A high throughput antiviral screening platform for alphaviruses based on Semliki Forest virus expressing eGFP reporter gene

Alphaviruses,which contain a variety of mosquito-borne pathogens,are important pathogens of emerging/reemerging infectious diseases and potential biological weapons.Currently,no specific antiviral drugs are available for the treatment of alphaviruses infection.For most highly pathogenic alphaviruses are classified as risk group-3 agents,the requirement of biosafety level 3(BSL-3)facilities limits the live virus-based antiviral study.To facilitate the antiviral development of alphaviruses,we developed a high throughput screening(HTS)platform based on a recombinant Semliki Forest virus(SFV)which can be manipulated in BSL-2 laboratory.Using the reverse genetics approach,the recombinant SFV and SFV reporter virus expressing eGFP(SFV-eGFP)were successfully rescued.The SFV-eGFP reporter virus exhibited robust eGFP expression and remained relatively stable after four passages in BHK-21 cells.Using a broad-spectrum alphavirus inhibitor ribavirin,we demonstrated that the SFV-eGFP can be used as an effective tool for antiviral study.The SFV-eGFP reporter virus-based HTS assay in a 96-well format was then established and optimized with a robust Z0 score.A section of reference compounds that inhibit highly pathogenic alphaviruses were used to validate that the SFV-eGFP reporter virus-based HTS assay enables rapid screening of potent broad-spectrum inhibitors of alphaviruses.This assay provides a safe and convenient platform for antiviral study of alphaviruses.

Visualization of the Oncolytic Alphavirus M1 Life Cycle in Cancer Cells

Oncolytic alphavirus M1 has been shown to selectively target and kill cancer cells,but cytopathic morphologies induced by M1 virus and the life cycle of the M1 strain in cancer cells remain unclear.Here,we study the key stages of M1 virus infection and replication in the M1 virus-sensitive HepG2 liver cancer cell line by transmission electron microscopy,specifically examining viral entry,assembly,maturation and release.We found that M1 virus induces vacuolization of cancer cells during infection and ultimately nuclear marginalization,a typical indicator of apoptosis.Specifically,our results suggest that the endoplasmic reticulum participates in the assembly of nucleocapsids.In the early and late stage of infection,three kinds of special cytopathic vacuoles are formed and appear to be involved in the replication,maturation and release of the virus.Taken together,our data displayed the process of M1 virus infection of tumor cells and provide the structural basis for the study of M1 virus-host interactions.

In Vitro Inhibition of Alphaviruses by Lycorine

Chikungunya virus(CHIKV) is a mosquito-borne alphavirus. As an emerging virus, CHIKV imposes a threat to public health. Currently, there are no vaccines or antivirals available for the prevention of CHIKV infection. Lycorine, an alkaloid from Amaryllidaceae plants, has antiviral activity against a number of viruses such as coronavirus, flavivirus and enterovirus. In this study, we found that lycorine could inhibit CHIKV in cell culture at a concentration of 10 lmol/L without apparent cytotoxicity. In addition, it exhibited broad-spectrum anti-alphavirus activity, including Sindbis virus(SINV),Semliki Forest virus(SFV), and Venezuelan equine encephalomyelitis virus(VEEV). The time of addition studies indicated that lycorine functions at an early post-entry stage of CHIKV life cycle. The results based on two different CHIKV replicons provided further evidence that lycorine exerts its antiviral activity mainly by inhibiting CHIKV translation.Overall, our study extends the antiviral spectrum of lycorine.

