福建省莆田市2007-2015年登革病毒E基因序列分析

目的 测定登革病毒E基因序列,探讨病毒传播来源及基因型.方法 收集福建省莆田市2007-2015年登革热患者血清样品分离的毒株12份,用RT-PCR法扩增登革病毒E基因测定基因序列,并绘制系统发育树.结果 莆田市登革热4次暴发的病例血清型别均为DENV-2型Cosmopolitan基因型.BLAST分析表明,几次暴发的毒株与东南亚国家或广东有较高的一致性,相似度达99％.结论 结合BLAST和系统发育树分析推测,莆田市2007-2015年DENV-2可能由东南亚国家或广东省输入,应加强出入境人员的疾病监测.     

2014—2019年东莞市Ⅰ型登革病毒E基因进化特征分析

目的了解东莞市2014—2019年登革热流行情况和血清型Ⅰ型登革病毒(DENV-1)基因型及E基因特征。方法收集2014—2019年东莞市各镇街医院上送的经临床诊断为疑似登革热病例血清样本共962份,其中的729份样本应用酶联免疫吸附试验(ELISA)进行登革IgM抗体检测,全部962份血清样本采用实时荧光PCR进行检测,核酸阳性样本中的血清型Ⅰ型样本用细胞培养方法进行病毒分离鉴定,并对其E基因序列进行测序及系统进化树分析。结果2014—2019年东莞市登革热7—11月为流行期,9—10月为发病高峰。检出IgM抗体阳性样本261份,阳性率35.90%。962份急性期血清中登革病毒核酸阳性362份,阳性率37.6%,其中DENV-1阳性311份,占85.9%。分离获得35株DENV-1毒株,E基因序列比对和系统进化分析显示,35株DENV-1分离株间核苷酸相似度为89.8%~100.0%,分属基因Ⅰ型、Ⅱ型和Ⅴ型,分别为29、1和5株。所有35株分离株与广州、浙江等地及越南、菲律宾、印度等东南亚国家当年或往年流行株同源性较高。结论2014—2019年东莞市流行的DENV-1优势型别为基因Ⅰ型。流行方式可能为广州等周边城市及东南亚国家输入病例引起的本地暴发流行。须警惕基因Ⅴ型DENV-1输入引起登革热暴发流行。     

重庆口岸输入性登革热病例的分子流行病学调查

目的对2014年重庆口岸首起输入性登革热疫情进行分子流行病学调查,确定病毒的基因型别及感染来源。方法采集2014年重庆口岸首起聚集性输入登革热疑似病例血清样本,进行IgM/IgG抗体检测、核酸检测(实时荧光RTPCR)以及病毒分离;用RT-PCR方法扩增病毒分离株E基因,进行核苷酸序列测定和进化分析。结果共采集21份血清样本,其中4份样本IgM抗体阳性,阳性率为19%,IgG均阴性;9份样本为登革热病毒1型核酸阳性。分离得到4株登革病毒,4株病毒的E基因序列同源性为100%,与印度尼西亚的毒株进化关系最近,同源性为99.1%。结论输入性登革病毒与印度尼西亚的登革病毒1型亲缘关系最近,提示其传染源极有可能在印度尼西亚。     

2013-2016年广东省登革病毒血清I型分布特征

目的 对2013-2016年广东的登革病毒血清I型序列进行分子流行病学分析,研究血清I型登革病毒在广东省的流行特征。方法 分离获得2013-2016年登革病毒血清I型序列,建立全球登革病毒血清I最大似然树,预测第2簇病毒的进化速率、共祖位置;使用PAML软件计算选择压力。结果 2013-2016年的广东序列分别聚类在11簇分支中,其中8簇为基因I型毒株、1簇基因IV型毒株、2簇基因V型毒株,且与东南亚国家分离的序列聚类在一起。第2簇病毒表现出一定的独特性,最大置信树以及时空动态图显示这簇株系最早是由泰国传入广州,之后又在广东地区流行。第2簇病毒的相对进化速率要比文献报道的DENV-1基因I型的进化速率要大,但该簇病毒的序列不存在正选择压力。结论 广东地区流行的登革病毒主要是从东南亚国家输入,同时发现一簇疑似本地化的株系,具有部分本地化的特征,包括:跨年传播、独立成簇,但还需要更多的证据去判定登革热是否已形成本地化。     

