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Made available in DSpace on 2022-04-28T19:50:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-05-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Human respiratory syncytial virus (hRSV) is an infectious agent in infants and young children which there are no vaccines or drugs for treatment. Neutrophils are recruited for airway, where they are stimulated by hRSV to release large amounts of neutrophil extracellular traps (NETs). NETs are compound by DNA and proteins, including microbicidal enzymes. They constitute a large part of the mucus accumulated in the lung of patients, compromising their breathing capacity. In contrast, NETs can capture/inactivate hRSV, but the molecules responsible for this effect are unknown. Objectives: We selected microbicidal NET enzymes (elastase, myeloperoxidase, cathepsin-G, and proteinase-3) to assess their anti-hRSV role. Methods and Results: Through in vitro assays using HEp-2 cells, we observed that elastase, proteinase-3, and cathepsin-G, but not myeloperoxidase, showed virucidal effects even at non-cytotoxic concentrations. Elastase and proteinase-3, but not cathepsin-G, cleaved viral F-protein, which is responsible for viral adhesion and fusion with the target cells. Molecular docking analysis indicated the interaction of these macromolecules in the antigenic regions of F-protein through the active regions of the enzymes. Conclusions: Serine proteases from NETs interact and inactive hRSV. These results contribute to the understanding the role of NETs in hRSV infection and to designing treatment strategies for the inflammatory process during respiratory infections. São Paulo State University (UNESP) Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) School of Sciences Humanities and Languages Graduate Program in Applied and Computational Mathematics – PGMAC - State University of Londrina Department of Medical Microbiology and Infectious Diseases University of Manitoba Biosystems Collaborative Nucleus Institute of Exact Sciences Federal University of Jatai Virology Laboratory Center for Microbiology Studies Department of Parasitology Microbiology and Immunology Federal University of Juiz de Fora, Minas Gerais São Paulo State University (UNESP) Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) School of Sciences Humanities and Languages FAPESP: 2018/09021-4

Background Acute respiratory infections caused by viruses are among the leading causes of morbidity and mortality. The inflammatory response that follows viral infection is important for the control of virus proliferation. However, if overwhelming, may be associated with complicated outcomes. Objectives We assessed the clinical characteristics of patients with severe acute respiratory illness (SARI) evolving to acute respiratory distress syndrome (ARDS) and the factors related to death. Study design. Prospective study in 273 adult patients with SARI performed in a university-affiliated 800-bed hospital serving an area of epidemiologic vigilance of 102 municipalities and more than 2 million inhabitants. Influenza A (H1N1) 2009 (A/H1N1), influenza A H3N2, and influenza B were tested in all patients by RT-PCR. Results The overall hospital mortality rate was 17.6%. A total of 30.4% of patients tested positive for influenza A/H1N1. Patients with SARI that evolved to ARDS took significantly longer to take the first dose of oseltamivir (6.0 vs 1.0 days, p=0.002). Patients with H1N1 positive tests had almost 3 times higher probability of death, despite having significantly less comorbidities (p=0.027). The influenza A/H1N1 pdm09 vaccine reduced the odds of death by 78%. Nonsurvivors had a more intense inflammatory response than did survivors at 48 h (C-reactive protein: 31.0 ± 17.5 vs. 14.6 ± 8.9 mg/dl, p=0.001) as well as a more positive fluid balance. Conclusions Hospital mortality associated with influenza H1N1-associated SARI and ARDS continued to be high years after the 2009 pandemic in a population with low vaccine coverage. Antiviral treatment started more than two days after onset of symptoms was more frequently associated with ARDS and death and, having had vaccine against influenza A (H1N1) was a factor independently related to survival.

