Edison Luiz Durigon

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.

Molecular characterization of canine parvovirus in Brazil by polymerase chain reaction assay.

Canine parvovirus (CPV) was first isolated in 1978 in the USA. Analysis of CPV isolates by monoclonal antibodies and restriction enzymes have shown that after the first emergence of CPV (CPV-2) it evolved to give rise to new antigenic types, which were designated CPV type 2a and type 2b. These new types have replaced the original CPV type 2, although the proportions of each of the new antigenic types vary in different countries. In Brazil, CPV-like infections were first observed in 1979, however, there has been no information concerning the antigenic types of CPV prevailing in South America. In this study, we designed a PCR assay to type canine parvovirus strains in fecal samples collected from symptomatic dogs during 1980 through 1986 and 1990 through 1995. Our data showed that the CPV epizootic in Brazil followed the same pattern observed in the USA of emergence of CPV-2 followed by replacement by the variants CPV-2a and 2b. The predominant strain found during 1980 was CPV-2a, which was substantially replaced by CPV-2b from 1990 to 1995.

Surveillance of eight respiratory viruses in clinical samples of pediatric patients in Southeast Brazil

OBJECTIVE Detection of the eight most common respiratory viruses: human respiratory syncytial virus (HRSV), influenza virus A and B (IA and IB), parainfluenza viruses 1, 2 and 3 (HPIV1, 2 and 3), adenovirus (Ad) and human metapneumovirus (HMPV), in order to establish the etiology of acute respiratory infections (ARIs) and the epidemiology of these viruses in young children seen at Hospital Universitário, Universidade de São Paulo, in São Paulo, Brazil, during 2003. METHODS The epidemiological surveillance was conducted in all children younger than 5 years hospitalized at the Hospital for lower respiratory tract infections (LRTI) from January 1, 2003 to December 30, 2003. Nasal and throat samples were scanned for respiratory viruses by polymerase chain reaction and detected by the GeneScan assay. RESULTS Of 336 samples collected from 336 patients, 187 (55.6%) were positive for at least one of the respiratory viruses studied. Of all the children, HRSV was identified in 24.1%, HMPV in 17.8%, HPIV3 in 8.3%, Ad in 6.8%, IA in 5%, HPIV1 in 0.6%, but no virus could be detected in 44.1%. Dual virus infections were detected in 7.1% of all samples (12.8% of positive samples). HPIV2 and IB were not detected in the present study. CONCLUSIONS This study confirms that children younger than 5 years and particularly younger than 1 year have a high hospitalization rate due to HRSV, HMPV, HPIV, influenza and adenovirus. We were able to determine the etiology and epidemiology of most ARIs and trace the seasonal profile of the commonest respiratory viruses among young children.

Detection of porcine parvovirus DNA by the polymerase chain reaction assay using primers to the highly conserved nonstructural protein gene, NS-1

Porcine parvovirus (PPV) infection is associated with reproductive losses in swine and its causative agent, the PPV, has been isolated worldwide. Serological surveys and virus isolation studies throughout Brazil confirm the occurrence of PPV infection in this country. The most common methods to detect PPV infection are fluorescent antibody staining of fetal tissues, hemagglutination assay of tissue extracts and virus isolation from fetal tissues. Non-specificity and low sensitivity are the major drawbacks of these techniques. The development of a polymerase chain reaction (PCR) and nested-PCR assays for PPV DNA detection from infected cell lines and clinical samples is described. The primers were designed to a highly conserved region of the PPV genome which codes for the non-structural protein, NS-1. Results showed that PCR could detect PPV in titres at least 10(6) higher than the hemagglutination assay. The PCR and nested-PCR assays were used to detect successfully PPV DNA in clinical samples.

Vigilância de oito vírus respiratórios em amostras clínicas de pacientes pediátricos no sudeste do Brasil

