广州市2010 年新发现一株基孔肯雅病毒基因特征研究

基孔肯雅热（CHIK）是由CHIK病毒（CHIKV）经伊蚊传播的一种急性传染病。2010 年广州市疾病预防控制中心监测到1 名疑似登革热发热症状的患者,血样经实验室检测显示登革病毒核酸为阴性,CHIKV核酸为阳性,继而分离获得1 株CHIKV。采用RT-PCR 扩增此毒株的全基因并测序,与GenBank 中的中国地区和全球代表株进行全基因进化分析,以了解其序列特征和可能的传播来源。     

广东省1990--2012年登革热和基孔肯雅热感染状况及流行风险分析

目的　掌握1990--2012年广东省登革热和基孔肯稚热感染状况和流行态势。方法　采用回顾性调查及文献研究相结合的方法获取1990--2012年广东省登革热和基孔肯雅热疫情及病原学监测资料,采集健康人群血清进行登革病毒（DENv）和基孔肯雅病毒（CHIKV）-IgG特异性抗体检测。结果　1995、2002、2006年广东省出现3个登革热流行高峰,发病率分别为9．75／10万、1．76／10万、1．25／10万。1990～1994年优势毒株为DENV-2和DENV-4型;1995年开始DENV-1型主导的流行模式持续近13年;2009年开始多种血清型合并流行。2003--2012年从7 718份健康人血清中检出DENV-IgG特异性抗体阳性标本180份,阳性率2．33％;2012年对采集的2132份健康人血清进行CHIKV-IgG特异性抗体检测,结果均为阴性。结论　广东省登革热感染水平总体较低,近年来由DENV-1型主导的流行模式逐步向多型并存的局面过渡,部分地区开始呈现地方化迹象。人群对CHIKV缺乏基本的免疫屏障,应加强监测与防控。     

回溯1例输入性基孔肯雅热疫情处置

目的回溯分析深圳市罗湖区1例输入性基孔肯雅热病例流行病学特征,完善罗湖区伊蚊传播传染病多部门协作防控模式,为深圳市罗湖区防控基孔肯雅热防控工作提供科学依据。方法采用描述性流行病学方法分析深圳市罗湖区1例输入性基孔肯雅热病例的诊疗过程及密切接触者信息,对感染来源进行流行病学调查,并采集生物标本进行实验室检测。结果经过流行病学调查及实验室诊断确认病例为罗湖区1例境外输入性基孔肯雅热病例。结论深圳市罗湖区1例基孔肯雅热病例经过及时诊断,科学治疗,详细的流行病学调查,有效的疫点处置,未发生疫情传播风险,未发生二代病例。     

1例输入性基孔肯雅病例的实验室检测

目的通过系统的实验室检测发现并确认1例输入性基孔肯雅病例。方法根据基孔肯雅病毒基因序列设计引物和荧光标记探针,利用实时荧光RT-PCR和常规RT-PCR检测方法对广东出入境检验检疫局发现的1例入境发热患者的血清样本进行检测,并对RT-PCR扩增产物进行核苷酸序列测定。结果通过实时荧光RT-PCR和常规RT-PCR 2种实验方法在该病例标本中检出基孔肯雅病毒核酸;对RT-PCR扩增产物进行序列测定,测出的521个碱基与GeneBank数据库公布的基孔肯雅病毒核酸序列进行比对,结果显示同源性高达99%。结论结合该患者临床症状、实验室检测结果及流行病学调查结果,判断该病例为输入性基孔肯雅病例。     

基孔肯雅热的流行现况及其研究进展

基孔肯雅病毒（chikungunyavirus，CHIKV）是1种动物源性、昆虫媒介传播的病原体．自20世纪50年代被发现以来导致了大规模的基孔肯雅热的暴发。掌握该病流行特点，防止该病在我国广泛流行是当前面临的重要任务。现就基孔肯雅热全球流行情况以及临床学、媒介生物学和基孔肯雅病毒的分子流行病学等方面的研究进展作一综述。     

2例马来西亚籍输入性基孔肯雅热病例的实验室诊断

[目的]通过系统的实验室检测发现并确认2例输入性基孔肯雅病例。[方法]采用实时荧光逆转录（Realtime RT-PCR）、逆转录PCR（RT—PCR）检测方法对病人血清进行检测,并对RT-PGR扩增产物进行核苷酸序列测定。[结果]通过实时荧光RT—PCR、RT—PCR 2种实验方法在该病例标本中检出基孔肯雅病毒核酸;对RT-PCR扩增产物进行测序,测出的521个碱基与基孔肯雅病毒核酸序列比对,结果同源性高达99％。[结论]2病例为输入性基孔肯雅实验室确诊病例。     

