In vitro study on the antiviral activity of 9 extracts of traditional Chinese herbal medicine against the human respiratory syncytial virus

Objective:To study the in vitro virucidal activity of 9 extracts of traditional Chinese herbal medicine(the water extracts of Evodia lepta,Clausena lansium,Clerodendrum cyrtophyllum,Callicarpa nudiflora,Nauclea officinalis and Elaeagnus gonyanthes,the alcohol extracts of Nauclea officinalis,Elaeagnus gonyanthes and Zanthoxylumarmatum)on human respiratory syncytial virus(HRSV).Methods:The cytotoxic effect of the extracts on cells was evaluated by a cell viability assay using the CCK-8 method,a concentration of the extracts with cell viability greater than 50%was selected for the follow-up anti-HRSV effect assay,the 50%effective concentration(EC50)was assessed by an in vitro cell infection model.Results:The EC50s of the water extract from Clerodendrum cyrtophyllum,Callicarpa nudiflora and Elaeagnus gonyanthes were 0.05 mg/mL,0.03 mg/mL and 0.05 mg/mL,and the therapeutic index(TI)of them were 18.60,21.67 and 56.80 respectively.Conclusion:The water extracts of Clerodendrum cyrtophyllum,Callicarpa nudiflora and Elaeagnus gonyanthes possess the activity of anti-HRSV virus.

Genetic Characterization of Fusion Protein of Human Respiratory Syncytial Virus: Beijing

Objective Fusion protein is a subunit of the human respiratory syncytial virus(HRSV)and a potential vaccine candidate.Thus,a study on the genetic characteristics of F protein was considered important for further investigations in this field.The aim of this study was to determine the prevalence and genetic diversity of the F gene of HRSV infections in hospitalized pediatric patients in Beijing with acute lower respiratory tract infections and to compare the circulating genotypes that are currently found worldwide.Methods HRSV particles were amplified by RT-PCR and the PCR products were purified for sequencing.Further analysis was carried out by Bioedit and MEGA 3.0 biological software programs.Results Seventy-six samples(23.1%)were positive for HRSV.The percentage of cases in patients younger than 1year was 84.21%.Among the six Beijing isolates,four belonged to subgroup A,whose respective F genes shared97.0%-97.4%nucleotide sequence identity and 92.1%-93.0%amino acid sequence identity.The other two isolates belonged to subgroup B.Here,97.3%and 98.2%sequence identity were found at nucleotide and amino acid levels,respectively.Conclusions Phylogenetic analysis of nucleotide sequences revealed that those four isolates within subgroup A were monophyletic and closely related to each other,but those two within subgroup B distributed in two distinct clusters.Subgroup A and B strains co-circulated,indicating that two different transmission chains occurred in Beijing from 2003-2004.

Antiviral Effect of Emodin from Rheum palmatum against Coxsakievirus B_5 and Human Respiratory Syncytial Virus In Vitro

Summary： Viral infections are the major causes of morbidity and mortality in elderly people and young children throughout the world. The most common pathogens include coxsackie virus （CV） and respira- tory syncytial virus （RSV）. However, no antiviral agents with low toxicity and drug resistance are cur- rently available in clinic therapy. The present study aimed to examine the antiviral activities of emodin （an ingredient of Rheum palmatum） against CVB5 and RSV infections, in an attempt to discover new antiviral agents for virus infection. The monomer emodin was extracted and isolated from Rheum pal- matum. The antiviral activities of emodin on HEp-2 cells were evaluated, including virus replication in- hibition, virucidal and anti-absorption effects, by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-2H-tet- razolium bromide （MTT） assay and plaque reduction assay （PRA）. The kinetics of virus inhibition by emodin in a period of 14 h was further determined by plaque assay and quantitative real time PCR （qPCR）. Cytokine （IFN-γ, TNF-α） mRNA expressions after emodin treatment （7.5, 15, 30 μmol/L） were also assessed by qPCR post-infection. The results showed that emodin had potent inhibitory activities against CVB5 and RSV, with the 50% effective concentration （EC50） ranging from 13.06 to 14.27 μmol/L and selectivity index （SI） being 5.38-6.41 μmol/L. However, emodin couldn＇t directly inacti- vate the viruses or block their absorption to cells. It acted as a biological synthesis inhibitor against CVB4 and RSV in a concentration- and time-dependent manner, especially during the first 0-4 h post-infection. Moreover, emodin could decrease the mRNA expression of IFN-α but enhance TNF-γ expression significantly compared to the viral controls in vitro. Our results provide a molecular basis for development of emodin as a novel and safe antiviral agent for human enterovirus and respiratory virus infection in the clinical therapy.

