深圳市输入性寨卡病毒分离株的进化分析及生物学特性研究

目的 对从深圳宝安国际机场口岸入境的1例自柬埔寨归国发热人员进行寨卡病毒实验室检测,研究寨卡病毒分离株的遗传进化和生物学特征,为寨卡病毒病的预防控制提供参考依据。方法 采集患者血液、唾液和尿液样本,采用荧光RT-PCR方法进行寨卡病毒核酸检测;选用多种组织培养细胞分离病毒,分别观察病毒株的致病变效应、空斑形成及病毒滴度等,并对两株病毒基因序列进行分子遗传进化研究,分析输入性病例来源。结果 荧光RT-PCR结果显示,该病例血清、唾液以及尿液样本寨卡病毒核酸阳性并持续一段时间。首次成功从唾液和尿液标本中分离到寨卡病毒株,将其分别命名为ZIKV/S/SZ1901和ZIKV/U/SZ1901。两分离株均可引起Vero细胞病变,不引起BHK-21细胞病变;均可在Vero细胞中形成空斑,滴度分别为3.4×10⁵ pfu/mL、1.4×10³ pfu/mL。RT-PCR扩增出预期大小约10 272 bp片段,测序结果表明该片段与ZIKV/Hu/Thai/KngSG/17-D501株相应序列的同源性为99.6%。系统进化树显示该病毒属亚洲谱系,与输入至我国的其它寨卡病毒株位于不同的进化分支。寨卡病毒编码区氨基酸位点分析提示,SZ1901毒株在结构基因的氨基酸位点(D683E、V763M、T777M)和非结构基因NS1基因的(A188V)变异与近几年流行株完全相同。结论 根据患者流行病学史、临床表现和实验室检测结果,确诊该病例为深圳市从柬埔寨输入性寨卡病毒感染病例,且寨卡病毒分离株具备与2016年以来流行的寨卡病毒大部分相同的分子基础。     

中国首例输入性基孔肯雅热临床分析

目的 了解2008年我国首例输入性基孔肯雅热病例的流行病学、临床、病原学特点及预后转归.方法 对1例成年男性患者流行病学及临床资料进行回顾性分析,并采用ELISA和胶体合法检测患者血清基孔肯雅病毒IgM(CHIKV-IgM)抗体,实时荧光PCR法检测基孔肯雅病毒核酸(CHIKV RNA).结果 该患者临床表现为发热起病,有头痛、全身肌肉及关节酸痛,全身充血性斑丘疹,WBC及PLT减少.发病第9天CHIKV-IgM阳性;第3、5,7、9天CHIKV RNA阳性;RT-PCR扩增产物为575个核苷酸碱基片段,经序列测序结果与基孔肯雅病毒核酸序列同源性高达99%.患者经对症支持等治疗痊愈出院.结论 该患者为我国首例输入性基孔肯雅热确诊病例,本病临床表现与登革热相似.     

山东半岛地区贝类中诺如病毒污染状况与病毒鉴定初报

目的调查研究山东半岛沿海地区双壳贝类中诺如病毒污状况并对阳性株进行鉴定。方法应用常规RT-PCR与实时荧光定量RT-PCR,对2007年9月到2008年8月间在山东半岛沿海8个地区收集的186份双壳贝类样品进行了诺如病毒的定性和定量检测,并对阳性株核酸片段进行克隆、转化和序列分析,使用DNAStar软件进行核酸序列同源性比较,经MEGA4绘制系统进化树。结果 186份样品中检出5份诺如病毒阳性,检出率为2.67%,诺如病毒含量为大于102拷贝RNA,序列分析与遗传进化树显示5份诺如病毒阳性株均为GGII型NVs,与国内报道的GGII型毒株同源性达到98%以上。结论山东半岛地区贝类中含有的诺如病毒以GGII型为主,冬季检出率较高。     