Introdução: O vírus SARS-CoV-2 invade a célula hospedeira através da ligação entre a proteína viral SPIKE e o receptor ECA-2. A resposta antiviral ativa vias inflamatórias do sistema imune. Entretanto, uma resposta exacerbada com hiper-citocinemia pró-inflamatória, na chamada Tempestade de Citocinas (TC), pode evoluir com Síndrome do Desconforto Respiratório Agudo (SRDA) e falência múltipla dos órgãos. Objetivos: Analisar a relação entre a tempestade de citocinas na COVID-19 e a evolução da doença. Material e métodos: Realizou-se revisão narrativa com busca de artigos publicados entre 2020 e 2021, na base de dados PubMed, partindo dos descritores boleanos “Cytokine Storm and COVID-19”. Contabilizou-se 2359 artigos e foram filtradas revisões e revisões sistemáticas cujo título apresentasse os descritores, totalizando 70 trabalhos científicos. Após avaliação criteriosa 11 artigos foram selecionados por convergirem com a temática proposta. Resultados: A TC inicia-se com a expressão de fatores de transcrição indutores de citocinas pró-inflamatórias, como NF-kB. Com isso, padrões sinalizadores resultam na secreção das citocinas de fase aguda (TNF-α, IL1-β e IL-6), responsáveis pela hiperinflamação encontrada na COVID-19. Deficiências na lise de células apresentadoras de antígenos infectadas prolongam as interações entre células do sistema imune, provocando secreção exacerbada de citocinas pró-inflamatórias, como TNF, interferon-γ, interleucinas 1 e 6. TNF e IL-1 induzem o fator de tecido pró-coagulante (TF), ocasionando trombose imunomediada. Já a IL-6 promove uma síndrome de ativação macrofágica, desencadeando hiper-citocinemia pró-inflamatória e danos ao epitélio pulmonar.Conclusão: A rápida replicação viral nos primeiros estágios da infecção resulta em resposta pró-inflamatória exacerbada, com aumento da liberação de IL-6, IL-1 e TNFα e outras citocinas, gerando um quadro grave de hiperinflamação secundária à infecção por COVID-19, frequentemente associado a um pior prognóstico. Surge, assim, o dilema das terapias imunomoduladoras, pois optar pela repressão da atividade do sistema imunológico poderia prejudicar a eliminação viral e produção de anticorpos, além de favorecer o surgimento de infecções oportunistas.

Background The respiratory syncytial virus (RSV) is recognized as an important cause of respiratory tract infections. Immunocompromised patients, healthcare workers (HCWs) and children contacts are at increased risk of acquiring the infection. However, the impact of asymptomatic infection in transmission has not been well studied. Objectives: this study evaluated the frequency and viral load (VL) of RSV in nasal swab samples of individuals with different risk factors for acquiring infection in a university hospital in Sao Paulo, Brazil. Methods We included 196 symptomatic children and their 192 asymptomatic caregivers, 70 symptomatic and 95 asymptomatic HCWs, 43 samples from symptomatic HIV‐positive outpatients, and 100 samples of asymptomatic HIV patients in the period of 2009‐2013. Results RSV infection was detected in 10.1% (70/696) of samples, 4.4% (17/387) of asymptomatic patients, and 17.1% (53/309) from symptomatic patients. (P < .0001). The VL of symptomatic patients (4.7 log copies/mL) was significantly higher compared to asymptomatic patients (2.3 log copies/mL). RSV detection among asymptomatic caregivers (6.8%; 13/192) was significantly higher compared to other asymptomatic adults, HIV and HCWs (2.0%; 4/195; P = .0252). A close contact with an infected child at home was an important risk to RSV acquisition [OR 22.6 (95% CI 4.8‐106.7)]. Children who possibly transmitted the virus to their asymptomatic contacts had significantly higher viral load than children who probably did not transmit (P < .0001). Conclusions According to our results, it is important to know if people circulating inside the hospital have close contact with acute respiratory infected children.

Human bocavirus (HBoV) is commonly associated with acute respiratory tract illness and gastroenteritis. We report six complete genomic sequences of HBoV strains from patients with gastroenteritis in Belém do Pará and Tocantins in the North Region of Brazil. Phylogenetic analysis indicated that the six HBoV strains belong to genotypes 1, 2, and 3.