Human Milk Oligosaccharides Enhance Innate Immunity to Respiratory Syncytial Virus and Influenza <i>in Vitro</i>

Human milk oligosaccharides (HMO) contribute to innate immunity by enhancing growth of beneficial bacteria, epithelial cell maturation and mucosal barrier integrity. They have immunomodulatory effects and can block pathogen binding to host cell surface glycans or receptors. We investigated the effects of 2’-fucosyllactose (2’FL), 6’-sialyllactose (6’SL), 3’-sialyllactose (3’SL) and lacto-N-neoTetraose (LNnT) on human respiratory epithelial cell lines or peripheral blood mononuclear cells (PBMCs) following respiratory viral infectionin vitro. Expression of cytokines and viral load were monitored in infected cells. These biomarkers of innate immunity were selected since viral load and cytokine levels (IP-10, MIP-1α, IL-6, IL-8, TNF-α) have been correlated with disease severity in respiratory syncytial virus (RSV) and influenza (IAV) virus infectionin vivo. 2’FL significantly decreased RSV viral load and cytokines associated with disease severity (IL-6, IL-8, MIP-1α) and inflammation (TNF-α, MCP-1) in airway epithelial cells. LNnT and 6’SL significantly decreased IAV viral load in airway epithelial cells. 6’SL dose-dependently down-regulated IP-10 and TNF-α in RSV infected PBMCs. HMO at or below levels found in breast milk enhance innate immunity to respiratory viruses in vitro and may interact directly with cells to modulate biomarkers of innate immunity.

A multi-center study on Molecular Epidemiology of Human Respiratory Syncytial Virus from Children with Acute Lower Respiratory Tract Infections in the Mainland of China between 2015 and 2019

Human respiratory syncytial virus(RSV) is a major pathogen of acute lower respiratory tract infection among young children. To investigate the prevalence and genetic characteristics of RSV in China, we performed a molecular epidemiological study during 2015–2019. A total of 964 RSV-positive specimens were identified from 5529 enrolled patients during a multi-center study. RSV subgroup A(RSV-A) was the predominant subgroup during this research period except in2016. Totally, 535 sequences of the second hypervariable region(HVR-2) of the G gene were obtained. Combined with182 Chinese sequences from GenBank, phylogenetic trees showed that 521 RSV-A sequences fell in genotypes ON1(512),NA1(6) and GA5(3), respectively;while 196 RSV-B sequences fell in BA9(193) and SAB4(3). ON1 and BA9 were the only genotypes after December 2015. Genotypes ON1 and BA9 can be separated into 10 and 7 lineages, respectively. The HVR-2 of genotype ON1 had six amino acid changes with a frequency more than 10%, while two substitutions H258 Q and H266 L were co-occurrences. The HVR-2 of genotype BA9 had nine amino acid substitutions with a frequency more than10%, while the sequences with T290 I and T312 I were all from 2018 to 2019. One N-glycosylation site at 237 was identified among ON1 sequences, while two N-glycosylation sites(296 and 310) were identified in the 60-nucleotide duplication region of BA9. To conclusion, ON1 and BA9 were the predominant genotypes in China during 2015–2019. For the genotypes ON1 and BA9, the G gene exhibited relatively high diversity and evolved continuously.

