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1.
Int J Legal Med ; 134(4): 1271-1274, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32458044

RESUMO

In the setting of the coronavirus disease 2019 (COVID-19) pandemic, only few data regarding lung pathology induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is available, especially without medical intervention interfering with the natural evolution of the disease. We present here the first case of forensic autopsy of a COVID-19 fatality occurring in a young woman, in the community. Diagnosis was made at necropsy and lung histology showed diffuse alveolar damage, edema, and interstitial pneumonia with a geographically heterogeneous pattern, mostly affecting the central part of the lungs. This death related to COVID-19 pathology highlights the heterogeneity and severity of central lung lesions after natural evolution of the disease.


Assuntos
Betacoronavirus , Infecções por Coronavirus/patologia , Pulmão/patologia , Pneumonia Viral/patologia , Adenoviridae/genética , Adenoviridae/isolamento & purificação , Adulto , Autopsia , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Bocavirus/genética , Bocavirus/isolamento & purificação , Proteína C-Reativa/análise , Coronavirus/genética , Coronavirus/isolamento & purificação , Feminino , Humanos , Influenzavirus A/genética , Influenzavirus A/isolamento & purificação , Influenzavirus B/genética , Influenzavirus B/isolamento & purificação , Macrófagos/patologia , Megacariócitos/patologia , Metapneumovirus/genética , Metapneumovirus/isolamento & purificação , Neutrófilos/patologia , Obesidade Mórbida , Pandemias , Pró-Calcitonina/sangue , Reação em Cadeia da Polimerase em Tempo Real , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhinovirus/genética , Rhinovirus/isolamento & purificação , Suíça , Linfócitos T/patologia
2.
Zhonghua Yu Fang Yi Xue Za Zhi ; 54(3): 294-300, 2020 Mar 06.
Artigo em Chinês | MEDLINE | ID: mdl-32187935

RESUMO

Objective: To understand the genetic variation and epidemiological characteristics of human respiratory syncytial virus (HRSV) in Guangzhou. Methods: Nasopharyngeal swabs specimens were collected from 0-6 year old children hospitalized with acute respiratory infection, then HRSV was tested and genotyped by RT-PCR. Phylogenetic tree was bulit using MEGA 6.0 software. NetNGlyc 1.0 server was used to predict the potential N-linked glycosylation sites. Results: A total of 1 225 nasopharyngeal specimens were collected, including 783 males and 442 females. The median (P(25), P(75)) age was 8 (3, 24) months. Among the 209 HRSV-positive cases (17.06%), 117 cases (55.98%) were HRSV-A and 92 cases (44.02%) were HRSV-B. The two distinct subgroups (HRSV-A and HRSV-B) alternately played dominant role to cause HRSV infection and exchange almost once every two years. The HRSV prevalence rate decreased with age. The HRSV-positive rate among children under 2 years old was 18.83% (196 cases), accounting for 93.78% of the total positive cases. There were 32 HRSV positive cases co-infected with at least one respiratory virus, with the co-infection rate of 15.31%. Phylogenetic tree analysis of the second hypervariable region (HVR2) of the G protein classified the HRSV-A specimens into ON1 (n=62) and NA1 (n=2) genotypes while all HRSV-B specimens belonged to BA genotype (n=53). The HVR2 of the G protein varied in using stop condon, amino acid substitutions, glycosylation sites. Conclusion: Children under 2 years old were the high risk population of HRSV infection in Guangzhou. ON1 genotype turned into a primary genetype of the HRSV-A subgroup while BA genotype dominated the HRSV-B subgroup. A greater diversification of amino acid substitutions, and some deletion and insertion of glycosylation sites embodied the polymorphism of G protein as main protective antigen.


Assuntos
Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Criança , Criança Hospitalizada , Pré-Escolar , China/epidemiologia , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Epidemiologia Molecular , Filogenia , Infecções por Vírus Respiratório Sincicial/diagnóstico
3.
Arch Virol ; 165(5): 1069-1077, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32144544