OBJECTIVE: Adenoviruses play an important role in the etiology of severe acute lower respiratory infection, especially in young children. The aim of the present study was to evaluate the Human Adenovirus (HAdV) detection by different methods (Direct Fluorescence Assay DFA and Nested Polymerase Chain Reaction nested PCR), among samples collected from different groups of pediatric patients. METHODS: Collection of samples was made in children with congenital heart disease (CHD 123 nasal aspirates collected in the years of 2005, 2007 and 2008) and in community children (CC 165 nasal aspirates collected in 2008). Children were eligible if they presented acute respiratory infection (ARI) of probable viral etiology, within up to 7 days of symptoms' onset. All studied samples were evaluated by DFA and nested PCR assay. RESULTS: Of the 290 samples included during the study period, 43 (14.8%) were positive on at least one test: 17/165 (10.3%) of the CC and 26/125 (20.8%) of the CHD children. The nested PCR detection rates in the community children were 15/165 (9.1%), and for children with CHD, 24/125 (19.2%). Molecular method showed higher detection rates when compared to the DFA test (p

Rotavirus is the main global cause of severe childhood diarrhoea among children. In 2006, Rotarix® (G1P[8]) was introduced into Brazil’s National Immunization Program. The vaccine coverage rate was 84.4% in 2009. Evidences of increasing G2P[4] after 2006 opened up the discussion about the vaccine effectiveness to non-G1 strains. The aim of this study was to identify the circulating rotavirus genotypes in two Brazilian regions during 2009. A total of 223 positive samples by immunochromatography and latex agglutination assay from the Northeast (Bahia/Pernambuco States) and Southeast (São Paulo/Rio de Janeiro States) regions were included in the study. The samples were submitted to genotyping by nested-PCR according to VP7(G) and VP4(P) and 175 samples (78.5%) were able to be characterized. Considering the characterization of VP7, the G-types detected were G1, G2, and G4 in the Northeast, and G2, G3, G5, and G9 in the Southeast. Considering the characterization of VP4, the P-types detected were P[4], P[8], and P[6]/P[9] in the Northeast and the Southeast. The most frequent mixed types found were G2P[4]/G2P[NT](81.4%), G2P[6](5.2%), G1P[6](5.2%) in the Northeast, and G2P[4]/G2P[NT](78.8%), G2P[6](8.2%), G9P[8](4.7%) in the Southeast. Among immunized individuals whose age ranged from 0-4 years, the G2P[4]/G2P[NT] genotype was identified in 91,0% of cases, and among non-immunized individuals of the same age, the G2P[4]/G2P[NT] genotype was identified in 85.7% of the cases. In accordance with the high level of vaccine coverage, the data suggest that the circulation of G2P[4] in these regions had a considerable increase after the introduction of Rotarix®.

Human rhinoviruses (HRVs) are the major cause of the common cold. HRVs were recently reclassified into the Enterovirus genus (HEV) in the Picornaviridae family. HRVs and other members of the HEV genus share many common features, including sense RNA genomes and partial nucleotide sequence identity. The aim of this study was to evaluate different HRV detection strategies. Samples from adults with acute respiratory infection (n = 291) who were treated in Sao Paulo Hospital (2001-2003) were tested using three assays. The first assay detected picornaviruses by RT-PCR and hybridization, the second detected rhinoviruses using RT-PCR/sequencing, and the third differentiated HRV from HEV using duplex semi-nested-RT-PCR. Analysis of the results obtained from the first two strategies revealed 83% concordance. Discordant samples were then evaluated by the third protocol, and 82% were negative. The picornavirus detection protocol was more sensitive but less specific than the rhinovirus detection protocols. The semi-nested protocol utilized in the present study was less sensitive and was not useful in differentiating HRV from HEV. Sequencing assays examining different genes would address the best strategy of confirming rhinovirus and enterovirus infections.

