Preparation and immunization effect evaluation of recombinant F protein vaccine of respiratory syncytial virus / 中国生物制品学杂志

@#Objective To prepare recombinant F protein vaccine of respiratory syncytial virus(RSV) and evaluate its immunization effect.Methods Two RSV vaccines based on RSV F protein were prepared:one was a mucosal vaccine with bacterial like particle(BLP)as adjuvant and the other was an injectable vaccine with aluminium hydroxide as adjuvant.Forty female BALB/c mice were randomly divided into four groups:BLP-F,BLP control,AL-F and AL control group,with 10mice in each group.BLP-F and BLP control group were inoculated intranasally,and AL-F and AL control group were inoculated subcutaneously.The mice were immunized once each at day 0,14 and 28,respectively.Two weeks after the last immunization,the titers of serum IgG antibody and IgA antibody in nasal lotion were detected by ELISA,and the titers of neutralizing antibody were detected by plaque test.Results Both vaccines induced high levels of serum binding antibodies and neutralizing antibodies,and the induction capacity of injected vaccine was stronger than that of mucosal vaccine.The injected vaccine induced the increase of IgG in serum,which was about 10 times higher than the mucosal immune response,but could not induce the increase of IgA.However,the mucosal vaccine induced the high level of mucosal IgA,but the serum IgG antibody was relatively low.Conclusion Both vaccines based on RSV F protein are promising candidates,and each vaccine has its own advantages.Follow-up studies will evaluate the feasibility of these two vaccines as immunogens using a combination immunization approach to simultaneously enhance systemic and mucosal immune responses against RSV.

Evaluation of Severe Acute Respiratory Syndrome surveillance caused by respiratory viruses in a pediatric unit, 2013 to 2019 / Avaliação da vigilância da Síndrome Respiratória Aguda Grave causada por vírus respiratórios em uma unidade pediátrica, 2013 a 2019

ABSTRACT Objective: To evaluate severe acute respiratory syndrome surveillance in a pediatric unit. Methods: Descriptive study of reported severe acute respiratory syndrome cases with the detection of respiratory viruses in the nasopharyngeal sample of patients hospitalized between 2013 and 2019, in a reference hospital in the Federal District, Brazil. Results: A total of 269 children had one or more viruses detected, resulting in 280 viruses, of which 152 (54%) were respiratory syncytial virus. The detection of respiratory syncytial virus was higher during the autumn-winter period. Children´s median age was 6.9 months, 156 (58%) were male, 104 (39%) had comorbidity, 197 (73%) required mechanical ventilation, 241 (90%) received antibiotics, and 146 (54%) oseltamivir. There were 19 (7%) deaths. The median time from symptom onset to sample collection was 5 days and the median time from sample collection to final results was 6 days. Conclusions: The system needs to reduce the time to deliver results so that inappropriate use of antibiotics and antivirals can be avoided. Moreover, the burden of viral pneumonia was relevant and the system must be flexible enough to include emerging viruses in order to be useful in responding to public health emergencies caused by respiratory viruses.

RESUMO Objetivo: Avaliar a vigilância da síndrome respiratória aguda grave em uma unidade pediátrica. Métodos: Estudo descritivo dos casos de síndrome respiratória aguda grave, notificados e com a detecção de vírus respiratório em amostra de nasofaringe de pacientes internados entre 2013 e 2019, em um hospital de referência do Distrito Federal. Resultados: Um total de 269 crianças tiveram algum vírus detectado, resultando em 280 vírus, sendo 152 (54%) vírus sincicial respiratório. A detecção do vírus sincicial respiratório foi maior durante o período de outono-inverno. A mediana da idade das crianças foi de 6,9 meses, 156 (58%) eram do sexo masculino, 104 (39%) tinham comorbidade, 197 (73%) necessitaram de ventilação mecânica, 241 (90%) receberam antibióticos e 146 (54%) oseltamivir. Ocorreram 19 (7%) óbitos. A mediana do tempo desde o início dos sintomas até a coleta da amostra foi de 5 dias, e do tempo da coleta até o resultado foi de 6 dias. Conclusões: O sistema necessita reduzir o tempo do resultado final para que seja possível evitar o uso inadequado de antibióticos e antivirais. Ademais, o impacto das pneumonias virais foi relevante e o sistema deve ser flexível suficiente para incluir vírus emergentes, para ser útil na resposta às emergências de saúde pública causada por vírus respiratórios.

Vigilancia de Infecciones Respiratorias Agudas en niños y adolescentes en un Servicio de Pediatría

Introducción: Las infecciones agudas de vías respiratorias inferiores, de etiología viral, constituyen la principal causa de hospitalización y mortalidad infantil. El objetivo del presente estudio es conocer la frecuencia de las infecciones respiratorias agudas grave (IRAG) y los virus prevalentes en pacientes ≤15 años. Materiales y Métodos: Estudio observacional, descriptivo, producto de la vigilancia de IRAG, periodo enero/2019-mayo/2023. Se incluyeron todos los pacientes ≤15 años hospitalizados con diagnóstico de IRAG. Se analizaron datos demográficos, características clínicas y evolutivas. Resultados: Fueron hospitalizados 256 pacientes ≤15 años con diagnóstico de IRAG, 54,3%(139/256) de sexo masculino. El 69,1% correspondían a <2años, 15,6% de 2 a 4 años y 15,2% de 5 a 19 años. El 17,5% presentaban factores de riesgo. El 3,5% (9/256) tenía vacuna anti-influenza. El mayor número de casos se objetivó en 2019. En 2020 y 2021 se registró el menor número de casos, relacionado a la política de encierro durante la pandemia de COVID-19. El virus prevalente fue el Virus Sincitial Respiratorio (VSR). En el 2019, el VSR fue el virus más frecuente (p<0.0001.RR=1,56.IC95%= 1,18 - 2,06); el SARS - CoV2 fue el más frecuente en 2020. Según grupo etario el VSR fue más frecuente en <2 años (p<0.0001.RR=1,70.IC95%=1,18-2,06) y el SARS - CoV2 en los ≥2 años (p<0.0001.RR=2,50.IC95%=2,53-3,53). El VSR no fue asociado a factores de riesgo. El 39,4%(101/256) requirió ingreso a la UCIP, situación más frecuente en pacientes con factores de riesgo (21,7%vs14,8%,NS). El ingreso a UCI fue más frecuente en los <2 años (44,1% vs 29,1%;p<0,02.RR=1,51.IC95%=1,03-2,22). No hubo diferencias significativas del requerimiento de UCI y los virus aislados. La letalidad fue del 7,8%(20/256), más frecuente en el sexo femenino (p<0.02.RR=0,36.IC95%=0,1-0,9), no asociado a la edad ni etiología. Conclusion: La IRAG por VSR constituye una causa importante de hospitalización en <2 años, más frecuente en el sexo masculino. El ingreso a UCIP fue más frecuente en los <2años. La letalidad fue más frecuente en el sexo femenino.