RESUMO

We investigated the molecular epidemiology of respiratory syncytial virus (RSV) isolated from children during 28 consecutive seasons (1990-2018) and the genetic variability of the duplication region of RSV genotypes ON1 and BA in South Korea. RSV was identified using culture-based methods in Hep-2 cells and was grouped as RSV-A or RSV-B by an immunofluorescence assay. The second hypervariable region of the G gene was sequenced for genotyping. The nucleotide and deduced amino acid sequences of the duplication region of RSV ON1 and BA were analyzed. A total of 670 RSV-A and 233 RSV-B isolates were obtained. For RSV-A, the NA1 genotype predominated during the 2004/2005-2011/2012 seasons. The ON1 genotype was first detected in 2011 and has since replaced all other genotypes. For RSV-B, the GB3 genotype predominated during the 1999/2000-2005/2006 seasons, but the BA genotype also replaced all other genotypes of RSV-B after the first season in which it was isolated (2005/2006). In ON1 and BA genotype RSV strains, novel sequence types of the duplication region of the G gene were identified in 50-95% and 33-80% of the isolates, respectively, in each season. The ON1 and BA9 genotypes are responsible for the current epidemics of RSV infection in South Korea. The sequences in the duplication region of the G gene have evolved continuously and might be sufficient for the identification of specific strains of the RSV-A ON1 and RSV-B BA genotypes.


Assuntos
Variação Genética , Genótipo , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/classificação , Proteínas Virais de Fusão/genética , Duplicação Gênica , Humanos , Epidemiologia Molecular , República da Coreia/epidemiologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Análise de Sequência de DNA
4.
BMC Infect Dis ; 20(1): 168, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32087697

RESUMO

BACKGROUND: Respiratory tract infection (RTI) in young children is a leading cause of morbidity and hospitalization worldwide. There are few studies assessing the performance for bronchoalveolar lavage fluid (BALF) versus oropharyngeal swab (OPS) specimens in microbiological findings for children with RTI. The primary purpose of this study was to compare the detection rates of OPS and paired BALF in detecting key respiratory pathogens using suspension microarray. METHODS: We collected paired OPS and BALF specimens from 76 hospitalized children with respiratory illness. The samples were tested simultaneously for 8 respiratory viruses and 5 bacteria by suspension microarray. RESULTS: Of 76 paired specimens, 62 patients (81.6%) had at least one pathogen. BALF and OPS identified respiratory pathogen infections in 57 (75%) and 49 (64.5%) patients, respectively (P > 0.05). The etiology analysis revealed that viruses were responsible for 53.7% of the patients, whereas bacteria accounted for 32.9% and Mycoplasma pneumoniae for 13.4%. The leading 5 pathogens identified were respiratory syncytial virus, Streptococcus pneumoniaee, Haemophilus influenzae, Mycoplasma pneumoniae and adenovirus, and they accounted for 74.2% of etiological fraction. For detection of any pathogen, the overall detection rate of BALF (81%) was marginally higher than that (69%) of OPS (p = 0.046). The differences in the frequency distribution and sensitivity for most pathogens detected by two sampling methods were not statistically significant. CONCLUSIONS: In this study, BALF and OPS had similar microbiological yields. Our results indicated the clinical value of OPS testing in pediatric patients with respiratory illness.


Assuntos
Líquido da Lavagem Broncoalveolar/microbiologia , Líquido da Lavagem Broncoalveolar/virologia , Criança Hospitalizada , Testes Diagnósticos de Rotina/métodos , Orofaringe/microbiologia , Orofaringe/virologia , Infecções Respiratórias/diagnóstico , Criança , Pré-Escolar , Feminino , Hospitalização , Humanos , Lactente , Recém-Nascido , Masculino , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/microbiologia , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação
5.
PLoS One ; 15(2): e0228329, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32017782