广州市2019年登革热确诊病例空间聚集性及登革病毒E基因进化特征分析

目的 分析2019年广州市登革热流行情况,评估登革病毒4种血清型对流行的影响。方法 在传染病报告信息管理系统中收集2019年广州市登革热确诊病例信息,使用ArcGIS 10.2软件进行空间自相关性和聚集性分析,使用荧光定量PCR对血清标本进行核酸检测,将结果为阳性的血清标本进行病毒分离并测定E基因序列,用PhyML 3.1软件绘制基因进化树并分析。结果 2019年广州市共报告登革热确诊病例1 655例,发病率11.10/10万,本地病例1 382例,输入病例273例,发病具有空间聚集性,发现18个高-高聚集性街道,输入病例来源以东南亚国家（86.08%,235/273）和非洲国家（2.56%,7/273）为主。荧光定量PCR检测确诊病例血清标本749例,阳性率93.06%（697/749）,分离毒株464株。同往年基因树相比,登革病毒未发现基因型的转换。登革病毒血清型1型仍然是广州市的优势毒株,血清型2型主要在白云区和荔湾区流行。结论 2019年广州市登革热疫情累及全市,范围向城乡接合部扩大和转移,应进一步重视城乡接合部的防控工作。加强来自东南亚和非洲国家的国境检疫。登革病毒血清型2型的流行和聚集性暴发风险不容忽视。多种血清型在广州市同时出现,提示需预防重症登革热的暴发和流行。     

广州市2010-2019年4型登革热病例流行特征及其病毒E基因

  目的  了解2010-2019年广州市4型登革热病例的流行病学特征和4型登革病毒的分子生物学特征。  方法  2010-2019年,收集广州市登革热病例相关资料及血清标本,使用RT-PCR法检测血清标本并分型,对4型登革热病例标本进行登革病毒包膜(envelope, E)蛋白基因序列测定,并进行进化树构建及基因重组分析。  结果  2010-2019年,广州市共报告43 174例登革热病例,其中23例为4型登革热病例。4型登革热病例仅分布在5个区,曾在2010年出现本地流行。广州市的4型登革热输入病例主要来自东南亚国家,病毒E基因序列与东南亚国家的序列有较高相似性,病毒基因型归属于印度尼西亚基因型和东南亚基因型,未检测到基因重组。  结论  应加强对广州市4型登革热病例流行状况和输入病例的监测和关注,并进一步加强对东南亚回国人员登革热常识和个人防护的健康宣传。某些区需要预防出现4型登革病毒的扩散和蔓延。登革病毒基因型转换并未引起登革热在广州市流行强度的变化,有待进一步的观察和研究其是否会发生重组。     

广州市2009-2010年DENV-3型病毒分子生物学特征分析

目的探讨广州市DENV-3型病毒的基因变异及传播模式,为登革热疫情防控提供科学建议。方法对2009-2010年采集的疑似登革热病例早期血标本进行检测RT-PCR,阳性标本血清接种至C6/36细胞进行病毒分离培养。对登革病毒E基因进行测序,并与基因库中全球和中国其他省份DENV-3代表病毒株进行比对,对广州病毒分离株的来源和同源性进行分析。结果共分离获得13份DENV-3型病毒样本(2009年7份,2010年6份),基因树系谱分析显示DENV-3型病毒包括3个基因型(Ⅰ,Ⅲ,Ⅴ),其中基因Ⅰ型来源于印度尼西亚,基因Ⅲ型的2个节点族中,A节点族来源于科特迪瓦,B节点族来源于坦桑尼亚,基因型Ⅴ来源于菲律宾。2009年和2010年病毒株E基因核酸分别存在1.3%~9.0%和0.5%~3.9%的差异。结论 2010年分离病毒株并非2009年的延续,不同基因型的DENV-3型病毒可能是通过不同的地理路径传入。     

