Human bocavirus detection and quantification in fecal and serum specimens from recipients of allogeneic hematopoietic stem cell transplantation: A longitudinal study.

OBJECTIVES: The aim of this study was to evaluate the occurrence of human bocavirus (HBoV) and to determine viral loads in samples of patients admitted for allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: Fecal and serum samples were collected from 19 patients, during a 24-month period. Samples were screened by quantitative polymerase chain reaction TaqMan assay, with specific probe and primers targeting the NP1 gene of all HBoVs genotypes (HBoV-1 to - 4), and viral loads were determined using serial dilutions of a recombinant plasmid. RESULTS: HBoV DNA was detected in 42.1% (8 of 19) of the patients in at least one type of sample (feces and/or serum) during the study period, with 75% (6 of 8) of the patients being positive in both types of sample. Viral shedding in feces had a median of 26 days (range, 5 to 121) and viremia was detected in 87.5% (7 of 8) of the patients. The HBoV loads in fecal samples were higher than in sera and, in most cases, HBoV was detected earlier in fecal than in sera samples. In six HBoV-positive patients (6 of 8) diarrhea was observed concomitantly to viral detection in fecal samples. CONCLUSIONS: A high frequency and loads of HBoV in allo-HSCT recipients was observed, especially in fecal samples. Positivity in fecal samples was an early predictor of HBoV presence.

Sapovirus detection and quantification in fecal samples from allogeneic hematopoietic stem cell transplant recipients.

Sapovirus are important agents of acute gastroenteritis (AGE) and they are associated with outbreaks and sporadic cases worldwide. They infect people of all ages, but mainly children, the elderly and immunocompromised individuals are affected. The aim of this study was investigate sapovirus and to determine viral loads in fecal samples from patient undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fecal samples were submitted to extraction of the genetic material using a commercial kit, and RT-qPCR TaqMan was used for sapovirus screening and determination of viral loads, using a standard curve with serial dilutions of a recombinant plasmid. Positive samples were sequence by Sanger method. Sapovirus was detected in one patient, 5.3% (1/19). Viral excretion lasted for 16 days. Viral load varied from 1.73 × 106 to 8.97 × 106 GC/g. One of the positive samples was characterized as GI.1 genotype. This is the first study to determine sapovirus loads in samples from allo-HSCT and to identify GI.1 genotype in immunocompromised patients.

Immunoinformatic construction of an adenovirus-based modular vaccine platform and its application in the design of a SARS-CoV-2 vaccine.

The current SARS-CoV-2 pandemic has imposed new challenges and demands for health systems, especially in the development of new vaccine strategies. Vaccines for many pathogens were developed based on the display of foreign epitopes in the variable regions of the human adenovirus (HAdV) major capsid proteins (hexon, penton and fiber). The humoral immune response against the HAdV major capsid proteins was demonstrated to play a role in the development of an immune response against the epitopes in display. Through the immunoinformatic profiling of the major capsid proteins of HAdVs from different species, we developed a modular concept that can be used in the development of vaccines based on HAdV vectors. Our data suggests that different immunomodulatory potentials can be observed in the conserved regions, present in the hexon and penton proteins, from different species. Using this modular approach, we developed a HAdV-5 based vaccine strategy for SARS-CoV-2, constructed through the display of SARS-CoV-2 epitopes indicated by our prediction analysis as immunologically relevant. The sequences of the HAdV vector major capsid proteins were also edited to enhance the IFN-gamma induction and antigen presenting cells activation. This is the first study proposing a modular HAdV platform developed to aid the design of new vaccines by inducing an immune response more suited for the epitopes in display.

Circulation profile of respiratory viruses in symptomatic and asymptomatic children from Midwest Brazil.

