2015-2016年深圳市登革Ⅲ、Ⅳ型分离病毒株E/NS1基因序列特征

目的 了解2015-2016年深圳市分离的登革Ⅲ、Ⅳ型病毒E/NS1基因序列特征及登革Ⅲ、Ⅳ型病毒流行规律,探究其可能的传播来源。方法 收集2015-2016年深圳市登革热患者病例资料及急性期血清,用C6/36细胞培养分离登革病毒,用FQ-PCR对其进行血清分型,分离成功的病毒株用反转录-聚合酶链反应(PCR)方法扩增E基因和NS1基因,进行序列分析,绘制系统进化树,氨基酸比对分析4个不同血清型NS1蛋白。结果 从5份Ⅲ型的登革病毒标本中成功分离4份病毒株,3份Ⅳ型登革病毒标本中成功分离2份登革病毒;BLAST分析保守E基因结果表明,与DENV-3分离株同源性最高(99%)的毒株主要是印度尼西亚2010和2015年分离株、菲律宾2015年分离株。与DENV-4分离株同源性最高(99%)的毒株主要是菲律宾2013年分离株、印度尼西亚2010分离株、意大利2009年分离株。NS1基因序列分析显示所选4株不同血清型的病毒株氨基酸相似性为77.69%,4个不同血清型的登革热NS1抗原存在5-7个氨基酸保守区域。结论 2015-2016年深圳市输入的登革Ⅲ、Ⅳ型病毒可能来自印度尼西亚和菲律宾,应加强出入境人员的监测工作,避免输入引起本地感染的风险。     

广州市2018年输入性登革病毒4型全基因组序列特征分析

目的 本文对广州市2018年输入性DENV-4型病毒的生物学来源进行分析,为本地登革热的防控提供科学依据并累积基线数据。方法 通过传染病监测网络收集广州市2018年输入性DENV-4病例, 对急性期患者血清进行病毒分离培养并对分离到的DENV-4毒株进行全基因组测序、系统发生树重建和生物信息学分析。结果 共分离得到5株DENV-4毒株,其中4株由泰国和柬埔寨输入的分离株均属于DENV-4基因Ⅰ型;菲律宾输入的病例分离株与其他4株亲缘性较低,属于DENV-4基因Ⅱa型;5株分离株间的核苷酸(氨基酸)同源性为90.8%(96.7%~96.9%);经过比对,5株分离株和6株参考株的E蛋白区域有9个位点出现差异,NS1蛋白区域有14个位点出现差异,NS5蛋白区域有25个位点出现差异。结论 本研究对广州地区输入性DENV-4毒株进行全基因组测定,间接地掌握周边国家地区DENV-4的基因特征,为本地登革热的防控和溯源提供基线数据,具有重要的公共卫生意义。     

重庆市库蚊标本中宜昌病毒的分离鉴定

目的 对2017年采集自重庆地区蚊虫标本携带的病毒进行分离鉴定。方法 采用组织细胞培养法进行病毒分离,应用高通量测序技术获得病毒分离物的全基因组序列,采用系统发育法进行种类及分子特征分析。结果 库蚊标本来源的病毒阳性分离物 CQFD2017能引起白纹伊蚊细胞C6/36病变,但不能引起金黄地鼠肾细胞BHK-21、非洲绿猴肾细胞Vero病变。 CQFD2017全基因组序列长度为20 893 bp, 包含6个开放阅读框。系统进化分析发现,该毒株位于Nidovirales病毒目Mesoniviridae病毒科的Yichang病毒所在的进化分支,与Yichang病毒(NC_040534)的核苷酸一致性为98.6%,ORF1a和ORF1b区氨基酸一致性分别为99.06%和99.15%,因此将该毒株命名为Yichang病毒CQFD2017株。结论 2017年重庆市库蚊标本分离的CQFD2017株为Yichang病毒。     

