深圳地区2014年柯萨奇病毒A组4型VP1区基因特征分析

目的 对2014年深圳市罗湖区一起疱疹性咽峡炎疫情的病原体进行鉴定,并对鉴定的柯萨奇A4病毒(CVA4)的VP1基因进行序列测定和遗传进化特征分析。方法 采集疫情中患儿的咽拭子样本8份,提取病毒核酸,利用荧光RT-PCR法检测样本总肠道病毒(EV)、人肠道病毒71型(EV71)、柯萨奇病毒A组16型(CVAl6)、CVA4、CVA6和CVAl0等,对CVA4阳性样本采用RT-PCR方法扩增其VP1区全长序列,并进行核苷酸序列测定和遗传进化分析。结果 引起此次疱疹性咽峡炎暴发的病原体为GIB亚型的CVA4病毒。同源性分析显示,深圳2014年CVA4病毒株与云南2004年分离株(AB268278)、以及台湾2008年分离株(AB571570)核苷酸同源性达94.1%~94.8%,而与CVA4原型株HIGHPOINT同源性最低,为84.3%。在氨基酸序列上与台湾2006年分离株(AB571563)、吉林2006年分离株(JQ715709)同源性最高,达99.3%;而与山东省2006年分离株(GQ253375)同源性最低,为97.1%。相比深圳2009年分离株(HQ728260),共有6个氨基酸突变位点:N22S,T34A,N63S,A165D,T200A和V126A;而进化树分析显示,深圳2014年CVA4病毒株虽属于CVA4的GIB亚型,但在进化走势上,已经不同于GIB亚型中其他地区早期的分离株。结论 2014年深圳地区CVA4病毒属于GIB亚型,但在VP1区变异较大。     

手足口病柯萨奇病毒A10和A6型的分子鉴定及其VP1区3′端序列分析

目的鉴定手足口病患儿咽拭子病原体柯萨奇病毒A10(CVA10)和A6(CVA6),并对其VP1编码区3′端序列进行分析。方法合成对肠道病毒VP1区3′端序列具有较高特异性的兼并引物040-011,RT-PCR方法测定其VP1区3′端基因序列,通过BLAST程序比对,鉴定病毒的基因型;与GenBank中其他已知病毒基因型序列进行同源性分析,构建遗传进化树。结果 38例非EV71非CVA16引起的手足口病患儿咽拭子,5例检测到CVA10,1例检测到CVA6。在进化关系上CVA10与D基因型最为密切,核苷酸和氨基酸同源性分别为92.7%~94.2%、91.7%~100%;CVA6与2003~2009年报告的部分毒株核苷酸同源性为86.8%~90.2%,其中与2008年台湾EU908148的毒株同源性最高;氨基酸同源性为85.2%~89.0%。结论兼并引物040-011对CVA10和CVA6有良好的基因扩增特异性;引起手足口病的肠道病毒除EV71和CVA16外,CVA10和CVA6也是重要的病原;鉴于2008年芬兰和2009年山东省文登市CVA10引起的手足口病暴发,应加强对手足口病病原的检测和分型,以了解柯萨奇病毒...     

2013-2022年江苏省柯萨奇病毒A6型全基因组序列特征分析

目的 研究江苏省2013-2022年分离到的柯萨奇病毒A6型(CV-A6)全基因组序列特征及对全基因组各编码区进行遗传进化分析,以期从分子流行病学特征的角度解释CV-A6取代肠道病毒A71和柯萨奇病毒A16成为江苏省引起手足口病(HFMD)主要病原的原因。方法 选取2013-2022年间江苏省地区流行的35株CV-A6毒株进行全基因组测序,采用DNASTAR、MEGA7.0和Similarity plots3.5.1等软件对获取的全基因组序列进行比对、相似性分析、系统进化分析和基因重组分析,对P1编码区和3D区主要氨基酸变异位点进行分析。结果 35株CV-A6江苏毒株的全基因组核苷酸和氨基酸序列相似性分别为87.5%～99.6%和97.0%～99.8%,与CV-A6原型株Gdula/USA/1949的全基因组核苷酸序列相似性仅为80.3%～81.0%,氨基酸序列相似性为94.7%～95.3%。基于VP1序列的系统进化树结果显示,2013-2022年江苏省有34株CV-A6分离毒株分属于D3a基因亚型,1株分属于D2基因亚型;基于3D区进化树划分不同重组模式,2013-2022年江苏省流...     