广州市2011年登革病毒流行状况及E基因进化特征分析

目的 了解2011年广州市登革热的流行情况,分析新分离毒株的E基因分子特征.方法 收集2011年广州市登革热的流行病学资料和血清标本.采用荧光定量PCR检测并确定血清型,C6/36细胞进行病毒分离,测定新分离毒株的E基因序列,利用Mega 4.0软件分析.结果 2011年广州市登革热发病高峰在9-11月.在患者血清中检测出登革热病毒1、2、4型,分离出5株登革热1型毒株.在基因型上,4株属于亚洲型,1株属于美洲/非洲型.结论 广州市登革病毒与东南亚地区的毒株有较高同源性,且存在登革热暴发的潜在风险,该病毒在广州市可能已出现本地化趋势.     

2013年广州市登革热流行病学特征及病毒E基因进化特征分析

目的了解登革热流行病学特征,分析分离毒株E基因分子进化特征。方法采用描述性流行病学方法分析2013年广州市登革热疫情,采用酶联免疫法(ELISA)对登革热疑似病例血清进行抗体检测,阳性病例的急性期血清标本以C6/36细胞进行病毒分离培养;采用RT-PCR扩增分离毒株的E基因,并对扩增产物进行序列分析,应用MEGA 5.05进行进化特征分析。结果 2013年广州市累计报告登革热确诊病例1 270例,发病率为9.96/10万,以本地病例为主(占98.66%),输入病例以东南亚国家为主(占88.24%)。发病高峰为10-11月(占85.28%)。169份登革热病例急性期血清共分离48株登革病毒(Ⅰ型47株,Ⅱ型1株),与近年来广州、东南亚分离株高度同源。结论广州市登革热发病率呈上升趋势,暴发风险较大,须加强监测力度,强化蚊媒的控制,降低登革热传播风险。     

河北省1例输入性登革热病例的病毒分离及其分子特征分析

目的从疑似登革热病例血清中分离病毒,获取全基因组数据并分析分子进化特征。方法采集河北省1例由柬埔寨输入的登革热疑似病例的血清样本,用胶体金法检测登革病毒IgM和IgG抗体以及NS1抗原;利用Vero E6细胞从血清中分离登革病毒,并采用Real-time PCR对病毒进行分型;扩增病毒全基因片段,将序列与国内外分离株序列进行比对,构建系统发生树。结果胶体金检测显示该病例血清登革病毒IgM、IgG抗体和NS1抗原均为阳性,从早期血清中分离到登革病毒,鉴定为I型登革病毒,分子进化分析揭示该分离株与东南亚地区毒株最为相似,病毒全基因组和E基因核苷酸序列同源性最高达99.99%,而与近年国内其他地区分离株进化关系较远。结论国内登革热的防控依旧需要加强输入性病例的监测。     

Phylogenetic analysis of dengue virus types 1 and 3 isolated in Jakarta,Indonesia in 1988

Dengue viruses are mosquito-borne viruses that cause dengue fever and dengue hemorrhagic fever, both of which are globally important diseases. These viruses have evolved in a transmission cycle between human hosts and mosquito vectors in various tropical and subtropical environments. We previously isolated three strains of dengue type 1 virus (DENV1) and 14 strains of dengue type 3 virus (DENV3) during an outbreak of dengue fever and dengue hemorrhagic fever in Jakarta, Indonesia in 1988. Here, we compared the nucleotide sequences of the entire envelope protein-coding region among these strains. The isolates were 97.6–100% identical for DENV1 and 98.8–100% identical for DENV3. All DENV1 isolates were included in two different clades of genotype IV and all DENV3 isolates were included in a single clade of genotype I. For DENV1, three Yap Island strains isolated in 2004 were the only strains closely related to the present isolates; the recently circulated Indonesian strains were in different clades. Molecular clock analyses estimated that ancestors of the genotype IV strains of DENV1 have been indigenous in Indonesia since 1948. We predict that they diverged frequently around 1967 and that their offspring distributed to Southeast Asia, the Western Pacific, and Africa. For DENV3, the clade containing all the present isolates also contained strains isolated from other Indonesian regions and other countries including Malaysia, Singapore, China, and East Timor from 1985–2010. Molecular clock analyses estimated that the common ancestor of the genotype I strains of DENV3 emerged in Indonesia around 1967 and diverged frequently until 1980, and that their offspring distributed mainly in Southeast Asia. The first dengue outbreak in 1968 and subsequent outbreaks in Indonesia might have influenced the divergence and distribution of the DENV1 genotype IV strains and the DENV3 genotype I strains in many countries.     

Detection of Dengue viruses among febrile patients in Lagos,Nigeria and phylogenetics of circulating Dengue serotypes in Africa

Dengue fever, a mosquito borne viral disease, is caused by Dengue virus. This virus and its vector is endemic in most tropical countries including Nigeria. Dengue presents with febrile symptoms and is a major cause of morbidity and mortality in affected countries. The infection presently has no licensed drugs and vaccine is only available for previously exposed individuals. Despite the endemicity of Dengue in Nigeria, very few studies have identified circulating Dengue genotypes in the country. There is also sparse information on the occurrence, distribution and temporal patterns of circulating dengue virus serotypes as well as genotypes in Africa. This situation creates barriers to effective control of the infection in the continent.This study identified Dengue serotypes and genotypes among febrile patients in two health centers in Lagos, Nigeria. Phylogenetic analysis of Dengue sequences previously collected from African countries and submitted to GenBank database from 1944 till date was also performed. One hundred and thirty febrile persons were recruited for the study between April and August 2018. Eleven (8.5%) persons were Dengue virus positive. Dengue virus serotypes 1 (genotype I) and 3 (genotype I) were identified as actively circulating in Lagos, Nigeria. DENV 1 genotype V, DENV 2 cosmopolitan genotype and DENV 3 genotype III has over the years been the predominant circulating Dengue strains in Africa. Relative genotypic stability of circulating Dengue serotypes in Africa occurred over the past five decades. This may be due to limited investigations on circulating Dengue serotypes among asymptomatic individuals in the region as most studies focused on disease outbreaks and imported cases.There is the need to describe circulating Dengue genotypes in northern Africa, southern Africa as well as among asymptomatic individuals in other parts of Africa as this will provide further information on the diversity of Dengue genotypes circulating in the region.     

Characterization of the 2013 dengue epidemic in Myanmar with dengue virus 1 as the dominant serotype

In 2013 in Myanmar, dengue epidemic occurred with 20,255 cases including 84 deaths. This study aimed to determine the serological and molecular characteristics of dengue virus (DENV) infection among children with clinical diagnosis of dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) during this period. Single acute serum samples were collected from 300 children in Mandalay Children Hospital, Mandalay, Myanmar. Out of the 300 children, 175 (58.3%) and 183 (61%) were positive for anti-dengue IgM and anti-dengue IgG, respectively. Among the IgM positives, 41 (23.4%) had primary DENV infection. Thirty-nine DENV strains (23 DENV-1, 10 DENV-2 and 6 DENV-4) were successfully isolated after inoculation of the patient serum samples onto C6/36 cells. DENV 1 was the dominant serotype in the 2013 epidemic. There was no correlation between the infecting serotypes and clinical severities. The DENV-1 strains belonged to three lineages of the genotype 1; the DENV-2 strains were of the Asian I genotype and were separated into two lineages; and DENV-4 strains belonged to the same lineage of genotype I. It is of interest to note the diversity of DENV-1 and -2 circulating in the same location during June–August 2013. These DENV isolates were genetically close (98%–100%) to the other previously reported isolates from Myanmar and its neighboring countries, namely China, Thailand, Sri Lanka, Cambodia and Vietnam. Primary DENV infection was still high among the severe dengue cases. Different serotypes of DENV were co-circulating in 2013, however, genotype shift was not observed. Additionally, amino acid mutations were detected in the study strains not seen in the previously reported strains from other countries and Myanmar. This paper provided information on the circulating serotypes for the last 15 years and the recent dengue situation in Mandalay, Myanmar after 2006.     

