森林脑炎自然疫源地样本的监测及病毒的分离研究

为了解森林脑炎疫源地的分布变化趋势及样本分离病毒的特性,采集了森林脑炎高发区周边的森林全沟硬蜱、血蜱样本及森林脑炎患者的脑组织样本,用小白鼠脑内接种法检测、分离病毒分离的病毒经鉴别试验证明为森林脑炎病毒：蜱、脑两种标本检测的阳性率分别为50％和100％、结果表明森林脑炎的疫区有从林区向农业区扩散的趋势,且全沟硬蜱的带毒率较高;森脑患者的脑组织样本与蜱标本病毒的性状育差异     

发热伴血小板减少综合征病毒灭活病毒的纯化及免疫原性初步研究

为了解纯化后灭活发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome bunyavirus,SFTSV)的免疫原性,本研究通过超滤浓缩和分子筛层析相结合的方法对灭活的SFTSV进行纯化,采用Western blot和SDS-PAGE对灭活病毒的纯化样品进行分析和鉴定,采用双抗夹心ELISA方法对其中糖蛋白(Glycoprotein,GP)和核蛋白(Nucleoprotein,NP)的抗原含量进行检测。浓缩纯化的SFTSV灭活病毒,经电镜观察并通过BCA法测定其总蛋白浓度。然后,将纯化的灭活SFTSV按两种不同的免疫程序免疫新西兰兔,评价免疫原性和比较免疫效果。结果显示,SFTSV灭活并超滤浓缩后,分子筛层析可观察到两个洗脱峰,其中之一为灭活病毒颗粒,SDS-PAGE、Western blot和ELISA法分析均可检测到GP和NP抗原,而另一洗脱峰主要成分则为NP。浓缩后的纯化病毒纯度达90%以上,总蛋白浓度约为1.1mg/mL,且具有典型的布尼亚病毒电镜形态。纯化的SFTSV灭活病毒免疫实验动物后,可在血清中检出高滴度病毒特异性IgG和中和抗体,但抗体滴度受免疫程序的影响,0d、14d和28d天三针程序免疫动物的效果明显优于0d、7d和28d免疫程序组。本研究显示了灭活SFTSV培养液中除完整病毒颗粒外,还含有大量游离的NP,经分子筛层析纯化可获得高纯度灭活病毒,同时明确了纯化的SFTSV灭活病毒具有良好的免疫原性,可诱导产生高滴度中和抗体和病毒特异IgG抗体,可为灭活病毒疫苗的进一步研究提供重要的线索。     

人源抗发热伴血小板减少综合征布尼亚病毒-Gn蛋白重组抗体的研究

为研制人源抗发热伴血小板减少综合征布尼亚病毒(Severe fever with thrombocytopenia syndrome virus,SFTSV)Gn蛋白重组抗体,本研究利用噬菌体表面展示技术,以SFTSV全病毒颗粒和重组表达SFTSV-Gn蛋白为抗原,从人源抗SFTSV Fab噬菌体抗体库中筛选抗SFTSV-Gn蛋白的重组Fab抗体,通过ELISA对Fab抗体的结合特异性进行检测。将Fab抗体基因克隆入哺乳动物细胞表达载体HL51-14,瞬时转染293T细胞获得分泌表达的IgG抗体。通过ELISA、IFA和Western-blotting检测IgG抗体的结合特异性。采用亲和层析纯化IgG抗体,并用微量中和试验检测IgG抗体的中和活性。结果表明经过三轮富集筛选,以SFTSV病毒颗粒为抗原筛选出364株针对SFTS病毒核蛋白Fab抗体,没有筛选出特异性结合Gn蛋白的阳性克隆,而通过Gn蛋白筛选得到8株特异结合Gn蛋白的Fab抗体,其中5株来自Lambda库,3株来自Kappa库。ELISA、IFA和Western-blotting检测证实这8株IgG抗体均能特异性结合Gn蛋白。微量中和试验显示8株新筛抗体没有中和活性,但仍可为后续SFTSV人源单克隆抗体的研究提供借鉴和参考。     