Qingfei oral liquid downregulates TRPV1 expression to reduce airway inflammation and mucus hypersecretion injury caused by respiratory syncytial virus infection and asthma in mice

Objective:Qingfei oral liquid(QF),an experimental Chinese medicine prescription developed from the ancient priscription of traditional Chinese medicines Ma Xin Shi Gan decoction and Tingli Dazao Xie Fei decoction,has been effectively used since decades to treat patients with viral pneumonia and asthma.In our previous study,we had demonstrated that QF can significantly reduce airway hyperresponsiveness,hyperemia,lung tissue edema,inflammatory lung tissue infiltration in mice,airway mucus secretion,and peripheral airway collagen hyperplasia;however,its mechanism of action is unknown.Methods:Fifty 6–8-week-old male BALB/c mice were equally and randomly divided into five groups:the control,ovalbumin(OVA),OVA+respiratory syncytial virus(RSV),QF,and dexamethasone(Dxms)groups.The QF group was administered QF at 1.17 g·kg−1·d−1,the Dxms group received dexamethasone injections at 0.2 mg·kg−1·d−1,and the remaining groups were administered PBS.Inflammation in the lung tissue was assessed by hematoxylin and eosin(HE),periodic acid–Schiff(PAS),and Van Gieson staining.ELISA was used to evaluate the IL-13,IL-25,and IL-33 in the mice.Western blotting was used to examine changes in the proteins levels of transient receptor potential vanilloid-1(TRPV1)and mucin 5AC(MUC5AC)in the lung tissues of mice.Results:Histopathological evaluation revealed that the OVA and OVA+RSV groups exhibited lung tissue edema and inflammatory lung tissue infiltration in the HE staining and airway secretions in the PAS staining;collagen hyperplasia around the airway was increased in these two groups compared with the control group.The QF group exhibited significantly reduced lung tissue edema,inflammatory lung tissue infiltration,airway secretions,and collagen hyperplasia around the airway compared with the OVA+RSV group.We analyzed the serum levels of IL-13,IL-25,and IL-33 in the mice and found that these levels were higher in the OVA and OVA+RSV groups than in the control group(P<0.05 in the OVA group,P<0.01 in the OVA+RSV group).The QF group exhibited significantly decreased serum levels of IL-13,IL-25,and IL-33 compared with the OVA+RSV group(all P<0.05).The Dxms group also exhibited significant decreases in the serum levels of IL-13 and IL-33(all P<0.05)but no significant decrease in the serum levels of IL-25 compared with the RSV+OVA group.Finally,we examined the protein levels of TRPV1 and MUC5AC in the lung tissues of mice using Western blotting.After identifying RSV infection in the mice with asthma,the protein levels of TRPV1 and MUC5AC in the lung tissues of mice were significantly higher than those in the control group(P<0.05,P<0.01).We found that compared with RSV+OVA,QF can significantly downregulate the protein level of TRPV1;further,the protein level of MUC5AC was also significantly reduced(all P<0.001).Conclusion:QF can inhibit RSV replication and reduce airway inflammation and mucus hypersecretion injury caused by RSV infection and asthma,and its mechanism of action may be associated with the downregulation of TRPV1 expression and a decrease in airway mucus hypersecretion injury.

The Association of Respiratory Syncytial Virus Infection and Childhood Asthma: A Meta-Analysis

Objective: To explore the close relationship between respiratory syncytial virus (RSV) infection and acute attack of childhood asthma. Methods: A computer-based search of database from Pumbed, CNKI, Wanfang, Baidu Scholar, Chongqing VIP, GeenMedica was performed to screen the articles about respiratory syncytial virus infection and childhood asthma. Then the literatures were screened out by the selection criteria. The RevMan5.3 software was used to test the heterogeneity and effect values of each study, analyze the sensitivity and publication bias of the literature, and draw on Meta forest plot and Funnel plot. Results: 5 articles conformed to the selection criteria. There were totally 881 cases in the case group, 826 cases in the control group. The results of heterogeneity test showed no heterogeneity between each study (P > 0.05). The fixed-effects model showed the 6.68 (5.06 - 8.82), (Z = 13.38, P < 0.00001). Conclusion: The infection rate of respiratory syncytial virus in children with acute asthma attack is higher than that in remission period. Respiratory syncytial virus infection rate can be used as an indicator of the severity of asthma in children.

Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

Human respiratory syncytial virus(RSV)infection is the leading cause of lower respiratory tract illness(LRTI),and no vaccine against LRTI has proven to be safe and effective in infants.Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice.The constructed recombinant plasmids harbored(5′to 3′)a T7 promoter,hammerhead ribozyme,RSV Long strain antigenomic cDNA with cold-passaged(cp)mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain(A2cpts)or further combined with SH gene deletion(A2cptsΔSH),HDV ribozyme(δ),and a T7 terminator.These vectors were subsequently co-transfected with four helper plasmids encoding N,P,L,and M2-1 viral proteins into BHK/T7-9 cells,and the recovered viruses were then passaged in Vero cells.The rescued recombinant RSVs(rRSVs)were named rRSV-Long/A2cp,rRSV-Long/A2cpts,and rRSV-Long/A2cptsΔSH,respectively,and stably passaged in vitro,without reversion to wild type(wt)at sites containing introduced mutations or deletion.Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed temperature-sensitive(ts)phenotype in vitro and in vivo,all rRSVs were significantly attenuated in vivo.Furthermore,BALB/c mice immunized with rRSVs produced Th1-biased immune response,resisted wtRSV infection,and were free from enhanced respiratory disease.We showed that the combination ofΔSH with attenuation(att)mutations of cpts contributed to improving att phenotype,efficacy,and gene stability of rRSV.By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains,we have laid an important foundation for the development of RSV live attenuated vaccines.

Cytokine responses in infants infected with respiratory syncytial virus

Introduction: Variability in severity of Respiratory Syncytial Virus (RSV) infection is reportedly due to differences in inflammatory response. Objective: To characterize the cytokine response in RSV+ infants aged 0 - 36 months and to relate their responses to disease severity. Methods: Nasopharyngeal aspirations (NPAs) were analyzed for RSV and IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-1RA, IL-4R, IFN-γ, sTNFR1, sTNFR2, and TNF-α. Clinical data were collected from the medical records. Results: We included 331 infants of whom 214 were RSV+. In comparison to RSV- infants, they had significantly higher levels of TNF-α, IL-6, IL-1β, and IFN-γ (p α, IL-6, and IL-1β. sTNFR1/2 were significantly increased in RSV+ infants. Hospitalized patients had significantly higher levels of TNF-α, sTNFR2, and IL-10 (p < 0.05) than non-hospitalized patients. The cytokine response could not be related to disease severity. We found no evidence of a skewed Th1/Th2 immune profile. Conclusion: In acute RSV disease, infected infants’ NPAs contain a significant amount of pro-inflammatory cytokines. Whether this response is beneficial or deleterious remains unanswered. Interpersonal variations in cytokine responses might be linked to an inherited tendency to variations in disease severity.

Respiratory syncytial virus infection in immunocompetent and immunocompromised murine