RESUMO

BACKGROUND: In Bangladesh, pneumonia has a higher mortality among malnourished children aged <5 years. Evaluating pneumonia etiology among malnourished children may help improve empiric treatment guidelines. METHODS: During April 2015-December 2017, we conducted a case-control study among severe acute malnourished (SAM) children aged <5 years admitted to the Dhaka hospital of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). We enrolled hospital admitted SAM children with clinical or radiological pneumonia as cases (during April 2015 to March 2017) and hospital admitted SAM children without any respiratory symptom in the past 10 days before admission as controls (during February 2016 to December 2017). We tested nasopharyngeal wash from both case and control for respiratory syncytial virus (RSV), human metapneumovirus (HMPV), influenza viruses, human parainfluenza viruses (HPIV), rhinovirus and adenovirus by singleplex real-time reverse transcriptase polymerase chain reaction. To identify the independent association of pneumonia with viral pathogens during February 2016 to March 2017, we used multivariable logistic regression for calculating adjusted odds ratios. RESULTS: We enrolled 360 cases and 334 controls. For case and control the median age was 8 months (IQR: 5-13) and 11 months (IQR: 6-18) (p = 0.001) respectively. Weight/age Z-score was -4.3 (SD ±0.7) for cases and -4.1 (SD ±1.1) for controls (p = 0.01). Among cases 68% had both clinical and radiological pneumonia, 1% had clinical pneumonia and 31% had only radiological pneumonia. Respiratory virus detection was high in cases compared to controls [69.9% (251) vs. 44.8% (148), p = 0.0001]. The most frequently detected viruses among cases were rhinoviruses (79, 22.0%) followed by RSV (32, 8.9%), adenovirus (23, 6.4%), HPIV (22, 6.1%), influenza virus (16, 4.5%), and HMPV (16, 4.5%). Among the controls, rhinoviruses (82, 24.8%) were most commonly detected one followed by adenovirus (26,7.9%), HMPV (5, 1.5%), HPIV (4, 1.2%), RSV (3, 0.9%), and influenza virus (2, 0.6%). RSV (OR 13.1; 95% CI: 1.6, 106.1), influenza virus (OR 8.7; 95% CI: 1.0, 78.9), HPIV (3.8; 95% CI: 1.0, 14.8), and HMPV (2.7; 95% CI: 1.3, 5.5) were independently associated with pneumonia while compared between 178 cases and 174 controls. CONCLUSION: Viral etiology of pneumonia in SAM children were mainly attributable to RSV, influenza, HPIV and HMPV. Our study findings may help in planning further studies targeting vaccines or drugs against common respiratory viruses responsible for pneumonia among SAM children.


Assuntos
Desnutrição/patologia , Pneumonia/diagnóstico , Adenoviridae/genética , Adenoviridae/isolamento & purificação , Bangladesh , Estudos de Casos e Controles , Pré-Escolar , Feminino , Hospitais Urbanos , Humanos , Lactente , Modelos Logísticos , Masculino , Desnutrição/complicações , Nasofaringe/microbiologia , Nasofaringe/virologia , Razão de Chances , Pneumonia/complicações , Pneumonia/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Respirovirus/genética , Respirovirus/isolamento & purificação , Rhinovirus/genética , Rhinovirus/isolamento & purificação , Índice de Gravidade de Doença
6.
PLoS One ; 15(1): e0227558, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923213

RESUMO

Respiratory syncytial virus (RSV) is a nonsegmented negative-strand RNA virus (NSV) and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3' promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription extending across the genome, with the highest level of mRNA coming from the most promoter-proximal gene, the first nonstructural (NS1) gene, and mRNA levels from subsequent genes dropping until reaching a minimum at the most promoter-distal gene, the polymerase (L) gene. However, recent reports show non-gradient levels of mRNA, with higher than expected levels from the attachment (G) gene. It is unknown to what extent different transcript stabilities might shape measured mRNA levels. It is also unclear whether patterns of RSV gene expression vary, or show strain- or genotype-dependence. To address this, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and in cotton rats infected with RSV isolates belonging to four genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were non-gradient and genotype-specific, where mRNA levels from the G gene exceeded those from the more promoter-proximal nucleocapsid (N) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. Our results indicate that gene expression from a small but diverse set of RSV genotypes is non-gradient and genotype-dependent. We propose novel models of RSV transcription that can account for non-gradient transcription.


Assuntos
RNA Viral/metabolismo , Vírus Sincicial Respiratório Humano/genética , Transcrição Genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Linhagem Celular , Feminino , Genótipo , Meia-Vida , Humanos , Masculino , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Sigmodontinae , Transcrição Genética/efeitos dos fármacos , Replicação Viral
7.
Nat Commun ; 11(1): 368, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953395

RESUMO

The respiratory syncytial virus (RSV) RNA polymerase, constituted of a 250 kDa large (L) protein and tetrameric phosphoprotein (P), catalyzes three distinct enzymatic activities - nucleotide polymerization, cap addition, and cap methylation. How RSV L and P coordinate these activities is poorly understood. Here, we present a 3.67 Å cryo-EM structure of the RSV polymerase (L:P) complex. The structure reveals that the RNA dependent RNA polymerase (RdRp) and capping (Cap) domains of L interact with the oligomerization domain (POD) and C-terminal domain (PCTD) of a tetramer of P. The density of the methyltransferase (MT) domain of L and the N-terminal domain of P (PNTD) is missing. Further analysis and comparison with other RNA polymerases at different stages suggest the structure we obtained is likely to be at an elongation-compatible stage. Together, these data provide enriched insights into the interrelationship, the inhibitors, and the evolutionary implications of the RSV polymerase.