福州地区2012年柯萨奇A6型肠道病毒分子特征分析

目的对2012年福州地区分离的其他肠道病毒CVA6进行分子鉴定,并对其VP1部分序列进化分析。方法肛拭子标本采用RD细胞进行病毒分离,通过RT—PCR对分离株的VP1部分序列进行扩增、克隆和测序;经过Blast程序比对来确定病毒的基因型;通过Mega4．0软件对不同病毒株VP1部分序列的差异进行比较。结果从10份其他肠道病毒患者的肛拭子标本中分离到6株CVA6病毒。测序结果显示,该6株CVA6病毒核苷酸序列具有高度一致性,但与福州周边国家或地区CVA6的核苷酸一致性为91．8％～97．3％。结论2012年福州市HFMD的病源除Ev71和CVA16外,还包含CVA6病毒。序列分析表明,因此,应加强对手足口病例中其他肠道病原体的检测,并掌握其流行以及病毒遗传进化特征,为HFMD的防控提供科学依据。     

2014-2018年昆明市重症手足口病病原柯萨奇病毒A6型的流行与遗传变异特点北大核心CSCD

目的了解2014-2018年昆明市重症手足口病患者感染的柯萨奇病毒A6(Coxsackievirus A6,CV-A6)型流行情况及其全长VP1区基因特征。方法收集2014-2018年昆明市重症手足口病病例资料及标本1064份,采用Real Time RT-PCR方法进行肠道病毒核酸检测,分析其流行特征。利用随机数表随机选取63例重症CV-A6阳性标本,对其进行完整VP1区基因序列测定,使用Mega6.0软件进行序列分析并构建系统进化树,并比较其核苷酸、氨基酸同源性及氨基酸变异情况。结果2014-2018年昆明市重症手足口病例中共检出肠道病毒阳性标本666份,其中CV-A6阳性195份。5年中重症手足口病例报告数呈下降趋势,但每年均有重症CV-A6病例报告,发病高峰期在4~7月,呈单峰模式,6月达最高峰。感染病例中男女性别比为2.55∶1,≤2岁儿童居多(73.33%),且重点集中在官渡和西山两区。63个CV-A6毒株均位于D3基因亚型D3a进化分支,与国内多地区CV-A6处于共循环状态。该分支分为3个进化小分支,D3a.1包含2014-2016年的所有毒株及少量2017年、2018年毒株;D3a.2包含绝大多数2017年及2018年毒株,D3a.3只有1株2014年昆明株。VP1区氨基酸在多个位点上与原型株存在特异突变,氨基酸变异已呈现年度间规律性。结论CV-A6是引起昆明市重症手足口病主要抗原之一,持续开展CV-A6分子流行病学检测,对昆明市手足口病的防控具有重要意义。     

2011-2020年福建省手足口病相关柯萨奇病毒A组8型和12型分子流行病学特征分析

目的 研究福建省2011-2020年手足口病(hand foot and mouth disease, HFMD)相关柯萨奇病毒A组8型(CVA8)和12型(CVA12)型的流行病学及全基因组序列特征。方法 对2011-2020年福建省疾病预防控制中心实验室收集的HFMD相关CVA8和CVA12病例进行流行病学特征分析;采用MegaⅩ和Simplot 3.5.1等软件对病毒VP1区和全基因组序列遗传进化分析和重组分析。结果 2011-2020年福建省HFMD相关CVA8和CVA12感染各7例。获得4株CVA8完整VP1基因福建分离株,其中2株属于D基因型,2株属于E基因型;获得CVA12完整VP1基因福建分离株6株,其中2株属于B1基因亚型,4株属于B2基因亚型。比对CVA8和CVA12全基因组序列与其原型株核苷酸相似性分别为68.7%～77.7%和69.1%～78%。结合P2和P3区进化树和重组分析结果显示,CVA12-2011FJQZ168在P3区与EVA71可能发生重组,CVA8-2011FJQZ146在P2-P3区与CVA7、在P3区与CVA12-2011FJQZ168可能发生...     