全国首例输入性基孔肯雅病的实验室诊断

目的:通过系统的实验室检测发现并确认全国首例输人性基孔肯雅病例.方法:利用实时荧光RT-PCR、RT-PCR检测方法,并对RT-PCR扩增产物进行核苷酸序列测定.结果:实时荧光RT-PCR、RT-PCR两种实验方法,在同一病例不同时间采集的多份标本中,都检测到基孑L肯雅病毒核酸;对RT-PCR扩增产物进行测序,测出的521个碱基与基孔肯雅病毒核酸序列比对,结果同源性高达99%.结论:该病例为全国首例输入性基孔肯雅病实验室确诊病例.     

基孔肯雅热的流行现况及其研究进展

基孔肯雅病毒(chikungunya virus,CHIKV)是1种动物源性、昆虫媒介传播的病原体,自20世纪50年代被发现以来导致了大规模的基孔肯雅热的暴发.掌握该病流行特点,防止该病在我国广泛流行是当前面临盏的重要任务.现就基孔肯雅热全球流行情况以及临床学、媒介生物学和基孔肯雅病毒的分子流行病学等方面的研究进展作一...     

基孔肯雅病研究进展

基孔肯雅病是由基孔肯雅病毒(Chikungunya virus,CHIKV)感染所致.该病主要经蚊虫吸血传播,其主要临床表现为发热、关节剧烈疼痛、皮疹和轻度出血体征,称为"基孔肯雅热".人群对基孔肯雅热普遍易感,任何年龄均可感染发病,当基孔肯雅热初次暴发时,可使大量人群发病[1].     

基孔肯雅病毒全基因组的多序列比对及遗传进化分析

目的 了解基孔肯雅病毒(Chikungunya virus,CHIKV)毒株的时间、地区及基因型的分布情况,不同时间和地区分离毒株的相似性,核苷酸位点的变异情况以及病毒的遗传进化趋势,为基孔肯雅热的预防和控制奠定基础.方法 对美国国立生物技术信息中心(NCBI)中收录的CHIKV毒株全基因组序列共133条用DNAStar7.1软件进行核苷酸和氨基酸相似性比较及变异分析,用Mega 6.06进行遗传进化分析.结果 CHIKV组内核苷酸序列相似性在97.7％ ～ 100.0％之间,CHIKV组内氨基酸序列相似性在80.3％ ～ 100.0％之间,组内相似性较高,变异率较低;CHIKV碱基间的转换较为常见占碱基突变的81.02％;分离时间、地区相近的毒株遗传距离较近,不同谱系的东/中/南非型毒株已蔓延至欧亚各地区.结论 CHIKV毒株存在一定的变异,但具有较高的遗传稳定性.     

Genetic characterization of Chikungunya virus in the Central African Republic

Chikungunya virus (CHIKV) is an alphavirus transmitted by the bite of mosquito vectors. Over the past 10 years, the virus has gained mutations that enhance its transmissibility by the Aedes albopictus vector, resulting in massive outbreaks in the Indian Ocean, Asia and Central Africa. Recent introduction of competent A. albopictus vectors into the Central African Republic (CAR) pose a threat of a Chikungunya fever (CHIKF) epidemic in this region. We undertook this study to assess the genetic diversity and background of CHIKV strains isolated in the CAR between 1975 and 1984 and also to estimate the ability of local strains to adapt to A. albopictus. Our results suggest that, local CHIKV strains have a genetic background compatible with quick adaptation to A. albopictus, as previously observed in other Central African countries.Intense surveillance of the human and vector populations is necessary to prevent or anticipate the emergence of a massive CHIKF epidemic in the CAR.     

Genetic characterization of E2 region of Chikungunya virus circulating in Odisha,Eastern India from 2010 to 2011