OBJECTIVE: Detection of the eight most common respiratory viruses: Human respiratory syncytial virus (HRSV), influenza virus A and B (IA and IB), parainfluenza viruses 1, 2 and 3 (HPIV1, 2 and 3), adenovirus (Ad) and human metapneumovirus (HMPV), in order to establish the etiology of acute respiratory infections (ARIs) and the epidemiology of these viruses in young children seen at Hospital Universitario, Universidade de Sao Paulo, in Sao Paulo, Brazil, during 2003. METHODS: The epidemiological surveillance was conducted in all children younger than 5 years hospitalized at the Hospital for lower respiratory tract infections (LRTI) from January 1, 2003 to December 30, 2003. Nasal and throat samples were scanned for respiratory viruses by polymerase chain reaction and detected by the GeneScan assay. RESULTS: Of 336 samples collected from 336 patients, 187 (55.6%) were positive for at least one of the respiratory viruses studied. Of all the children, HRSV was identified in 24.1%, HMPV in 17.8%, HPIV3 in 8.3%, Ad in 6.8%, IA in 5%, HPIV1 in 0.6%, but no virus could be detected in 44.1%. Dual virus infections were detected in 7.1% of all samples (12.8% of positive samples). HPIV2 and IB were not detected in the present study. CONCLUSIONS: This study confirms that children younger than 5 years and particularly younger than 1 year have a high hospitalization rate due to HRSV, HMPV, HPIV, influenza and adenovirus. We were able to determine the etiology and epidemiology of most ARIs and trace the seasonal profile of the commonest respiratory viruses among young children.

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.

Isolation of Rickettsia felis in the mosquito cell line C6/36.

ABSTRACT We report the isolation and establishment of Rickettsia felis in the C6/36 cell line. Rickettsial growth was intense, always with 90 to 100% of cells being infected after few weeks. The rickettsial isolate was confirmed by testing infected cells by PCR and sequencing fragments of three major Rickettsia genes (gltA, ompB, and the 17-kDa protein gene).

Severity of viral coinfection in hospitalized infants with respiratory syncytial virus infection

OBJECTIVE To compare the severity of single respiratory syncytial virus (RSV) infections with that of coinfections. METHODS A historical cohort was studied, including hospitalized infants with acute RSV infection. Nasopharyngeal aspirate samples were collected from all patients to detect eight respiratory viruses using molecular biology techniques. The following outcomes were analyzed: duration of hospitalization and of oxygen therapy, intensive care unit admission and need of mechanical ventilation. Results were adjusted for confounding factors (prematurity, age and breastfeeding). RESULTS A hundred and seventy six infants with bronchiolitis and/or pneumonia were included in the study. Their median age was 4.5 months. A hundred and twenty one had single RSV infection and 55 had coinfections (24 RSV + adenovirus, 16 RSV + human metapneumovirus and 15 other less frequent viral associations). The four severity outcomes under study were similar in the group with single RSV infection and in the coinfection groups, independently of what virus was associated with RSV. CONCLUSION Virus coinfections do not seem to affect the prognosis of hospitalized infants with acute RSV infection.

Incidence and clinical characteristics of the infection by the Respiratory Syncytial Virus in children admitted in Santa Casa de São Paulo Hospital

The purpose of this study was to identify the rate of infections due to RSV and other viruses in children. In addition we have analyzed demographic data and clinical characteristics of the RSV-positive patients comparing with patients infected by other respiratory viruses. We also described the seasonality of the RSV occurrence in a hospital in São Paulo. Children below 5 years old admitted in Santa Casa de São Paulo Hospital between February 2005 and September 2006 due to acute respiratory infections (ARI) were included. A nasopharyngeal specimens were obtained with sterile No. 5 French feeding catheters as soon as possible (usually within 24 h). Specimens were kept refrigerated at 4 degrees C and transported to Adolfo Lutz Institute, where the indirect immunofluorescent assay was performed. Virus identified by these assay included RSV, Adenovirus, Influenza A and B virus and Parainfluenza 1, 2, and 3. Clinical data from each group was compared. Four hundred and fifty five cases were included in the study, with 30% positive for some type of virus. Viruses that were identified included Respiratory Syncytial Virus (73.03%), Influenza (8.42%), Parainfluenza (8.42%) and Adenovirus (3.37%). We divided the subjects in 3 groups: Group 1 RSV-Positive, Group 2 Other Positive Viruses and Group 3 Negative for Respiratory Virus. Mean age (months) was of 7.5 for RSV-positive children, 7.6 for other viruses, and 8 for negative for respiratory virus. The RSV-Positive Group was significantly younger than the Group Negative for Respiratory Virus (p<0.05). Signs of UAI were more present in the Positive RSV Group (p<0.05). General mortality was of 2.41%. There was a higher incidence of RSV between the months of March and August in the two years of the study. Our study indicates RSV as the most prevalent viral agent in children admitted due to (ARI), especially in infants below 3 months old. We have also found that infections due to RSV can occur in months others than the classic seasonal period.