南京都市圈肾综合征出血热疑似病例血清学检测及基因亚型分析北大核心CSCD

目的通过检测肾综合征出血热疑似患者血清标本中特异性抗体和病毒基因组RNA,了解其基因型别及序列特征。方法采用胶体金法、荧光定量PCR法、巢式PCR法进行样本检测,对巢式PCR扩增后的阳性产物进行双向测序获得M基因部分片段序列,与各亚型代表株对应序列进行同源性分析并构建系统进化树,确定其基因亚型。结果122份标本中,IgM阳性27份,阳性率22.1%,IgG阳性57份,阳性率46.7%;荧光定量PCR法检测汉坦病毒核酸阳性13份,阳性率10.7%;15份样本经巢式PCR扩增测序后获得目的片段,其中13株分别属于汉滩病毒H5、H6、H9亚型和汉城病毒S3亚型,另有两株病毒株已形成新的亚型。结论南京都市圈存在汉滩病毒和汉城病毒流行,其中汉滩病毒存在多种亚型。     

一起聚集性发热伴血小板减少综合征病原S基因分子进化分析

目的 对2020年4月南京鼓楼医院送检的一起疑似聚集性发热伴血小板减少综合征(SFTS)开展病原检测,对其S基因进行测序,分析基因分型和系统发育特征。方法 采用荧光定量PCR法对6例疑似患者血清标本进行新型布尼亚病毒(SFTSV)核酸定性检测, RT-PCR扩增SFTSV S基因片段并测序。分析核苷酸同源性,构建系统进化树。结果 6例血清样本中,SFTSV核酸阳性5例。5例核苷酸高度同源,其中4例S片段基因核苷酸序列同源性100%,1例与其他4例间同源性为99.7%;最大相似株为江苏2014年分离株JS2014-06、2015年分离株JS2015-26,同源性100%。5例均聚集在C2亚型分支上。结论 聚集性疫情SFTSV基因型为C2型,与近年来国内分离株亲缘关系近。需加强监测和宣教,关注病毒的进化与变异。     

深圳市一起输入性登革热疫情的分子流行病学研究

摘 要：目的 对2007年深圳市报告的一起输入性疑似登革热病例查明病因。分离鉴定病原体,从分子水平分析分离株的生物学特征。方法 对疑似患者血清标本采用ELISA、胶体金免疫层析法和实时荧光定量PCR方法分别检测登革病毒IgM、IgG抗体和病毒核酸,并用C6/36细胞分离登革病毒。采用RT-PCR方法扩增病毒PrM/M-E基因后进行序列测定,并与不同国家和地区的登革毒株进行同源性比较和进化树分析。结果 从患者血清标本中检测到登革病毒IgM、IgG抗体和登革3型病毒核酸,并首次成功分离到登革3病毒,将其命名为DEN3-SZ0739。深圳市登革3型病毒分离株SZ0739与登革3型国际标准株H87株、国内外流行株80-2、GWL-25株在PrM/M-E基因上核苷酸同源性分别为94.1%、93.9%、96.9%,而与登革1、2、4型国际标准株HAWAII、NGC、H241同源性分别为66.1%、59.1%、58.2%。进化树显示SZ0739株与2007年新加坡分离株SGEHI(D3)0235Y07亲缘关系最近,在进化树的同一分支上,和1416株(India 1984)、1326株(Sri Lanka 1981)、1696株(Samoa 1986)等同属基因Ⅲ亚型。结论 从病原学、血清学和分子生物学特征上均证实该输入病例是由登革3型病毒引起,该毒株最有可能来源于新加坡。     

一种汉滩病毒和汉城病毒双重实时荧光定量RT-PCR检测方法的建立

目的 建立一种汉滩病毒(Hantaan virus,HTNV)和汉城病毒(Seoul virus,SEOV)双重实时定量荧光RT-PCR检测方法。方法 根据HTNV和SEOV S基因设计引物和探针、优化反应条件,建立2种汉坦病毒的双重实时荧光定量RT-PCR方法。以甲型流感病毒、登革热病毒、新布尼亚病毒、寨卡病毒、新冠病毒阳性核酸为模板验证方法的特异性,将建立的方法与巢氏RT-PCR比较,确定方法对临床样本的适用性。结果 建立了一种HTNV和SEOV 双重实时定量荧光RT-PCR检测方法,该方法对2种型别病毒的最低检测限均为10 copies/μL,不同浓度标准品Ct值批内和批间差异均小于2%。与登革热病毒、新布尼亚病毒、寨卡病毒、新型冠状病毒、甲型流感病毒均无交叉反应。对10份肾综合征出血热急性期血清样本进行检测,结果9份为HTNV、1份为SEOV,与巢式RT-PCR方法结果一致。结论 建立的双重实时荧光定量RT-PCR方法可以快速对HTNV和SEOV 进行分型和定量检测,适用于肾综合征出血热临床早期诊断。     