The purpose of this study is to distinguish respiratory syncytial virus （RSV） infection and immunology between immunocompetent and immunocompromised murine and to explore immune mechanism of RSV infection. At various time points after RSV infection of BALB/c mice and nude mice, pulmonary viral titers were assayed, RSV antigen was tested by direct immunofluorescent assay and immunohistochemistry. Pulmonary mRNA expressions of Toll like receptor （TLR）2 and TLR4 were assayed by RT-PCR. CD4^＋ ceils and CD8 ^＋ ceils in peripheral blood were examined by flow cytometry and plasma total IgE was assayed by ELISA. Leukocytes in bronchoalveolar lavage fluid （BALF） and pulmonary histology were identified to reflect airway inflammation. It was found that RSV titers of both mice peaked on the 3rd day post infection with a much higher level of viral titer in nude mice than in BALB/c mice and a longer viral duration in nude mice （over 9 days post infection） than in BALB/c mice （6 days post infection）. RSV infection induced higher viral antigen expression in nude mice （0.267 ±0.045） than in BALB/c mice （0. 168 ±0.031）. RSV infection enhanced pulmonary TLR4 expression of BALB/c mice （51.96% ±11.34%） and nude mice （48.96% ± 12.35%） compared with each control （34.04% ±10.06% and 32.37% ±9.87% respectively）. CD4^＋ peripheral blood ceils increased in RSV infected BALB/c mice （66.51% ±2.09% ） compared with the control BALB/c mice （51.63% ±5.90%）, and CD4^＋ ceils and CD8^＋ ceils were deficient in nude mice. RSV infection increased plasma total IgE in both mice, and BALB/c mice had a larger amount of IgE on the 7th day post infection （9.02 ng/ml ±2.90 ng/ml） and on the 14th day post infection （12.76 ng/ml ±4.15 ng/ml） than corresponding nude mice （3.72 ng/ml ±1.06 ng/ml and 7.62 ng/ml ±3.08 ng/ml respectively on the 7th and 14th day post infection）. RSV infected nude mice had more severe airway inflammation than infected BALB/c mice. It is concluded that BALB/c mice and nude mice presented similar RSV infectious characteristics. However, infection of nude mice showed higher viral titer with longer duration and more severe airway inflammation, lower level of plasm total IgE and CD4^＋ peripheral blood cells, but the similar pulmonary TLR4 expression with BALB/c mice.

Use of flow cytometry to investigate the cytokine response pattern in infants with respiratory syncytial virus infection and bronchiolitis

Objective: To investigate the cytokine response pattern (IL 4/IFN γ ) in infants with RSV infections and bronchiolitis during the acute phase. Methods: Four color flow cytometry was used to measure intracellular IL 4 and IFN γ expressions in peripheral blood CD3+ and CD8+ lymphocytes from RSV infected and bronchiolitis infants. Serum IL 4 and IFN γ levels were also determined. Results: RSV infected and bronchiolitis infants showed no statistical differences from not RSV infected or pneumonia infants and control in the frequency of IL 4 and IFN γ expressions in CD3+CD8 lymphocytes, showed no obvious Th1/Th2 imbalance, while IFN γ was expressed much more frequently in CD3+CD8+ lymphocytes. Systematically, RSV infected and bronchiolitis infants showed much lower levels of serum IL 4 and IL 4/IFN γ ratios and much higher serum IFN γ levels than control. However, there were no statistical differences in the above three indices between RSV infected and not RSV infected infants or between bronchiolitis and pneumonia infants, except that bronchiolitis infants had a higher level of serum IFN γ than pneumonia infants statistically. Conclusions: There is no type 2 cytokine response predominance in the acute phase of RSV infection and bronchiolitis. IL 4 production is suppressed and IFN γ production upregulated, the latter being most prominent in bronchiolitis infants.

Surfactant Protein D for Pathological Evaluation of Infant Acute Respiratory Distress Syndrome Caused by Respiratory Syncytial Virus Infection

Pediatric respiratory syncytial viral infection (RS) usually shows relatively good outcome;however, when it accompanies acute respiratory distress syndrome (ARDS), this becomes fatal. We experienced three pediatric patients with RS + ARDS, with all showing good outcome with steroid pulse therapy. We wish to emphasize;1) steroid pulse therapy may become an option for this condition, and 2) plasma KL-6 and surfactant protein D levels may become a biomarker reflecting the disease progression/condition. Patients were, aged 1 month, 1 year 5 months, and 1 year 11 months. In all three, the respiratory condition deteriorated rapidly, requiring invasive ventilator management. Although the effectiveness of steroid treatment for ARDS is controversial, very severe condition prompted us to employ steroid pulse therapy, after which, oxygenation rapidly improved without adverse events. Plasma KL-6 and surfactant protein D levels were measured during exacerbations of ARDS, steroid pulse therapy, and recovery. Surfactant protein D levels were closely associated with oxygenation, suggesting this substance level might be a biomarker of ARDS caused by the disruption of the alveolar epithelial lining and to understand oxygenation without time lag.