广东省阿卡斑病毒和OYAV的检测及分子特征研究

阿卡斑病毒(Akabane virus,AKV)和Oya病毒(Oya virus,OYAV)均属于泛布尼亚病毒科、正布尼亚病毒属的两种病毒,可引起动物疾病,目前广东省尚不清楚是否存在这两种病毒。为了解这两种病毒在广东省的存在状况及分子特征,2018年4月至7月,分别在广东省的佛山、信宜、河源和阳江四个地市利用人诱法和诱蚊灯法采集吸血昆虫标本。采集的标本分类后,按20～50只/批,蠓虫按200～500只/批分装,液氮保存运输。标本破碎离心后,上清用于接种细胞和病毒核酸提取。Real-time RT-PCR检测AKV和OYAV阳性的样本用于S基因的扩增、测序和系统进化分析。本次研究共采集各类吸血昆虫421批次,Real-time RT-PCR检测AKV阳性10份,均来自蠓虫标本,RT-PCR检测AKV阳性5份,其中4份成功测序;Real-time RT-PCR检测OYAV阳性15份,其中蠓虫13份、中华按蚊和三带喙库蚊各1份,RT-PCR检测OYAV阳性6份,其中4份成功测序。病毒分离结果均为阴性。进化分析结果显示,所有序列共分为5个血清群,本次研究的8条序列均位于Simbu群,其中4个新毒株与OYAV代表株NIV86209的核苷酸同源性最高可达96.87%,氨基酸同源性最高为100%;另外4株新病毒与AKV代表株GXLCH02的核苷酸同源性最高达98.60%,氨基酸同源性最高为99.57%。本研究结果揭示,AKV和OYAV在广东省的虫媒中分布广泛,主要以蠓虫分布为主,来自蠓虫中的新OYAV和AKV与其它媒介或宿主中的AKV和OYAV高度同源,未显示明显媒介差异的分子特征。     

山东省柯萨奇病毒B1型VP1区全基因序列分析

为了解柯萨奇病毒B1型(Coxsackievirus B1,CV-B1)山东地方株的分子流行病学特征,本研究对1994年至2015年山东省急性弛缓性麻痹(Acute flaccid paralysis,AFP)监测系统、环境污水监测和无菌性脑膜炎病例标本中分离到的CV-B1病毒进行了VP1序列测定、系统发生学分析和同源性分析。共分离到CV-B1病毒53株,其中AFP监测、污水和及脑炎标本各分离到41株、4株和8株。基于VP1完整编码区序列的系统发生学分析显示CVB1山东株与国内其他分离株属于一个大的分支,该分支内无国外分离株,国外分离株构成了其他两个分支。山东株之间的VP1核苷酸同源性为84.4%~100.0%,与其他国家分离株的同源性为77.9%~85.0%。研究结果表明,中国CV-B1分离株与国外株相比有较大的遗传差异,需要加强相关手足口病病毒学监测,关注不同传播链的新的基因亚型的肠道病毒的输入。     

发热伴血小板减少综合征病毒感染人源巨噬细胞形成包涵体结构

发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome virus,SFTSV)是在我国首次发现的布尼亚病毒科白蛉病毒属的一种新型病毒,是新发传染病发热伴血小板减少综合征的致病病原。本研究通过免疫荧光共聚焦实验观察不同感染剂量和不同感染时相SFTSV核衣壳蛋白(Nucleocapsid protein,NP)在THP-1细胞内的动态定位特点,并应用电镜方法在超微结构水平观察不同感染时相THP-1细胞的胞内形态结构特征,发现SFTSV感染THP-1在胞内形成包涵体结构。研究进一步显示感染后的第3天核衣壳蛋白NP所形成包涵体的形态大小与感染后第7天培养上清中的病毒载量之间存在相关。本研究揭示SFTSV感染人源巨噬细胞THP-1形成的包涵体结构可能是SFTSV利用宿主细胞形成的特殊胞内结构以利于大量合成和产生新的病毒颗粒。     