中缅边境地区不明原因发热病人蚊媒病毒性疾病分子流行病学调查北大核心CSCD

目的了解中缅边境地区不明原因发热病人感染的蚊媒病毒种类,为当地蚊媒传染病防控提供科学依据。方法采用实时荧光RT-PCR法对2014-2015年在瑞丽市采集的1738份登革病毒NS1抗原检测阴性的不明原因发热病人血清进行Dengue virus(DENV)、Chikungunya virus(CHIKV)、Zika virus(ZIKV)、Sindbis virus(SINV)、Banna virus(BAV)、Batai virus(BATV)和Tahyna virus(TAHV)的检测,并将阳性标本接种于C6/36白纹伊蚊细胞进行病毒分离和鉴定,对阳性血清或新分离病毒株提取病毒RNA进行E基因RT-PCR扩增,测序后进行同源性和进化分析。结果从2015年采集的血清标本中检出1份ZIKV阳性,17份DENV阳性(6份DENV-1,6份DENV-2,未分型5份),从2014年采集的标本中检出1份DENV-1阳性。病毒分离得到1株ZIKV,2株DENV-1和2株DENV-2。对新分离ZIKV病毒株进行E基因测序,属于亚洲基因型,与2019年输入云南的缅甸株(2019YNZIKV02)进化关系较近。对2015年DENV阳性血清PCR产物测序,获得3条DENV-1E基因序列(本地病例),均属于基因1型,与2015年缅甸输入株进化关系较近;3条DENV-2均属于亚洲基因型(Asian Genotype),其中2条来源于本地病例序列和1条缅甸病例序列均与2015年缅甸输入的DENV-2亲缘关系较近。结论从中缅边境地区不明原因发热病人血清中检测和分离到ZIKV,并发现登革热漏检病例,提示亟待提高当地临床医生对寨卡病毒病的诊断意识并积极开展寨卡病毒病的常规监测,对疑似登革热病人需采用抗原和DENV、ZIKV病毒核酸检测方法联检以避免漏检。     

中缅边境地区不明原因发热病人蚊媒病毒性疾病分子流行病学调查

目的了解中缅边境地区不明原因发热病人感染的蚊媒病毒种类,为当地蚊媒传染病防控提供科学依据。方法采用实时荧光RT-PCR法对2014-2015年在瑞丽市采集的1 738份登革病毒NS1抗原检测阴性的不明原因发热病人血清进行Dengue virus(DENV)、Chikungunya virus(CHIKV)、Zika virus(ZIKV)、Sindbis virus(SINV)、Banna virus(BAV)、Batai virus(BATV)和Tahyna virus(TAHV)的检测,并将阳性标本接种于C6/36白纹伊蚊细胞进行病毒分离和鉴定,对阳性血清或新分离病毒株提取病毒RNA进行E基因RT-PCR扩增,测序后进行同源性和进化分析。结果从2015年采集的血清标本中检出1份ZIKV阳性,17份DENV阳性(6份DENV-1,6份DENV-2,未分型5份),从2014年采集的标本中检出1份DENV-1阳性。病毒分离得到1株ZIKV,2株DENV-1和2株DENV-2。对新分离ZIKV病毒株进行E基因测序,属于亚洲基因型,与2019年输入云南的缅甸株(2019YNZIKV02)进化关系较近。对2015年DENV阳性血清PCR产物测序,获得3条DENV-1 E基因序列(本地病例),均属于基因1型,与2015年缅甸输入株进化关系较近;3条DENV-2均属于亚洲基因型(Asian Genotype),其中2条来源于本地病例序列和1条缅甸病例序列均与2015年缅甸输入的DENV-2亲缘关系较近。结论从中缅边境地区不明原因发热病人血清中检测和分离到ZIKV,并发现登革热漏检病例,提示亟待提高当地临床医生对寨卡病毒病的诊断意识并积极开展寨卡病毒病的常规监测,对疑似登革热病人需采用抗原和DENV、ZIKV病毒核酸检测方法联检以避免漏检。     

寨卡病毒病研究进展

寨卡病毒病是由寨卡病毒引起的一种自限性急性传染病。寨卡病毒主要通过埃及伊蚊和白纹伊蚊叮咬传播,有证据表明也可通过性传播和母婴传播。临床表现主要为皮疹、发热、关节痛或结膜炎等非特异性症状,但寨卡病毒感染与新生儿小头畸形、格林-巴利综合征等存在密切关系。实验室检测方法包括实时荧光定量PCR检测病毒核酸和ELISA检测Ig M抗体。该疾病目前无有效的抗病毒药物和疫苗。预防措施主要为预防蚊虫叮咬和采取虫媒控制措施。     

Introduction of Zika virus in Bangladesh: An impending public health threat

Objective: To explore the presence of Zika virus(ZIKV) in Bangladesh and to understand the associated risk factors.Methods: A retrospective sero-surveillance was performed on stored serum samples of dengue surveillance conducted from 2013 to 2016. Real time RT-PCR was performed on randomly selected acute serum samples to detect the Zika virus nucleic acid.Results: Of 200 samples screened, one was found positive for ZIKV by real time RTPCR and further confirmed by genome sequencing. The case was a 65 years old male from a metropolitan city of Bangladesh who had no history of travel outside Bangladesh.Phylogenetic analysis of partial E gene sequences from Bangladeshi isolates demonstrated a close relationship with ZIKV from Brazil and current South American strains clustering within a monophyletic clade distinct from African lineage.Conclusions: Presence of ZIKV raises serious public health concerns in Bangladesh owing to its association with congenital anomalies/neurological-manifestations. We,therefore, recommend every suspected viral fever patient, particularly pregnant women be screened for ZIKV infection to rule out yet another emerging infection in Bangladesh.     

Update on the Transmission of Zika Virus Through Breast Milk and Breastfeeding: A Systematic Review of the Evidence

We systematically searched regional and international databases and screened 1658 non-duplicate records describing women with suspected or confirmed ZIKV infection, intending to breastfeed or give breast milk to an infant to examine the potential of mother-to-child transmission of Zika virus (ZIKV) through breast milk or breastfeeding-related practices. Fourteen studies met our inclusion criteria and inform this analysis. These studies reported on 97 mother–children pairs who provided breast milk for ZIKV assessment. Seventeen breast milk samples from different women were found positive for ZIKV via RT-PCR, and ZIKV replication was found in cell cultures from five out of seven breast milk samples from different women. Only three out of six infants who had ZIKV infection were breastfed, no evidence of clinical complications was found to be associated with ZIKV RNA in breast milk. This review updates our previous report by including 12 new articles, in which we found no evidence of ZIKV mother-to-child transmission through breast milk intake or breastfeeding. As the certainty of the present evidence is low, additional studies are still warranted to determine if ZIKV can be transmitted through breastfeeding.     

Lack of Association between Adverse Pregnancy Outcomes and Zika Antibodies among Pregnant Women in Thailand between 1997 and 2015

Data about Zika virus infection and adverse pregnancy outcomes in Southeast Asia are scarce. We conducted an unmatched case-control study of Zika virus (ZIKV) serology in pregnant women enrolled in human immunodeficiency virus (HIV) or hepatitis B virus (HBV) perinatal prevention trials between 1997 and 2015 in Thailand. Case and control groups included women with and without adverse pregnancy outcomes. Plasma samples collected during the last trimester of pregnancy were tested for ZIKV IgG/IgM and Dengue IgG/IgM (Euroimmun, AG, Germany). Case newborn plasma samples were tested for ZIKV IgM and ZIKV RNA (Viasure, Spain). The case group included women with stillbirth (n = 22) or whose infants had microcephaly (n = 4), a head circumference below the first percentile (n = 14), neurological disorders (n = 36), or had died within 10 days after birth (n = 11). No women in the case group were positive for ZIKV IgM, and none of their live-born neonates were positive for ZIKV IgM or ZIKV RNA. The overall ZIKV IgG prevalence was 29%, 24% in the case and 34% in the control groups (Fisher’s exact test; p = 0.13), while the dengue IgG seroprevalence was 90%. Neither neonatal ZIKV infections nor ZIKV-related adverse pregnancy outcomes were observed in these women with HIV and/or HBV during the 18-year study period.     

Zika virus spreading in South America: evolutionary analysis of emerging neutralizing resistant Phe279Ser strains

Objective: To investigate the genetic diversity of Zika virus(ZIKV) and the relationships existing among these circulating viruses worldwide. To evaluate the genetic polymorphisms harboured from ZIKV that can have an influence on the virus circulation. Methods: Three different ZIKV dataset were built. The first dataset included 63 E gene sequences, the second one 22 NS3 sequences and the third dataset was composed of 108 NS5 gene sequences. Phylogenetic and selective pressure analysis was performed. The edited nucleic acid alignment from the Envelope dataset was used to generate a conceptual translation to the corresponding peptide sequences through UGene software. Results: The phylogeographic reconstruction was able to discriminate unambiguously that the Brazilian strains are belonged to the Asian lineage. The structural analysis reveals instead the presence of the Ser residue in the Brazilian sequences(however already observed in other previously reported ZIKV infections) that could suggest the presence of a neutralization-resistant population of viruses. Conclusions: Phylogenetic, evolutionary and selective pressure analysis contributed to improve the knowledge on the circulation of ZIKV.     

Zika Virus Antibody Titers Three Years after Confirmed Infection

Background: In 2015–2016, a large Zika virus (ZIKV) outbreak occurred in the Americas. Although the exact ZIKV antibody kinetics after infection are unknown, recent evidence indicates the rapid waning of ZIKV antibodies in humans. Therefore, we aimed to determine the levels of ZIKV antibodies more than three years after a ZIKV infection. Methods: We performed ZIKV virus neutralization tests (VNT) and a commercial ZIKV non-structural protein 1 (NS1) IgG ELISA in a cohort of 49 participants from Suriname who had a polymerase-chain-reaction-confirmed ZIKV infection more than three years ago. Furthermore, we determined the presence of antibodies against multiple dengue virus (DENV) antigens. Results: The ZIKV seroprevalence in this cohort, assessed with ZIKV VNT and ZIKV NS1 IgG ELISA, was 59.2% and 63.3%, respectively. There was, however, no correlation between these two tests. Furthermore, we did not find evidence of a potential negative influence of DENV immunity on ZIKV antibody titers. Conclusions: ZIKV seroprevalence, assessed with two commonly used serological tests, was lower than expected in this cohort of participants who had a confirmed previous ZIKV infection. This can have implications for future ZIKV seroprevalence studies and possibly for the duration of immunological protection after a ZIKV infection.     

Real-time PCR analysis of dog cerebrospinal fluid and saliva samples for ante-mortem diagnosis of rabies

The use of a 10-day observation to determine whether a dog is rabid is standard practice. This study was conducted in order to look for evidence of rabies vius in saliva and cerebrospinal fluid (CSF) of suspected live rabid dogs at the time of quarantine by using a SYBR Green real-time RT-PCR based assay for the detection of rabies virus RNA. Saliva and CSF of dogs were collected once on the day of admission for the 10-day quarantine. All test dogs were or became ill and died of rabies within the observation period. Thirteen of 15 dogs (87%) had saliva samples that were positive for rabies RNA. Two dogs with furious rabies had negative saliva samples. Positive CSF samples were found in 4 of 15 dogs (27%) whose saliva samples were positive. The time from sample collection to result was less than 5 hours. Because virus may be absent or present at very low level in both clinical fluids, samples taken for ante-mortem diagnosis cannot definitively rule out rabies.     

一例暴露因素不明的狂犬病病例的分子流行病学溯源

目的 对一例暴露因素不明的人狂犬病病例进行实验室确诊,通过分子流行病学分析探索其可能的感染来源。方法 通过对存活疑似人狂犬病病例的唾液、脑脊液、血清标本采用直接免疫荧光试验、反转录-聚合酶链式反应和快速荧光灶抑制试验进行实验室确诊;通过时空进化分析探索该病例可能的感染来源。结果 病例唾液标本直接免疫荧光试验结果呈阳性,病例血清标本经快速荧光灶抑制试验检测抗狂犬病病毒中和抗体呈阳性。病例唾液标本经反转录-聚合酶链式反应获得狂犬病病毒核蛋白基因预期扩增片段,序列测定进一步证实为狂犬病病毒。该狂犬病病毒株与2011年安徽省一株犬源狂犬病病毒株核苷酸序列同源性最高,为98.4%。结论 该疑似狂犬病病例可确诊为狂犬病病例。其发病源于至少2011年以来的某次不自觉的狂犬病病毒感染。