Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

Objective Newly identified human rhinovirus C （HRV-C） and human bocavirus （HBoV） cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.Methods A platform for culturing human airway epithelia in a three-dimensional （3D） pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction {PCR）. Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-2, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial （HAE） cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.Conclusion Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Association of Different Human Rhinovirus Species with Asthma in Children： A Preliminary Study

Background： Human rhinoviruses （HRVs） are divided into three genetic species： HRV-A, HRV-B, and HRV-C. The association of different HRV species with asthma in children in China has not yet been evaluated. This preliminary study aimed to assess the associations between different HRV species, particularly HRV-C, and asthma in young children in China. Methods： A total of 702 nasopharyngeal aspirates were obtained from 155 children with asthma （asthma group）, 461 children with acute respiratory infection （ARI） without asthma （nonasthma ARI group）, and 86 children from the control group. Semi-nested polymerase chain reaction （PCR） was used to detect HRVs, and PCR products were sequenced for species identification. Epidemiological characteristics of HRV-positive cases were analyzed. Results： HRVs were the most common pathogen （ 15.4%; 108/702） in the patients in this study. The prevalence of HRV was significantly different （F = 20.633, P = 0.000） between tile asthma （25.8%） and nonasthma ARI groups （11.1%）. Phylogenetic analysis indicated that in the 108 cases positive for HRVs, 41 were identified as HRV-A, 8 as HRV-B, and 56 as HRV-C, Comparing the asthma with the nonasthlna ARI group, Spearman＇s rank correlation analysis revealed an association between HRV-A （P 〈 0.05） and C （P 〈 0.01） and asthma, confirmed by regression analysis, with odds ratios of 2.2 （HRV-A） and 4.2 （HRV-C）. Conclusions： Our data revealed a high prevalence of HRVs in children in China, regardless of clinical status. HRV-C was the dominant species and may be one of the key factors in the association of HRVs with asthma.

Human rhinovirus C infection is associated with asthma in children determined by x TAG respiratory viral panel FAST

Dear Editor,Cumulative evidence supports the role of early-life viral infections,especially respiratory syncytial virus(RSV)and human rhinovirus(HRV),as major antecedents of childhood asthma(Lemanske,2002;Jackson et al.,2008).In this study,the x TAG respiratory viral panel FAST(RVP FAST)assay,a multiplex polymerase chain reaction(PCR)-based method(Arens et al.,2010;BaladaLlasat et al.,2011;Gharabaghi et al.,2011;Selvaraju,2012),was used to investigate the association of infec-

The role of human rhinovirus in immunology, COPD, and corresponding treatments

The common cold is most often a result of human rhinovirus （HRV） infection. Common cold symptoms including rhinorrhea and nasal obstruction frequently occur during HRV infection of the upper respiratory tract. Conversely, HRV may also infect the epithelial cells of the lower respiratory tract. Symptom severity associated with HRV infection ranges from mild to potentially serious depending on a person＇s susceptibility and preexisting condition, such as chronic obstructive pulmonary disease. An over active host immune response is believed to be the primary contributor to HRV pathogenesis. Enhanced activity of various host cell cytokines and granulocytes mediate specific cellular pathways inducing many of the symptoms associated with HRV infection. There are over 100 serotypes of HRV which can be further categorized based on the specific characteristics of each type. The two main categories of HRV consist of the major and minor groups. The unique host cell receptor is the distinguishing factor between these two groups. Yet, these viruses may also differ in mechanism of infection and replication. Due to the high frequency of hospital and clinical visits and the corresponding economic burden, novel therapies are of interest. Several different treatment options varying from herbal remedies to anti-viral drugs have been studied. However, the vast number of HRV serotypes complicates the progress of developing a universal treatment for attenuating HRV infection.

Effect of human rhinovirus infection in pediatric patients with influenza-like illness on the 2009 pandemic influenza A（H1N1） virus

Background Some research groups have hypothesized that human rhinoviruses （HRVs） delayed the circulation of the 2009 pandemic influenza A（H1N1） virus （A（H1N1）pdm09） at the beginning of Autumn 2009 in France.This study aimed to evaluate the relationship between HRV and A（H1N1）pdm09 in pediatric patients with influenza-like illness in Beijing,China.Methods A systematic analysis to detect A（H1N1）pdm09 and seasonal influenza A virus （FLU A） was performed on 4 349 clinical samples from pediatric patients with influenza-like illness during the period June 1,2009 to February 28,2010,while a one-step real-time RT-PCR （rRT-PCR） assay was used to detect HRV in 1 146 clinical specimens selected from those 4 349 specimens.Results During the survey period,only one wave of A（H1N1）pdm09 was observed.The percentage of positive cases for A（H1N1）pdm09 increased sharply in September with a peak in November 2009 and then declined in February 2010.Data on the monthly distribution of HRVs indicated that more HRV-positive samples were detected in September （2.2％） and October （3.3％）,revealing that the peak of HRV infection in 2009 was similar to that of other years.Among the 1 146 specimens examined for HRVs,21 （1.8％） were HRV-positive,which was significantly lower than that reported previously in Beijing （15.4％ to 19.2％） （P ＜0.01）.Overall,6 samples were positive for both A（H1N1）pdm09 and HRV,which represented a positive relative frequency of 1.60％ and 2.08％ HRV,considering the A（H1N1）pdm09-positive and-negative specimens,respectively.The odds ratio was 0.87 （95％ CI 0.32; 2.44,P=0.80）.Conclusions HRVs and A （H1N1）pdm09 co-circulated in this Chinese population during September and October 2009,and the HRV epidemic in 2009 did not affect A（H1N1）pdm09 infection rates in Beijing,China as suggested by other studies.However,the presence of A（H1N1）pdm09 might explain the unexpected reduction in the percentage of HRV positive cases during the period studied.