福建省2015年本地登革热病例的病原学特征

目的 分析2015年福建省本地登革热病例部分登革病毒流行株的基因组序列,调查相关病毒株之间的遗传联系,为福建省登革热防控提供病原学证据。方法 采集急性期患者血清,实时荧光RT-PCR检测登革病毒RNA,阳性者分离病毒,扩增病毒基因组并测序,以病毒全长编码区序列进行种系发生分析。结果 2015年福建省先后出现登革1型(DENV1)和登革2型病毒(DENV2)引起的本地暴发疫情,共发生4起聚集性病例,报告41例。分离到DENV1毒株9株,DENV2毒株8株。种系发生分析显示,9株DENV1高度相似,同属于基因1型(G1),提示其共同的输入来源可能为斯里兰卡;而8株DENV2病毒虽然同属于大都市基因型,却分为差异较大的两簇,表明莆田与福州两地疫情相关毒株的遗传关联度较低,可能分别来自马来西亚和印度。结论 2015年共出现4起聚集性本地登革热疫情,部分患者病毒分离株的病原学特征表明,福建省2015年本地登革热暴发疫情存在多个输入来源。     

2001-2016年广州市登革3型病毒E基因序列及系统进化树分析

目的 了解2001-2016年广州市登革3型病毒的流行情况,掌握毒株的进化情况和趋势。方法 将登革热确诊病例的血清用荧光PCR检测,阳性血清用C6/36细胞进行病毒分离,测定分离毒株的E基因序列,与NCBI的毒株序列相比较,利用Mega 4.0软件构建系统进化树。结果2001-2016年共分离到登革3型病毒24株,从基因型上分类属于基因亚型I、II、III和V型,基因亚型III在流行年份和分离毒株数上稍占优势。流行病学调查和序列分析均显示与东南亚国家流行的登革热相关度较高。结论 广州市登革3型病毒以输入为主,随着输入压力增大、基因亚型增多和转换,可能会使广州市登革热流行传播更为复杂,流行风险进一步增高。     

呼肠孤病毒空斑检测方法建立

目的 建立呼肠孤病毒空斑检测方法,为进一步研究该病毒奠定基础。方法 通过Western Blot、免疫荧光检测纳尔逊海湾正呼肠孤病毒Miyazaki病毒株感染L929细胞和BSR细胞情况,病毒RNA水平凝胶电泳验证Miyazaki病毒株核酸成分。建立呼肠孤病毒空斑检测方法,计算病毒滴度。结果 Miyazaki病毒株能成功感染L929细胞和BSR细胞,水平凝胶电泳验证病毒株核酸成分正确,病毒株感染的L929细胞出现空斑,计算得出病毒滴度结果为2.2×10⁸ PFU/mL。结论 呼肠孤病毒空斑检测方法成功建立,该方法可以形成清晰可见的空斑,能稳定地对病毒滴度进行定量测定,为进一步研究呼肠孤病毒提供帮助。     

广东首例苏里南输入性寨卡病毒感染病例的实验室检测

目的对从广州白云国际机场口岸入境的一例自苏里南归国发热人员进行寨卡病毒实验室检测。方法采集发热人员的血液和唾液样本,同时使用两种寨卡病毒实时荧光RT-PCR试剂进行寨卡病毒核酸检测。RT-PCR扩增寨卡病毒部分基因片段,并进行序列测定和同源性比较。基于扩增片段构建系统进化树,分析输入性病例的来源。结果实时荧光RT-PCR结果显示,该病例血清样本寨卡病毒核酸弱阳性、唾液样本寨卡病毒核酸阳性。RT-PCR扩增出预期大小约1.5Kb片段,测序结果表明该片段与巴西寨卡病毒株(GenBank号KX197250)相应序列的同源性为99%。系统进化树显示该病毒属亚洲谱系。结论根据患者流行病学史、临床表现和病例标本核酸检测情况,确诊该病例为广东首例从苏里南地区输入性寨卡病例。     