柯萨奇病毒A组19型VP1蛋白生物信息学分析

目的 分析柯萨奇病毒A组19型(coxsackievirus A19,CVA-19)VP1蛋白的理化性质、结构功能并预测其线性B细胞表位和T细胞表位。方法 应用ProtParam分析CVA-19 VP1蛋白的理化性质;ProtScale预测其亲疏水性;使用SignalP 6.0、DeepTMHMM、NetPhos-3.1和Motif Scan分别预测CVA-19 VP1蛋白的信号肽、跨膜结构域、磷酸化位点和脂酰化位点;利用NetCGlyc-1.0、NetNGlyc-1.0和NetOGlyc-4.0分别预测CVA-19 VP1蛋白的C-甘露糖基化位点、N-糖基化位点和O-N-乙酰半乳糖胺(N-acetylgalactosamine, GalNAc)(粘蛋白型)糖基化位点;通过SOPMA和SWISS-MODEL在线工具分别预测CVA-19 VP1蛋白的二级结构和三级结构;使用网络服务器IEDB、Bepipred 3.0、ABCpred和SVTMrip联合预测其线性B细胞表位;应用IEDB和SYFPEITHI综合预测其T细胞表位。结果 CVA-19 VP1蛋白的分子质量为33.099 ku,...     

锌硒宝治疗疱疹性咽峡炎疗效观察

疱疹性咽峡炎好发于夏秋季,主要与柯萨奇A组病毒感染有关,常表现为发热、流涎、拒食、咽峡部疱疹、溃疡,部分患儿可出现呕吐、腹泻。2005年9月～2006年8月,我们采用锌硒宝辅助治疗题疹性咽峡炎,疗效较好。现报告如下。     

北京市西城区手足口病柯萨奇病毒A5型VP4区基因特征分析

目的分析2009年北京市西城区手足口病柯萨奇病毒A5型(CoxA5)的VP4区基因特征,了解其变异情况。方法采集手足口病患儿咽试子,提取病毒RNA,用肠道病毒VP4区分型引物进行半巢式RT-PCR(RT-nPCR),通过测序和GenBank Blast比对确定肠道病毒型别,并对其VP4区进行序列和进化分析。结果共鉴定出5株CoxA5,GenBank登录号为JN981017-JN981021。在VP4区,5株CoxA5与中国株HQ728261核苷酸及氨基酸序列同源性分别为96%～99%和98%～100%。进化分析表明,5株CoxA5与中国株HQ728261遗传距离为0.005～0.045,共同构成一个独立的进化树分支。5株CoxA5在VP4区无氨基酸的缺失和插入,未发现特有的氨基酸置换。结论 2009年北京市西城区手足口病病原体CoxA5VP4区与中国株HQ728261在进化上密切相关,其序列未发生明显变异。     

中国大陆地区柯萨奇病毒A组4型VP1区基因特征分析

目的 分析中国大陆地区柯萨奇病毒A组4型(Coxsackievirus A4,CV-A4)流行毒株基因特征和进化规律。方法 利用Mega6.0软件对GenBank核苷酸数据库收录的分离于中国大陆地区CV-A4毒株VP1区基因序列进行分析,构建系统进化树,并计算分离毒株核苷酸和氨基酸同源性。结果 纳入中国大陆地区CV-A4毒株合计116株,中国大陆地区16个省份CV-A4流行毒株潜在的优势基因型为F。与原型株High Point相比,中国大陆地区CV-A4毒株核苷酸和氨基酸同源性分别为79.9%~84.1%和92.5%~98.7%,收录中国大陆地区CV-A4毒株间核苷酸和氨基酸同源性分别为82.6%~100.0%和91.5%~100.0%。结论 针对中国大陆地区CV-A4毒株流行特征和进化规律,采取有效措施防控手足口病。     

Oncolytic Coxsackievirus A21 as a novel therapy for multiple myeloma

Oncolytic viruses are attractive biological agents for the control of human malignancy. This study assessed the capacity of Coxsackievirus A21 (CVA21) to target and destroy multiple myeloma (MM) and precursor aberrant plasma cells in vitro using established MM cell lines and 15 patient bone marrow (BM) biopsies [n = 10 MM and five monoclonal gammopathy of undetermined significance (MGUS)]. Cell surface analysis revealed that all tumour cells lines expressed high levels of intercellular adhesion molecule-1 (ICAM-1) and decay-accelerating factor (DAF), the receptor molecules to which CVA21 can bind, leading to subsequent cell-entry and infection. MM cell lines were remarkably susceptible to CVA21 lytic infection, producing 100-1000-fold increases in viral progeny within 24 h. In contrast, normal peripheral blood cells were refractile to CVA21 infection. Furthermore, challenge of patient BM biopsies with CVA21 for 48 h resulted in specific purging of up to 98.7% of CD138+ plasma cells, with no significant decrease in progenitor cell function. Data generated in this study suggests that CVA21 virotherapy may have potential applications as a systemic anti-tumour agent for MM, or in the ex vivo purging of malignant plasma cells prior to autologous stem cell transplantation.     