Disruption of predicted dengue virus type 3 major outbreak cycle coincided with switching of the dominant circulating virus genotype

Dengue is hyperendemic in most of Southeast Asia. In this region, all four dengue virus serotypes are persistently present. Major dengue outbreak cycle occurs in a cyclical pattern involving the different dengue virus serotypes. In Malaysia, since the 1980s, the major outbreak cycles have involved dengue virus type 3 (DENV3), dengue virus type 1 (DENV1) and dengue virus type 2 (DENV2), occurring in that order (DENV3/DENV1/DENV2). Only limited information on the DENV3 cycles, however, have been described. In the current study, we examined the major outbreak cycle involving DENV3 using data from 1985 to 2016. We examined the genetic diversity of DENV3 isolates obtained during the period when DENV3 was the dominant serotype and during the inter-dominant transmission period. Results obtained suggest that the typical DENV3/DENV1/DENV2 cyclical outbreak cycle in Malaysia has recently been disrupted. The last recorded major outbreak cycle involving DENV3 occurred in 2002, and the expected major outbreak cycle involving DENV3 in 2006–2012 did not materialize. DENV genome analyses revealed that DENV3 genotype II (DENV3/II) was the predominant DENV3 genotype (67%–100%) recovered between 1987 and 2002. DENV3 genotype I (DENV3/I) emerged in 2002 followed by the introduction of DENV3 genotype III (DENV3/III) in 2008. These newly emerged DENV3 genotypes replaced DENV3/II, but there was no major upsurge of DENV3 cases that accompanied the emergence of these viruses. DENV3 remained in the background of DENV1 and DENV2 until now. Virus genome sequence analysis suggested that intrinsic differences within the different dengue virus genotypes could have influenced the transmission efficiency of DENV3. Further studies and continuous monitoring of the virus are needed for better understanding of the DENV transmission dynamics in hyperendemic regions.     

Genotype replacement of dengue virus type 3 and clade replacement of dengue virus type 2 genotype Cosmopolitan in Dhaka,Bangladesh in 2017

Dengue is a mosquito-borne disease that has spread to >100 countries and is caused by the dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. DENV comprises 4 serotypes (DENV-1 to -4), and each serotype is further divided into distinct genotypes. In India, it is reported that all 4 serotypes of DENV co-circulate. Although Bangladesh is a neighboring country of India, very few reports have published DENV sequence data for the country, especially after 2012. To understand the current distribution of DENV genotypes in Bangladesh, we determined the nucleotide sequences of envelope regions obtained from 58 DENV-positive patients diagnosed at Apollo Hospitals Dhaka during the period between September 2017 and February 2018. We found 5 DENV-1, 47 DENV-2, and 6 DENV-3 serotypes. A phylogenetic analysis of the obtained viral sequences revealed that DENV-3 genotype I was present instead of DENV-3 genotype II, which was predominant in Bangladesh between 2000 and 2009. Furthermore, we found two distinct lineages of the Cosmopolitan genotype of DENV-2, one of which was closely related to strains from Southeast Asia and has never been reported previously in Bangladesh. These results indicated that DENVs in Bangladesh have increased in genotypic diversity and suggest that the DENV genotypic shift observed in other Asian countries also might have been taking place in Bangladesh.     