Acute respiratory infection (ARI) is a major cause of morbidity and mortality worldwide. Most of these infections are caused by viruses. Infections pose as important triggers of acute episodes of chronic respiratory diseases (CRD). This study sought to evaluate the frequency and circulation profile of respiratory viruses among ARI symptomatic patients and completely asymptomatic children in Midwest Brazil. The study enrolled symptomatic children with and without ARI symptoms. During 1 year, 225 nasal respiratory samples were obtained from patients aged 4-14 years old. The samples were screened by multiplex nested-PCR for 16 common respiratory viruses. From 225 samples, 42 had at least one virus detected. Samples from four different patients had multiple viruses detected. The viral detection rate in symptomatic (20.1%) and asymptomatic patients (14.8%) showed no significant difference. The most frequent viruses detected were rhinovirus (28.6%), FLUA (11.9%), adenovirus (11.9%), human bocavirus (HBoV) (11.9%), and respiratory syncytial virus (RSV) antigenic group A (9.5%). Monthly detection rate was higher during the rainy season. RSVs were detected during the months with higher rainfall indexes and higher air humidity, while FLU and HBoV were detected during the winter months. The obtained results reinforce the importance of viral pathogens in pediatric population, emphasizing similar viral occurrence in symptomatic and asymptomatic children.

Quantitative proteomic analysis of A549 cells infected with human adenovirus type 2.

Human adenovirus (HAdV-2) is considered a common agent of respiratory tract infection in the human, especially in children. Virus infection is believed to modify host cell expression necessary for its replication and therefore cell proteome can reflect the changes of specific cellular pathways during infection. This study aims to identify differentially expressed proteins of A549 cells in response to HAdV-2 infection using a label-free liquid chromatography-high-resolution tandem mass spectrometry strategy (LC-MS/MS) at 24 and 48 hpi. A total of 248 and 216 proteins were deregulated by 1.35-fold at 24 and 48 hpi, respectively. Among them, 155 were upregulated at 24 hpi and 86 at 48 hpi, whereas 93 and 130 were downregulated at 24 and 48 hpi, respectively. The identified proteins were involved in different pathways as energy, transcription, protein synthesis, cytoskeleton, rescue and defense, cell cycle, DNA processing, transportation, and metabolism. Glycolytic pathway and histone deregulated proteins were further confirmed by chemical testing and immunofluorescence, respectively. The results suggest that the identified proteins influenced HAdV-2 infection in the context of viral replication and propagation. This study complement proteomic data obtained from previous studies and reinforce the understanding of the relationship between HAdV and host cell.

Asthma exacerbations in a subtropical area and the role of respiratory viruses: a cross-sectional study.

BACKGROUND: Multiple factors are involved in asthma exacerbations, including environmental exposure and viral infections. We aimed to assess the association between severe asthma exacerbations, acute respiratory viral infections and other potential risk factors. METHODS: Asthmatic children aged 4-14 years were enrolled for a period of 12 months and divided into two groups: those with exacerbated asthma (group 1) and non-exacerbated asthma (group 2). Clinical data were obtained and nasopharyngeal samples were collected through nasopharyngeal aspirate or swab and analysed via indirect fluorescent immunoassays to detect influenza A and B viruses, parainfluenza 1-3, adenovirus and respiratory syncytial virus. Rhinovirus was detected via molecular assays. Potential risk factors for asthma exacerbation were identified in univariate and multivariate analyses. RESULTS: In 153 children (group 1: 92; group 2: 61), median age 7 and 8 years, respectively, the rate of virus detection was 87.7%. There was no difference between groups regarding the frequency of virus detection (p = 0.68); however, group 1 showed a lower frequency (19.2%) of inhaled corticosteroid use (91.4%, p < 0.01) and evidence of inadequate disease control. In the multivariate analysis, the occurrence of three or more visits to the emergency room in the past 12 months (IRR = 1.40; p = 0.04) and nonadherence to inhaled corticosteroid (IRR = 4.87; p < 0.01) were the only factors associated with exacerbation. CONCLUSION: Our results suggest an association between asthma exacerbations, poor disease control and nonadherence to asthma medication, suggesting that viruses may not be the only culprits for asthma exacerbations in this population.