In 2010, 96 patients suspected of being infected with the influenza A (H1N1) virus were hospitalized at the Hospital Sao Paulo, located in the city of Sao Paulo, Brazil. Of those 96 patients, 4 (4.2%) were found to be infected with influenza A virus-3 with influenza A (H1N1) and 1 with seasonal influenza A-and 2 patients (2.1%) were found to be infected with influenza B virus. Most (63.5%) of the suspected cases occurred in children, as did half of the positive cases. The second wave of influenza A (H1N1) infection was weaker in Sao Paulo. The decrease in the number of hospitalizations for H1N1 infection in 2010 might be attributable to vaccination.

INTRODUCTION: Human adenoviruses (HAdV) play an important role in the etiology of severe acute lower respiratory infection, especially in immunocompromised individuals. The aim of the present study was detect the HAdV through different methods: direct fluorescence assay (DFA) and nested-polymerase chain reaction (PCR-nested) from patients with acute respiratory infection (ARI) up to 7 days of symptoms onset. METHODS: Samples (n=643) were collected from different risk groups during from 2001 to 2010: 139 adults attended in an Emergency Room Patients (ERP); 205 health care workers (HCW); 69 from Renal Transplant Outpatients (RTO); 230 patients in hematopoietic stem cell transplantation (HSCT) program. RESULTS: Among all patients (n=643) adenovirus was detected on 13.2% by DFA and/or PCR: 6/139 (4.3%) adults from ERP, 7/205 (3.4%) from HCW samples, 4/69 (5.8%) from RTO and 68/230 (29.5%) from HSCT patients. Nested PCR showed higher detection (10%) compared to DFA test (3.8%) (p < 0.001). HSCT patients presented significantly higher prevalence of HAdV infection. CONCLUSIONS: Adenovirus detection through nested-PCR assay was higher. However the inclusion of molecular method in laboratorial routine diagnostic should be evaluated considering the reality of each specific health service. Universidade Federal de São Paulo (UNIFESP) Departamento de Medicina Disciplina de Infectologia Universidade do Centro-Oeste Departamento de farmacologia Clínica UNIFESP, Depto. de Medicina Disciplina de Infectologia SciELO

Human respiratory syncytial virus (HRSV) causes severe infections among children and immunocompromised patients. We compared HRSV infections among Haematopoietic Stem Cell Transplant program (HSCT) patients and children using direct immunofluorescence (DFA), point-of-care RSV Bio Easy(r) and a polymerase chain reaction (PCR) assay. Overall, 102 samples from HSCT patients and 128 from children obtained positivity rate of 18.6% and 14.1% respectively. PCR sensitivity was highest mainly on samples collected after five days of symptoms onset. A combination of both DFA and reverse transcriptase-PCR methods for HSCT high-risk patients is the best diagnostic flow for HRSV diagnosis among these patients.