Acute enteritis associated with Coxsackievirus A19 in a kidney transplant patient

Diarrhea is a frequent complication after kidney transplantation, with an incidence rate between 22% and 51%. In many cases, the cause remains unknown. We describe here the first case, to our knowledge, of persistent diarrhea associated with Coxsackievirus A19 (CVA19) in a kidney transplant recipient. The patient was a 46‐year‐old man who received a deceased‐donor kidney. He experienced delayed graft function because of donor kidney donation after circulatory determination of death. Maintenance immunosuppression consisted of low‐dose cyclosporine, high‐dose mycophenolate mofetil (MMF) (3 g/day), and prednisone (10 mg/day). He had severe diarrhea for 2 weeks associated with acute renal failure. No pathogens were found in the stool cultures. Enterovirus detection was positive by real‐time polymerase chain reaction, and sequence analysis found CVA19 (from Enterovirus C group). Area under the curve of MMF was 48 mg.h/L. Because of the persistence of diarrhea, MMF was stopped and replaced by azathioprine. The diarrhea disappeared, but serum creatinine did not return to baseline. CVA19 rarely causes gastroenteritis. This case illustrates that MMF is not always the direct cause of diarrhea, and that new clinical infectious diseases will be detected with the expansion of molecular‐based DNA diagnostics.     

Acute gastroenteritis outbreak caused by a GII.6 norovirus

AIM:To report an acute gastroenteritis outbreak caused by a genogroup 2 genotype 6(GII.6) strain norovirus in Shanghai,China.METHODS:Noroviruses are responsible for approximately half of all reported gastroenteritis outbreaks in many countries.Genogroup 2 genotype 4 strains are the most prevalent.Rare outbreaks caused by GII.6 strains have been reported.An acute gastroenteritis outbreak occurred in an elementary school in Shanghai in December of 2013.Field and molecular epidemiologic investigations were conducted.RESULTS:The outbreak was limited to one class in an elementary school located in southwest Shanghai.The age of the students ranged from 9 to 10 years.The first case emerged on December 10,2013,and the last case emerged on December 14,2013.The cases peaked on December 11,2013,with 21 new cases.Of 45 students in the class,32 were affected.The main symptom was gastroenteritis,and 15.6%(5/32) of the cases exhibited a fever.A field epidemiologic investigation showed the pathogen may have been transmitted to the elementary school from employees in a delicatessen via the first case student,who had eaten food from the delicatessen one day before the gastroenteritis episodes began.A molecular epidemiologic investigation identified the cause of the gastroenteritis as norovirus strain GII.6;the viral sequence of the student cases showed 100% homology with that of the shop employees.Genetic relatedness analyses showed that the new viral strain is closely related to previously reported GII.6 sequences,especially to a strain reported in Japan.CONCLUSION:This is the first report to show that norovirus strain GII.6 can cause a gastroenteritis outbreak.Thus,the prevalence of GII.6 noroviruses requires attention.     

柯萨奇病毒B3m重复感染致病毒性心肌炎的心脏形态学改变

目的探讨柯萨奇病毒B3m(CVB3m)重复感染致病毒性心肌炎的心脏形态学改变。方法用梯度倍增法连续4次经腹腔注射CVB3m病毒液感染昆明小鼠,分别于末次感染后10、30、60d处死动物,HE和VG染色观察心肌病变和纤维增生情况;免疫组化法检测心肌中Ⅰ、Ⅲ型胶原。结果(1)HE染色显示末次感染后10d心肌损伤严重,30d时仍可见小灶坏死,60d时未检出心肌的炎性损伤。(2)VG染色表明末次感染后10d心肌即出现较弱的胶原染色,30d时胶原明显可见,60d时心肌组织的胶原显著增加,心肌组织血管周围胶原面积(PCVA)和心肌胶原容积分数(CVF)均显著增加。(3)心肌胶原的免疫组化进一步显示心肌胶原的增加以Ⅰ型为主,Ⅲ型胶原在末次感染后10、30d未见表达,60d时表达量亦较少,而Ⅰ型胶原的表达与胶原染色结果基本一致。结论CVB3m重复感染可导致心肌纤维化和心室重构,使室壁僵硬而影响心脏的收缩和舒张功能,是心力衰竭发生的重要原因之一。     

Efficacy of a Trivalent Hand,Foot, and Mouth Disease Vaccine against Enterovirus 71 and Coxsackieviruses A16 and A6 in Mice