Proteomic analysis of A-549 cells infected with human adenovirus 40 by LC-MS.

Human Adenoviruses (HAdVs) are etiological agents of different syndromes such as gastroenteritis, cystitis, ocular, and respiratory diseases, and infection by these viruses may cause alterations in cellular homeostasis. The objective of the study was the proteomic analysis of A-549 cells infected with HAdV-40 using LC-MS. At 30 h of infection, the quantitative analysis revealed 336 differentially expressed proteins. From them, 206 were induced (up-regulated) and 130 were suppressed (down-regulated). The majority of up-regulated proteins were related to energy, cellular organization, stress response, and apoptosis pathways. It was observed alteration of cell metabolism with increase of the glycolytic pathway, ß-oxidation, and respiratory chain. Also, the results suggest cytoskeleton reorganization and apoptosis induction. The data can improve knowledge about the replication of HAdV-40 in cell culture considering the proteins related to distinct metabolic pathways induced by viral infection in A-549 cells.

Identification of Human Bocavirus type 4 in a child asymptomatic for respiratory tract infection and acute gastroenteritis - Goiânia, Goiás, Brazil

Abstract Human Bocavirus (HBoV) has been identified from feces and respiratory samples from cases of both acute gastroenteritis and respiratory illness as well as in asymptomatic individuals. The aim of this study was to detect and characterize HBoV from fecal samples collected from hospitalized children aged less than five years old with no symptoms of respiratory tract infection (RTI) or acute gastroenteritis (AGE). The study involved 119 children and one fecal sample was collected from each participant between 2014 and 2015. HBoV was detected using Nested-PCR, and the viral type identified by genomic sequencing. HBoV-4 was identified from one sample obtained from a hospitalized child with soft tissue tumor of the submandibular region. This is the first report of HBoV-4 identification in Brazil, but we consider that this type may be circulating in the country similar to the other types and new investigations are necessary.

Identification of Human Bocavirus type 4 in a child asymptomatic for respiratory tract infection and acute gastroenteritis - Goiânia, Goiás, Brazil.

Human Bocavirus (HBoV) has been identified from feces and respiratory samples from cases of both acute gastroenteritis and respiratory illness as well as in asymptomatic individuals. The aim of this study was to detect and characterize HBoV from fecal samples collected from hospitalized children aged less than five years old with no symptoms of respiratory tract infection (RTI) or acute gastroenteritis (AGE). The study involved 119 children and one fecal sample was collected from each participant between 2014 and 2015. HBoV was detected using Nested-PCR, and the viral type identified by genomic sequencing. HBoV-4 was identified from one sample obtained from a hospitalized child with soft tissue tumor of the submandibular region. This is the first report of HBoV-4 identification in Brazil, but we consider that this type may be circulating in the country similar to the other types and new investigations are necessary.

Circulating profile of rotavirus a, g and p genotypes before and after vaccine introduction in the Brazilian mid-west 1986-2015

The purpose of this study was to perform a comparative analysis of Rotavirus A (RVA) G and P genotypes circulating in the Brazilian Mid-West in the period 1986-2015. Seven studies conducted from 1986 to 2009 were included, as well as fecal samples obtained in the period 2014-2015. RVA was screened by ELISA and/or PAGE; genotyping by conventional RT-PCR and/or genomic sequencing. A temporal variation in the predominance of G genotypes mainly G1 and G2 with G9 and G12 emergence was observed. Even with vaccination, RVA continues to circulate in the population, requiring continuous virus monitoring

Adenovirus infection among allogeneic stem cell transplant recipients.