INTRODUÇÃO E OBJETIVOS: O bocavírus humano (HBoV) tem sido descrito desde 2005 como agente etiológico de infecções respiratórias virais. O presente estudo tem como objetivo investigar a etiologia da infecção respiratória pelo HBoV em pacientes adultos e crianças de diferentes grupos de risco para complicação de infecções respiratórias agudas desde 2001 até 2008 em um hospital terciário no Brasil. PACIENTES E MÉTODOS: O HBoV foi investigado, através de reação em cadeia da polimerase, em 598 amostras respiratórias coletadas de pacientes hospitalizados e não hospitalizados. RESULTADOS: Das 598 amostras testadas o HBoV foi detectado em 2,44% (8/328) das crianças, incluindo cinco crianças portadoras de cardiopatia congênita, e 0,4% (1/270) dos adultos em programa de transplante de células tronco hematopoiéticas. CONCLUSÃO: Os dados do presente estudo sugerem baixa freqüência de detecção do HBoV entre pacientes de risco, e destaca a necessidade de novos estudos para um melhor entendimento do verdadeiro papel desse agente em infecções respiratórias agudas em pacientes sintomáticos. BACKGROUND AND OBJECTIVES: Human Bocavirus (HBoV) has been described since 2005 as an etiological agent of respiratory virus infections. From 2001 to 2008 we investigated the etiology of HBoV among adults and children in different groups at risk of presenting complications arising from acute respiratory infection, the investigation was carried out in a tertiary hospital health care system in Brazil. METHODS: HBoV DNA was assayed in 598 respiratory samples from community and hospitalized patients by PCR. RESULTS: Of the 598 tested samples, 2.44% (8/328) of children, including five children with heart disease, and 0.4% (1/270) of adult bone-marrow-transplant were HBoV positive. CONCLUSIONS: These data suggested lower HBoV frequency among different at-risk patients and highlights the need to better understand the real role of HBoV among acute respiratory symptomatic patients. LEAL, E. S. Pós-Dr. Docente da Universidade Federal do Pará, Instituto de Ciências Biológicas, Faculdade de Biotecnologia

INTRODUCTION: Influenza A H1N1 2009 is associated with a high morbidity rate among children around the world, including Brazil. This survey was conducted on samples of symptomatic children (< 12 years) to investigate the influenza virus as the etiological agent of respiratory infections in a day care school in a health facility during the first and second pandemic wave of H1N1 (2009-2010) in São Paulo, Brazil. METHODS: Influenza infections were determined by real-time PCR in 34% (47/137) of children with a median age of 5 years (8 months - 12 years), from June to October 2009 and in 16% (14/85) of those with median age of 6 years (1-12 years), from March to November 2010. RESULTS: In general, most positive cases (64%) occurred in children aged 5-12 years, this age group was significantly the most affected (39.8%, p = 0.001, OR = 8.3, CI 95% 1.9-36.9). Wheezing was reported by 31% (19/61) and dyspnea by 23% (14/61) of the studied patients. An outbreak of influenza H1N1 with an attack rate of 35.7% among children (median age 6 years) was documented in April 2010, before the vaccination campaign against the pandemic virus was extended for children up to 5 years in Brazil. CONCLUSIONS: Therefore, the study reinforces the recommendation to immunize school children to reduce the incidence of the disease. INTRODUÇÃO: Influenza A H1N1 2009 está associado com uma alta taxa de morbidade entre crianças ao redor do mundo, incluindo o Brasil. Esta pesquisa foi realizada em amostras de crianças sintomáticas (< 12 anos) em uma creche escola para filhos de funcionários do hospital durante a primeira e segunda onda pandêmica (2009-2010) em São Paulo, Brasil. MÉTODOS: Infecções pelo vírus influenza foram determinadas por PCR em tempo real em 34% (47/137) em crianças com idade mediana de 5 anos (8 meses -12 anos), entre junho e outubro de 2009 e em 16% (14/85) daquelas com mediana de idade de 6 anos (1-12 anos), de março a novembro de 2010. RESULTADOS: Em geral, a maioria dos casos positivos (64%) ocorreu em crianças com idade entre 5-12 anos, esta faixa etária foi significativamente a mais afetada (39,8%, p = 0,001, OR = 8,3, CI 95%: 1,9-36,9). Chiado foi relatado em 31% (19/61) e dispnéia em 23% (14/61) dos pacientes estudados. Um surto de gripe H1N1 com uma taxa de ataque de 35,7% entre as crianças (mediana de idade de 6 anos) foi documentado em abril de 2010, antes da extensão da campanha de vacinação contra o vírus pandêmico para crianças até 5 anos no Brasil. CONCLUSÕES: Portanto, neste estudo reforça a recomendação para imunizar crianças em idade escolar para reduzir a incidência da doença.

