wirksamkeit attenuierter Masern-Lebendvakzine wurde an 7–12 Monate alten Kleinkindern untersucht. Im Hämagglutinations-hemmungstest (HAH) wurde die Antikörpertiter im Serum vor und nach der Impfung bestimmt. Bei 20 (= 46%) der 43 untersuchten Kleinkinder wurden niedrige Antikörpertiter festgestellt, d. h. bei fünf von sechs Kleinkindern im Alter von 7 Monaten und bei zwei von neun Kleinkindern im Alter von 12 Monaten. Die serologische Konversionsrate der Kleinkinder mit diaplazentarem mütterlichem Antikörper betrug nach der Impfung ganze 38% im Gegensatz zur 100%igen Serumkonversion der Kleinkinder ohne meßbare mütterliche Antikörper. Die HAH-Antikörpertiter waren bei den 7–9 Monate alten Kleinkindern niedrig und spiegelten eine mangelhafte Antikörperreaktion auf den verabreichten Impfstoff wider. Bei den 12 Monate alten Kleinkindern betrug der geometrische mittlere Antikörpertiter 1 : 52. Es wird gefolgert, daß der mütterliche Antikörper selbst in niedriger Konzentration die Bildung des HAH-Antikörpers bei den Geimpften hemmt und demzufolge die Masernimpfung zur Erzielung einer erfolgreichen Immunisierung nach dem 12. Lebensmonat vorgenommen werden muß. Kleinkinder, die den Impfstoff vor Vollendung des ersten Lebensjahres erhielten, müssen im oder nach dem 15. Lebensmonat erneut geimpft werden.

Viral infections in hospitalized children affected by acute lower respiratory tract disease

OBJECTIVE: To evaluate the frequency of the main respiratory viruses in hospitalized children affected by acute lower respiratory tract disease at a university hospital. METHODS: This is a prospective trial that included two cohorts of hospitalized children in the period from April to July 1996. The groups were selected according to the presence of lower respiratory tract disease on admission: Group A- with acute disease (history of less than 7 days) and B- without present or recent respiratory disease. The parameters for defining lower respiratory tract disease included physical and/or radiological pulmonary changes. Clinical and radiological criteria were established for the classification of lower respiratory tract diseases in group A. Nasopharyngeal swab was collected from all children on admission for viral detection by cellular cultures and direct immunofluorescence. RESULTS: 201 cases were selected, 126 in group A and 75 in group B. Viruses were identified in 71 children from groupA(56.4%) and only in 3 from group B (4.0%). The predominant agent in group A was respiratory syncytial virus, identified in 66 cases; adenovirus (4) and influenza (1) were detected in other patients. In group B two patients with respiratory syncytial virus and one with adenovirus were identified. The patients from group A affected by respiratory syncytial virus were younger (median age 3 months versus 13 months) and more wheezy on physical examination (78.7%) than the other patients of the group (33.3%). This virus was associated to most of the bronchiolitis cases (84%) and to half of the pneumonia cases (46.4%). CONCLUSION: The authors found a significant presence of viruses in the majority of children hospitalized with acute lower respiratory tract disease. The respiratory syncytial virus was the predominant agent identified. These results are similar to others previously reported both in developed and some developing countries. The authors emphasize that the present study evaluated only partially the possibility of simultaneous infection by other pathogens and that the present protocol was conducted during the season with the highest incidence of respiratory syncytial virus.

Ocorrência de astrovírus humanos na cidade de São Paulo, Brasil

Foram detectados astrovirus humanos durante estudo longitudinal de 13 meses sobre a incidencia de virus diarreicos em 146 criancas menores de 2 anos de idade, hospitalizadas em clinica pediatrica de um hospital universitario, na cidade de Sao Paulo, SP, Brasil. Das 67 criancas internadas com diarreia aguda, 3% foram positivas para astrovirus, por ocasiao de sua admissao, pelo Ensaio Imunoenzimatico Monoclonal Amplificado (ASTROVIRUS BIOTIN-AVIDIN ELISA, CDC, USA). As 79 criancas sem diarreia, admitidas durante o mesmo periodo por outra causa (controles), foram negativas para astrovirus, por ocasiao de seu internamento. Entretanto, 4,8% do total de criancas hospitalizadas sofreram infeccoes por astrovirus durante sua permanencia no hospital. Este e o primeiro estudo sobre a ocorrencia de astrovirus humanos no Brasil, que assim participam significativamente na etiologia da gastroenterite infantil em nosso meio.