The human adenovirus (HAdV) infection can cause severe disease in immunocompromised patients, such as those undergoing allogeneic hematopoietic stem cell transplant (ASCT). The main objective of this study was to prospectively monitor ASCT recipients for HAdV occurrence in a reference center in Brazil, and also to correlate viral positivity, viral load, molecular variant, clinical symptoms, and patients' prognosis. From October/2012 to October/2014, blood and feces of 21 ASCT recipients were screened for HAdV by Nested-PCR. Viral loads were determined by real-time PCR. In total, 57% of the patients had at least one positive sample (serum or feces) for HAdV. Patients presented significantly higher viral load in feces when compared to serum. Positive samples were characterized as HAdVs of species HAdV-C, -D, and -F. The main clinical symptom presented by infected patients was diarrhea, and Graft-versus-host disease (GVHD) was the main intercurrence. An association was observed between HAdV-positivity and diarrhea and also between HAdV-positivity and GVHD. Results from this study may contribute to a better understanding of the HAdV infection pattern in patients submitted to ASCT. Data therein highlight the importance of including HAdV testing during all routine laboratory exams performed on ASCT patients. J. Med. Virol. 89:298-303, 2017. © 2016 Wiley Periodicals, Inc.

Phylodynamics of DENV-1 reveals the spatiotemporal co-circulation of two distinct lineages in 2013 and multiple introductions of dengue virus in Goiás, Brazil.

Dengue virus type 1 (DENV-1) was the first serotype introduced in Brazil, during in the 1980s. Since then, this virus has spread in the Brazilian territory, causing several outbreaks. In 2013 the highest number of dengue cases was notified, when compared to the previous years in Brazil, and the state of Goiás reported over 160 thousand cases. In this study, we aimed to present the Phylodynamics of DENV-1 isolates from the state of Goiás, Brazil, during 2013 outbreak, based on the envelope gene (E) sequences. Phylogenetic analysis revealed that Brazilian DENV-1 isolates are grouped together with viruses from genotype V in two distinct lineages (lineage I and lineage II) reflecting co-circulation. Phylogeographic analyses showed that these lineages were introduced in different moments in Goiás, Brazil, using distinct routes, likely originated from the Caribbean. Lineage I was first introduced coming from Rio de Janeiro (2007-2012), followed by the introduction from Argentina (2010-2013). Lineage II was introduced in a single moment from Rio de Janeiro and this clade has existed since 2007-2010. The different viral introduction events demonstrate the viral dispersion process with neighboring regions, which is essential for the maintenance of outbreaks and introduction of new emerging viruses. In conclusion, obtained data reveals the importance of continuous molecular surveillance of this virus in different regions, providing a better understanding of DENV-1 circulation, considering the evolutionary and virus spread patterns.

Prospective study on Norovirus infection among allogeneic stem cell transplant recipients: prolonged viral excretion and viral RNA in the blood.

BACKGROUND: Human caliciviruses (Norovirus and Sapovirus) are important acute gastroenteritis agents. The Norovirus (NoV) disease is usually self-limited; however, prolonged viral excretion and complications have been reported, mainly in immunosuppressed individuals. OBJECTIVES: In this prospective study, we have monitored allogeneic stem cell transplant (ASCT) patients for human calicivirus infection. STUDY DESIGN: Ten ASCT patients were monitored for NoV and sapoviruses (SaV) infection, for a period of five months to a maximum of one year. Prolonged NoV excretion and long term viral RNA in the blood were assessed by multiplex RT-PCR targeting region C of the viral capsid. Secretor status of the patients was determined by enzyme immunoassay using Ulex Europaeus agglutinin. Partial genomic sequencing and phylogenetic analysis were performed to characterize the viral genotypes. RESULTS: NoV was detected in six out of ten patients (60%). Prolonged viral excretion in feces (mean of 61.6 days) and long term presence of NoV RNA in the sera (mean of 33.6 days) of the patients were observed. SaV was not detected in any of the samples. All patients had diarrhea, vomiting and fever during NoV positivity. All NoV-positive samples were characterized as GI.3 NoV. Three Nov-infected patients presented with acute intestinal graft versus host disease. CONCLUSIONS: This study brings important information on NoV course of infection in ASCT patients. It also provides evidence for long term viral RNA in the blood highlighting the importance of the inclusion of NoV screening in the routine testing performed before transplantation and during follow-up of these patients.