The incidence and clinical features of human coronaviruses (HCoVs) among Brazilian patients with respiratory illness are not well known. We investigated the prevalence of HCoVs among Brazilian outpatients and hospitalised patients with respiratory illnesses during 2009 and 2010. To identify the HCoVs, pancoronavirus and species-specific reverse-transcriptase polymerase chain reaction assays were performed. Five of 394 samples were positive for HCoVs (1.2%): 1/182 (0.5%) outpatients and 4/212 (1.8%) hospitalised patients. The OC43 and NL63 HCoVs were identified. Two patients were admitted to the intensive care unit. Underlying chronic disease was reported in cases and one diabetic adult died. HCoVs can cause lower respiratory infections and hospitalisation. Patients with pre-existing conditions and respiratory infections should be evaluated for HCoV infections.

This study assessed the occurrence of human rhinovirus (HRV) species in outpatient children attending day-care in Sao Paulo, Brazil. HRV reverse transcriptase polymerase chain reaction and amplicon sequencing were done in 120 samples collected in 2008. HRV was detected in 27.5% of samples. HRV C was detected in 60.7% of wheezers, a frequency not different from that observed in nonwheezers (69.6%).

A duplex reverse transcriptase polymerase chain reaction-restriction fragment length polymorphism for influenza virus subtyping was applied to 412 patient samples. The assay was able to discriminate all 47 influenza A H1N1 and H3N2 viruses. This rapid technique assessed if positive samples were current circulating strains or an emergent one and could be used as the 1st test in prepandemic stages.

ResumoObjetivoOs adenovírus desempenham um papel importante na etiologia da infecção aguda grave do trato respiratório inferior, especialmente entre crianças. O objetivo do estudo foi avaliar a detecção do adenovírus humano (HAdV) por diferentes métodos (imunofluorescência direta – DFA e reação em cadeia da polimerase nested – nested PCR) em amostras coletadas de diferentes populações de pacientes pediátricos.MétodosO material foi coletado de crianças portadoras de doença cardíaca congênita (DCC – 123 aspirados nasais coletados em 2005, 2007 e 2008) e de crianças da comunidade (CC – 165 aspirados nasais coletados em 2008). As crianças eram consideradas elegíveis se apresentassem infecção respiratória aguda (IRA) de provável etiologia viral, com até sete dias de início dos sintomas. Todas as amostras coletadas no estudo foram avaliadas por meio de DFA e nested PCR.ResultadosDe 209 amostras incluídas, 43 (14,8%) foram positivas em pelo menos um dos testes feitos: 17/165 (10,3%) das crianças da comunidade e 26/125 (20,8%) das crianças cardiopatas. As taxas de detecção por nested PCR foram 15/165 (9,1%) em crianças da comunidade e 24/125 (19,2%) em crianças cardiopatas. O método molecular mostrou maiores taxas de detecção quando comparado com a DFA (p