An RNA polymerase I-driven human respiratory syncytial virus minigenome as a tool for quantifying virus titers and screening antiviral drug

Human respiratory syncytial virus（RSV） is an important pediatric pathogen of lower respiratory tract worldwide. No vaccines and antiviral drugs are available. Herein the use of an RNA polymerase I-driven RSV minigenome for analyzing RSV replication and screening anti-RSV drugs was investigated. The RNA polymerase I（Pol I） was used to transcribe RSV minigenome from the constructed plasmid, designated p HM-RSV-Gluc, of minigenome c DNA which comprised trailer region, gene start sequence（GS）, reverse complementary copy of Gaussia luciferase（Gluc） gene, gene end sequence（GE）, and leader region in the direction of 5＇–3＇end and was flanked by promoter and terminator of Pol I. The expression of Gluc was confirmed in p HM-RSV-Gluc transfected HEp-2 cells following RSV infection and had the characteristics of dose-dependent, which provided a rapid, sensitive, and quantitative method for quantifying virus titers and screening antiviral drugs.

Burden of respiratory syncytial virus infection in young children

Respiratory syncytial virus(RSV) is the most frequent and important cause of lower respiratory tract infection in infants and children. It is a seasonal virus, with peak rates of infection occurring annually in the cold season in temperate climates, and in the rainy season, as temperatures fall, in tropical climates. High risk groups for severe RSV disease include infants below six mo of age, premature infants with or without chronic lung disease, infants with hemodynamically significant congenital heart disease, infants with immunodeficiency or cystic fibrosis, and infants with neuromuscular diseases. Mortality rates associated with RSV infection are generally low in previous healthy infants(below 1%), but increase significantly in children with underlying chronic conditions and comorbidities. Following early RSV lower respiratory tract infection, some patients experience recurrent episodes of wheezing mimicking early childhood asthma with persistence of lung function abnormalities until adolescence. There is currently no RSV vaccine available, but promising candidate vaccines are in development. Palivizumab, a monoclonal RSV antibody that is the only tool for immunoprophylaxis in high-riskinfants, lowers the burden of RSV infection in certain carefully selected patient groups.

Prevalence of respiratory syncytial virus infection among children hospitalized with acute lower respiratory tract infections in Southern India

BACKGROUND Respiratory syncytial virus(RSV) is a leading cause of lower respiratory infections among children.AIM To investigate the proportion of RSV and non-RSV respiratory viral infections among hospitalized children ≤ 5 years.METHODS Hospitalized children aged < 5 years, with a diagnosis of acute lower respiratory infections(ALRI), admitted between August 2011-August 2013, were included.Cases were defined as laboratory-confirmed RSV and non-RSV respiratory viruses by direct fluorescence assay from the nasopharyngeal wash.RESULTS Of 383 1-59 mo old children hospitalized with an acute lower respiratory infection, 33.9%(130/383) had evidence of viral infection, and RSV was detected in 24.5%(94/383). Co-infections with RSV and other respiratory viruses(influenza A or B, adenovirus, para influenza 1, 2 or 3) were seen in children 5.5%(21/383). Over 90% of the RSV-positive children were under 2 years of age. RSV was detected throughout the year with peaks seen after the monsoon season.Children hospitalized with RSV infection were more likely to have been exposed to a shorter duration of breastfeeding of less than 3 mo. RSV positive children had a shorter hospital stay, although there were significant complications requiring intensive care. Use of antibiotics was high among those with RSV and non-RSV viral infections.CONCLUSION Our study provides evidence of a high proportion of RSV and other virusassociated ALRI among hospitalized children in India. RSV infection was associated with fewer days of hospital stay compared to other causes of lower respiratory infections. A high level of antibiotic use was seen among all respiratory virus-associated hospitalizations. These results suggest the need for implementing routine diagnostics for respiratory pathogens in order to minimize the use of unnecessary antibiotics and plan prevention strategies among pediatric populations.

RELATIONSHIP BETWEEN HYPERSENSITIVITY TYPE I AND WHEEZING BY RESPIRATORY SYNCYTIAL VIRUS (RSV) INFECTION

We studied the RSV specific IgE antibody, histamine and basophil from infants with RSV bronchiolitis and found during the acute phase either the titers of RSV-IgE or the concentration of histamine increased significantly, the number of basophil and basophil degranulation in the presence of RSV antigen also increased. In vitro studies revealed hypersensitivity participates in the pathogenesis of RSV bronchiolitis. We also found that infants with RSV bronchiolitis, the RSV-IgE persisted for a long time presumably this plays an important role in recurrent wheezing after RSV infection for years.

Prevalence,variation,and transmission patterns of human respiratory syncytial virus from pediatric patients in Hubei,China during 2020–2021

Human respiratory syncytial virus(RSV)is a severe threat to children and a main cause of acute lower respiratory tract infections.Nevertheless,the intra-host evolution and inter-regional diffusion of RSV are little known.In this study,we performed a systematic surveillance in hospitalized children in Hubei during 2020–2021,in which 106 RSV-positive samples were detected both clinically and by metagenomic next generation sequencing(mNGS).RSV-A and RSV-B groups co-circulated during surveillance with RSV-B being predominant.About 46 high-quality genomes were used for further analyses.A total of 163 intra-host nucleotide variation(iSNV)sites distributed in 34 samples were detected,and glycoprotein(G)gene was the most enriched gene for iSNVs,with non-synonymous substitutions more than synonymous substitutions.Evolutionary dynamic analysis showed that the evolutionary rates of G and NS2 genes were higher,and the population size of RSV groups changed over time.We also found evidences of inter-regional diffusion from Europe and Oceania to Hubei for RSV-A and RSV-B,respectively.This study highlighted the intra-host and inter-host evolution of RSV,and provided some evi-dences for understanding the evolution of RSV.

A protective human antibody against respiratory syncytial virus by targeting a prefusion epitope across sites IV and V of the viral fusion glycoprotein

Respiratory syncytial virus(RSV)is one of the leading pathogens that cause lower respiratory tract infections in infants and the elderly.Passive immunoprophylaxis with monoclonal antibody(mAb)has been approved to prevent morbidity and mortality from RSV infection in infants.Here we report the isolation of two neutralizing mAbs against RSV from convalescent children by prefusion form of fusion(F)glycoprotein as bait.One mAb RV11 exhibited good potency in neutralization of RSV strains from both A and B subtypes in cell-based assay,and protected mice from RSV infection in vivo.An RV11 escape mutant was identified,which contains an S443P mutation in F protein.Crystal structure showed the RV11 bound to a conserved prefusion epitope across the antigenic sites IV and V of the F glycoprotein.RV11 showed a strong synergistic effect when combined with two RSV antivirals,an F-targeting small molecular inhibitor ziresovir and a siteØneutralizing mAb D25(the parental mAb for nirsevimab).The study extended our knowledge to the neutralizing and protective epitopes of RSV,and the mAb RV11 deserves further development for clinical translation.