Screening of fecal samples from asymptomatic children, for norovirus detection, using a third generation enzyme immunoassay commercial kit

Norovirus is the leading cause of non-bacterial acute gastroenteritis outbreaks worldwide. Recently, third generation Enzyme Immunoassay (EIA) commercial kits have been developed, and controversial results have been obtained by different studies regarding the sensitivity and specificity of these assays. Therefore, the aim of this study was to test 60 fecal samples, previously tested as positive by RT-PCR for caliciviruses (40 norovirus-positive and 20 sapovirus-positive samples), for qualitative determination of genogroup I and II noroviruses by a commercial EIA kit (RIDASCREEN® Norovirus (C1401) 3rd Generation, R-Biopharm, Darmstadt, Germany). The samples were obtained from 30 children aged less than five years, mostly asymptomatic, who attend a day-care center in Goiânia, Goiás, Brazil. The results conferred a positivity rate for NoV of 35 percentand a specificity rate of 100 percent for the EIA, when compared to the RT-PCR. The test also failed to detect samples that were positive for GI.1 and GI.4 norovirus. The presumably lower viral load of asymptomatic children might be related to the poor sensitivity. Our results reinforce the notion that screening of samples by molecular assays, especially of samples that might have a low number of viral particles such as those obtained from asymptomatic patients, should not be replaced by the use of EIA kits...

Triagem de amostras fecais de crianças assintomáticas utilizando-se um kit comercial de Elisa 3a geração determinação qualitativa de norovírus dos genogrupos I e II por meio de kit comercial de EIE (RIDASCREEN® Norovirus (C1401) 3rd Generation, R-Biopharm, Darmstadt, Germany). Previamente testadas, elas se mostraram positivas para calicivírus por RT-PCR (40 positivas para norovirus e 20 positivas para sapovirus). As amostras foram obtidas de 30 crianças menores de 5 anos de idade, predominantemente assintomáticas, que frequentavam uma creche em Goiânia, Goiás, Brasil. Os resultados revelaram índices de 35 porcento de positividade para os norovírus e de 100 porcento de especificidade para o EIE quando comparado a RT-PCR. O teste também falhou em detectar amostras que eram positivas para norovírus GI.1 e GI.4. A carga viral, presumidamente mais baixa, das crianças assintomáticas pode estar relacionada com a baixa sensibilidade. Os resultados reforçam o entendimento de que a triagem de amostras por ensaios moleculares não deve ser substituída pelo uso de kits de EIE, especialmente quando se tratar de amostras que, presumidamente, apresentem um baixo número de partículas virais como as obtidas de pacientes assintomáticos...

Human bocavirus 1 and 3 infection in children with acute gastroenteritis in Brazil

To determine the positivity rate of human bocavirus (HBoV) 1 and 3 among children who presented with acute gastroenteritis symptoms during the period of 1994-2004 in the Central-West Region of Brazil, 762 faecal samples were tested using polymerase chain reaction (PCR) for the detection of HBoV DNA. Primers for a segment of the non-structural viral protein 1 (NS1) gene of HBoV-1 and HBoV-3 were used. Twelve HBoV-positive samples were further characterised via genomic sequencing and phylogenetic analysis. Of the samples tested, 5.8% (n = 44) were positive for HBoV-1 or HBoV-3 and co-infection was observed in 14 (31.8%) of the 44 HBoV-positive samples. Nine of the 14 samples were also positive for Rotavirus A and five were positive for Aichi virus. The genomic sequencing of the NS1 partial sequence of 12 HBoV-samples showed that 11 samples were characterised as HBoV-1 and that one was characterised as HBoV-3. The phylogenetic analysis showed that the HBoV-1 samples had a high sequence homology to others previously identified in China, Sweden and Brazil. This is the first study conducted in the Central-West Region of Brazil to detect HBoV-1 and HBoV-3 in faecal samples from children with acute gastroenteritis. Further studies are required to define the role of HBoVs as aetiological agents of gastroenteritis.

