黄热病病毒检测技术研究进展

本文综述了黄热病病毒检测技术的研究进展。黄热病病毒的检测技术对于黄热病爆发的快速诊断、流行病学研究以及接种黄热病疫苗后的血清抗体监测都有重要的意义。     

呼吸道病毒检测方法的进展及意义

呼吸道病毒是指一类能侵犯呼吸道引起呼吸道局部病变或仅以呼吸道为入侵门户,主要引起呼吸道组织器官病变的病毒.呼吸道病毒感染是全球范围内人类发病和死亡的主要原因之一,多数急性呼吸道感染是由病毒引起的.在急性呼吸道感染中,成人病毒阳性率约为61.8％,儿童为46.6％～66.9 ％[1].急性下呼吸道感染是儿童住院的主要原因,病毒感染率为32.3％～52.3％[2].呼吸道病毒主要包括:呼吸道合胞病毒(RSV)、甲型流感病毒(FluA)、乙型流感病毒(FluB)、副流感病毒1、2、3、4(PIV1、2、3、4),腺病毒3、7(ADV 3、7)、以及最近几年新发现的人类偏肺病毒(Hmpv)、人类博卡病毒(Hbov)等.     

腹泻病毒检测方法的研究进展

小儿腹泻病是世界范尉内影响童健康的常见病。随着抗生素的广泛应用，人们生活水平的提高，卫生条件的改善，细菌性腹泻得到了有效控制，病毒引起的腹泻逐渐受到人们的重视。目前，能引起人类腹泻的病毒主要包括：轮状病毒（rotavirus，RV）、人类杯状病毒（human caliciviruses，HuCVs）、星状病毒（Astrovirus，Astv）、肠腺病毒（enteric adenovirus，Ad）等。     

广州国境口岸出入境人员黄热病病毒血清学检测及感染风险预测

黄热病是由黄热病病毒引起的蚊媒传染病,主要发生在非洲亚撒哈拉地区和南美洲热带地区,是我国《国境卫生检疫法》规定的3种检疫传染病之一.为了解广州国境口岸入境人员黄热病病毒感染情况,本研究进行了黄热病毒抗原、抗体检测及其感染风险预测.     

水病毒检测方法的研究及黄浦江源水病毒检测

以Ａｌｃｌ３－滤膜洗脱法、Ａｌ２（ＳＯ４）３－滤膜洗脱法及Ａｌｃｌ３沉淀法浓缩实验性污染水样中的病毒，并以单层细胞病变法检测病毒，３法的病毒平均回收率分别为３６．４３％、５０．５３％和２１．８２％，并以第二法和第三法检测黄浦江水中的病毒，４０价水样有８份检测出ＣｏｘＢ５病毒、ＥＣＨＯ４和ＥＣＨＯ１２病毒共９株，文中叙述了所用方法的优点。     

黄热病现状分析及研究进展

黄热病是黄病毒科、黄病毒属的黄热病病毒引起的急性传染病,经蚊媒传播,主要发生在非洲和美洲地区,严重威胁人类健康。目前该病尚无特效药治疗,接种疫苗是最好的预防途径。虽然亚洲地区尚未发现黄热病,但随着全球经济的发展,国际间的人群交往更为便利,全球贸易一体化和都市化生活等因素使得病毒和传播媒介更易在各个国家之间传播。我国应长期保持警惕,及时预防、控制该病通过国境口岸传入。本文就黄热病流行病学、预防控制,以及疫苗研究等情况进行了综述。     

流行性感冒病毒检测方法的研究进展

近几年,流行性感冒（流感）在全球范围频繁暴发,2009年暴发了甲型流感（甲流）,2013年又暴发了H7N9的禽流感疫情,势必会威胁着动物和人类的生存和社会的经济的发展,从而使医学工作者关注的焦点转移到流感病毒的鉴定上,所以建立快速、高效的流感病毒检测方法,对于流感患者的临床诊断、有效及时的治疗有着非常重大的意义。作者综述了国内外流感病毒的检测方法,主要有血清免疫学检测、流感病毒分离培养、流感病毒核酸检测、环介导等温扩增法、基因芯片技术等。但每一种方法都有一定的优点和不足。随着科学技术水平的提高,更多的方法将应用到流感病毒检测中,同时方法间的互相融合更加提升了现有的检测水平,从而一定会在流感的监测、控制和预防工作中发挥更为积极有效的作用。     

黄热病毒实时荧光定量PCR方法的建立

目的 建立黄热病毒的实时荧光定量PCR检测技术,以实现黄热病毒感染早期的筛选和检测.方法 以带有黄热病毒基因质粒为阳性模板,采用TaqMan探针法,制作标准曲线,以实现对黄热病毒的简单快速的高灵敏度、高特异性实时检测.结果 该方法检测的最低拷贝数可以达到3.457 copies/μl.组内及组间变异系数均低于5％,证明该方法具有良好的敏感性及稳定性.以1～4型登革病毒和乙脑病毒基因组为模板,检测结果为阴性,证明该方法有良好的特异性.结论 该方法可快速、灵敏、特异、定量检测黄热病毒,能用于黄热病毒感染的早期诊断.     