我国新疆首次检出西伯利亚型蜱传脑炎病毒

对新疆地区蜱传病毒类群进行调查,本研究利用Illumia深度测序技术对新疆全沟硬蜱体内的病毒小RNA分子进行筛查,从提取的蜱小RNA组份中共测得32 631条病毒特异性读序,77条读序与蜱传黄病毒基因组序列匹配,核苷酸数合计约占病毒基因组的3.8%~2.4%,其中32条读序与国内尚未发现的西伯利亚型蜱传脑炎病毒一致。为对上述结果进行确证,利用RT-PCR对此型病毒核酸进行检测,从蜱总RNA中分别扩增获得病毒包膜基因与基因组3′末端片段,测序比对表明上述片段序列与西伯利亚南部检出病毒高度近似,核苷酸与氨基酸似然率分别为97.2%~95.5%与99.4%~98.3%。综上所述,本研究利用基于深度测序的病毒筛查方法,首次从我国新疆蜱传脑炎疫源地检出西伯利亚型病毒,为下一步针对病毒的分离与流行病学调查提供了重要线索。     

柯萨奇病毒A组16型河南省分离株基因组序列分析

为了解河南省手足口病患者标本中分离柯萨奇A组的16型(CoxAl6)病毒基因组特征,对2010年采集的手足口病患者临床标本406份进行RT-PCR扩增和病毒分离鉴定;通过10对引物分段扩增和拼接CoxAl6分离株基因组序列,利用生物信息学软件对序列分析,构建序列遗传发育树。测序获得河南省CoxAl6分离株HN1162/HN/CHN/2010基因组全长序列7 411bp,5′非编码区(5′UTR)、P1、P2、P3、3′非编码区(3′UTR)区域核苷酸序列与GenBank公布的其它分离株相似性分别为87.0%～97.9%、77.0%～95.4%、80.3%～96.9%、77.9%～96.2%、80.5%～100%;VP1区核苷酸相似性为91.4%～96.4%,氨基酸相似性为99.3%～99.7%;遗传发育树分析表明与我国深圳、广州、福建分离株处于同一分支。河南省手足口病患者标本CoxAl6病毒分离株属于C2基因亚型/B-2基因亚型,对加强该病毒变异检测,预防控制手足口病疫情具有重要意义。     

中国广西龙虎山野生猕猴粪便的病毒宏基因组学分析

非人灵长类携带的病毒种类繁多,其中部分对人具有致病性。为深入了解我国野生猕猴携带病毒的状况,本研究应用MiSeq高通量测序及生物信息学分析技术,对从广西采集的280份猕猴粪便标本进行了病毒宏基因组学的分析。高通量测序共获得了233 726 79条读长(Reads),其中4641条序列与病毒相关,进一步注释到27个病毒科(包含细小病毒科中的细小病毒亚科和浓核病毒亚科),包括其中5种脊椎动物病毒(占78.2%)、6种昆虫病毒(占5.5%)、11种植物病毒(占10.4%)、其他的病毒(占9.8%);遗传进化分析结果显示,被注释为萨佩罗病毒、肠道病毒、细小病毒、腺相关病毒等序列与已知病毒相似,部分序列呈现明显的差异;应用PCR扩增进一步证实了病毒序列的真实存在。本研究初步确定了广西地区猕猴粪便中病毒的病毒谱,为深入分析和研究其中有潜在公共卫生意义的病毒奠定了基础。     

Experimental and Natural Infections of Goats with Severe Fever with Thrombocytopenia Syndrome Virus: Evidence for Ticks as Viral Vector

Background

Severe fever with thrombocytopenia syndrome virus (SFTSV), the causative agent for the fatal life-threatening infectious disease, severe fever with thrombocytopenia syndrome (SFTS), was first identified in the central and eastern regions of China. Although the viral RNA was detected in free-living and parasitic ticks, the vector for SFTSV remains unsettled.

