Concurrent infection with dengue virus type-2 and DENV-3 in a patient from Ceará, Brazil

Dengue outbreaks have occurred in several regions in Brazil and cocirculating dengue virus type 1 (DENV-1), DENV-2, and DENV-3 have been frequently observed. Dual infection by DENV-2 and DENV-3 was identified by type-specific indirect immunofluorescence assay and confirmed by reverse transcription polymerase chain reaction in a patient in Ceará with a mild disease. This is the first documented case of simultaneous infection with DENV-2 and DENV-3 in Brazil. Sequencing confirmed DENV-2 and DENV-3 (South-East/American) genotype III and (SriLanka/India), genotype III respectively.

Astrovirus infection in children living in the Central West region of Brazil

This study presents data regarding the circulation of astrovirus in Goiânia-GO and Brasília-DF. These viruses were detected in fecal samples from hospitalized children up to five years old with and without acute gastroenteritis. A total of 1244 fecal samples were collected in two periods, 1994 to 1996 (Brasília) and 1998 to 2002 (Goiânia and Brasília), and were analyzed for viral RNA using the reverse transcription-polymerase chain reaction (RT-PCR). Positivity rates of 4.3 and 0.5% for astrovirus were observed in children with acute gastroenteritis and those without gastroenteritis, respectively. Among children with gastroenteritis no statistically significant difference was seen with regards to viral positivity rates in relation to gender and age. However, a higher incidence rate was observed for children from Brasília aged 36 months or more. Overall, astroviruses occurred predominantly from September to March in the two cities, suggesting a seasonal pattern for these viruses which coincides with the highest relative air humidity period. The results of this study highlight the importance of astrovirus as an etiologic agent of acute gastroenteritis in children of the Central West region of Brazil.

Hepatitis A virus subgenotypes dissemination during a community outbreak in a surrounding region of Rio de Janeiro

From December 1999 to December 2001, many cases of hepatitis A were notified in the county of Belford Roxo involving individuals aged 0 to 79 years. Serum samples were collected to evaluate the prevalence of anti-hepatitis A virus (HAV) antibodies, to detect HAV-RNA and to correlate with possible risk factors of HAV infection. Serum samples were screened by commercial IgM and total anti-HAV antibody ELISA and HAV-RNA was isolated and subsequently amplified by reverse transcription-polymerase chain reaction (RT-PCR) at VP1/2A region, sequenced and analyzed. Total anti-HAV prevalence was 87.9% (203/231) and IgM anti-HAV prevalence was 38.7% (89/231). Multivariate analysis showed that individuals under 20 years old are risks groups to acquire the infection suggesting that hygienic habits of young subjects are the principal factor of transmission and so they could be the target for vaccine programs. HAV-RNA was amplified from 29 (32.5%) IgM anti-HAV positive patients and 26 samples were sequenced and classified into subgenotypes IB (8 isolates) and IA (18 isolates). Isolates classified into subgenotype IB were identical representing one distinct strain. We could observe both subgenotypes circulating during the study which suggests different sources of infection. Prophylactic measures as vaccination strategies added to improvements in hygienic and sanitary conditions would be highly effective to reduction of infection.

Prevalence and clinical aspects of respiratory syncytial virus A and B groups in children seen at Hospital de Clínicas of Uberlândia, MG, Brazil

Respiratory syncytial virus (RSV) is well recognized as the most important pathogen causing acute respiratory disease in infants and young children, mainly in the form of bronchiolitis and pneumonia. Two major antigenic groups, A and B, have been identified; however, there is disagreement about the severity of the diseases caused by these two types. This study investigated a possible association between RSV groups and severity of disease. Reverse transcription-polymerase chain reaction was used to characterize 128 RSV nasopharyngeal specimens from children less than five years old experiencing acute respiratory disease. A total of 82 of 128 samples (64.1%) could be typed, and, of these, 78% were group A, and 22% were group B. Severity was measured by clinical evaluation associated with demographic factors: for RSV A-infected patients, 53.1% were hospitalized, whereas for RSV B patients, 27.8% were hospitalized (p = 0.07). Around 35.0% of the patients presented risk factors for severity (e.g., prematurity). For those without risk factors, the hospitalization occurred in 47.6% of patients infected with RSV A and in 18.2% infected with RSV B. There was a trend for RSV B infections to be milder than those of RSV A. Even though RSV A-infected patients, including cases without underlying condition and prematurity, were more likely to require hospitalization than those infected by RSV B, the disease severity could not to be attributed to the RSV groups.