Human bocavirus: Current knowledge and future challenges

Human bocavirus(HBoV) is a parvovirus isolated about a decade ago and found worldwide in both respiratory samples, mainly from early life and children of 6-24 mo of age with acute respiratory infection, and in stool samples, from patients with gastroenteritis. Since then, other viruses related to the first HBoV isolate(HBoV 1), namely HBoV 2, HBoV 3 and HBoV 4, have been detected principally in human faeces. HBo Vs are small nonenveloped single-stranded DNA viruses of about 5300 nucleotides, consisting of three open reading frames encoding the first two the non-structural protein 1(NS1) and nuclear phosphoprotein(NP1) and the third the viral capsid proteins 1 and 2(VP1 and VP2). HBoV pathogenicity remains to be fully clarified mainly due to the lack of animal models for the difficulties in replicating the virus in in vitro cell cultures, and the fact that HBo V infection is frequently accompanied by at least another viral and/or bacterial respiratory and/or gastroenteric pathogen infection. Current diagnostic methods to support HBoV detection include polymerase chain reaction, real-time PCR, enzymelinked immunosorbent assay and enzyme immunoassay using recombinant VP2 or virus-like particle capsid proteins, although sequence-independent amplification techniques combined with next-generation sequencing platforms promise rapid and simultaneous detection of the pathogens in the future. This review presents the current knowledge on HBoV genotypes with emphasis on taxonomy, phylogenetic relationship and genomic analysis, biology, epidemiology, pathogenesis and diagnostic methods. The emerging discussion on HBoV s as true pathogen or innocent bystander is also emphasized.

Network pharmacology, molecular docking, and untargeted metabolomics reveal molecular mechanisms of multi-targets effects of Qingfei Tongluo Plaster improving respiratory syncytial virus pneumonia

Objective: Qingfei Tongluo Plaster(QFP), an improved Chinese medicine hospital preparation, is an attractive treatment option due to its well clinical efficacy, convenience, economy, and patient compliance in the treatment of respiratory syncytial virus(RSV) pneumonia. The aim of this study was to investigate the efficacy mechanism of QFP on RSV rats from the perspective of alleviating lung inflammation and further explore the changes of serum metabolites and metabolic pathways in RSV rats under the influence of QFP.Methods: This study used network pharmacological methods and molecular docking combined with molecular biology and metabolomics from multi-dimensional perspectives to screen and verify the therapeutic targets. Open online databases were used to speculate the gene targets of efficient ingredients and diseases. Then, we used the String database to examine the fundamental interaction of common targets of drugs and diseases. An online enrichment analysis was performed to predict the functional pathways. Molecular docking was applied to discover the binding modes between essential ingredients and crucial gene targets. Finally, we demonstrated the anti-inflammatory ability of QFP in the RSV-evoked pneumonia rat model and explained the mechanism in combination with the metabolomics results.Results: There were 19 critical targets defined as the core targets: tumor necrosis factor(TNF), inducible nitric oxide synthase 2(NOS2), mitogen-activated protein kinase 14(MAPK14), g1/S-specific cyclin-D1(CCND1), signal transducer and activator of transcription 1-alpha/beta(STAT1), proto-oncogene tyrosine-protein kinase Src(SRC), cellular tumor antigen p53(TP53), interleukin-6(IL6), hypoxiainducible factor 1-alpha(HIF1A), RAC-alpha serine/threonine-protein kinase(AKT1), signal transducer and activator of transcription 3(STAT3), heat shock protein HSP 90-alpha(HSP90AA1), tyrosine-protein kinase JAK2(JAK2), cyclin-dependent kinase inhibitor 1(CDKN1A), mitogen-activated protein kinase 3(MAPK3), epidermal growth factor receptor(EGFR), myc proto-oncogene protein(MYC), protein c-Fos(FOS) and transcription factor p65(RELA). QFP treated RSV pneumonia mainly through the phosphatidylinositol 3-kinase(PI3K)/RAC AKT pathway, HIF-1 pathway, IL-17 pathway, TNF pathway, and MAPK pathway. Animal experiments proved that QFP could effectively ameliorate RSV-induced pulmonary inflammation. A total of 28 metabolites underwent significant changes in the QFP treatment, and there are four metabolic pathways consistent with the KEGG pathway analyzed by network pharmacology,suggesting that they may be critical processes related to treatment.Conclusion: These results provide essential perspicacity into the mechanisms of action of QFP as a promising anti-RSV drug.