Rotavirus associated diarrhoea during infancy in the city of S. Luís (MA), Brazil: a two-year longitudinal study

A total of 479 diarrhoeic children and 337 children without diarrhoea (controls) less than 5 years old were investigated in a two-year study in the city of S. Luís (MA), with the purpose to determine the incidence, the age distribution and the seasonality of rotaviruses, as well as to establish the severity of the disease in this region between the North and the Northeast of Brazil. rotavirus incidence was highest in children of the 1st. year of life, showing an average of 25% per year among the diarrhoeic patients attending the two main hospitals and three health units at the periphery of the city. It was shown that rotaviruses are significant enteropathogens in children less than 18 months old. Frequency of rotaviruses dropped in diarrhoeic patients 18 to 23 months old to only 4%, the same percentage observed in children of the control group. A typical seasonal distribution of rotaviruses was not seen during the two years of study. There was a peak in the incidence of rotaviruses in 1986, during the rainy season, and two peaks in 1987, one in the rainy season and one in the dry season. It was also shown that severity of diarrhoea in rotavirus positive cases was higher than in the negative cases. Rotavirus diarrhoeic patients had more loose stools per day, and higher frequencies of vomiting and fever, resulting more often (> 2 times) in moderate or severe dehydration. Finally, it is concluded that the introduction of immunoprophylaxis may reduce significantly the high mortality rates in early childhood observed in S. Luís.

Estado imunitário relativo à poliomielite das crianças de 0-12 anos, residentes no município de São Paulo, Brasil e assistidas pelo Hospital Menino Jesus

A prevalencia dos anticorpos neutralizantes contra os tres tipos de poliovirus e os niveis de imunidade para diferentes grupos etarios foram determinados, atraves de um inquerito soro-epidemiologico, numa populacao de criancas de 0-12 anos de idade, residentes no municipio de Sao Paulo, Brasil e assistidas pelo Hospital Menino Jesus. Os resultados mostraram um numero elevado de criancas suscetiveis a infeccao por poliovirus no primeiro ano de vida, particularmente no grupo etario de 9-12 meses, em que a proporcao de criancas completamente desprotegidas (triplo-suscetiveis) alcancou 42,5%. Neste grupo, a prevalencia de anticorpos dos tipos 1, 2 e 3 foi apenas em torno de 40%. Dentre as criancas do grupo etario de 0-5 anos, que receberam tres ou mais doses de vacina oral trivalente, verificou-se a baixa proporcao de 60% de duplo mais triplo-imunes. Os resultados mostraram que o estado imunitario das criancas deste grupo foi o mesmo nas tres zonas geograficas da Capital, sendo em torno de apenas 50% a proporcao de criancas duplo mais triplo-imunes. Estes resultados indicam niveis precarios de imunidade, particularmente nas criancas do primeiro ano de vida. Existe, pois, uma necessidade evidente de realizar novos inqueritos sorologicos, alem de intensificar e melhorar a vacinacao de manutencao contra a poliomielite em nosso meio.A prevalencia dos anticorpos neutralizantes contra os tres tipos de poliovirus e os niveis de imunidade para diferentes grupos etarios foram determinados, atraves de um inquerito soro-epidemiologico, numa populacao de criancas de 0-12 anos de idade, residentes no municipio de Sao Paulo, Brasil e assistidas pelo Hospital Menino Jesus. Os resultados mostraram um numero elevado de criancas suscetiveis a infeccao por poliovirus no primeiro ano de vida, particularmente no grupo etario de 9-12 meses, em que a proporcao de criancas completamente desprotegidas (triplo-suscetiveis) alcancou 42,5%. Neste grupo, a prevalencia de anticorpos dos tipos 1, 2 e 3 foi apenas em torno de 40%. Dentre as criancas do grupo etario de 0-5 anos, que receberam tres ou mais doses de vacina oral trivalente, verificou-se a baixa proporcao de 60% de duplo mais triplo-imunes. Os resultados mostraram que o estado imunitario das criancas deste grupo foi o mesmo nas tres zonas geograficas da Capital, sendo em torno de apenas 50% a proporcao de criancas duplo mais triplo-imunes. Estes resultados indicam niveis precarios de imunidade, particularmente nas criancas do primeiro ano de vida. Existe, pois, uma necessidade evidente de realizar novos inqueritos sorologicos, alem de intensificar e melhorar a vacinacao de manutencao contra a poliomielite em nosso meio.