Adenovirus, calicivirus and astrovirus detection in fecal samples of hospitalized children with acute gastroenteritis from Campo Grande, MS, Brazil

We analyzed fecal samples from hospitalized children up to three years of age with acute gastroenteritis at Campo Grande, Mato Grosso do Sul, Brazil, from May 2000-January 2004. Astrovirus and calicivirus were detected by Reverse Transcription-Polymerase Chain Reaction and adenovirus was detected using the Rotavirus and Adenovirus combined immunoenzyme assay. Astrovirus, adenovirus and calicivirus were detected at rates of 3.1%, 3.6% and 7.6%, respectively. These results re-emphasize the need for the establishment of regional vigilance systems to evaluate the impact of enteric viruses on viral gastroenteritis.

The circulation of human astrovirus genotypes in the Central West Region of Brazil

Out of 1,588 faecal samples of children taken from three locations of the Central West Region of Brazil, 57 were positive for astroviruses (HAstVs) using reverse transcription-polymerase chain reaction (RT-PCR). They were genotyped by nested RT-PCR and/or genomic sequencing. HAstV-1 (42.8%), HAstV-2 (23.2%), HAstV-3 (3.6%), HAstV-4 (14.3%) and HAstVs -5, -6, -7 and -8 (1.8% each) were detected. In Goiânia and Campo Grande, HAstV-1 was the most frequently detected genotype while in Brasília (DF) it was HAstV-2. Shifts in the circulation of astrovirus genotypes were observed in DF and Campo Grande. All samples collected by rectal swabs were viral negative. The astrovirus genotypes were detected in all age groups and there was no correlation between genotype and age group.

Impact of human metapneumovirus infection on in and outpatients for the years 2006-2008 in Southern Brazil

The human metapneumovirus (hMPV), member of the Paramyxoviridae family, has been reported as an important agent involved with acute respiratory infections (ARIs). The aim of this study is to identify hMPV as the etiological agent of ARIs on in and outpatients in the city of Curitiba, Southern Brazil, and describe clinical data of hMPV subtyping. A retrospective study was performed in 1,572 respiratory samples over a period of three years. hMPV was detected by reverse transcription-polymerase chain reaction and subtyping was performed by nucleotide sequencing. hMPV was present in 61 (3.9%) samples and subtypes A1, A2a, B1 and B2 were detected. The incidence of hMPV was higher in outpatients (5.9%), whose mean age was 19.7 years (range 6 months-75 years old), than in inpatients (3%), whose mean age was 7.6 months (range 1 month-26 years old). The outpatients had upper respiratory tract infections with flu-like symptoms and all hospitalized children had lower respiratory tract infections. A pediatric patient died from complications associated with hMPV A2a infection. hMPV has been reported as a respiratory pathogen in all age groups. No correlation was observed between viral subtype and disease severity in the samples of this study.

Modulation of expression and activity of cytochrome P450s and alteration of praziquantel kinetics during murine schistosomiasis

In this study, we investigated the expression and activity of liver cytochrome P450s (CYPs) and praziquantel (PZQ) kinetics in mice infected with Schistosoma mansoni. Swiss Webster (SW) mice of both genders were infected (100 cercariae) on postnatal day 10 and killed on post-infection days (PIDs) 30 or 55. Non-infected mice of the same age and sex served as controls. Regardless of mouse sex, infection depressed the activities of CYP1A [ethoxy/methoxy-resorufin-O-dealkylases (EROD/MROD)], 2B9/10 [pentoxy/benzyloxy-resorufin-O-dealkylases (PROD, BROD)], 2E1 [p-nitrophenol-hydroxylase (PNPH)] and 3A11 [erythromycin N-demethylase (END)] on PID 55 but not on PID 30. On PID 55, infection decreased liver CYP mRNA levels (real-time reverse transcription-polymerase chain reaction). On PID 30, whereas mRNA levels remained unaltered in males, they were depressed in females. Plasma PZQ (200 and 400 mg/kg body weight intraperitoneally) levels were measured (high-performance liquid chromatography) at different post-treatment intervals. In males and females, infection delayed the PZQ clearance on PID 55, but not on PID 30. Therefore, it can be concluded that schistosomiasis down-modulated CYP expression and activity and delayed PZQ clearance on PID 55, when a great number of parasite eggs were lodged in the liver. On PID 30, when egg-laying was initiated by the worms, no change of CYP expression and activity was found, except for a depression of CYP1A2 and 3A11 mRNAs in female mice.