Chikungunya virus (CHIKV) infection has caught attention yet again as it rages around the globe affecting millions of people. The virus caused epidemic outbreaks affecting more than 15,000 people in Odisha, Eastern India since 2010. In this study, complete genetic characterization of E2 gene of CHIKV circulating in Odisha from 2010 to 2011 was performed by virus isolation, RT-PCR, molecular phylogenetics and bioinformatics methods. Phylogenetic analyses revealed the circulation of Indian Ocean Lineage (IOL) strains of ECSA genotype of CHIKV in Odisha. Several mutations were detected in the E2 gene, viz. E2-R82G, E2-L210Q, E2-I211T, E2-V229I and E2-S375T which had various adaptive roles during the evolution of CHIKV. The CHIKV E2 peptide ⁵⁷KTDDSHD⁶³ was predicted to be the most probable T-cell epitope and peptide ⁸⁴FVRTSAPCT⁹² predicted to be the common T and B cell epitope having high antigenicity. The amino acid positions 356–379 and 365–385 were predicted to be transmembrane helical domains and indicated E2 protein anchorage in intracellular membranes for effective interaction with the host receptors. Positive selection pressure was observed in five specific sites, 210, 211, 318, 375, and 377 which were observed to be fixed advantageously in most viral isolates. Structural modeling revealed that E2 gene of CHIKV was composed of 3 domains and the major adaptive mutations were detected in domain B, which can modulate binding of CHIKV to host cells, while the transmembrane domain in domain C and the epitopes were located in domain A, which was found to be most conserved. This is the first report from Eastern India demonstrating a predictive approach to the genetic variations, epitopic regions and the transmembrane helices of the E2 region. The results of this study, combined with other published observations, will expand our knowledge about the E2 region of CHIKV which can be exploited to develop control measures against CHIKV.     

Differential regulation of TLR mediated innate immune response of mouse neuronal cells following infection with novel ECSA genotype of Chikungunya virus with and without E1:A226V mutation

Chikungunya virus (CHIKV) has received global attention due to the series of large-scale outbreaks in different parts of the world including Africa, Indian Ocean Islands, India and South-East Asia. The appearance of many unusual severe manifestations including neurological disorders was reported in post resurgence epidemics with implication of novel East Central South African (ECSA) genotype with E1:A226V mutation. The molecular mechanism of CHIKV neuropathogenesis is not yet understood and very little is known about the host–pathogen interactions. In the present study replication kinetics and innate immune response of ECSA genotype of CHIKV with and without A226V mutation were determined in mouse neuroblastoma cell line (N2a). The 226V mutant strain was more replication competent in N2a cells with a peak titer of 10⁸ PFU/ml compared to 10⁶ PFU/ml for A226 virus. Besides, the 226V mutant virus showed relatively less induction of antiviral genes i.e. IFN-β, OAS-3, MX-2, ISG-15 and Toll like receptors 3 and 7 as compared to non mutant strain (A226). Further pretreatment of N2a cells either with Poly I: C, IFN-β or TNF-α resulted in inhibition of CHIKV replication hence confirming the role of TLR mediated innate immune response in CHIKV pathogenesis. Differential regulation of TLRs and associated down stream antiviral genes might have attributed for increased pathogenesis of the 226V mutant novel ECSA genotype of CHIKV during the recent epidemics.     

Chikungunya Fever Outbreak,Bhutan, 2012

In 2012, chikungunya virus (CHIKV) was reported for the first time in Bhutan. IgM ELISA results were positive for 36/210 patient samples; PCR was positive for 32/81. Phylogenetic analyses confirmed that Bhutan CHIKV belongs to the East/Central/South African genotype. Appropriate responses to future outbreaks require a system of surveillance and improved laboratory capacity.     

Co-circulation of Chikungunya and Dengue viruses in Dengue endemic region of New Delhi,India during 2016

Co-circulation of Chikungunya and Dengue viral infections (CHIKV and DENV) have been reported mainly due to transmission by common Aedes vector. The purpose of the study was to identify and characterise the circulating strains of CHIKV and DENV in DENV endemic region of New Delhi during 2016. CHIKV and DENV were identified in the blood samples (n = 130) collected from suspected patients by RT-PCR. CHIKV was identified in 26 of 65 samples (40%). Similarly, DENV was detected in 48 of 120 samples (40%). Co-infection with both the viruses was identified in five (9%) of the samples. Interestingly, concurrent infection with DENV, CHIKV and Plasmodium vivax was detected in two samples. CHIKV strains (n = 11) belonged to the ECSA genotype whereas DENV-3 sequences (n = eight) clustered in Genotype III by phylogenetic analysis. Selection pressure of E1 protein of CHIKV and CprM protein of DENV-3 revealed purifying selection with four and two positive sites, respectively. Four amino acids of the CHIKV were positively selected and had high entropy suggesting probable variations. Co-circulation of both viruses in DENV endemic regions warrants effective monitoring of these emerging pathogens via comprehensive surveillance for implementation of effective control measures.Key words: Chikungunya virus, co-circulation, co-infection, Dengue virus, phylogenetic analysis     