Monitoring the circulation of rotavirus among children after the introduction of the RotarixTM vaccine in Goiânia, Brazil

The epidemiological features of rotavirus A (RVA) infection differ between children from developing and developed countries which could result in differences in vaccine efficacy around the world. To evaluate the impact of RotarixTM on RVA prevalence, we monitored RVA genotypes circulating in Goiânia by monitoring virus in faecal samples from children that had or had not been previously vaccinated. From February-November of 2008, 220 faecal samples were collected from children in seven day-care centres. RVA detection was performed by two methodologies and the results were confirmed by polyacrylamide gel electrophoresis. From the 220 samples, eight were RVA-positive (3.6 percent) and five were from children that had received either one or two doses of the vaccine. All positive samples were collected from children with diarrhoea during August and September. Genotyping of the RVA characterised five of the viral samples as genotype G2P[4] and one as G8P[4], suggesting that G2P[4] was the predominant circulating genotype in Goiânia during the study. The fact that vaccinated children were also infected by RVA suggests that the vaccine does not fully protect against infection by the G2[P4] RVA genotype.

Molecular characterization of adenovirus detected from fecal samples obtained from children in the Central West region of Brazil.

The adenoviruses are frequently associated with sporadic gastroenteritis outbreaks in different parts of the world. This study aimed at the molecular characterization of human adenoviruses (HAdV) species and serotypes, in fecal samples from children, by multiplex-PCR and by PCR-RFLP, respectively, followed by genomic sequencing. Of 39 adenovirus-positive samples, 30 (76.9%) were classified as species F, six (15.4%) as species C, and two (5.1%) as species A, and one (2.6%) had a mixed F/C pattern. The serotyping showed that 14 (41.2%) were HAdV-41, 15 (44.1%) were HAdV-40, five (14.7%) were HAdV-5, and five samples could not be serotyped. This is the first study to molecularly characterize HAdV in the Central West region of Brazil, and the results highlight the circulation of the HAdV-5 among children with acute gastroenteritis in this region.

Gastroenteric virus detection in fecal samples from women in Goiânia, State of Goiás, Brazil.

INTRODUCTION: This was a prospective study that included women seen in the obstetrics and gynecology sector of Hospital das Clínicas, Federal University of Goiás, in Goiânia, State of Goiás, with the aim of detecting rotaviruses, adenoviruses, caliciviruses and astroviruses. Eighty-four women participated in the study and from these, 314 fecal samples were collected. Out of all of the women, 29 were seropositive for HIV and 55 were seronegative, and 45 and 39 were pregnant and non-pregnant, respectively. METHODS: Fecal samples were collected from each woman once every two months over the period from July 2006 to June 2007, and they were screened for rotaviruses by means of polyacrylamide gel electrophoresis and immunoenzymatic assays, for caliciviruses and astroviruses by means of RT-PCR and for adenovirus by means of immunoenzymatic assays. The astroviruses were genotyped using nested PCR. RESULTS: Among the 84 patients, 19 (22.6%) were positive for either calicivirus (14/19) or astrovirus (6/19), while one women was positive for both viruses in fecal samples collected on different occasions. Most of the positive samples were collected during the months of July and August (astrovirus) and September and October (calicivirus). None of the samples analyzed was positive for rotavirus or adenovirus. Gastroenteric viruses were detected in 13/19 (68.4%) of the pregnant women, whether HIV-seropositive or not. CONCLUSIONS: The results from the present study showed that neither pregnancy nor HIV-seropositive status among the women increased the risk of infection by any of the gastroenteric viruses studied. This study presents data on gastroenteric virus detection among pregnant and/or HIV-positive women.