Human rhinovirus in the lower respiratory tract infections of young children and the possible involvement of a secondary respiratory viral agent

Human rhinoviruses (HRV) are usually associated with mild respiratory symptoms in children. However, some studies have found that HRV can cause severe disease, especially when the patient is co-infected with a second virus. In this study, 532 nasopharyngeal aspirates (NPAs) were collected over a nine-year period from children at the Clinics Hospital of Uberlândia. The collected NPAs were then tested for HRV RNA using the reverse transcription-polymerase chain reaction. Eighty-three specimens from children diagnosed with lower respiratory tract illness (LRTI) were positive for HRV RNA and were then tested for the presence of eight other respiratory viruses. A second virus was detected in 37.3% (31/83) of the samples. The most frequent clinical diagnosis was bronchiolitis, followed by other LRTI and then pneumonia. The frequency of severe disease in children infected with more than one virus was not significantly different from the frequency of severe disease in children infected with HRV alone. Children infected with both HRV and parainfluenza virus (1.5 m.o.) were significantly younger than those infected by HRV alone (5.0 m.o.) (p = 0.0454). Overall, these results suggest that infection with a second virus does not lead to a higher frequency of severe syndromes in children presenting with LRTI.

The infection of microvascular endothelial cells with ExoU-producing Pseudomonas aeruginosa triggers the release of von Willebrand factor and platelet adhesion

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

Hepatitis E virus in liver and bile samples from slaughtered pigs of Brazil

The objective of this study was to detect and identify hepatitis E virus (HEV) strains in liver and bile samples from slaughtered pigs in the state of Paraná, Brazil. Liver and bile samples were collected from 118 asymptomatic adult pigs at a slaughterhouse in a major Brazilian pork production area. The samples were assayed using a nested reverse transcription-polymerase chain reaction protocol with primer sets targeting open reading frames (ORF)1 and 2 of the HEV genome. HEV RNA was detected in two (1.7%) liver samples and one (0.84%) bile sample using both primers sets. The HEV strains were classified as genotype 3b on the basis of their nucleotide sequences. These data suggest that healthy pigs may be a source of HEV infection for consumers of pig liver and slaughterhouse workers in Brazil.

Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.

Influenza A virus infection of healthy piglets in an abattoir in Brazil: animal-human interface and risk for interspecies transmission

Asymptomatic influenza virus infections in pigs are frequent and the lack of measures for controlling viral spread facilitates the circulation of different virus strains between pigs. The goal of this study was to demonstrate the circulation of influenza A virus strains among asymptomatic piglets in an abattoir in Brazil and discuss the potential public health impacts. Tracheal samples (n = 330) were collected from asymptomatic animals by a veterinarian that also performed visual lung tissue examinations. No slaughtered animals presented with any noticeable macroscopic signs of influenza infection following examination of lung tissues. Samples were then analysed by reverse transcription-polymerase chain reaction that resulted in the identification of 30 (9%) influenza A positive samples. The presence of asymptomatic pig infections suggested that these animals could facilitate virus dissemination and act as a source of infection for the herd, thereby enabling the emergence of influenza outbreaks associated with significant economic losses. Furthermore, the continuous exposure of the farm and abattoir workers to the virus increases the risk for interspecies transmission. Monitoring measures of swine influenza virus infections and vaccination and monitoring of employees for influenza infection should also be considered. In addition regulatory agencies should consider the public health ramifications regarding the potential zoonotic viral transmission between humans and pigs.

Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silicoana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and SmBAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.

The prevalence of norovirus, astrovirus and adenovirus infections among hospitalised children with acute gastroenteritis in Porto Velho, state of Rondônia, western Brazilian Amazon

Although viruses are well-established causes of acute gastroenteritis, few data on the circulation of these pathogens in Porto Velho, state of Rondônia, Brazil, are available. Thus, faecal samples from hospitalised diarrhoeic children, under six years of age, were collected and tested for the presence of norovirus (NoV), adenovirus (AdV) and astrovirus (AstV) from February 2010-February 2012. Specimens were screened by reverse-transcription polymerase chain reaction and viruses were found in 10.7% (63/591) of the cases. NoV, AdV and AstV were detected in 7.8%, 2% and 0.8% of the samples, respectively. NoV infection was observed at all ages and was most prevalent in zero-18-month-old children (84.7%; p = 0.002). A higher incidence of NoV was detected from February-April 2010, when it was found in 52.2% of the cases. Co-infections involving these viruses, rotavirus and enteropathogenic bacteria were detected in 44.4% (28/63) of the children with viral diarrhoea. Nosocomial infections were demonstrated in 28.6% (18/63) of the cases in which viruses were detected. The present paper reports, for the first time, the circulation of NoV and AstV among the paediatric population of Porto Velho and it contributes to our understanding of the roles of these pathogens in gastrointestinal infections.