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。     

2016年福建省B型流感病毒基因特征分析

目的 分析2016年福建省B型流感病毒(Influenza Virus)血凝素(Hemagglutinin,HA)和神经氨酸酶(Neuramidinase,NA)的基因变异。方法 从中国疾病预防控制信息系统采集日期2016.1.1至2016.12.31的福建省流感监测数据。毒株来源2016年福建省流感网络监测流感样病例,提取病毒(犬肾传代细胞或鸡胚培养分离)核酸进行RT-PCR扩增HA和NA基因片段并测序,MEGA5.0分析基因特征。结果 测序51株B型流感病毒HA、NA片段,与疫苗株核苷酸同源性在97.8%~100%。 研究发现2株重配株(BY-NA/BV-NA)、耐药株1株、Victoria系HA片段新增一个糖基化位点(197),酶活性中心和周围的相关位点氨基酸仍保持高度保守。结论 2016年福建省B型流感毒株少数发生变异与疫苗株不匹配。     

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.     

Comparison of biological properties of St. Louis encephalitis and Rio bravo viruses

St. Louis encephalitis (SLE) virus, an arbovirus, and Rio Bravo (RB) virus, a non-arthropod-borne virus, are flaviviruses which cross-react in neutralization tests. Several of their biological properties were compared. Viral growth curves revealed that greater than 99% of infectious SLE (Parton) virus remained cell-associated in Vero cells but was released slowly into the medium of infected BHK-21 cells. In contrast, RB (M-64) virus was released upon maturation into the fluids of Vero and BHK-21 cell cultures. Fortyfold more SLE virus than RB virus adsorbed to monolayer cultures of Aedes dorsalis cells. SLE but not RB virus replicated and reached high titers (10(8) pfu/ml) in Ae. dorsalis cell cultures maintained in media which were expected to enhance recovery of RB virus. Further, SLE but not RB virus replicated in cultured bat epithelial cells and primary duck embryo cells incubated at 42 degrees C. Clearly, the prototype strains of RB and SLE viruses are distinct viruses. Both the classification of RB virus in the genus flavivirus and the antigenic relationship between RB and SLE viruses require further clarification.     

Clinical and laboratory findings of acute Zika virus infection in patients from Salvador during the first Brazilian epidemic

Several major epidemics of Zika fever, caused by the ZIKA virus (ZIKV), have emerged in Brazil since early 2015, eventually spreading to other countries on the South American continent. The present study describes the clinical manifestations and laboratory findings of patients with confirmed acute ZIKV infection during the first epidemic that occurred in Salvador, Brazil. All included patients were seen at the emergency room of a private tertiary hospital located in Salvador, Brazil from 2015 through 2017. Patients were considered eligible if signs of systemic viral febrile disease were present. All individuals were tested for ZIKV and Chikungunya infection using PCR, while rapid test was used to detect Dengue virus antibodies or, alternatively, the NS1 antigen. A diagnosis of acute ZIKV infection was confirmed in 78/434 (18%) individuals with systemic viral febrile illness. Positivity was mainly observed in blood, followed by saliva and urine. Coinfection with Chikungunya and/or Dengue virus was detected in 5% of the ZIKV-infected patients. The most frequent clinical findings were myalgia, arthralgia and low-grade fever. Laboratory analysis demonstrated normal levels of hematocrit, platelets and liver enzymes. In summary, in acute settings where molecular testing remains unavailable, clinicians face difficulties to confirm the diagnosis of ZIKV infection, as they rely only on clinical examinations and conventional laboratory tests.     

Can Zika Virus Infection in High Risk Pregnant Women Be Differentiated on the Basis of Symptoms?

Zika virus (ZIKV) infection in pregnancy is associated with congenital neurological abnormalities. Our understanding of the full clinical spectrum of ZIKV infection is incomplete. Using data from this prospective cohort study consisting of 650 women attending a high-risk pregnancy clinic during the Zika virus outbreak in Brazil, we investigated the extent to which specific symptoms can be utilized to differentiate ZIKV-infected pregnant women from those with other pregnancy-related problems. All were tested for ZIKV in urine by RT–qPCR. Demographic and clinical data including physical symptoms during follow-up were recorded and analyzed with respect to Zika virus exposure status. Forty-eight (7.4%) women were positive for ZIKV by RT–qPCR. The majority (70.8%) were asymptomatic, and only four ZIKV-positive women (8.3%) reported symptoms during pregnancy that met the WHO case definition. Zika-positive and -negative women reported similar frequencies of ZIKV-like symptoms (as per the WHO definition): fever (16.7% vs. 13.6%), arthralgia/arthritis (10.4% vs. 11.3%), rash (4.2% vs. 5.3%), and conjunctivitis (2.1% vs. 3.2%). Most pregnant women positive for ZIKV in urine are asymptomatic and do not deliver a baby with microcephaly. Physical symptoms alone did not differentiate between high-risk pregnant women positive or negative for ZIKV.     