The distinct distribution and phylogenetic characteristics of dengue virus serotypes/genotypes during the 2013 outbreak in Yunnan,China: Phylogenetic characteristics of 2013 dengue outbreak in Yunnan,China

Since 2000, sporadic imported cases of dengue fever were documented almost every year in Yunnan Province, China. Unexpectedly, a large-scale outbreak of dengue virus (DENV) infection occurred from August to December 2013, with 1538 documented cases. In the current study, 81 dengue-positive patient samples were collected from Xishuangbanna, the southernmost prefecture of the Yunnan province, and 23 from Dehong, the westernmost prefecture of the Yunnan province. The full-length envelope genes were amplified and sequenced. Phylogenetic analysis revealed that nine strains (39.1%) and 14 strains (60.9%) from the Dehong prefecture were classified as genotype I of DENV-1 and Asian I genotype of DENV-2, respectively. All strains from Xishuangbanna were identified as genotype II of DENV-3. Bayesian coalescent analysis indicates that the outbreak originated from bordering southeastern Asian countries. These three epidemic genotypes were predicted to originate in Thailand and then migrate into Yunnan through different routes.     

Molecular investigation of the dengue outbreak in Karnataka,South India,reveals co-circulation of all four dengue virus serotypes

The growing incidence of dengue outbreaks in the state of Karnataka prompted us to study the circulating dengue virus (DENV) and their proportion among the suspected cases of dengue patients during the disease outbreak at Mysuru district of Southern India. The presence of the DENV in a patient's serum sample was identified by RT-PCR using previously published primer pairs targeting CprM gene. DENV serotyping was carried out by semi-nested multiplex PCR using serotype-specific primers and nucleotide sequencing. Three hundred fifty-five samples of serum from suspected dengue cases were collected, and 203 samples (57.18%) were found positives. In 2016, DENV-4 (97.87%) was found to be the most dominant DENV serotype either alone or as co-infection, followed by DENV-2 (8.51%) and DENV-3 (4.25%). In 47 positive cases, co-infection with more than one serotype was detected in 4 cases (8.51%). The analysis of the dengue cases in 2017, DENV-4 was dominating serotype (33.97%), followed by the emergence of DENV-2 (32.05%), DENV-3 (25.64%), and DENV-1 (25.00%). Our study also reports the circulation of all four DENV serotypes in the Mysuru district of Southern India, with concurrent infections rate of 16.66% in 2017. The present study provides information regarding the genetic variation among the circulating DENV serotype in an Indian state of Karnataka. The need for the studying genetic diversity of DENV will be useful during the continuous monitoring for disease burden as well as the development of appropriate prophylactic measures to control the spread of dengue infection.     

2019年广西7份本地感染登革病毒E基因特征分析

目的 了解2019年广西本地感染登革病毒(Dengue virus,DENV)E基因特性,探索可能的输入来源。方法 采用实时荧光定量PCR（real - time fluorescence quantitative PCR,RT - qPCR）方法对广西本地感染登革热病例急性期血清样本进行病毒核酸检测并分型,扩增登革病毒 E基因后测序,测序结果与不同地区和国家的参考株进行同源性比较和系统进化分析。结果 53份登革热病例血清标本经RT - qPCR分型,结果42份（79.2%,42/53）登革病毒核酸阳性,其中南宁9份、梧州10份、玉林17份、崇左6份,均为DENV - 1型,其余11份为登革病毒核酸阴性,42份DENV - 1型阳性核酸样本经过E基因扩增共得到7份,其中南宁市1份、梧州市1份、玉林市2份、崇左市3份,进化分析结果显示7份样本均为DENV - 1型Genotype Ⅰ基因型,其中DENV1/GXNN/007/2019、DENV1/GXWZ/009/2019、DENV1/GXYL/011/2019、DENV1/GXYL/012/2019与2019年广东株（序列号:MN921500）同源性99.94%～100.00%,DENV1/GXCZ/015/2019、DENV1/GXCZ/016/2019、DENV1/GXCZ/017/2019与 2015年印度尼西亚株（序列号:MG894852）同源性达99.60%,与1945年夏威夷参考株（序列号:AF425619）比较存在12处氨基酸位点变异。结论 2019年广西登革病毒是Genotype Ⅰ基因型,推测病毒从广东和东南亚国家输入导致的本地流行,应加强登革热跨省、跨境传播的防控。