Introduction: Acute lower respiratory tract infections, of viral etiology, are the main cause of hospitalization and infant mortality. The objective of the present study is to know the frequency of severe acute respiratory infections (SARI) and prevalent viruses in patients ≤15 years of age. Materials and Methods: Observational, descriptive study, product of SARI surveillance, period January/2019-May/2023. All patients ≤15 years of age hospitalized with a diagnosis of SARI were included. Demographic data, clinical and evolutionary characteristics were analyzed. Results: 256 patients ≤15 years old with a diagnosis of SARI were hospitalized, 54.3% (139/256) male. 69.1% corresponded to <2 years, 15.6% from 2 to 4 years and 15.2% from 5 to 19 years. 17.5% had risk factors. 3.5% (9/256) had an anti-influenza vaccine. The highest number of cases was observed in 2019. In 2020 and 2021, the lowest number of cases was recorded, related to the confinement policy during the COVID-19 pandemic. The prevalent virus was Respiratory Syncytial Virus (RSV). In 2019, RSV was the most common virus (p<0.0001.RR=1.56.95%CI= 1.18 - 2.06); SARS - CoV2 was the most frequent in 2020. According to age group, RSV was more frequent in <2 years (p<0.0001.RR=1.70.CI95%=1.18-2.06) and SARS - CoV2 in those ≥2 years (p<0.0001.RR=2.50.95%CI=2.53-3.53). RSV was not associated with risk factors. 39.4% (101/256) required admission to the PICU, a more frequent situation in patients with risk factors (21.7% vs 14.8%,NS). Admission to the ICU was more frequent in those <2 years (44.1% vs 29.1%; p<0.02.RR=1.51.95%CI=1.03-2.22). There were no significant differences in the ICU requirement and the isolated viruses. The fatality rate was 7.8% (20/256), more frequent in females (p<0.02.RR=0.36.95%CI=0.1-0.9), not associated with age or etiology. Conclusion: SARI due to RSV is an important cause of hospitalization in <2 years, more frequent in males. Admission to the PICU was more frequent in those <2 years old. Fatality was more frequent in females.

Características clínico-etiológicas de pacientes pediátricos hospitalizados con diagnóstico de bronquiolitis. Ciudad de Santa Fe, Argentina

Introducción: La bronquiolitis constituye una de las principales causas de Infecciones Respiratorias Agudas Bajas en Pediatría, y es responsable de una proporción significativa de hospitalizaciones en lactantes, fundamentalmente, en menores de 1 año. Su diagnóstico es clínico, caracterizado por síntomas y signos de amplio grado de gravedad. Objetivo: Describir las características clínico-etiológicas de casos de bronquiolitis en menores de 1 año, internados en un hospital de niños de la ciudad de Santa Fe, durante un periodo de un año. Materiales y Métodos: Estudio observacional, transversal. Se analizaron datos demográficos, clínicos y de laboratorio de fichas médicas de vigilancia epidemiológica. Las técnicas diagnósticas usadas según el agente viral fueron RT-PCR en tiempo real, PCR-punto final e Inmunofluorescencia Indirecta. Resultados: Sobre un total de 108 casos, 90,7% tuvo de 1 a 6 meses de edad. La mediana de internación fue de 5 días, y el nacimiento prematuro fue la condición médica previa más frecuente. Un 78,7% (85) tuvo diagnóstico viral positivo, siendo Rinovirus (hRV) y Virus Sincicial Respiratorio (VSR) los agentes más prevalentes, tanto en infección única como en coinfección. El tiempo de oxigenoterapia fue mayor en los pacientes más graves (p<0,001). El 32,4% (35) recibió alguna medicación que fue, en mayor frecuencia, antibiótico. Se encontró asociación positiva y significativa entre la edad menor de 3 meses y una hospitalización mayor a 5 días (OR=2,5; IC: 1,1-5,8; p=0,02); y entre un diagnóstico VSR positivo y un cuadro grave (OR: 7,7; IC: 1,95-39,6; p<0,001). Conclusión: Las características y condiciones médicas consideradas por la literatura como factores de riesgo para el padecimiento y la gravedad de una IRAB, fueron halladas con mayor frecuencia en la población de estudio. El hRV y el VRS fueron los agentes de mayor rescate viral, encontrándose una asociación positiva entre la infección por VSR y la gravedad del cuadro.

Introduction: Bronchiolitis is one of the main causes of Acute Lower Respiratory Infections in pediatrics, and is responsible for a significant proportion of hospitalizations in infants, mainly in children under 1 year of age. Its diagnosis is clinical. The disease is characterized by a wide variety and degree of signs and symptoms. Objective: To describe the clinical-etiological characteristics of cases of bronchiolitis in children under 1 year of age, admitted to a children's hospital in the city of Santa Fe, over a period of one year. Materials and Methods: This was an observational and cross-sectional study. Demographic, clinical and laboratory data from epidemiological surveillance medical records were analyzed. The diagnostic techniques used, based on the viral agent, were real-time RT-PCR, end-point PCR and Indirect Immunofluorescence. Results: Out of a total of 108 cases, 90.7% were between 1 and 6 months old. The median hospital stay was 5 days, and premature birth was the most common prior medical condition. 78.7% (85) had a positive viral diagnosis, with Rhinovirus (hRV) and Respiratory Syncytial Virus (RSV) being the most prevalent agents, both in single infection and coinfection. Oxygen therapy time was longer in the most seriously ill patients (p<0.001). 32.4% (35) received some medication, which was, most frequently, antibiotics. A positive and significant association was found between age less than 3 months and hospitalization longer than 5 days (OR=2.5; CI: 1.1-5.8; p=0.02); and between a positive RSV diagnosis and a severe condition (OR: 7.7; CI: 1.95-39.6; p<0.001). Conclusions: The characteristics and medical conditions considered by the literature as risk factors for the morbidity and severity of a lower respiratory tract infection were found more frequently in the study population. hRV and RSV were the most commonly-detected viral agents. We found a positive association between RSV infection and the severity of the condition.

Modelado matemático del efecto de las medidas de cuidado por COVID-19 sobre otras enfermedades respiratorias / Mathematical modelling of the impact of COVID-19 care measures on other respiratory diseases

RESUMEN INTRODUCCIÓN Los modelos matemáticos de la transmisión de enfermedades infecciosas permiten estudiar distintos mecanismos que afectan su comportamiento temporal. Este trabajo analizó el efecto sobre la dinámica de la influenza y el virus sincitial respiratorio (VSR) de la disminución de la transmisibilidad debida a las medidas de cuidado adoptadas para reducir la circulación de COVID-19. MÉTODOS Se empleó un modelo determinista tipo SIRS (susceptible-infectado-recuperado-susceptible) con modulación estacional para representar la influenza y el VSR, en ambos casos con inmunidad de corta duración y ciclo anual. Los cambios en la transmisibilidad de la enfermedad se modelaron reduciéndola durante dos años y planteando distintos escenarios. RESULTADOS En el modelo planteado, la reducción en la transmisibilidad genera cambios que se sostienen en los años siguientes: eventos epidémicos muy pronunciados con alargamiento del intervalo interbrote. Este efecto resulta dominante respecto del comportamiento estacional. El escenario de una reducción inicial de la transmisibilidad del 40% resulta compatible con el comportamiento de influenza y VSR reportados actualmente para Argentina. DISCUSIÓN El modelo general propuesto, en condiciones de disminución transitoria en la transmisibilidad, exhibe una epidemiología compatible con la observada recientemente en Argentina para ambas enfermedades e ilustra el modelado como herramienta útil en la comprensión de efectos no intuitivos.

ABSTRACT INTRODUCTION Mathematical models of infectious diseases transmission allow to study different mechanisms which affect their temporal behavior. This work analyzed the impact of the decrease in transmissibility, as a result of measures of personal care adopted to reduce circulation of COVID-19, on the dynamics of influenza and respiratory syncytial virus (RSV). METHODS A deterministic SIRS (susceptible-infected-recovered-susceptible) model with seasonal modulation was used to represent two diseases with short-term immunity and annual cycle: influenza and RSV. Changes in disease transmissibility were modeled by reducing it for two years and analyzing different scenarios. RESULTS In the proposed model, transmissibility reduction brings changes which sustain in the following years: very pronounced epidemic events with lengthening of the inter-outbreak interval. This effect prevails over the seasonal behavior. The scenario of 40% initial reduction in transmissibility is compatible with the behavior of influenza and RSV currently reported in Argentina. DISCUSSION The general model proposed here, under conditions of temporary reduced transmissibility, shows an epidemiology compatible with recently reported data of influenza and RSV in Argentina. This result illustrates modeling as a useful tool to understand non-intuitive effects.

Current strategies and perspectives for active and passive immunization against Respiratory Syncytial Virus in childhood

Abstract Objectives: Despite the global impact of the Respiratory Syncytial Virus (RSV) infection in children, only one monoclonal antibody (Palivizumab) has been approved for clinical use. However, advances in the knowledge of RSV immunology may enable the development of safe and effective new vaccines and monoclonal antibodies in a few years. The purpose of this review is to summarize available data on approved and developing passive and active immunizations against RSV in childhood and pregnancy. Data source: A non-systematic review of RSV immunoprophylaxis in childhood and pregnancy was carried out in PubMed, path.org and clinical trial registries, without language restrictions, up to September 2022. Data synthesis: Three monoclonal antibodies and 17 active immunization candidates are under development in phase 1 to 3 clinical studies. Regarding the first group, Nirsevimab is a monoclonal antibody with a prolonged half-life whose approval for clinical use is expected in the next months. Among the vaccines under development, six techniques are being used: protein subunit, viral particles, live attenuated virus, recombinant viral vector, chimeric, and mRNA. The first two approaches are being tested primarily in pregnancy, while the others are being developed for the pediatric population. Conclusion: The approval of extended half-life monoclonal antibodies is the next expected advance in RSV prevention, although the costs may be a barrier to the implementation. Regarding active immunizations, maternal and infant vaccination are complementary strategies and there are many promising candidates in clinical studies using different platforms.

Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro / Efeitos das redes extracelulares dos neutrófilos durante a infecção in vitro pelo vírus sincicial respiratório

The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5–16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

O vírus sincicial respiratório humano (hRSV) é a causa mais comum de doenças graves do trato respiratório inferior em crianças pequenas em todo o mundo, resultando em grande número de hospitalizações e gastos significativos para os sistemas de saúde. Neutrófilos são recrutados em massa para o tecido pulmonar de pacientes com doenças respiratórias agudas. No local da infecção, eles liberam armadilhas extracelulares de neutrófilos (NETs) que podem capturar e/ou inativar diferentes tipos de microrganismos, incluindo vírus. Evidências demonstraram que o acúmulo de NETs resulta em efeitos citotóxicos diretos nas células endoteliais e epiteliais. Os neutrófilos estimulados pela proteína F do vírus sincicial respiratório (hRSV-F) geram NETs que são capazes de capturar partículas virais, reduzindo assim sua transmissão. No entanto, a produção maciça de NETs obstrui as vias aéreas e aumenta a gravidade da doença. Assim, um maior conhecimento sobre os efeitos das NETs durante as infecções por hRSV é essencial para o desenvolvimento de novos tratamentos específicos e eficazes. Este estudo avaliou os efeitos das NETs no contato prévio ou posterior à infecção de células Hep-2 com hRSV. As células Hep-2 foram infectadas com diferentes quantidades de hRSV (multiplicidade de infecção ou MOI 0,5 ou 1,0), antes ou após a incubação com NETs (0,5–16 μg/mL). Células infectadas e não tratadas mostraram redução da viabilidade celular e intensa coloração com azul de tripano, que foi acompanhada pela formação de sincícios numerosos e grandes. O contato prévio entre as NETs e as células não resultou em efeito protetor. As células em monocamadas mostraram um número e área de sincícios reduzidos, mas a morte celular foi semelhante àquela apresentada por células infectadas e não tratadas. A adição de NETs aos tecidos infectados manteve taxa de morte celular e formação de sincícios [...].

Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro

Abstract The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.516 g/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

Resumo O vírus sincicial respiratório humano (hRSV) é a causa mais comum de doenças graves do trato respiratório inferior em crianças pequenas em todo o mundo, resultando em grande número de hospitalizações e gastos significativos para os sistemas de saúde. Neutrófilos são recrutados em massa para o tecido pulmonar de pacientes com doenças respiratórias agudas. No local da infecção, eles liberam armadilhas extracelulares de neutrófilos (NETs) que podem capturar e/ou inativar diferentes tipos de microrganismos, incluindo vírus. Evidências demonstraram que o acúmulo de NETs resulta em efeitos citotóxicos diretos nas células endoteliais e epiteliais. Os neutrófilos estimulados pela proteína F do vírus sincicial respiratório (hRSV-F) geram NETs que são capazes de capturar partículas virais, reduzindo assim sua transmissão. No entanto, a produção maciça de NETs obstrui as vias aéreas e aumenta a gravidade da doença. Assim, um maior conhecimento sobre os efeitos das NETs durante as infecções por hRSV é essencial para o desenvolvimento de novos tratamentos específicos e eficazes. Este estudo avaliou os efeitos das NETs no contato prévio ou posterior à infecção de células Hep-2 com hRSV. As células Hep-2 foram infectadas com diferentes quantidades de hRSV (multiplicidade de infecção ou MOI 0,5 ou 1,0), antes ou após a incubação com NETs (0,516 g/mL). Células infectadas e não tratadas mostraram redução da viabilidade celular e intensa coloração com azul de tripano, que foi acompanhada pela formação de sincícios numerosos e grandes. O contato prévio entre as NETs e as células não resultou em efeito protetor. As células em monocamadas mostraram um número e área de sincícios reduzidos, mas a morte celular foi semelhante àquela apresentada por células infectadas e não tratadas. A adição de NETs aos tecidos infectados manteve taxa de morte celular e formação de sincícios semelhantes àqueles induzidos pelo vírus em células não tratadas, indicando danos citotóxicos e deletérios. Nossos resultados corroboram achados relatados anteriormente de que as NETs contribuem para a imunopatologia desenvolvida por pacientes infectados com hRSV.

Effects of neutrophil extracellular traps during human respiratory syncytial virus infection in vitro / Efeitos das redes extracelulares dos neutrófilos durante a infecção in vitro pelo vírus sincicial respiratório

Abstract The human respiratory syncytial virus (hRSV) is the most common cause of severe lower respiratory tract diseases in young children worldwide, leading to a high number of hospitalizations and significant expenditures for health systems. Neutrophils are massively recruited to the lung tissue of patients with acute respiratory diseases. At the infection site, they release neutrophil extracellular traps (NETs) that can capture and/or inactivate different types of microorganisms, including viruses. Evidence has shown that the accumulation of NETs results in direct cytotoxic effects on endothelial and epithelial cells. Neutrophils stimulated by the hRSV-F protein generate NETs that are able to capture hRSV particles, thus reducing their transmission. However, the massive production of NETs obstructs the airways and increases disease severity. Therefore, further knowledge about the effects of NETs during hRSV infections is essential for the development of new specific and effective treatments. This study evaluated the effects of NETs on the previous or posterior contact with hRSV-infected Hep-2 cells. Hep-2 cells were infected with different hRSV multiplicity of infection (MOI 0.5 or 1.0), either before or after incubation with NETs (0.5-16 μg/mL). Infected and untreated cells showed decreased cellular viability and intense staining with trypan blue, which was accompanied by the formation of many large syncytia. Previous contact between NETs and cells did not result in a protective effect. Cells in monolayers showed a reduced number and area of syncytia, but cell death was similar in infected and non-treated cells. The addition of NETs to infected tissues maintained a similar virus-induced cell death rate and an increased syncytial area, indicating cytotoxic and deleterious damages. Our results corroborate previously reported findings that NETs contribute to the immunopathology developed by patients infected with hRSV.

Resumo O vírus sincicial respiratório humano (hRSV) é a causa mais comum de doenças graves do trato respiratório inferior em crianças pequenas em todo o mundo, resultando em grande número de hospitalizações e gastos significativos para os sistemas de saúde. Neutrófilos são recrutados em massa para o tecido pulmonar de pacientes com doenças respiratórias agudas. No local da infecção, eles liberam armadilhas extracelulares de neutrófilos (NETs) que podem capturar e/ou inativar diferentes tipos de microrganismos, incluindo vírus. Evidências demonstraram que o acúmulo de NETs resulta em efeitos citotóxicos diretos nas células endoteliais e epiteliais. Os neutrófilos estimulados pela proteína F do vírus sincicial respiratório (hRSV-F) geram NETs que são capazes de capturar partículas virais, reduzindo assim sua transmissão. No entanto, a produção maciça de NETs obstrui as vias aéreas e aumenta a gravidade da doença. Assim, um maior conhecimento sobre os efeitos das NETs durante as infecções por hRSV é essencial para o desenvolvimento de novos tratamentos específicos e eficazes. Este estudo avaliou os efeitos das NETs no contato prévio ou posterior à infecção de células Hep-2 com hRSV. As células Hep-2 foram infectadas com diferentes quantidades de hRSV (multiplicidade de infecção ou MOI 0,5 ou 1,0), antes ou após a incubação com NETs (0,5-16 μg/mL). Células infectadas e não tratadas mostraram redução da viabilidade celular e intensa coloração com azul de tripano, que foi acompanhada pela formação de sincícios numerosos e grandes. O contato prévio entre as NETs e as células não resultou em efeito protetor. As células em monocamadas mostraram um número e área de sincícios reduzidos, mas a morte celular foi semelhante àquela apresentada por células infectadas e não tratadas. A adição de NETs aos tecidos infectados manteve taxa de morte celular e formação de sincícios semelhantes àqueles induzidos pelo vírus em células não tratadas, indicando danos citotóxicos e deletérios. Nossos resultados corroboram achados relatados anteriormente de que as NETs contribuem para a imunopatologia desenvolvida por pacientes infectados com hRSV.

Modified Wuhutang Alleviates Airway Inflammation and Down-regulates Expression of Muc5AC， STAT3， NF-κB， and NLRP3 in Lung Tissues of RSV-infected Asthmatic Mice / 中国实验方剂学杂志

ObjectiveTo explore effect of modified Wuhutang on airway inflammation and expression of mucin （Muc） 5AC， signal transducer and activator of transcription 3 （STAT3）， nuclear factor kappa B （NF-κB）， and NOD-like receptor thermal protein domain associated protein 3 （NLRP3） in respiratory syncytial virus （RSV）-infected asthmatic mice. MethodSeventy male BALB/c mice of 6-8 weeks old were randomized into normal control （CON）， asthma （ovalbumin， OVA）， RSV infection-induced asthma （OVA+RSV）， high-， medium-， and low-dose （4.08， 2.04， 1.02 g·kg-1·d-1， respectively） modified Wuhutang， and dexamethasone （Dxms， 0.1 g·kg-1d-1） groups （n=10）. The model of asthma was established by sensitization and atomization inhalation with OVA. The RSV infection-induced asthma model was established by three consecutive RSV nasal infusions （1.0 × 106 PFU·mL-1， 50 μL）. Wuhutang was administrated by gavage， and Dxms by intraperitoneal injection. The CON group was given the same amount of normal saline by gavage. The mice were anesthetized with 2.5% pentobarbital sodium 24 h after the last administration， and then the lung tissue was stained by hematoxylin-eosin （HE） and Van Gieson （VG） for observation of airway inflammation. The immunohistochemical assay was employed to detect the expression of Muc5AC. Western blot was employed to determine the protein levels of phosphorylated （p）-STAT3， STAT3， p-NF-κB， NF-κB， and NLRP3. ResultCompared with the CON group， the OVA group presented airway inflammatory cell infiltration， tissue hyperemia and edema， and collagen fiber deposition. The OVA+RSV group showed severer airway inflammatory cell infiltration and tissue hyperemia and edema than the OVA group. Compared with the OVA+RSV group， modified Wuhutang alleviated the airway inflammatory cell infiltration， tissue hyperemia and edema， and collagen fiber deposition， and the high-dose group had the best performance. Compared with the CON group， the OVA group and the OVA+RSV group showed increased expression level of Muc5AC （P<0.01）. Compared with the OVA+RSV group， modified Wuhutang reduced the expression level of Muc5AC， and the reduction was significant in the high-dose group （P<0.05）. Compared with the high-dose modified Wuhutang group， Dxms lowered the expression level of Muc5AC （P<0.05）. Compared with the CON group， the OVA and OVA+RSV groups showed up-regulated protein levels of p-STAT3， p-NF-κB， and NLRP3 （P<0.05， P<0.01）. Compared with the OVA+RSV group， modified Wuhutang down-regulated the protein levels of p-STAT3， p-NF-κB， and NLRP3 （P<0.01）. Compared with the high-dose modified Wuhutang group， the Dxms group showed up-regulated levels of p-STAT3， p-NF-κB proteins （P<0.01）. ConclusionModified Wuhutang can reduce airway inflammation and down-regulate the expression of Muc5AC， p-STAT3， p-NF-κB， and NLRP3 in RSV-infected asthmatic mice， which suggests that Wuhutang reduces airway inflammation in RSV-infected asthma by regulating the STAT3/NF-κB signaling pathway.

Research progress on vaccines against human respiratory syncytial virus / 中国生物制品学杂志

@#Human respiratory syncytial virus(hRSV)is one of the main pathogens that cause lower respiratory tract infection in infants and the elderly.hRSV genome contains 10 genes with a full length of 15 222 bp,encoding 11 proteins(9structural proteins and 2 non-structural proteins).Different proteins play different roles in the pathogenesis of hRSV.With the in-depth research on the biological and structural characteristics of hRSV,various types of hRSV vaccines have been developed,making rapid progress.For example,hRSV attenuated live vaccine hRSV ?NS2/?1313/I1314L has entered Phase II clinical trial,and hRSV subunit protein vaccine Pre-F-GCN4t has entered Phase III clinical trial.In this paper,the biological characteristics of hRSV and the types of hRSV vaccines with rapid progress are reviewed so as to provide a reference for the development of hRSV vaccines in China.

Establishment of UPLC fingerprint and study on anti-respiratory syncytial virus spectrum-effective relationship of ethanol extract from Callicarpa nudiflora / 中国药房

OBJECTIVE To establish the fingerprint of the ethanol extract from Callicarpa nudiflora， analyze its anti- respiratory syncytial virus （RSV） activity in vitro， and study the relationship between spectrum and effect. METHODS Using 10%， 30%， 50%， 70% and 90% ethanol as solvent， 20 batches of ethanol extracts from 4 batches of C. nudiflora were prepared. The fingerprints for 20 batches of ethanol extracts from C. nudiflora were mapped by ultra-high-performance liquid chromatography （UPLC）， and the similarity evaluation was conducted by using the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints （2012 edition）. The cytopathic effect method and MTT method were used to investigate the in vitro inhibitory activity of the ethanol extracts from C. nudiflora on RSV. Pearson correlation analysis， grey correlation degree and orthogonal partial least squares （OPLS） analysis were used to study the spectrum-effect relationship. RESULTS There were 25 common peaks in 20 batches of ethanol extracts from C. nudiflora， and the similarities ranged from 0.912 to 0.998， and the RSDs of common peak areas were 33.54%-162.28%. The average values of IC50 for RSV of 20 batches of ethanol extracts from C. nudiflora were 9.55-272.23 μg/mL. The results of Pearson correlation analysis， grey correlation analysis and OPLS analysis showed that the Pearson correlation coefficients （P＜0.05） of the common peaks 8， 10， 12， 16， 18-19， 22-24 with pharmacodynamic indicators and regression coefficients were all negative， the correlation coefficients were all greater than 0.6， and the values of variable importance in projection were all greater than 1. CONCLUSIONS Twenty batches of ethanol extracts from C. nudiflora have similar components but significant differences in content， and exhibit different degrees of anti-RSV activity in vitro. The corresponding components of common peaks 8， 10， 12， 16， 18-19， 22-24 may be the characteristic components of anti-RSV of C. nudiflora.

Research progress of m 6A methylation modification in respiratory viruses replication and host immune response / 中华临床感染病杂志

N 6-methyladenosine (m 6A) modification is the most prevalent internal modification of eukaryotic mRNA and is dynamically regulated by a variety of m 6A modifying enzymes, including methylation transferases, demethylases and specific binding proteins. Respiratory viral infections have received much attention in recent years, and the process of virus replication and metabolism in host cells is regulated by m 6A. This article reviews the mechanism of m 6A-regulated enzymes, the roles of m 6A modifications in respiratory viruses replication and the host immune response to viruses, including adenovirus, influenza A virus, severe acute respiratory syndrome coronavirus 2, respiratory syncytial virus, and human metapneumovirus. It would provide a reference for exploring the regulatory role of viral episodic transcriptome modifications and antiviral targets or vaccine development.

Expression of TLR3 in human adenocarcinoma of the lung cells induced by respiratory syncytial virus and its significance in the diagnosis of pneumonia in children / 中国医师杂志

Objective:To study the expression of Toll like receptor 3 (TLR3) in human adenocarcinoma of the lung cells induced by respiratory syncytial virus (RSV) and its significance in the diagnosis of pneumonia in children.Methods:A549 cells were divided into RSV infection group [added 1 μg/ml Lipopolysaccharide (TLR3 agonist) transfected RSV virus after 150 μl intervention], Lipopolysaccharide stimulation group (added 1 μg/ml Lipopolysaccharide 150 μl intervention) and normal control group (normal culture). The mRNA expressions of tumor necrosis factor-α, interleukin 8, TLR3 protein and TLR3 in A549 Cells of different groups were compared. We prospectively selected 80 children with RSV infectious pneumonia admitted to Baoding Second Central Hospital from August 2019 to October 2021 as the RSV pneumonia group, and sixty children with common pneumonia were taken as the common pneumonia group, and 60 healthy children in our hospital were taken as the control group. The mRNA expression of serum TLR3 in different groups was compared, and the diagnostic efficacy of serum TLR3 in RSV pneumonia was evaluated by receiver operating characteristic.Results:There was a statistically significant difference in the expression of TLR3 protein among different groups of A549 cells ( P<0.001). The expression differences of TLR3 mRNA in different groups of A549 cells at different time points were statistically significant(all P<0.001). There was significant difference in the expression of tumor necrosis factor-α and interleukin 8 of A549 cells at different time points in different groups (all P<0.05). There was a statistically significant difference in the expression of serum TLR3 mRNA among the three groups of subjects ( F=155.237, P<0.001). The critical value for TLR3 gene diagnosis was 66.87, with corresponding sensitivity of 73.75%, specificity of 70.83%, and the area under curve (AUC) of 0.803(95% CI: 0.753-0.855). Conclusions:Respiratory syncytial virus induces human lung cancer cells and promotes disease progression through TLR3 expression; Serum TLR3 can be used for the diagnosis of RSV pneumonia.

Influencing factors of severe pneumonia in children with respiratory syncytial virus infection / 中国医师杂志

Objective:To explore the influencing factors of severe pneumonia in children with respiratory syncytial virus (RSV) infection.Methods:A retrospective case-control study was used to collect 210 children with RSV infected pneumonia admitted to Hebei Children′s Hospital from October 2017 to October 2020. Among them, 70 children with severe pneumonia were included in the severe pneumonia group, and 140 children with common pneumonia were included in the common pneumonia group; the baseline data and relevant laboratory indicators of the two groups were compared; Logistic regression was used to analyze the influencing factors of severe pneumonia in children infected with RSV.Results:The proportions of wheezing, congenital heart disease, respiratory failure, heart failure and pleural effusion of children in severe pneumonia group were higher than those in common pneumonia group, and the forced vital capacity (FVC) and forced expiratory volume in the first second (FEV 1) were lower than those in common pneumonia group (all P<0.05); the levels of C-reactive protein (CRP), CD8 + cells, RSV load and Beclin-1 in severe pneumonia group were higher than those in common pneumonia group, and the levels of CD4 + cells and 1, 25-dihydroxyvitamin D [1, 25-(OH) 2D] were lower than those in common pneumonia group (all P<0.05). After treatment, the levels of CRP, CD8 + cells and Beclin-1 in children with severe pneumonia were lower than those before treatment, and the levels of CD4 + cells and 1, 25-(OH) 2D were higher than those before treatment (all P<0.05). Multiple regression model analysis was established. The results showed that congenital heart disease, high CRP level, high CD8 + cells, high RSV load and high Beclin-1 level were risk factors for severe pneumonia in children with RSV infected pneumonia (all OR>1, P<0.05), and high CD4 + cells and 1, 25-(OH) 2D level were protective factors (all OR<1, P<0.05). Conclusions:Severe pneumonia in children with RSV infected pneumonia may be affected by congenital heart disease, CRP, CD4 + cells, CD8 + cells, 1, 25-(OH) 2D, RSV load and Beclin-1.

The detection and clinical characteristics of respiratory syncytial virus and human rhinovirus in children in eastern Guangdong from 2019 to 2020 / 中国小儿急救医学

Objective:To analyze the detection rate of respiratory syncytial virus(RSV)and human rhinovirus(HRV), in different months and age groups, and the clinical characteristics in children in eastern Guangdong from 2019 to 2020.Methods:Pharyngeal swabs were collected from 6 658 children with respiratory tract infections hospitalized in the Second Affiliated Hospital of Shantou University Medical College from January 2019 to December 2020, and respiratory pathogen nucleic acid was detected.The detection rate, month distribution, age group distribution, and clinical characteristics of single RSV as well as single HRV positive cases were analyzed and compared.Results:There were 416 single RSV positive cases(6.25%)and 341 single HRV positive cases(5.12%).The detection rates of RSV was higher than those of HRV, and the difference was statistically significant( χ2=7.880, P<0.05).The detection rates of HRV in March, April, November and December were higher than those of RSV, and the detection rates of RSV in July, August and September were higher than those of HRV, with statistically significant difference( P<0.05).The highest detection rate of RSV was in the age group of ≤6 months with a detection rate of 13.47%(192/1 425), which gradually decreased with age, and the difference was statistically significant( P<0.01).The detection rates of HRV fluctuated between 4.21% and 6.13% in each age group, and the differences among the detection rates of each age group were not statistically significant( P>0.05).All RSV-positive cases showed cough, while 77.13%(263/341)of HRV-positive cases showed cough, with a statistically significant difference( P<0.001).The incidence of wheezing in RSV-positive cases was 37.26%(155/416)compared with 28.45%(97/341)in HRV-positive cases, with a higher incidence of wheezing in RSV than that in HRV, and the difference was statistically significant( P<0.05).In terms of indicators to assess severe pneumonia, RSV-positive cases showed a higher proportion of increased respiratory rate, decreased oxygen saturation or dyspnea than HRV-positive cases, and all differences were statistically significant( P<0.05). Conclusion:The detection rate of single RSV is higher than that of single HRV in children with respiratory infections in eastern Guangdong from 2019 to 2020.The epidemic season of RSV is mainly in autumn, and the epidemic season of HRV is mainly in winter and spring.RSV is more susceptible up to 6 months of age, and the detection rate decreases gradually with age, and there is no significant difference in the detection rate of HRV by age.RSV-positive cases are more likely to have cough and wheeze.RSV-positive cases are more likely to have increased respiratory rate, decreased oxygen saturation, or respiratory distress.

A multicenter clinical study of the impact of COVID-19 pandemic on hospitalization of children with bronchiolitis / 国际儿科学杂志

Objective:In order to explore the impact of corona virus disease 2019(COVID-19)on the hospitalization of children with bronchiolitis and to improve clinicians′ understanding of the characteristics of bronchiolitis during the COVID-19 epidemic.Methods:This was a multicenter clinical study, and the data have been collected from 23 children′s medical centers in China.All the clinical data were retrospectively collected from children with bronchiolitis who were hospitalized at each study center from January 1, 2019 to December 31, 2021.The results included gender, age at hospitalization, length of stay, respiratory syncytial virus(RSV) test results, severity rating, ICU treatment, and the total number of children hospitalized with respiratory tract infection during the same period.The clinical data of children with bronchiolitis in 2019 before COVID-19 epidemic and in 2020、2021 during COVID-19 epidemic were statistically analyzed and compared.Results:According to a summary of data provided by 23 children′s medical centers, there were 4 909 cases of bronchiolitis in 2019, 2 654 cases in 2020, and 3 500 cases in 2021.Compared with 2019, the number of bronchiolitis cases decreased by 45.94% in 2020 and 28.70% in 2021.In 2019, 2020 and 2021, there were no significant differences in gender ratio, age, and duration of hospitalization.Compared with 2019, the ratio of bronchiolitis to the total number of hospitalizations for respiratory tract infection decreased significantly in 2020 and 2021( χ2=12.762, P<0.05; χ2=84.845, P<0.05).The proportion of moderate to severe bronchiolitis cases in both 2020 and 2021 was lower than that in 2019, and the difference was statistically significant ( χ2=4.054, P<0.05; χ2=8.109, P<0.05).There was no statistically significant difference in the proportion of bronchiolitis cases requiring ICU treatment between 2019, 2020, and 2021 ( χ2=1.914, P>0.05).In 2019, a total of 52.60%(2 582/4 909) of children with bronchiolitis underwent RSV pathogen testing, and among them, there were 708 cases with RSV positive, accounting for 28.00%.In 2020, 54.14%(1 437/2 654) of children with bronchiolitis underwent RSV pathogen testing, and there were 403 cases with RSV positive, accounting for 28.04%.In 2021, 66.80%(2 238/3 500) of children with bronchiolitis underwent RSV pathogen testing, and there were 935 cases with RSV positive, accounting for 41.78%.Compared with 2019 and 2020, the RSV positive rate in 2021 showed a significant increase( χ2=99.673, P<0.05; χ2=71.292, P<0.05). Conclusion:During the COVID-19 epidemic, the implementation of epidemic prevention and control measures reduced the hospitalization rate and severity of bronchiolitis, but did not reduce the positive rate of RSV detection.

Cellular metabolomic study of baicalin against respiratory syncytial virus / 中国药理学通报

Aim To explore the effect of baicalin on respiratory syncytial virus in vitro and its effect on cell metabolism. Methods The anti-RSV effect of baicalin in vitro was verified by antiviral cell experiment, and the cellular metabolic mechanism of baicalin against RSV was explored by cell metabolomics. Results Baicalin had an inhibitory effect on all stages of RSV infection, and the condition of CPE was significantly improved, which may mainly play a role in the adsorption and proliferation of RSV. A total of 19 differential metabolites were screened by cell metabolomics, which were mainly glycerol phospholipids, nucleosides and fatty acids. Seven metabolic pathways were obtained by enrichment analysis, which were mainly related to glycerol-phospholipid metabolism, fatty acid metabolism (arachidonic acid metabolism, α-linolenic acid metabolism, linoleic acid metabolism), amino acid metabolism and purine metabolism. Conclusions Baicalin has significant inhibitory effect on the adsorption and proliferation of RSV, which may be related to fatty acid metabolism, glycerol phospholipid metabolism, amino acid and purine metabolism.

Effects of Dayuanyin decoction on lung injury and gut microbiota in mice with RSV-Hanshi yufei syndrome / 中国药房

OBJECTIVE To study the effects of Dayuanyin decoction （DYY） on lung injury and gut microbiota in mice with RSV-Hanshi yufei syndrome. METHODS Thirty-six BALB/c mice were divided into the normal group （NC group）， model group （MC group）， positive control group （LBM group， 57.59 mg/kg ribavirin）， DYY low-dose， medium-dose and high-dose groups （LDYY，MDYY，HDYY groups，1.67，3.34，6.68 g/kg，calculated by crude drug）， with 6 mice in each group. Except for NC group， RSV-Hanshi yufei syndrome model was induced by “Hanshi modeling+RSV infection”； after the second cold and wet stimulation， RSV solution was dripped into the nasal cavity at 50% tissue culture infectious dose， once a day， for 3 consecutive days； each administration group was given corresponding solution intragastrically 4 hours after each nasal drip， once a day， for 5 consecutive days. The levels of motilin （MTL） and gastrin （GAS） in serum， the levels of IL-6 and IL-1β in supernatant of lung tissue， and the percentage of peripheral blood lymphocytes were all determined. The pathological changes of lungs were observed， and the changes of gut microbiota were analyzed. RESULTS Compared with MC group， the levels of MTL in serum （except for HDYY group）， and the levels of IL-6 and IL-1β in supernatant of lung tissue were all decreased significantly in DYY groups， while the level of GAS （except for HDYY group） in serum was increased significantly （P＜0.05 or P＜0.01）. The pathology of lung tissue was improved， the percentage of T lymphocytes in LDYY group and the percentages of T lymphocyte and B lymphocyte in MDYY group were all increased significantly （P＜0.05 or P＜0.01）. Compared with the NC group， Bacteroidetes， Actinobacteria， Bacteroides， Alistipes， Bacteroidota， Bacteroides acidfaciens and Alloprevotella were expressed in high abundance， while Firmicutes， Proteobacteria， Lachnospiraceae NK4A136 group， Kineothrix and Clostridiales unclassified were expressed in low abundance. After the intervention of medium-dose DYY， the relative abundance of each microbiota tended to be adjusted back， with different species including Lachnospiraceae_UCG-Ruminococcus， etc. CONCLUSIONS Dayuanyin decoctioncan reduce the lung injury caused by RSV， the mechanism of which may be associated with relieving inflammation， regulating gastrointestinal hormone levels， the percentage of lymphocytes and the abundance of beneficial and harmful bacteria in the intestinal tract.

Development progress and application of monoclonal antibody drugs against respiratory virus / 中国生物制品学杂志

@#Abstract：In recent years，the outbreak and prevalence of respiratory infectious diseases in the world seriously endanger human health，among which the respiratory infectious diseases caused by viral infection account for a large proportion. The use of vaccines and common antiviral drugs is an effective way to fight viral infection，but there are also problems such as lag and drug resistance. Monoclonal antibodies against respiratory viral infections provide a new strategy for clinical treatment. This paper reviews the development of monoclonal antibody against respiratory virus and its application in respiratory viral infectious diseases. Keywords：Respiratory viral infectious diseases；Respiratory syncytial virus（RSV）；Influenza virus（IFV）；Coronavirus （CoV）；Monoclonal antibody