Methodology/Principal Findings

Firstly, an experimental infection study in goats was conducted in a bio-safety level-2 (BSL-2) facility to investigate virus transmission between animals. The results showed that infected animals did not shed virus to the outside through respiratory or digestive tract route, and the control animals did not get infected. Then, a natural infection study was carried out in the SFTSV endemic region. A cohort of naïve goats was used as sentinel animals in the study site. A variety of daily samples including goat sera, ticks and mosquitoes were collected for viral RNA and antibody (from serum only) detection, and virus isolation. We detected viral RNA from free-living and parasitic ticks rather than mosquitoes, and from goats after ticks’ infestation. We also observed sero-conversion in all members of the animal cohort subsequently. The S segment sequences of the two recovered viral isolates from one infected goat and its parasitic ticks showed a 100% homology at the nucleic acid level.

Conclusions/Significance

In our natural infection study, close contact between goats does not appear to transmit SFTSV, however, the naïve animals were infected after ticks’ infestation and two viral isolates derived from an infected goat and its parasitic ticks shared 100% of sequence identity. These data demonstrate that the etiologic agent for goat cohort’s natural infection comes from environmental factors. Of these, ticks, especially the predominant species Haemaphysalis longicornis, probably act as vector for this pathogen. The findings in this study may help local health authorities formulate and focus preventive measures to contain this infection.     

Deep sequencing and phylogenetic analysis of severe fever with thrombocytopenia syndrome virus from the tick,Haemaphysalis longicornis,in Korea

The Asian longhorned tick, Haemaphysalis longicornis, is widely distributed in China, Japan, and Korea and may transmit infectious diseases. Severe fever with thrombocytopenia syndrome (SFTS) is an important tick‐borne disease caused by the SFTS virus (SFTSV). Deep sequencing to confirm the presence of SFTSV in ticks has not been reported in Korea. To detect SFTSV, RNA was extracted from tick samples and analyzed using high‐throughput deep sequencing. Based on BLASTN results, numerous SFTSV reads were identified. Moreover, a nearly complete genome of SFTSV (JNU‐1 isolate) was obtained using Sanger sequencing. The genome of the JNU‐1 isolate includes three segments of 6,286, 3,299 and 1,642 nucleotides (nt) termed large (L), medium (M), and small (S), respectively. Also, phylogenetic and recombination analyses for each segment of SFTSV were performed using the JNU‐1 isolate. The three segments of JNU‐1 isolate were closely related to the genotype B known human‐derived Korean SFTSV isolate; the JNU‐1 isolate showed no recombination sites with other isolates. This study is the first report of detection of SFTSV from ticks using deep sequencing in Korea and provides information on the genetic diversity of SFTSV in East Asia.     

Ecology of the Tick-Borne Phlebovirus Causing Severe Fever with Thrombocytopenia Syndrome in an Endemic Area of China

Background

Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a tick-borne phlebovirus in family Bunyaviridae. Studies have found that humans, domestic and wildlife animals can be infected by SFTSV. However, the viral ecology, circulation, and transmission remain largely unknown.

Methodology/Principal Findings

Sixty seven human SFTS cases were reported and confirmed by virus isolation or immunofluorescence assay between 2011 and 2014. In 2013–2014 we collected 9,984 ticks from either vegetation or small wild mammals in the endemic area in Jiangsu, China, and detected SFTSV-RNA by real-time RT-PCR in both questing and feeding Haemaphysalis longicornis and H. flava. Viral RNA was identified in larvae of H. longicornis prior to a first blood meal, which has never been confirmed previously in nature. SFTSV-RNA and antibodies were also detected by RT-PCR and ELISA, respectively, in wild mammals including Erinaceus europaeus and Sorex araneus. A live SFTSV was isolated from Erinaceus europaeus captured during the off tick-feeding season and with a high SFTSV antibody titer. Furthermore, SFTSV antibodies were detected in the migratory birds Anser cygnoides and Streptopelia chinensis using ELISA.

Conclusions/Significance

The detection of SFTSV-RNA in non-engorged larvae indicated that vertical transmission of SFTSV in H. longicornis might occur in nature, which suggests that H. longicornis is a putative reservoir host of SFTSV. Small wild mammals such as Erinaceus europaeus and Sorex araneus could be infected by SFTSV and may serve as natural amplifying hosts. Our data unveiled that wild birds could be infected with SFTSV or carry SFTSV-infected ticks and thus might contribute to the long-distance spread of SFTSV via migratory flyways. These findings provide novel insights for understanding SFTSV ecology, reservoir hosts, and transmission in nature and will help develop new measures in preventing its rapid spread both regionally and globally.     