Assuntos
Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/química , RNA Replicase/química , Vírus Sincicial Respiratório Humano/enzimologia , Proteínas Virais/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares , Fosfoproteínas/química , Conformação Proteica , Domínios Proteicos , RNA Replicase/genética , RNA Replicase/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Estruturas Virais
8.
Int J Infect Dis ; 90: 5-17, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31634614

RESUMO

BACKGROUND: Human respiratory syncytial virus (RSV) is one of the most important pathogens that cause acute respiratory infections in children and immunocompromised adults. This work was conducted to understand the epidemiological and phylogenetic features of RSV in southern China during 2011-2016. METHODS: A total of 16 024 nasopharyngeal swabs were collected from patients with respiratory infections in 14 hospitals, and screened for RSV and seven other respiratory viruses using real-time PCR. Six hundred and twenty-three RSV-positive samples from 13 hospitals were further analyzed for subtypes. G gene sequencing and phylogenetic analysis were performed based on 46 RSV-A and 15 RSV-B strains. RESULTS: RSV was detected in 9.5% of the 16 024 specimens, the highest among the eight respiratory viruses screened. Most of these specimens came from inpatients and children under 3 years of age. The incidence of RSV-A (9.4%) was higher than that of RSV-B (4.4%) in children (<15 years), but not in adults (0.64% vs. 0.58%). A 2-year RSV-A dominance followed by a 1-year RSV-B dominance pattern was found. The co-detection rate of RSV was 25.1%. The main prevalent genotypes were NA1, ON1, and BA9. The prevalent RSV-A genotype in 2011-2012 was NA1, close to Chongqing and Brazil, but a new Hong Kong ON1 genotype was introduced and became the prevalent genotype in Guangzhou in 2014-2015. Deduced amino acid sequence analysis confirmed the ongoing evolution and a high selection pressure of RSV-A and B strains, especially in RSV-A ON1 and NA1 genotypes. CONCLUSIONS: This study demonstrated the molecular epidemiological characteristics of RSV in patients with respiratory infections in southern China.


Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Adolescente , Adulto , Criança , Pré-Escolar , China/epidemiologia , Feminino , Genótipo , Hong Kong/epidemiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Filogenia , Prevalência , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/epidemiologia , Adulto Jovem
9.
Int J Infect Dis ; 90: 170-180, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31669592

RESUMO

OBJECTIVES: The present study provides a comprehensive review of the recently published data on RSV epidemiology in adults and the elderly in Latin America. METHODS: A systematic literature search was carried out in Medline, Scielo, Lilacs, and Cochrane Library. The search strategy aimed at retrieving studies focusing on RSV prevalence, burden, risk factors, and the routine clinical practice in the prevention and management of RSV infections in Latin American countries. Only articles published between January 2011 and December 2017 were considered. RESULTS: Eighteen studies were included. Percentages of RSV detection varied highly across included studies for adult subjects with respiratory infections (0% to 77.9%), influenza-like illness (1.0% to 16.4%) and community-acquired pneumonia (1.3% to 13.5%). Considerable percentages of hospitalization were reported for RSV-infected adults with influenza-like illness (40.9% and 69.9%) and community-acquired pneumonia (91.7%). CONCLUSIONS: Recent RSV data regarding adult populations in Latin America are scarce. RSV was documented as a cause of illness in adults and the elderly, being identified in patients with acute respiratory infections, influenza-like illness and community-acquired pneumonia. The studies suggest that RSV infections may be a significant cause of hospitalization in adult populations in Latin America, including younger adults.


Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Infecções Comunitárias Adquiridas/epidemiologia , Infecções Comunitárias Adquiridas/virologia , Feminino , Hospitalização , Humanos , América Latina/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/genética , Fatores de Risco , Adulto Jovem
10.
Braz J Infect Dis ; 23(6): 427-434, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31734172

RESUMO

To investigate the genetic variation and molecular epidemiology characteristics of Human Respiratory Syncytial Virus (HRSV) in Guizhou Province, nasopharyngeal aspirates were collected from patients with acute respiratory infection (ARI) in Guizhou Provincial People's Hospital, from December 2017 to March 2018, and inoculated to Hep-2 cells to isolate HRSV. Cells that showed cytopathic effect (CPE) were then confirmed by indirect immunofluorescence assay and reverse transcription. The sequence of the PCR products was determined for HRSV isolates, and the genetic variation was analyzed. Out of 196 nasopharyngeal aspirate samples, HRSV were isolated in 39. The second hypervariable region at the 3' terminal of glycoprotein gene (HVR2) sequence analysis showed that subgroup A was dominant. Seventy-nine percent of the isolates belonged to subgroup A, ON1 genotype, and 21 % belonged to subgroup B, BA9 genotype, which indicates that the dominant HRSV circulating in Guizhou Province was subgroup A, genotype ON1, co-circulating with a less prevalent subgroup B, genotype BA9.


Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/virologia , Pré-Escolar , China/epidemiologia , Genótipo , Humanos , Epidemiologia Molecular , Cavidade Nasal/virologia , Filogenia , Reação em Cadeia da Polimerase , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções Respiratórias/epidemiologia , Análise de Sequência de DNA
11.
Indian J Med Microbiol ; 37(2): 248-254, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31745027

RESUMO

Introduction: Human respiratory syncytial virus (HRSV) an RNA virus belonging to Pneumoviridae family, is an important cause of acute respiratory infections (ARIs) in young children. HRSV circulates as two subgroups A and B, which are further categorised into several genotypes. New genotypes may replace existing ones over successive epidemic seasons and multiple genotypes may cocirculate in the same community rendering it important to monitor them at the molecular level. The present study assessed the circulating genotypes of HRSV in Chennai. Materials and Methods: Two hundred and sixty-seven children with ARI were recruited during the study from April 2016 to March 2018 for detecting HRSV A and B by real-time reverse transcription-polymerase chain reaction. Phylogeny and selection pressure analysis were done. Results: Fifty-seven of the 267 samples (21.3%) were positive for HRSV, of which 7.1% and 14.2% were HRSV A and B, respectively, indicating that HRSV B was the major subgroup circulating in Chennai. Peak activity of HRSV was observed during the monsoon and winter months. Phylogenetic analysis of 2nd hypervariable region (HVR) of attachment glycoprotein gene (G gene) revealed that the HRSV A strains belonged to ON1 and HRSV B strains belonged to BA9 genotypes. Several unique amino acid substitutions were observed among the study strains. The Shannon entropy plot revealed that the HRSV A strains from our study have a high potential for amino acid substitutions in the 2nd HVR of G gene. Conclusion: This study underlines the genetic diversity of HRSV and emphasises the need for continued molecular surveillance for infection management and prevention strategies.


Assuntos
Variação Genética , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Criança , Pré-Escolar , Entropia , Evolução Molecular , Humanos , Índia/epidemiologia , Lactente , Mutação , Filogenia , Vigilância da População , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/classificação , Infecções Respiratórias/diagnóstico , Estações do Ano , Análise de Sequência de DNA
12.
Nat Commun ; 10(1): 4595, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31597913

RESUMO

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.


Assuntos
Adenosina/análogos & derivados , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Replicação Viral/imunologia , Células A549 , Adenosina/genética , Adenosina/imunologia , Adenosina/metabolismo , Animais , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Feminino , Células HeLa , Humanos , Masculino , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/patogenicidade , Sigmodontinae , Regulação para Cima/imunologia , Vacinas Atenuadas/imunologia , Células Vero , Virulência/genética , Virulência/imunologia , Replicação Viral/genética
13.
J Microbiol Biotechnol ; 29(12): 2006-2013, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31581384

RESUMO

The isolation of respiratory viruses, especially from clinical specimens, often shows poor efficiency with classical cell culture methods. The lack of suitable methods to generate virus particles inhibits the development of diagnostic assays, treatments, and vaccines. We compared three inoculation methods, classical cell culture, the addition of a JAK2 inhibitor AZD1480, and centrifugation-enhanced inoculation (CEI), to replicate human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV). In addition, a combined method using AZD1480 treatment and CEI was used on throat swabs to verify that this method could increase virus isolation efficiency from human clinical specimens. Both CEI and AZD1480 treatment increased HRSV and HMPV genome replication. Also, the combined method using CEI and AZD1480 treatment enhanced virus proliferation synergistically. The combined method is particularly suited for the isolation of interferon-sensitive or slowly growing viruses from human clinical specimens.


Assuntos
Centrifugação/métodos , Pneumovirus/isolamento & purificação , Pirazóis/farmacologia , Pirimidinas/farmacologia , Cultura de Vírus/métodos , Humanos , Metapneumovirus/efeitos dos fármacos , Metapneumovirus/genética , Metapneumovirus/crescimento & desenvolvimento , Metapneumovirus/isolamento & purificação , Pneumovirus/efeitos dos fármacos , Pneumovirus/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/isolamento & purificação , Manejo de Espécimes , Replicação Viral
14.
Biomedica ; 39(2): 415-426, 2019 06 15.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-31529826

RESUMO

Introduction: Human respiratory syncytial virus (hRSV) is the most frequent cause of acute respiratory infection of the lower respiratory tract in children under the age of five. The development of molecular techniques able to identify hRSV is one of the current challenges in the field of clinical research. Objective: To evaluate the ability of an isothermal amplification method to rapidly detect hRSV in children with acute respiratory infection. Materials and methods: We collected 304 nasopharyngeal swab samples from children with symptoms of acute respiratory infection who attended the emergency unit at Hospital de la Universidad del Norte in Barranquilla from April, 2016, to July, 2017. After extracting viral RNA from the samples, we evaluated the ability of the reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) M assay to rapidly detect hRSVA and hRSVB compared to other molecular techniques: quantitative PCR (qPCR), reverse transcriptase-LAMP L assay, and as a standard, the multiplex nested reverse transcriptase polymerase chain reaction (nested RT-PCR). Results: The RT-LAMP M assay had a sensitivity of 93.59% and a specificity of 92.92%, and a concordance of 0.83 ± 0.036 as compared with the nested RT-PCR test. While the Kappa index of the RT-LAMP M assay was higher than the values for the RT-LAMP L assay and the qPCR, the values of the latter two methods were in agreement (0.75 ± 0.043 and 0.71 ± 0.045, respectively). Conclusion: Due to the shorter running times, lower costs and better performance of the RT-LAMP M assay, it can be considered as a useful clinical tool for the detection of RSVA.


Assuntos
Técnicas de Amplificação de Ácido Nucleico , RNA Viral/isolamento & purificação , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Pré-Escolar , Estudos Transversais , Diagnóstico Precoce , Feminino , Humanos , Lactente , Masculino , Nasofaringe/virologia , Reação em Cadeia da Polimerase , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/genética , Sensibilidade e Especificidade
15.
Intervirology ; 62(3-4): 112-115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31542785

RESUMO

OBJECTIVE: Respiratory syncytial virus (RSV) infection causes lower respiratory tract infection primarily in infants and toddlers. RSV reinfection also occurs throughout life and can be a significant cause of pneumonia and mortality in the elderly. Surges in physician offices, emergency department visits, and hospitalization often result from RSV illness. Point-of-care (POC) testing reduces healthcare costs and permits informed decisions on treatment, further testing, or hospitalization to occur during the physician-patient encounter. Optimal POC assays must be sensitive, easy to perform, and provide rapid results. METHODS: In this study, 2 POC assays (Alere i; Abbot Rapid Diagnostics and cobas Liat, Roche Molecular, Inc.) and a laboratory-based assay (Solana; Quidel, Inc.) were evaluated using 133 patient nasopharyngeal specimens. RESULTS: Sensitivity/specificity values (%) of 94.7/96.1, 98.2/96.1, and 96.5/94.7 were obtained for the Alere i, Liat, and Solana assays, respectfully. These values approximated those stated in each assay's package insert. CONCLUSION: Rapid molecular assays for RSV are sensitive and accurate. The choice of assay should reflect each healthcare institution's specific testing needs with respect to the benefits and drawbacks of each product.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vírus Sincicial Respiratório Humano/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Nasofaringe/virologia , Testes Imediatos , Vírus Sincicial Respiratório Humano/genética , Sensibilidade e Especificidade , Adulto Jovem
16.
Infect Genet Evol ; 76: 104039, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31521788

RESUMO

Acute respiratory infection (ARI) is the most common infection in children under 5 years of age and it is frequently caused by two pneumoviruses, human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV). Epidemic seasons of these viruses overlap and disease manifestations are highly similar, including severe lower ARI such as bronchiolitis or pneumonia. Reinfections with pneumoviruses are frequent and limited prevention treatment is available. Genetic diversity of HRSV and HMPV strains circulating in Croatia was monitored during four consecutive years (2014-2017). Co-circulation of multiple lineages was observed for both viruses. Within HRSV group A, ON1 strains gained strong predominance during the 4-year period, while previously dominant genotype NA1 was detected only sporadically. Similarly, newly occurring HMPV genotype A2c gained predominance over genotype A2b during this period, resulting in all infection in 2017 being caused by A2c. Along with phylogenetic analysis based on the commonly used fragments for detection and genotyping of these viruses, full length G and SH genes were also analysed. Evolutionary dynamics showed that inferred substitution rates of HRSV and HMPV are between 2.51 × 10-3 and 3.61 × 10-3 substitutions/site/year. This study established presence of recently described HMPV strains containing large duplications in the G gene in Croatia. Viruses with either of the two duplications belong to a subcluster A2c, which has completely replaced all other group A subclusters in 2017.


Assuntos
Metapneumovirus/classificação , Infecções por Paramyxoviridae/epidemiologia , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/classificação , Infecções Respiratórias/virologia , Criança , Pré-Escolar , Croácia/epidemiologia , Substituição de Medicamentos , Evolução Molecular , Feminino , Humanos , Lactente , Masculino , Metapneumovirus/genética , Metapneumovirus/isolamento & purificação , Epidemiologia Molecular , Filogenia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação
17.
PLoS One ; 14(9): e0220908, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31479459

RESUMO

Acute respiratory tract infections are a major cause of respiratory morbidity and mortality in pediatric patients worldwide. However, accurate viral and immunologic markers to predict clinical outcomes of this patient population are still lacking. Droplet digital PCR assays for influenza and respiratory syncytial virus (RSV) were designed and performed in 64 respiratory samples from 23 patients with influenza virus infection and 73 samples from 19 patients with RSV infection. Samples of patients with hematologic malignancies, solid tumors, or sickle cell disease were included. Clinical information from institutional medical records was reviewed to assess disease severity. Samples from patients with fever or respiratory symptoms had a significantly higher viral loads than those from asymptomatic patients. Samples from patients with influenza virus and RSV infection collected at presentation had significantly higher viral loads than those collected from patients after completing a course of oseltamivir or ribavirin, respectively. RSV loads correlated positively with clinical symptoms in patients ≤5 years of age, whereas influenza viral loads were associated with clinical symptoms, irrespective of age. Patients receiving antivirals for influenza and RSV had a significant reduction in viral loads after completing therapy. Digital PCR offers an effective method to monitor the efficacy of antiviral treatment for respiratory tract infections in immunocompromised hosts.


Assuntos
Vírus da Influenza A/genética , Vírus da Influenza B/genética , Influenza Humana/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Carga Viral , Adolescente , Antivirais/uso terapêutico , Criança , Pré-Escolar , Coinfecção , Feminino , Humanos , Lactente , Influenza Humana/diagnóstico , Influenza Humana/tratamento farmacológico , Linfopenia/diagnóstico , Linfopenia/etiologia , Masculino , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Avaliação de Sintomas , Resultado do Tratamento , Adulto Jovem
18.
Rev Soc Bras Med Trop ; 52: e20180249, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365620

RESUMO

INTRODUCTION: Prevalence of influenza A virus (Flu-A), respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) was assessed in children with acute respiratory infections (ARIs). METHODS: Nasopharyngeal aspirates and throat swabs were subjected to real-time polymerase chain reaction (PCR) to detect RSV and Flu-A and to conventional PCR to detect hMPV. RESULTS: Of the 156 children assessed, 93 (59.6%) carried at least one virus, with 35.9% positive for RSV, 14.1% for hMPV, and 9.6% for Flu-A. The prevalence of co-infections was 2.6%. CONCLUSIONS: The high detection rate may reflect increased sensitivity of real-time PCR compared to traditional PCR and viral culture.


Assuntos
Influenza Humana/epidemiologia , Infecções por Paramyxoviridae/epidemiologia , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções Respiratórias/virologia , Adolescente , Criança , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Vírus da Influenza A/genética , Irã (Geográfico)/epidemiologia , Masculino , Metapneumovirus/genética , Nasofaringe/virologia , Reação em Cadeia da Polimerase em Tempo Real , Vírus Sincicial Respiratório Humano/genética , Infecções Respiratórias/epidemiologia
19.
PLoS Pathog ; 15(7): e1007944, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31306469

RESUMO

The respiratory syncytial virus (RSV) fusion (F) glycoprotein is a major target of neutralizing antibodies arising from natural infection, and antibodies that specifically bind to the prefusion conformation of RSV F generally demonstrate the greatest neutralization potency. Prefusion-stabilized RSV F variants have been engineered as vaccine antigens, but crystal structures of these variants have revealed conformational differences in a key antigenic site located at the apex of the trimer, referred to as antigenic site Ø. Currently, it is unclear if flexibility in this region is an inherent property of prefusion RSV F or if it is related to inadequate stabilization of site Ø in the engineered variants. Therefore, we set out to investigate the conformational flexibility of antigenic site Ø, as well as the ability of the human immune system to recognize alternative conformations of this site, by determining crystal structures of prefusion RSV F bound to neutralizing human-derived antibodies AM22 and RSD5. Both antibodies bound with high affinity and were specific for the prefusion conformation of RSV F. Crystal structures of the complexes revealed that the antibodies recognized distinct conformations of antigenic site Ø, each diverging at a conserved proline residue located in the middle of an α-helix. These data suggest that antigenic site Ø exists as an ensemble of conformations, with individual antibodies recognizing discrete states. Collectively, these results have implications for the refolding of pneumovirus and paramyxovirus fusion proteins and should inform development of prefusion-stabilized RSV F vaccine candidates.


Assuntos
Antígenos Virais/química , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Sítios de Ligação/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Prolina/química , Conformação Proteica , Vírus Sincicial Respiratório Humano/química , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais de Fusão/genética
20.
BMC Infect Dis ; 19(1): 613, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299924

RESUMO

BACKGROUND: The Respiratory Syncytial Virus (RSV) A genotype ON1, which was first detected in Ontario (Canada) in 2010/11, appeared in Germany in 2011/12. Preliminary observations suggested a higher clinical severity in children infected with this new genotype. We investigated spread and disease severity of RSV-A ON1 in pediatric in- and outpatient settings. METHODS: During 2010/11 to 2016/17, clinical characteristics and respiratory samples from children with acute respiratory tract infections (RTI) were obtained from ongoing surveillance studies in 33 pediatric practices (PP), one pediatric hospital ward (PW) and 23 pediatric intensive care units (PICU) in Germany. RSV was detected in the respiratory samples by PCR; genotypes were identified by sequencing. Within each setting, clinical severity markers were compared between RSV-A ON1 and RSV-A non-ON1 genotypes. RESULTS: A total of 603 children with RSV-RTI were included (132 children in PP, 288 in PW, and 183 in PICU). Of these children, 341 (56.6%) were infected with RSV-A, 235 (39.0%) with RSV-B, and one child (0.2%) with both RSV-A and RSV-B; in 26 (4.3%) children, the subtype could not be identified. In the 341 RSV-A positive samples, genotype ON1 was detected in 247 (72.4%), NA1 in 92 (26.9%), and GA5 in 2 children (0.6%). RSV-A ON1, rarely observed in 2011/12, was the predominant RSV-A genotype in all settings by 2012/13 and remained predominant until 2016/17. Children in PP or PW infected with RSV-A ON1 did not show a more severe clinical course of disease compared with RSV-A non-ON1 infections. In the PICU group, hospital stay was one day longer (median 8 days, inter-quartile range (IQR) 7-12 vs. 7 days, IQR 5-9; p = 0.02) and duration of oxygen treatment two days longer (median 6 days, IQR 4-9 vs. 4 days, IQR 2-6; p = 0.03) for children infected with RSV-A ON1. CONCLUSIONS: In children, RSV-A ON1 largely replaced RSV-A non-ON1 genotypes within two seasons and remained the predominant RSV-A genotype in Germany during subsequent seasons. A higher clinical severity of RSV-A ON1 was observed within the group of children receiving PICU treatment, whereas in other settings clinical severity of RSV-A ON1 and non-ON1 genotypes was largely similar.


Assuntos
Infecções por Vírus Respiratório Sincicial/patologia , Vírus Sincicial Respiratório Humano/genética , Infecções Respiratórias/patologia , Pré-Escolar , Feminino , Genótipo , Alemanha/epidemiologia , Hospitais Pediátricos , Humanos , Lactente , Unidades de Terapia Intensiva Pediátrica , Tempo de Internação , Masculino , Filogenia , RNA Viral/metabolismo , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Estações do Ano , Índice de Gravidade de Doença
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