Hand, foot, and mouth disease (HFMD) has recently emerged as a major public health concern across the Asian-Pacific region. Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the primary causative agents of HFMD, but other members of the Enterovirus A species, including Coxsackievirus A6 (CVA6), can cause disease. The lack of small animal models for these viruses have hampered the development of a licensed HFMD vaccine or antivirals. We have previously reported on the development of a mouse model for EV71 and demonstrated the protective efficacy of an inactivated EV71 vaccine candidate. Here, mouse-adapted strains of CVA16 and CVA6 were produced by sequential passage of the viruses through mice deficient in interferon (IFN) α/β (A129) and α/β and γ (AG129) receptors. Adapted viruses were capable of infecting 3 week-old A129 (CVA6) and 12 week-old AG129 (CVA16) mice. Accordingly, these models were used in active and passive immunization studies to test the efficacy of a trivalent vaccine candidate containing inactivated EV71, CVA16, and CVA6. Full protection from lethal challenge against EV71 and CVA16 was observed in trivalent vaccinated groups. In contrast, monovalent vaccinated groups with non-homologous challenges failed to cross protect. Protection from CVA6 challenge was accomplished through a passive transfer study involving serum raised against the trivalent vaccine. These animal models will be useful for future studies on HFMD related pathogenesis and the efficacy of vaccine candidates.     

Optimization and Characterization of Candidate Strain for Coxsackievirus A16 Inactivated Vaccine

Coxsackievirus A16 (CA16) and enterovirus 71 (EV71), both of which can cause hand, foot and mouth disease (HFMD), are responsible for large epidemics in Asian and Pacific areas. Although inactivated EV71 vaccines have completed testing in phase III clinical trials in Mainland China, CA16 vaccines are still under development. A Vero cell-based inactivated CA16 vaccine was developed by our group. Screening identified a CA16 vaccine strain (CC024) isolated from HFMD patients, which had broad cross-protective abilities and satisfied all requirements for vaccine production. Identification of the biological characteristics showed that the CA16CC024 strain had the highest titer (10^7.5 CCID₅₀/mL) in Vero cells, which would benefit the development of an EV71/CA16 divalent vaccine. A potential vaccine manufacturing process was established, including the selection of optimal time for virus harvesting, membrane for diafiltration and concentration, gel-filtration chromatography for the down-stream virus purification and virus inactivation method. Altogether, the analyses suggested that the CC-16, a limiting dilution clone of the CC024 strain, with good genetic stability, high titer and broad-spectrum immunogenicity, would be the best candidate strain for a CA16 inactivated vaccine. Therefore, our study provides valuable information for the development of a Vero cell-based CA16 or EV71-CA16 divalent inactivated vaccine.     

Polyamine Analog Diethylnorspermidine Restricts Coxsackievirus B3 and Is Overcome by 2A Protease Mutation In Vitro

Enteroviruses, including Coxsackievirus B3 (CVB3), are pervasive pathogens that cause significant disease, including cardiomyopathies. Unfortunately, no treatments or vaccines are available for infected individuals. We identified the host polyamine pathway as a potential drug target, as inhibiting polyamine biosynthesis significantly reduces enterovirus replication in vitro and in vivo. Here, we show that CVB3 is sensitive to polyamine depletion through the polyamine analog diethylnorspermidine (DENSpm), which enhances polyamine catabolism through induction of polyamine acetylation. We demonstrate that CVB3 acquires resistance to DENSpm via mutation of the 2A protease, which enhances proteolytic activity in the presence of DENSpm. Resistance to DENSpm occurred via mutation of a non-catalytic site mutation and results in decreased fitness. These data demonstrate that potential for targeting polyamine catabolism as an antiviral target as well as highlight a potential mechanism of resistance.     

柯萨奇病毒B组3型基因疫苗预防实验性心肌炎的研究

目的：以构建的柯萨奇病毒B组3型（CV3）VP1基因的真核表达系统pCEP4-CVB3VP1作为基因疫苗,评价其对CVB3实验性心肌炎的预防作用。方法：大量制备pCEP4-CVB3VP1,提纯后将其接种BALB／C小鼠,取血清进行ELISA,病毒中和试验和病毒攻击保护实验,结果：ELISA证明pCEP4-CVB3VP1表达产物可刺激机体产生抗CVB3的的特异性IgM,病毒中和试验证明其可诱导机体产生中和抗体。中和CVB3毒力,病毒攻击保护实验证明其可保护接种小鼠抵抗CVB3攻击,结论：pCEP4-CVB3VP1能诱导机产生免疫应答,可作为基因疫苗预防CVB3心肌炎。