黄热病疫苗接种所致不良反应及防控措施

目的探讨黄热病疫苗接种所致严重不良反应、引起不良反应的危险因子、危险人群及结合工作实际提出防控措施。方法通过对研究全球黄热病流行概况及其对人类的严重威胁、黄热病疫苗及接种所引起的严重不良反应、接种禁忌症、危险人群等,深入探讨目前黄热病疫苗接种现状和急需改进的措施。结果结合新《国际卫生条例》草案提出国境口岸应具备的预防、应对和处理黄热病疫苗接种所致突发事件的能力。结论国家应严格规范黄热病疫苗的生产,国际旅行保健中心应加强对黄热病疫苗使用的管理,卫生检疫部门应做好疫苗所致不良反应事件的预防和疫苗使用后的监测工作。     

实时荧光RT-PCR技术在黄热病快速检测中的应用

[目的]建立黄热病病毒的实时荧光RT-PCR检测方法并用于口岸黄热病的快速检测. [方法]体外转录黄热病病毒5-UTR的部分序列核酸作为阳性对照模板,设计实时荧光RT-PCR不同的反应程序和引物/探针浓度的组合,摸索最佳反应体系条件并用于黄热病疫苗和入境发热患者标本的检测.[结果]建立的黄热病病毒的实时荧光RT-PCR检测方法最佳反应体系为:2xRT-PCR缓冲液10 μl,正向引物5UTR-FP终浓度750 nM,反向引物5UTR-RP终浓度750 nM,探针5UTR-Probe终浓度500 nM,25xRT-PCR Enzyme Mix 0.8μl,RNA 5μl,用DEPC H2O补足到反应总体积20μl;Roche lightcycler仪器适用的扩增程序为:45℃10 min,95℃10 min;95℃5 s,56℃10 s(single),72℃15 s,45次循环.该方法最低检测限约为20copies病毒/反应,与登革病毒、西尼罗病毒、日本脑炎病毒、基孔肯雅病毒等蚊媒病毒无交叉反应.应用该方法对黄热病疫苗、媒介蚊虫和入境发热患者的血清标本进行检测,结果为黄热病疫苗核酸阳性,其他标本为阴性.[结论]该实时荧光RT-PER方法灵敏度高,特异性强,适用于黄热病病毒的快速检验,防止该病在国境口岸的传入.     

黄热病毒与甲型流感病毒及其分型集合检测芯片的初步研制

目的：研制黄热病毒与流感病毒及其分型集合检测芯片。方法：设计并合成60mer寡核苷酸（Oligo）探针用于制备检测芯片。提取黄热病、甲型流感病毒核酸,以限制性显示技术进行扩增标记,完成杂交后对芯片进行扫描和数据分析。结果：Oligo探针与相应的荧光标记样本杂交后,大部分能检测出阳性荧光信号,而空白对照和阴性对照均为阴性信号。结论：寡核苷酸芯片技术可应用于多种病毒的检测及分型,从而为多种病毒感染的早期鉴别诊断提供科学依据。     

Production of yellow fever virus in microcarrier-based Vero cell cultures

In this work, the propagation of the 17DD yellow fever virus in Vero cells grown on Cytodex-1 microcarriers was evaluated. After verifying that upon infection the virus adsorption step could be performed under continuous agitation, experiments were carried out in spinners and sparged lab-scale stirred-tank bioreactor to evaluate the use of a commercial serum-free medium (VP-SFM) and to investigate the effects of multiplicity of infection (MOI) and time of infection (TOI) on virus production. Virus titers as high as 8.4 × 10⁸ pfu/mL were obtained upon infection with MOI of 0.02 and TOI of 3 days, using the serum-free medium in the sparged bioreactor.     

Management and control of yellow fever virus: Brazilian outbreak January-April, 2018

ABSTRACT

Yellow fever virus (YFV) has a long history of causing human disease. Today, YFV is persevered in jungle environments with occasional sporadic human outbreaks in South America and periodic intermediate human transmissions with occasional urban outbreaks in sub-Saharan Africa. The ever-present risk of outbreak is primarily controlled for via vaccination coverage to vulnerable human populations. Global vaccine supplies have been strained in the setting of recent outbreaks in Africa and Brazil. The increasingly global community of today has placed an ever-growing tension on the management and control of YFV. A historic outbreak of YFV in Brazil is tracked from January to April 2018 using the International Society for Infectious Diseases’ (ISID) Program for Monitoring Emerging Diseases (ProMed). A narrative summary is generated from the review of 29 ProMed reports pertaining to the key words yellow fever and Brazil. Significant topics addressed include urban proximity, vaccination dose sparing with 1/5th standard dose, international travellers, epizootic trends, vaccine hesitancy, and mass immunisation campaigns. These topics are reviewed in detail for the current outbreak in comparison to previous outbreaks. Through close attention to these topics the degree and extent of the current outbreak was attenuated.     

Japanese encephalitis virus/yellow fever virus chimera is safe and confers full protection against yellow fever virus in intracerebrally challenged mice

Yellow fever (YF) is an acute viral haemorrhagic disease caused by the yellow fever virus (YFV), which remains a potential threat to public health. The live-attenuated YF vaccine (17D strain) is a safe and highly effective measure against YF. However, increasing adverse events have been associated with YF vaccinations in recent years; thus, safer, alternative vaccines are needed. In this study, using the Japanese encephalitis live vaccine strain SA14-14–2 as a backbone, a novel chimeric virus was constructed by replacing the pre-membrane (prM) and envelope (E) genes with their YFV 17D counterparts.The chimeric virus exhibited a reduced growth rate and a much smaller plaque morphology than did either parental virus. Furthermore, the chimera was much less neurovirulent than was YF17D and protected mice that were challenged with a lethal dose of the YF virus. These results suggest that this chimera has potential as a novel attenuated YF vaccine.     

Genetic divergence and dispersal of yellow fever virus, Brazil

An analysis of 79 yellow fever virus (YFV) isolates collected from 1935 to 2001 in Brazil showed a single genotype (South America I) circulating in the country, with the exception of a single strain from Rondonia, which represented South America genotype II. Brazilian YFV strains have diverged into two clades; an older clade appears to have become extinct and another has become the dominant lineage in recent years. Pairwise nucleotide diversity between strains ranged from 0% to 7.4%, while amino acid divergence ranged from 0% to 4.6%. Phylogenetic analysis indicated traffic of virus variants through large geographic areas and suggested that migration of infected people may be an important mechanism of virus dispersal. Isolation of vaccine virus from a patient with a fatal case suggests that vaccine-related illness may have been misdiagnosed in the past.     

Enzootic transmission of yellow fever virus in Peru

The prevailing paradigm of yellow fever virus (YFV) ecology in South America is that of wandering epizootics. The virus is believed to move from place to place in epizootic waves involving monkeys and mosquitoes, rather than persistently circulating within particular locales. After a large outbreak of YFV illness in Peru in 1995, we used phylogenetic analyses of virus isolates to reexamine the hypothesis of virus movement. We sequenced a 670-nucleotide fragment of the prM/E gene region from 25 Peruvian YFV samples collected from 1977 to 1999, and delineated six clades representing the states (Departments) of Puno, Pasco, Junin, Ayacucho, San Martin/Huanuco, and Cusco. The concurrent appearance of at least four variants during the 1995 epidemic and the genetic stability of separate virus lineages over time indicate that Peruvian YFV is locally maintained and circulates continuously in discrete foci of enzootic transmission.     

Increasing Vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium

In this work, changes in Vero cell cultivation methods have been employed in order to improve cell growth conditions to obtain higher viable cell densities and to increase viral titers. The propagation of the 17DD yellow fever virus (YFV) in Vero cells grown on Cytodex I microcarriers was evaluated in 3-L bioreactor vessels. Prior to the current changes, Vero cells were repeatedly displaying insufficient microcarrier colonization. A modified cultivation process with four changes has resulted in higher cell densities and higher virus titers than previously observed for 17DD YFV.     

流行性乙型脑炎病毒的分子生物学检测技术研究进展

流行性乙型脑炎(乙脑)病毒可引起严重的中枢神经系统疾病,致死率高,危害严重.建立快速准确的诊断方法对于控制乙脑的传播意义重大.此文就乙脑病毒的分子生物学检测技术最新进展进行综述.