Made available in DSpace on 2013-08-12T18:58:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-01 Made available in DSpace on 2013-09-30T18:38:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:51:06Z No. of bitstreams: 0 Made available in DSpace on 2014-05-20T13:51:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Introdução: Infecções agudas do trato respiratório estão entre as doenças mais comuns em todas as pessoas. Os rinovírus têm sido descritos como agente etiológico de mais de 50% das infecções do trato respiratório ao redor do mundo. O objetivo deste trabalho foi avaliar a ocorrência de rinovírus humano (HRV), influenza vírus A e B, vírus respiratório sincicial humano e metapneumovírus (hMPV) em uma população de idosos que apresentava sintomas de gripe ou resfriado, e que residiam na Cidade de Botucatu, Estado de São Paulo, Brasil. Métodos: Foram coletados swabs nasais de 47 idosos após visitas de inclusão, entre os anos de 2002 e 2003 e que foram testadas através de GeneScan RT-PCR. Resultados: HRV foi detectado em 28.6% (14/47) e hMPV em 2% (1/47). De 14 amostras positivas para HRV, 9 foram sequenciadas, mostrando a seguinte distribuição de grupos: grupo A: 6 amostras, grupo B: 1 amostra e grupo C: 2 amostras. Conclusões: A alta incidência de HRV durante os meses de ocorrência de gripe necessita de estudos posteriores para avaliar o impacto desse vírus entre os idosos. A alta frequência de HRV pode contribuir para o aumento da demanda por serviços de saúde durante a estação de influenza. Introduction: Acute respiratory tract infections are the most common illness in all individuals. Rhinoviruses have been reported as the etiology of more than 50% of respiratory tract infections worldwide. The study prospectively evaluated 47 elderly individuals from a group of 384 randomly assigned for acute respiratory viral infections (cold or flu) and assessed the occurrence of human rhinovirus (HRV), influenza A and B, respiratory syncytial virus and metapneumovirus (hMPV) in Botucatu, State of São Paulo, Brazil. Methods: Forty-nine nasal swabs collected from 47 elderly individuals following inclusion visits from 2002 to 2003 were tested by GenScan RT-PCR. HRV-positive samples were sequenced for phylogenetic analysis. Results: No sample was positive for influenza A/B or RSV. HRV was detected in 28.6% (14/47) and hMPV in 2% (1/47). of 14 positive samples, 9 isolates were successfully sequenced, showing the follow group distribution: 6 group A, 1 group B and 2 group C HRVs. Conclusions: The high incidence of HRV during the months of the influenza season requires further study regarding HRV infection impact on respiratory complications among this population. Infection caused by HRV is very frequent and may contribute to increasing the already high demand for healthcare during the influenza season. Univ Fed São Paulo, Dept Med, Infect Dis Unit, Clin Virol Lab, São Paulo, Brazil São Paulo State Univ, Biosci Inst, Human & Anim Virol Lab, Botucatu, SP, Brazil Univ Estadual Campinas, Med Sci Coll, Campinas, SP, Brazil Fed Univ Para, Inst Biotechnol, BR-66059 Belem, Para, Brazil São Paulo State Univ, Biosci Inst, Human & Anim Virol Lab, Botucatu, SP, Brazil