Clinical characteristics and predictive score of dengue vs. chikungunya virus infections

BackgroundChikungunya (CHIKV) and dengue viruses (DENV) are two arboviruses with epidemic potential and similar clinical presentations. The potential life-threatening risk associated with DENV justifies an immediate biological assessment and medical follow-up which may be delayed for CHIKV.ObjectivesTo compare the clinical variables that would help differentiate patients infected with CHIKV or DENV, and then to compute a predictive score.Patients and methodRetrospective case-control study comparing CHIKV-infected patients diagnosed by RT-PCR in 2014 with patients infected with DENV diagnosed by positive NS1 antigen test in 2013. Children aged < 15 years and pregnant women were excluded. Clinical and biological variables were compared, and a multivariate analysis was performed. A clinical score was developed using the β coefficients to differentiate the infections.ResultsOver the study period 168 patients infected with CHIKV were compared with 452 patients with DENV. The clinical variables independently associated with CHIKV was joint and back pain, and those associated with DENV were headache, muscle pain, nausea/vomiting, diarrhea, and hemorrhagic signs. The clinical score had 98% sensitivity for DENV and a ROC curve of 0.96.ConclusionThese two infections have a similar clinical presentation but the use of the proposed clinical score during the acute phase of the disease would make it possible to identify cases of DENV during a CHIKV epidemic to suggest adequate patient management.     

我国基孔肯雅热的流行状况

2010年10月,广东省东莞市暴发了我国首起基孔肯雅热社区聚集性疫情,打破了其长期以来以散在输入性病例为特征的流行现状。基孔肯雅热是一种由基孔肯雅病毒引起的急性传染病,伊蚊是其主要传播媒介。而我国大多数地区拥有其主要传播媒介埃及伊蚊和白纹伊蚊,一旦病原体侵入,可能暴发基孔肯雅热疫情。如何控制该疫情,防止疫情的进一步扩散,是摆在我们面前的当务之急。现就基孔肯雅病毒的病原学特征以及基孔肯雅热在我国历年的流行状况做一概述,以便更好地认识基孔肯雅热,为有效地监测和防治提供科学依据。     

Status of research and development of vaccines for chikungunya

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that during the last decade has significantly expanded its geographical range and caused large outbreaks of human disease around the world. Although mortality rates associated with CHIKV outbreaks are low, acute and chronic illnesses caused by CHIKV represent a significant burden of disease largely affecting low and middle income countries. This report summarizes the current status of vaccine development for CHIKV.     

Evaluation of recombinant E2 protein-based and whole-virus inactivated candidate vaccines against chikungunya virus

Objectives

With the re-emergence of chikungunya virus (CHIKV) in an explosive form and in the absence of a commercially available vaccine, we aimed to develop candidate vaccines employing recombinant E2 protein or chemically inactivated whole virus.

Design and methods

E2 gene of CHIKV isolate of ECSA genotype was cloned in pET15b vector, expressed and purified (rE2p). The virus was propagated in Vero cell line, purified and inactivated with formalin and BPL individually. Six to eight weeks old female BALB/c mice were immunized intramuscularly with two doses of 10 μg, 20 μg and 50 μg of vaccine formulations with or without adjuvants, 2 weeks apart. The adjuvants evaluated were alum, Mw, CadB (rE2p), alum/Mw (formalin inactivated CHIKV) and alum (BPL-inactivated CHIKV). Humoral immunity was assessed by ELISA and in vitro neutralization test using homologous and heterologous (Asian genotype) strains of CHIKV. Two cohorts of vaccinated mice were challenged separately via intranasal route with homologous virus two and 20 weeks after the 2nd dose. Viral load (CHIKV RNA by real time PCR) was determined in the serum and tissues (muscle, brain, spleen) of the mice challenged with the homologous virus.

Results

Anti-CHIK-antibody titres were dose dependent for all the immunogen formulations. BPL-inactivated vaccines led to the highest ELISA/neutralizing antibody (nAb) titres while alum was the most effective adjuvant. Asian genotype strain could be neutralized by the nAbs. In an adult mouse model, complete protection was offered by the alum-adjuvanted rE2p and both the inactivated vaccines as no virus was detected in the tissues and blood after challenge 2 weeks or 20 weeks-post-2nd dose. However, with rE2p-CadB, very low viremia was recorded on the 2nd day-post-challenge.

Conclusion

Both rE2p and BPL/formalin-inactivated virus are promising candidate vaccines deserving further evaluation.