A new luciferase immunoprecipitation system assay provided serological evidence for missed diagnosis of severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV) infection, was first reported in 2010 in China with an initial fatality of up to 30%. The laboratory confirmation of SFTSV infection in terms of detection of viral RNA or antibody levels is critical for SFTS diagnosis and therapy. In this study, a new luciferase immunoprecipitation system (LIPS) assay based on pREN2 plasmid expressing SFTSV NP gene and tagged with Renilla luciferase (Rluc), was established and used to investigate the levels of antibody responses to SFTSV. Totally 464 serum samples from febrile patients were collected in the hospital of Shaoxing City in Zhejiang Province in 2019. The results showed that 82 of the 464 patients (17.7%) had antibody response to SFTSV, which were further supported by immunofluorescence assays (IFAs). Further, qRT-PCR and microneutralization tests showed that among the 82 positive cases, 15 patients had viremia, 10 patients had neutralizing antibody, and one had both (totally 26 patient). However, none of these patients were diagnosed as SFTS in the hospital probably because of their mild symptoms or subclinical manifestations. All the results indicated that at least the 26 patients having viremia or neutralizing antibody were the missed diagnosis of SFTS cases. The findings suggested the occurrence of SFTS and the SFTS incidence were higher than the reported level in Shaoxing in 2019, and that LIPS may provide an alternative strategy to confirm SFTSV infection in the laboratory.     

Severe Fever with Thrombocytopenia Syndrome Virus Antigen Detection Using Monoclonal Antibodies to the Nucleocapsid Protein

Background

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease with a high case fatality rate, and is caused by the SFTS virus (SFTSV). SFTS is endemic to China, South Korea, and Japan. The viral RNA level in sera of patients with SFTS is known to be strongly associated with outcomes. Virological SFTS diagnosis with high sensitivity and specificity are required in disease endemic areas.

Methodology/Principal Findings

We generated novel monoclonal antibodies (MAbs) against the SFTSV nucleocapsid (N) protein and developed a sandwich antigen (Ag)-capture enzyme-linked immunosorbent assay (ELISA) for the detection of N protein of SFTSV using MAb and polyclonal antibody as capture and detection antibodies, respectively. The Ag-capture system was capable of detecting at least 350–1220 TCID₅₀/100 μl/well from the culture supernatants of various SFTSV strains. The efficacy of the Ag-capture ELISA in SFTS diagnosis was evaluated using serum samples collected from patients suspected of having SFTS in Japan. All 24 serum samples (100%) containing high copy numbers of viral RNA (>10⁵ copies/ml) showed a positive reaction in the Ag-capture ELISA, whereas 12 out of 15 serum samples (80%) containing low copy numbers of viral RNA (<10⁵ copies/ml) showed a negative reaction in the Ag-capture ELISA. Among these Ag-capture ELISA-negative 12 samples, 9 (75%) were positive for IgG antibodies against SFTSV.

Conclusions

The newly developed Ag-capture ELISA is useful for SFTS diagnosis in acute phase patients with high levels of viremia.     

A highly attenuated vaccinia virus strain LC16m8-based vaccine for severe fever with thrombocytopenia syndrome