Inhibition of Brazilian ZIKV strain replication in primary human placental chorionic cells and cervical cells treated with nitazoxanide

Zika virus (ZIKV) infection during pregnancy is associated with a congenital syndrome. Although the virus can be detected in human placental tissue and sexual transmission has been verified, it is not clear how the virus reaches the fetus. Despite the emerging severity caused by ZIKV infection, no specific prophylactic and/or therapeutic treatment is available. The aim of the present study was to evaluate the effectiveness antiviral of nitazoxanide (NTZ) in two important congenital transmission targets: (i) a primary culture of human placental chorionic cells, and (ii) human cervical epithelial cells (C33-A) infected with Brazilian ZIKV strain. Initially, NTZ activity was screened in ZIKV infected Vero cells under different treatment regimens with non-toxic drug concentrations for 48 h. Antiviral effect was found only when the treatment was carried out after the viral inoculum. A strong effect against the dengue virus serotype 2 (DENV-2) was also observed suggesting the possibility of treating other Flaviviruses. Additionally, it was shown that the treatment did not reduce the production of infectious viruses in insect cells (C6/36) infected with ZIKV, indicating that the activity of this drug is also related to host factors. Importantly, we demonstrated that NTZ treatment in chorionic and cervical cells caused a reduction of infected cells in a dose-dependent manner and decreased viral loads in up to 2 logs. Pre-clinical in vitro testing evidenced excellent therapeutic response of infected chorionic and cervical cells and point to future NTZ activity investigation in ZIKV congenital transmission models with the perspective of possible repurposing of NTZ to treat Zika fever, especially in pregnant women.     

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.     

Alternate paradigms on Zika virus-related complications: An analytical review

The proportion of the reported cases of Zika virus(ZIKV) infection reached the status of a pandemic. Numerous studies are being conducted on the isolation of ZIKV strains from various epidemics, diagnosis of the infections, various animal models and cell culture designs to study the pathogenesis of ZIKV in the attempts to find an effective ZIKV vaccine. This review focuses upon the 'Off-Spectrum' body of studies which analyses the epidemiology, pathogenesis and other attributes of ZIKV in the light of various dissident hypotheses.     

Biological Characteristics and Patterns of Codon Usage Evolution for the African Genotype Zika Virus

We investigated temporal trends of codon usage changes for different host species to determine their importance in Zika virus (ZIKV) evolution. Viral spillover resulting from the potential of codon adaptation to host genome was also assessed for the African genotype ZIKV in comparison to the Asian genotype. To improve our understanding on its zoonotic maintenance, we evaluated in vitro the biological properties of the African genotype ZIKV in vertebrate and mosquito cell lines. Analyses were performed in comparison to Yellow fever virus (YFV). Despite significantly lower codon adaptation index trends than YFV, ZIKV showed evident codon adaptation to vertebrate hosts, particularly for the green African monkey Chlorocebus aethiops. PCA and CAI analyses at the individual ZIKV gene level for both human and Aedes aegypti indicated a clear distinction between the two genotypes. African ZIKV isolates showed higher virulence in mosquito cells than in vertebrate cells. Their higher replication in mosquito cells than African YFV confirmed the role of mosquitoes in the natural maintenance of the African genotype ZIKV. An analysis of individual strain growth characteristics indicated that the widely used reference strain MR766 replicates poorly in comparison to African ZIKV isolates. The recombinant African Zika virus strain ArD128000*^E/NS5 may be a good model to include in studies on the mechanism of host tropism, as it cannot replicate in the tested vertebrate cell line.