Rinovírus (RV) é freqüentemente detectado nos tecidos tonsilares e nas secreções de nasofaringe de pacientes com doença adenotonsilar crônica, sem sintomas de infecção respiratória aguda (IRA). O objetivo deste estudo foi investigar a infecção por rinovírus em tonsilas humanas, com base em dois aspectos: infecção in vivo de células linfóides de tonsilas humanas naturalmente infectadas; e infecção ex vivo de células dissociadas desses tecidos inoculadas com rinovírus, visando a contribuir para elucidar possíveis mecanismos de infecção em amígdalas palatinas e adenóides humanas. De um total de 104 pacientes com doenças adenotonsilar crônicas, 21.1% (22/104) e 42.3% (44/104) apresentaram respectivamente amígdalas palatinas e adenóides positivas para RV por PCR. A replicação viral foi confirmada por hibridização in situ com sonda para intermediário replicativo nas regiões internas e externas aos folículos linfóides de amígdalas e adenóides, bem como em porções do epitélio ciliado de adenoides, e apenas raramente nas células epiteliais escamosas de tonsilas palatinas. A presença e distribuição de proteína estrutural do capsídeo viral foi detectada por imunohistoquímica (IHQ), utilizando anticorpos contra proteínas estruturais virais VP1 e VP2 nas tonsilas positivas para RV por qPCR. Os resultados indicaram marcação positiva tanto na superfície (epitélio), quanto em regiões extrafoliculares e centros germinativos. Em seguida, foi possível verificar a co-localização da marcação positiva da proteína estrutural VP2 de RV com marcadores linfocitários de membrana. Células CD4 + e CD20 + apresentaram marcação positiva para VP2 verificada usando estratégia de \'sequential immuno-peroxidase labelling and erasing\' (SIMPLE). Culturas primárias de células linfomononucleares (CLMN) de tonsilas sabidamente negativas para RV por PCR, foram infectadas in vitro, com RV (MOI=1). A replicação de RV foi titulada por TCID50, mostrando aumento inicial (24 h) e subsequente queda após 48 horas. Por IF observamos que os fenótipos de CLMN infectadas com RV in vitro foram células T CD4 + e B, mas também com células CD8 +, CD56 + e CD33 +. RV não infectou células CD123 +. RV foi isolado em WI- 38 e HeLa a partir de tecidos e secreções de nasofaringe de pacientes com hipertrofia tonsilar sem sintomas de infecção respiratória aguda. Nossos resultados confirmam que tonsilas de pacientes sem sintomas respiratórios agudos podem ser reservatórios de RV, que infecta não somente epitélio, mas também CLMN (frequentemente linfócitos T CD4 + e linfócitos B). A detecção de RNA intermediário replicativo e proteínas estruturais VP1 e VP2 nas tonsilas hipertróficas, além do isolamento de vírus infeccioso a partir de tecidos e secreções nasofaríngeas, classificam tonsilas hipertróficas como sítios de infecção e replicação de RV, e sugerem que esses indivíduos hipertróficos são portadores assintomáticos de RV persistente, e podem ser importantes fontes de transmissão de RV na comunidade. The chronic adenotonsillar diseases are frequent otorhinolaryngologic conditions caused by chronic inflammation of adenoids and palatine tonsils. Rhinovirus (RV) is highly frequently detected in secretions, and tonsillar tissues from patients experience chronic tonsillar hypertrophy without symptoms of ARI, and our goal is to full understanding of viral infections in hypertrophic tonsillar tissues by RV. Of 104 enrolled patients with adenotonsillar chronic diseases, 21.1% (22/104) and 42.3% (44/104) had palatine tonsils and adenoids positive for RV by qPCR, respectively. RV Viral replication was confirmed by in situ hybridizations. Minus-strand RNA were detected in all tested samples (7 tonsils and 9 adenoids), and positive reactions were seen inside and outside of lymphoid follicles from tonsils and adenoids, in the ciliated epithelium of the adenoids and rarely in positive squamous epithelium cells from tonsils. The presence of viral structural protein VP1 and VP2 was detected within and outside of the lymphoid follicles from tonsils and adenoids, and also in epithelial cells from adenoids by immunohistochemistry (IHC). Later, by sequential immuno-peroxidase labelling and erasing protocol (SIMPLE), we saw co-localization of RV VP2 capsid protein staining with CD4 positive cells and CD20 positive cells. We confirmed that RV could infect primary culture of tonsilar mononuclear cells (TMNCs). Additionally, intracellular replication of RV in TMNCs, measured by TCID50 in HeLa cells, had an initial increase in the first 24 hours, and dropped at 48 hours post infection. Immunolocalization staining with anti-RV and TMNCs surface markers indicated that phenotypes of susceptible cells were T-cells both CD4+ and CD20+, but also, we saw co-localization of VP-2 protein with CD8+ cells, CD56+ cells and CD33+ cells. RV-16 couldn\'t infect CD123+ cell in our experiments. Finally, we were able to recover 4 rhinoviruses by inoculating WI-38 fibroblast cells and HeLa cells, confirming by the cytopathic effect and immunofluorescence positive staining with anti-VP1 antibody. Taken together, our results indicate that tonsils and adenoids of patients without ARI may be reservoirs of replicating human rhinovirus, infecting manly Tcells CD4+ and CD20+ B-cells. The high-frequency detection of RNA (-) and VP1 expression in tissues from patients with chronic adenotonsillar diseases, plus the isolation of infectious viral particles, suggests that these detected agents replicate in the adenotonsillar tissues and this specific sites may be important sources of transmission of RV in the community.