Efficacy and mechanisms of Traditional Chinese Medicine prevention and treatment for respiratory viruses

Respiratory virus infection was the most common viral infection in clinical practice with the greatest impact,including the Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),posing a huge threat to the world's public health and human life safety.Commonly used antiviral drugs have obvious side effects and a narrow scope of application.Respiratory viruses are susceptible to infection,mutation,and prevalence,which also pose challenges to traditional antiviral drugs and vaccine development.Traditional Chinese Medicine(TCM)has a long history of treating infectious diseases,with many herbs and compounds.Its multi-component,multi-target and multi-path characteristics have made it have great advantages and potential in the development of new anti-respiratory virus drugs.This review summarized TCM for the prevention and treatment of common respiratory viruses,and provided new strategies for the research and development of new TCM antiviral drugs and for responding to infectious respiratory virus diseases.

热毒宁注射液对人鼻病毒(N36)的体外抑制作用

目的:研究中草药热毒宁注射液对人鼻病毒(HRV,N36)的抑制作用。方法:以利巴韦林为阳性对照药,采用细胞培养技术及MTT比色法,观察在热毒宁干预下,人胚胎上皮细胞的病变效应(CPE)及吸收度值(A),计算各组细胞的病毒抑制率。比较不同药物剂量及不同给药方式下热毒宁对HRV的抑制作用效果。结果:热毒宁注射液半数中毒浓度TC50为5.873g/L。预防、直接灭活及治疗给药方式下,CPE法测定的半数有效浓度EC50分别为0.576、0.836及0.640g/L,治疗指数(TI)分别为10.20、7.03及9.18;MTT法测定的EC50分别为0.551、2.310及1.230g/L,TI分别为10.66、2.54及4.78。结论:热毒宁对HRV(N36)有直接灭活及抑制增殖作用,对其感染靶细胞有阻断作用,且呈明显量效关系。热毒宁对HRV(N36)的抑制作用,以预防给药更明显。

基于结构设计的鼻病毒3C蛋白酶抑制剂的研究进展

人鼻病毒(HRVs)是引起人类普通感冒的主要致病因素,并可导致严重的后遗症。3C蛋白酶是HRVs复制和感染中重要的必需蛋白酶,是目前抗病毒药物的重要选择性靶标。本文对3C蛋白酶的结构与功能及基于结构设计的3C蛋白酶抑制剂研究进展进行了简要概述。

Innate and adaptive immune responses against picornaviruses and their counteractions: An overview

Picornaviruses, small positive-stranded RNA viruses, cause a wide range of diseases which is based on their differential tissue and cell type tropisms. This diversity is reflected by the immune responses, both innate and adaptive, induced after infection, and the subsequent interactions of the viruses with the immune system. The defense mechanisms of the host and the countermeasures of the virus significantly contribute to the pathogenesis of the infections. Important human pathogens are poliovirus, coxsackievirus, human rhinovirus and hepatitis A virus. These viruses are the beststudied members of the family, and in this review we want to present the major aspects of the reciprocal effects between the immune system and these viruses.

Surges of hospital-based rhinovirus infection during the 2020 coronavirus disease-19(COVID-19)pandemic in Beijing,China

Background A series of public health preventive measures has been widely implemented in Beijing to control the corona-virus disease-19(COVID-19)pandemic since January 2020.An evaluation of the effects of these preventive measures on the spread of other respiratory viruses is necessary.Methods Respiratory specimens collected from children with acute respiratory infections were tested by NxTAGTN,respiratory pathogen panel assays during January 2017 and December 2020.Specimens characterized as rhinoviruses(RVs)were sequenced to identify the RV species and types.Then,the epidemiology results of respiratory pathogens in 2020 were compared with those from 2017 to 2019 using SPSS statistics 22.0.Results The positive rates of adenovirus(ADV),influenza virus(flu),RVs,and respiratory syncytial virus(RSV)dropped abruptly by 86.31%,94.679c,94.59%,and 92.17%,respectively,from February to May 2020,compared with the average level in the same period during 2017-2019.Positive rates of RVs then steeply increased from June 2020(13.77%),to an apex(37.25%)in August 2020,significantly higher than the average rates(22.51%)in August 2017-2019(7^=0.005).The increase,especially in group>3 years,was accompanied by the reopening of schools and kindergartens after the 23rd and 24th week of 2020 in Beijing.Conclusions Whereas the abrupt drop in viral pathogen positive rates from February to May 2020 revealed the remarkable effects of the COVID-19 preventive measures,the sharp increase in positive rates of RVs from the 23rd week of 2020 might be explained by the reopening of schools and kindergartens in Beijing.

南京市鼻病毒感染暴发疫情流行病学及病原学特征

目的分析南京市鼻病毒(human rhinoviruses,HRV)感染暴发疫情的流行病学及病原学特征。方法2020年5—6月,南京市江北新区、雨花台区发生HRV感染暴发疫情3起,采集有HRV感染症状人群的咽拭子样本,排除新型冠状病毒及流感病毒感染后,采用实时荧光定量PCR方法检测HRV核酸,用巢式PCR扩增HRV的VP4/2的靶基因,测序后鉴定阳性HRV样本血清型。从GenBank数据库下载HRV各血清型参考株的序列资料,使用MEGA6和BioEdit等软件将测序结果与参考株进行比较分析。结果累计收集咽拭子样本33份,核酸阳性率60.00%~87.50%,共鉴定出13种血清型,对HRV各组内的核苷酸同源性进行分析,HRV-A组内核苷酸的同源性为75.20%~100.00%,HRV-C组内核苷酸的同源性为72.70%~100.00%。HRV-A组是造成的疫情的主要致病原,HRV-A11和HRV-A29是造成的个体感染数最多的血清型,存在同一起疫情检测出多种血清型的现象。结论HRV疫情存在多种血清型共流行,A组为主要病原,A11和A29为主要流行的血清型。在不同血清型之间没有交叉免疫,要不断对HRV导致的呼吸道疫情进行深入研究,加强对HRV感染的监测,为HRV的诊断和疫情防控提供科学依据。

Structure of the HRV-C 3C-Rupintrivir Complex Provides New Insights for Inhibitor Design

Human rhinoviruses(HRVs)are the predominant infectious agents for the common cold worldwide.The HRV-C species cause severe illnesses in children and are closely related to acute exacerbations of asthma.3C protease,a highly conserved enzyme,cleaves the viral polyprotein during replication and assists the virus in escaping the host immune system.These key roles make 3C protease an important drug target.A few structures of 3Cs complexed with an irreversible inhibitor rupintrivir have been determined.These structures shed light on the determinants of drug specificity.Here we describe the structures of HRV-C153C in free and inhibitor-bound forms.The volume-decreased S1'subsite and half-closed S2 subsite,which were thought to be unique features of enterovirus A 3C proteases,appear in the HRV-C 3C protease.Rupintrivir assumes an“intermediate”conformation in the complex,which might open up additional avenues for the design of potent antiviral inhibitors.Analysis of the features of the three-dimensional structures and the amino acid sequences of 3C proteases suggest new applications for existing drugs.

HRV16 Infection Induces Changes in the Expression of Multiple piRNAs

Human rhinovirus(HRV)is one of the most important cold-causing pathogens in humans.Piwi-interacting RNAs(piRNAs)are a recently discovered class of small non-coding RNAs whose best-understood function is to repress mobile element(ME)activity in animal germline.However,the profile of human/host piRNA during HRV infection is largely unknown.Here we performed high-throughput sequencing of piRNAs from H1-HeLa cells infected with HRV16 at 12 h,24 h,and 36 h.The results showed that 22,151,664,24,362,486 and 22,726,546 piRNAs displayed differential expression after HRV16 infection for three time points.A significant differential expression of 21 piRNAs was found in all time points and further verified by RT-qPCR,including 7 known piRNAs and 14 newly found piRNAs.In addition,piRNA prediction was performed on Piano using the SVM algorithm and transposon information.It found that novel_pir78110,novel_pir78107,novel_pir78097,novel_pir78094 and novel_pir76584 are associated with the DNA/hobo of Drosophila,Ac of maize and Tam3 of snapdragon(hAT)-Charlie transposon.The novel_pir97924,novel_pir105705 and novel_pir105700 recognize long interspersed nuclear elements 1(LINE-1).The novel_pir33182 and novel_pir46604 are related to the long terminal repeat(LTR)/(Endogenous Retrovirus1)ERV1 repetitive element.The novel_pir73855 is related to the LTR/ERVK repetitive element.Both novel_pir70108 and novel_pir70106 are associated with the LTR/ERVL-MaLR repetitive element.The novel_pir15900 is associated with the DNA/hAT-Tip100 repetitive element.Overall,our results indicated that rhinovirus infection could reduce the expression of some piRNAs to facilitate upregulation of LINE-1 transcription or retrotransposons'expression,which is helpful to further explore the mechanism of rhinovirus infection.