Severe fever with thrombocytopenia syndrome (SFTS) caused by a species Dabie bandavirus (formerly SFTS virus [SFTSV]) is an emerging hemorrhagic infectious disease with a high case-fatality rate. One of the best strategies for preventing SFTS is to develop a vaccine, which is expected to induce both humoral and cellular immunity. We applied a highly attenuated but still immunogenic vaccinia virus strain LC16m8 (m8) as a recombinant vaccine for SFTS. Recombinant m8s expressing SFTSV nucleoprotein (m8-N), envelope glycoprotein precursor (m8-GPC), and both N and GPC (m8-N+GPC) in the infected cells were generated. Both m8-GPC- and m8-N+GPC-infected cells were confirmed to produce SFTSV-like-particles (VLP) in vitro, and the N was incorporated in the VLP produced by the infection of cells with m8-N+GPC. Specific antibodies to SFTSV were induced in mice inoculated with each of the recombinant m8s, and the mice were fully protected from lethal challenge with SFTSV at both 10³ TCID₅₀ and 10⁵ TCID₅₀. In mice that had been immunized with vaccinia virus strain Lister in advance of m8-based SFTSV vaccine inoculation, protective immunity against the SFTSV challenge was also conferred. The pathological analysis revealed that mice immunized with m8-GPC or m8-N+GPC did not show any histopathological changes without any viral antigen-positive cells, whereas the control mice showed focal necrosis with inflammatory infiltration with SFTSV antigen-positive cells in tissues after SFTSV challenge. The passive serum transfer experiments revealed that sera collected from mice inoculated with m8-GPC or m8-N+GPC but not with m8-N conferred protective immunity against lethal SFTSV challenge in naïve mice. On the other hand, the depletion of CD8-positive cells in vivo did not abrogate the protective immunity conferred by m8-based SFTSV vaccines. Based on these results, the recombinant m8-GPC and m8-N+GPC were considered promising vaccine candidates for SFTS.     

The ecology, genetic diversity, and phylogeny of Huaiyangshan virus in China

Surveys were carried out to better understand the tick vector ecology and genetic diversity of Huaiyangshan virus (HYSV) in both regions of endemicity and regions of nonendemicity. Haemaphysalis longicornis ticks were dominant in regions of endemicity, while Rhipicephalus microplus is more abundant in regions of nonendemicity. HYSV RNA was found in human and both tick species, with greater prevalence in H. longicornis and lesser prevalence in R. microplus. Phylogenetic analyses indicate that HYSV is a novel species of the genus Phlebovirus.     

Prevalence and Molecular Phylogenetic Analysis of Severe Fever with Thrombocytopenia Syndrome Virus in Domestic Animals and Rodents in Hubei Province,China

<正>Dear Editor,In 2009, an outbreak of an unknown infectious disease occurred in rural areas of Hubei Province, China, affecting17 persons, five of whom died. It was initially suspected to be human anaplasmosis(Zhang et al. 2008). However, the laboratory evidence was insufficient to verify the diagnosis     

No Detection of Severe Fever with Thrombocytopenia Syndrome Virus from Ixodid Ticks Collected in Seoul

Larvae, nymphs, and adult stages of 3 species of ixodid ticks were collected by tick drag methods in Seoul during June-October 2013, and their infection status with severe fever with thrombocytopenia syndrome (SFTS) virus was examined using RT-PCR. During the period, 732 Haemaphysalis longicornis, 62 Haemaphysalis flava, and 2 Ixodes nipponensis specimens were collected. Among the specimens of H. longicornis, the number of female adults, male adults, nymphs, and larvae were 53, 11, 240, and 446, respectively. Ticks were grouped into 63 pools according to the collection site, species, and developmental stage, and assayed for SFTS virus. None of the pools of ticks were found to be positive for SFTS virus gene.     

The nucleoprotein of severe fever with thrombocytopenia syndrome virus processes a stable hexameric ring to facilitate RNA encapsidation

Severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the Phlebovirus genus from the Bunyaviridae family endemic to China, is the causative agent of life-threatening severe fever with thrombocytopenia syndrome (SFTS), which features high fever and hemorrhage. Similar to other negative-sense RNA viruses, SFTSV encodes a nucleocapsid protein (NP) that is essential for viral replication. NP facilitates viral RNA encapsidation and is responsible for the formation of ribonucleoprotein complex. However, recent studies have indicated that NP from Phlebovirus members behaves in inhomogeneous oligomerization states. In the present study, we report the crystal structure of SFTSV NP at 2.8 ? resolution and demonstrate the mechanism by which it processes a ringshaped hexameric form to accomplish RNA encapsidation. Key residues essential for oligomerization are identifi ed through mutational analysis and identifi ed to have a signifi cant impact on RNA binding, which suggests that correct formation of highly ordered oligomers is a critical step in RNA encapsidation. The fi ndings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection.