阿瓦朗病毒和休斯病毒实时荧光RT-PCR检测方法研究

目的建立可以检测阿瓦朗病毒(Avalon virus,AVAV)和休斯病毒(Hughes virus,HUGV)两种内罗病毒的实时荧光定量RT-PCR检测方法,并进行初步的评价。方法收集、整理、比对、分析在公共数据库发布的两种病毒基因组核苷酸序列,确定检测靶标,设计特异性引物、探针,优化检测程序,建立实时荧光定量RT-PCR检测方法。利用体外转录技术制备的模拟样本、其他病毒感染标本、病毒株和正常人血标本比较评价所建方法的检测限、特异性、重复性特征。结果所建实时荧光定量RT-PCR检测方法可有效扩增检测AVAV和HUGA靶标RNA,检测限分别约为20拷贝/μl和70拷贝/μl,检测科萨努尔森林病毒、乙型流感病毒BV和BY型、甲型流感病毒H3N2、黄热病毒、乙型脑炎病毒、克里米亚-刚果出血热病毒、发热伴血小板减少综合征、内罗毕羊病毒和塔西那病毒样本无非特异性扩增,两种内罗病毒相互间无交叉反应,重复性比较分析显示变异系数小于2%。结论本研究建立的检测AVAV和HUGV的实时荧光定量RT-PCR方法,可用于临床样本检测和媒介生物、宿主动物标本筛查,便于病原的快速识别和疾病诊断。     

实时RT-PCR检测西尼罗病毒

目的建立西尼罗病毒快速、敏感和特异的实时RT-PCR法,用于西尼罗病毒疾病的早期诊断。方法对Vero细胞增殖的西尼罗病毒分别进行常规RT-PCR和实时RT-PCR法扩增,以PFU／ml为参考标准,比较二者的敏感性和特异性。并用实时RT-PCR法检测西尼罗病毒感染的小鼠组织标本。结果采用所建立的实时RT-PCR法和常规RT-PCR法可分别检测到0．02PFU／ml和2PFU／ml的西尼罗病毒RNA,前者的敏感性比后者高100倍。而对其他黄病毒科成员的检测均为阴性,表明该方法是特异的。采用这种实时RT-PCR法可从西尼罗病毒感染小鼠的血液、脾脏、肾脏和脑组织标本中检测出病毒核酸,且在感染后第2天最先自血液和脾脏中检测到病毒,在感染后1周的脑组织中的病毒滴度最高。结论本研究建立的实时RT-PCR法具有很高的敏感性和特异性,因此该方法可用于西尼罗病毒疾病的早期监测和流行病学研究。     

H9N2禽流感病毒一步法双重荧光RT-PCR检测方法的建立

目的 建立一种可同时检测禽流感病毒H9N2的HA和NA基因一步法双重荧光RT-PCR方法.方法 针对H9N2禽流感病毒的HA和NA基因保守区,设计相应的特异性引物以及探针,优化检测体系及反应条件,建立一步法双重荧光定量RT-PCR方法.对该方法的灵敏度、特异性、稳定性进行验证与评估,并对家禽粪便标本进行应用检测,以单重实时荧光RT-PCR方法作为参照,检测结果不一致的样本采用测序进行验证.结果 该方法特异性强,与H1、H3、H5、H7亚型禽流感病毒、鸡新城疫及鸡传染性支炎病毒均无交叉反应,对HA和NA基因的最低检出限分别可达50拷贝/μL和25拷贝/μL,组间与组内的变异系数在0.20 ～0.79％之间.对82份粪便标本进行检测,H9N2禽流感病毒的阳性率为8.14％ (7/82),与单重实时荧光RT-PCR法检测结果一致.结论 该方法特异性强、灵敏度高、稳定性好,可应用于临床禽流感样本的检测.     

广西家鼠型肾综合征出血热病毒的发现和鉴定

目的 从广西各地捕获的鼠肺标本检测汉坦病毒并鉴定其型别.方法 结合广西鼠疫监测采集啮齿类宿主动物,用ELISA法检测鼠肺标本,对汉坦病毒抗原阳性标本通过RT-PCR法扩增部分M片段上的核苷酸序列并测序,将扩增片段的核苷酸序列与已知病毒序列进行比对并分型.结果 在306份鼠肺标本中检测到1株SEO型的出血热病毒,阳性标本为广西沿海地区钦州市捕获的褐家鼠.结论 在广西沿海地区检测到SEO型汉坦病毒,其流行病学意义有待进一步研究.     

一起由札如病毒引起的急性胃肠炎暴发疫情的分子流行病学研究

目的 调查深圳市某幼儿园发生的一起急性胃肠炎暴发疫情的病原体,并分析病原体的分子流行病学特征.方法 收集患病幼儿及相关人员的粪便标本16份,剩余食物6份,采用国家卫生行业标准规定方法检测肠道致病菌、实时荧光RT-PCR法检测诺如病毒、札如病毒、轮状病毒核酸,逆转录RT-PCR法对病毒衣壳区和聚合酶区序列进行扩增,对PCR产物进行序列测定.序列用Clustalx软件进行多重比对、Mega 5.0软件绘制系统进化树.结果 16份样本中检出11例札如病毒核酸阳性,对其中3例阳性标本进行札如病毒衣壳区和聚合酶区序列测定,3株札如病毒与GII.3型参考株AY603425衣壳区核苷酸同源性为86.3％,聚合酶区核苷酸同源性为90.2％,3株札如病毒与GII.3型参考株AY603425、AB455793同属于GII.3型遗传分支.结论 此起急性胃肠炎爆发是由GII.3型札如病毒导致,GII.3型引起的急性胃肠炎暴发属广东省首次报道.     

我国沈阳一起GI.6型札如病毒引起的胃肠炎暴发的分子病原学特征研究

目的明确我国沈阳一起急性胃肠炎暴发的病原学及其基因进化特征。方法采集2021年11月沈阳某高校暴发疫情中的学生病例肛拭子和环境涂抹标本共计57份, 采用实时荧光RT-PCR进行常规腹泻病毒基因检测, 并对札如病毒核酸阳性标本采用传统RT-PCR方法扩增、序列测定、拼接和遗传进化分析。结果实时荧光RT-PCR检测确定26份标本为札如病毒核酸阳性, 成功扩增15份标本的衣壳蛋白基因片段。遗传进化分析表明, 15个阳性标本札如病毒分子分型均为GI.6基因型, 各标本序列间同源性为99.99%, 与2005日本的LC380411毒株亲缘较近为98.84%。结论引起本次暴发的病原札如病毒在东北地区较少检出, 其基因型为GI.6亚型引起暴发全球罕见报道。     

56例神经系统疾病患者外周血尼帕病毒N基因片段检测

尼帕病毒(Nipah vires,NiV)为副黏液病毒,Henipavims亚属,单股负链RNA病毒,不分节段.人感染NiV后病死率达4|D%～70%,因此,NiV被列为最危险的生物安全4(P-4)级病原.NiV共有6个结构基因,其中N基因高度保守且高效表达,因此被广泛用于NiV感染的诊断和流行病学调查.     

扎伊尔型埃博拉病毒的实时荧光RT-PCR检测方法研究

目的 建立扎伊尔型埃博拉病毒的核酸检测方法,以期用于埃博拉出血热临床标本的检测.方法 针对扎伊尔型埃博拉病毒核蛋白和糖蛋白基因设计引物和探针,建立单重和双重实时荧光RT-PCR检测方法,利用体外转录病毒RNA和埃博拉病毒系列参考品RNA评价其敏感性,利用马尔堡病毒、健康人、登革热患者和发热伴血小板减少综合征患者血清评价其特异性.结果 所建立的实时荧光RT-PCR检测方法扩增效率在95％～105％,可特异性地检测扎伊尔型埃博拉病毒核蛋白和糖蛋白基因,与马尔堡病毒、登革热和发热伴血小板减少综合征病毒均无交叉反应,体外转录的病毒RNA可检出10～100拷贝/μl.双重检测方法通过细胞培养的扎伊尔型埃博拉病毒RNA验证,可检出100 pfu/ml病毒.结论 本研究建立的检测扎伊尔型埃博拉病毒的实时荧光RT-PCR方法具有良好的特异性和敏感性,可用于埃博拉出血热临床标本的检测.     

在兰州地区儿童粪便中检测Aichi virus

目的 从兰州腹泻和正常儿童粪便标本中检测 Aichi virus,同时探讨 Aichi virus 与婴幼儿腹泻之间的联系.方法 根据文献发表资料,采用RT-PCR方法扩增 Aichi virus 3CD 片段,阳性产物经测序确定,并与已发表的该病毒序列进行比对分析.结果 在46份腹泻住院患儿粪便标本和299份腹泻门诊就诊患儿标本中各检出1例 Aichi virus,总检出率为0.06%,正常对照儿童中未检测到 Aichi virus.2株病毒3CD区基因与已知参考株核苷酸序列同源性均为97%,系统进化分析显示这2株病毒属于B基因型.结论 我国存在B基因型的 Aichi virus,但要明确我国 Aichi virus 的病原学及流行病学特点需要更多研究.     

浙江省首次发现新型布尼亚病毒感染病例及分离病毒分子鉴定

目的 了解浙江省是否存在新型布尼亚病毒潜在自然疫源地,分离疑似病例血清中新型布尼亚病毒并进行鉴定.方法 免疫荧光法检测浙江省台州地区野生啮齿动物不同组织标本中新型布尼亚病毒抗原.实时荧光定量RT-PCR检测疑似病人血清中新型布尼亚病毒核酸,扩增产物进行序列测定.Vero细胞分离疑似病人血清中新型布尼亚病毒,以新型布尼亚病毒株核衣壳蛋白编码基因为靶基因,采用RT-PCR及扩增产物测序对分离的疑似新型布尼亚病毒株进行鉴定,另对该序列进行同源性分析和比较.结果 70只野生啮齿动物中,免疫荧光法检测阳性率为5.71%.实时荧光定量RT-PCR检测结果显示,4例疑似病人血清中有两例检测结果阳性.1例阳性血清样本中分离出1株疑似新型布尼亚病毒,RT-PCR和测序结果证实该病毒确为新型布尼亚病毒,其核衣壳蛋白编码基因序列与湖北省新型布尼亚病毒分离株相似性高达92.2%,但与国内其他地区新型布尼亚病毒分离株序列差异较大.结论 首次证实浙江省存在新型布尼亚病毒自然疫源地及感染病人,不同群新型布尼亚病毒分布可能存在一定的地理差异.     

Acute Hendra virus infection: Analysis of the pathogenesis and passive antibody protection in the hamster model

Hendra virus (HeV) and Nipah virus (NiV) are recently-emerged, closely related and highly pathogenic paramyxoviruses. We have analysed here the pathogenesis of the acute HeV infection using the new animal model, golden hamster (Mesocricetus auratus), which is highly susceptible to HeV infection. HeV-specific RNA and viral antigens were found in multiple organs and virus was isolated from different tissues. Dual pathogenic mechanism was observed: parenchymal infection in various organs, including the brain, with vasculitis and multinucleated syncytia in many blood vessels. Furthermore, monoclonal antibodies specific for the NiV fusion protein neutralized HeV in vitro and efficiently protected hamsters from HeV if given before infection. These results reveal the similarities between HeV and NiV pathogenesis, particularly in affecting both respiratory and neuronal system. They demonstrate that hamster presents a convenient novel animal model to study HeV infection, opening new perspectives to evaluate vaccine and therapeutic approaches against this emergent infectious disease.     

Nipah virus entry can occur by macropinocytosis

Nipah virus (NiV) is a zoonotic biosafety level 4 paramyxovirus that emerged recently in Asia with high mortality in man. NiV is a member, with Hendra virus (HeV), of the Henipavirus genus in the Paramyxoviridae family. Although NiV entry, like that of other paramyxoviruses, is believed to occur via pH-independent fusion with the host cell's plasma membrane we present evidence that entry can occur by an endocytic pathway. The NiV receptor ephrinB2 has receptor kinase activity and we find that ephrinB2's cytoplasmic domain is required for entry but is dispensable for post-entry viral spread. The mutation of a single tyrosine residue (Y304F) in ephrinB2's cytoplasmic tail abrogates NiV entry. Moreover, our results show that NiV entry is inhibited by constructions and drugs specific for the endocytic pathway of macropinocytosis. Our findings could potentially permit the rapid development of novel low-cost antiviral treatments not only for NiV but also HeV.     

Sites of phosphorylation of P and V proteins from Hendra and Nipah viruses: newly emerged members of Paramyxoviridae

Hendra (HeV) and Nipah (NiV) viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the subfamily Paramyxoviridae. The polymerase-associated phosphoproteins (P proteins) of paramyxoviruses have been shown, by direct and indirect methods, to be highly phosphorylated. In this study, a comprehensive comparison of in vivo phosphorylation of HeV and NiV P proteins, derived from virus particles, was achieved by a direct approach using electrospray ionization ion trap mass spectrometry (ESI-IT-MS). Phosphorylation sites for the P proteins were determined at Ser-224 and Thr-239 in HeV and at Ser-240 and Ser-472 in NiV. These phosphorylation patterns do not appear to be consistent with those reported for other paramyxoviruses. Protein V, a product of a frame shift in the P protein gene, was identified by specific antibodies in HeV preparations but not in NiV. HeV V protein was found to contain phosphoserine but not phosphothreonine. In addition, P proteins from both viruses were found to be modified by N-terminal acetylation.     

Functional studies of host-specific ephrin-B ligands as Henipavirus receptors

Hendra virus (HeV) and Nipah virus (NiV) are closely related paramyxoviruses that infect and cause disease in a wide range of mammalian hosts. To determine whether host receptor molecules play a role in species-specific and/or virus-specific infection we have cloned and characterized ephrin-B2 and ephrin-B3 ligands from a range of species, including human, horse, pig, cat, dog, bats (Pteropus alecto and Pteropus vampyrus) and mouse. HeV and NiV were both able to infect cells expressing any of the ephrin-B2 and ephrin-B3 molecules. There did not appear to be significant differences in receptor function from different species or receptor usage by HeV and NiV. Soluble ephrin ligands, their receptors and G-specific human monoclonal antibodies differentially blocked henipavirus infections suggesting different receptor affinities, overlapping receptor binding domains of the henipavirus attachment glycoprotein (G) and that the functional domains of the ephrin ligands may be important for henipavirus binding.     

Emerging trends of Nipah virus: A review

Since emergence of the Nipah virus (NiV) in 1998 from Malaysia, the NiV virus has reappeared on different occasions causing severe infections in human population associated with high rate of mortality. NiV has been placed along with Hendra virus in genus Henipavirus of family Paramyxoviridae. Fruit bats (Genus Pteropus) are known to be natural host and reservoir of NiV. During the outbreaks from Malaysia and Singapore, the roles of pigs as intermediate host were confirmed. The infection transmitted from bats to pigs and subsequently from pigs to humans. Severe encephalitis was reported in NiV infection often associated with neurological disorders. First NiV outbreak in India occurred in Siliguri district of West Bengal in 2001, where direct transmission of the NiV virus from bats‐to‐human and human‐to‐human was reported in contrast to the role of pigs in the Malaysian NiV outbreak. Regular NiV outbreaks have been reported from Bangladesh since 2001 to 2015. The latest outbreak of NiV has been recorded in May, 2018 from Kerala, India which resulted in the death of 17 individuals. Due to lack of vaccines and effective antivirals, Nipah encephalitis poses a great threat to public health. Routine surveillance studies in the infected areas can be useful in detecting early signs of infection and help in containment of these outbreaks.     

Neutralization assays for differential henipavirus serology using Bio-Plex protein array systems

Hendra virus (HeV) and Nipah virus (NiV) are related emerging paramyxoviruses classified in the genus Henipavirus. Both cause fatal disease in animals and humans and are classified as biosafety level 4 pathogens. Here we detail two new multiplexed microsphere assays, one for antibody detection and differentiation and another designed as a surrogate for virus neutralization. Both assays utilize recombinant soluble attachment glycoproteins (sG) whereas the latter incorporates the cellular receptor, recombinant ephrin-B2. Spectrally distinct sG(HeV)- and sG(NiV)-coupled microspheres preferentially bound antibodies from HeV- and NiV-seropositive animals, demonstrating a simple procedure to differentiate antibodies to these closely related viruses. Soluble ephrin-B2 bound sG-coupled microspheres in a dose-dependent fashion. Specificity of binding was further evaluated with henipavirus G-specific sera and MAbs. Sera from henipavirus-seropositive animals differentially blocked ephrin-B2 binding, suggesting that detection and differentiation of HeV and NiV neutralizing antibodies can be done simultaneously in the absence of live virus.     

Antigen capture ELISA system for henipaviruses using polyclonal antibodies obtained by DNA immunization

A novel antigen-capture sandwich ELISA system targeting the glycoproteins of the henipaviruses Nipah virus (NiV) and Hendra virus (HeV) was developed. Utilizing purified polyclonal antibodies derived from NiV glycoprotein-encoding DNA-immunized rabbits, we established a system that can detect the native antigenic structures of the henipavirus surface glycoproteins using simplified and inexpensive methods. The lowest detection limit against live viruses was achieved for NiV Bangladesh strain, 2.5 × 10(4) TCID(50). Considering the recent emergence of genetic variants of henipaviruses and the resultant problems that arise for PCR-based detection, this system could serve as an alternative rapid diagnostic and detection assay.     

Monoclonal antibodies against the nucleocapsid proteins of henipaviruses: production, epitope mapping and application in immunohistochemistry

Summary Four monoclonal antibodies (mAbs) were generated by immunizing BALB/C mice with recombinant nucleocapsid protein (N) of Nipah virus (NiV) and Hendra virus (HeV) expressed in E. coli. Two mAbs each were obtained for the HeV N and NiV N, respectively. All four mAbs displayed specific reactivity with the recombinant N proteins of both viruses by western blot, which was further confirmed by immunofluorescent antibody assay using fixed insect cells infected with recombinant baculoviruses expressing either the HeV or NiV N protein. Epitope mapping using a 12-mer random peptide phage display library revealed two linear antigenic sites of the henipavirus N proteins, KLxR (aa 17–20) and FKREM (aa 446–450), which have not been reported previously. Two of the mAbs were able to specifically recognize HeV antigens by immunohistochemical staining of lung tissue sections of a horse experimentally infected with HeV. These reagents will be a useful addition to the collection of tools essential for further research and improvement in diagnosis of henipaviruses. Correspondence: Changchun Tu, Institute of Veterinary Sciences, Academy of Military Medical Sciences, 1068 Qinglong Road, Changchun 130062, China     

Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase

Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2 μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.     

Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae

Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.