鲑鱼甲病毒E1基因高保守区融合表达及免疫特性的分析

根据Gen Bank中公布的鲑鱼甲病毒(salmonid alphavirus,SAV)SAV 1、SAV 2和SAV3三个基因型中E1基因,选择高保守序列702 bp(436-1137)合成基因,命名为SAV E1,将其克隆到原核表达载体p Cold TF中构建重组质粒。然后将重组质粒转化到大肠杆菌感受态细胞BL21中,经终浓度为1.0 mmol/L的IPTG诱导表达,SDSPAGE和Western blot鉴定,重组蛋白均获得了表达,表达E1重组蛋白约95 k D。用镍离子亲和层析柱纯化重组蛋白,制备抗血清。间接ELISA结果显示,鼠抗重组蛋白E1血清效价为1∶25 600;间接免疫荧光结果显示,鼠抗重组E1蛋白血清可与SAV发生特异反应,由此表明表达的E1重组蛋白具有良好的免疫原性和免疫反应性,为SAV检测方法的建立提供理论依据。

海南岛不明发热病人血虫媒病毒分离鉴定及抗体检测

目的从海南岛不明发热病人血中分离和鉴定虫媒病毒,并在当地人群进行抗体检测,以了解南方地区虫媒病毒的感染情况。方法采用微量法细胞培养对205份发热原因不明患者血标本进行病毒分离,应用生物学性状检测、间接免疫荧光试验(IFA)和血凝抑制试验(HI)等技术对分离病毒进行鉴定。采用IFA检测从当地人群血清中分离出的病毒的抗体水平。结果从发热早期患者血标本中分离出2株病毒,鉴定为甲病毒,命名为HF 7和HC 6。这2株病毒与8种甲病毒免疫腹水中的SIN的免疫腹水呈现最高的血凝抑制滴度(分别为1∶160和1∶320),与SIN免疫腹水的交叉荧光效价最高(分别为1∶320和1∶640),但HF 7和HC 6病毒的免疫腹水与8种甲病毒的血凝抑制试验和交叉免疫荧光试验无反应,表明HF 7和HC 6病毒虽与SIN病毒的免疫腹水血抑效价及荧光效价高,但HF 7和HC 6的免疫腹水与SIN病毒无抑制效价和免疫荧光效价。当地人群对这两株病毒感染的抗体阳性率分别为3.3%(15/451)和8.4%(38/451)。结论HF7和HC6为甲病毒的一个新种。海南岛人群存在甲病毒感染。

Chikungunya infection:A potential re-emerging global threat

Infectious diseases are indeed a lifelong threat to everyone irrespective of age, sex, lifestyle and socio-economic status. The infectious diseases have persisted among the prominent causes of death globally. Recently, re-emergence of Chikungunya viral infection harmed many in Asian and African countries. Chikungunya was considered as a major threat in developing and underdeveloped countries; the recent epidemiological outbreak of Chikungunya in La Reunion urges the global researchers to develop effective vaccine against this viral disease. In this review, Chikungunya, pathogenesis and epidemiology were briefly described.

Architecture and biogenesis of plus-strand RNA virus replication factories

Plus-strand RNA virus replication occurs in tight association with cytoplasmic host cell membranes. Both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replication factories. This compartmentalization allows coordination of the different steps of the viral replication cycle, highly efficient genome replication and protection of the viral RNA from cellular defense mechanisms. Electron tomography studies conducted during the last couple of years revealed the three dimensional structure of numerous plus-strand RNA virus replication compartments and highlight morphological analogies between different virus families. Based on the morphology of virusinduced membrane rearrangements, we propose two separate subclasses: the invaginated vesicle/spherule type and the double membrane vesicle type. This review discusses common themes and distinct differences in the architecture of plus-strand RNA virus-induced membrane alterations and summarizes recent progress that has been made in understanding the complex interplay between viral and co-opted cellular factors in biogenesis and maintenance of plus-strand RNA virus replication factories.

Drug Therapeutic in the Subacute and Chronic Phase of Chikungunya Virus Infection

Background: Chikungunya fever is an infectious disease that can evolve to a subacute or chronic condition, with changes in the daily activities of patients. Drugs that aim to reduce these symptoms are used, such as corticoids (acute phase) and disease-modifying anti-rheumatic drugs (chronic phase). Objective: To evaluate the clinical response to drug therapy in the subacute and chronic phase of infection by the Chikungunya virus. Methodology: A prospective and a retrospective study with patients with subacute and chronic Chikungunya infection, out at the infection and autoimmunity outpatient clinic at the Nucleus of Tropical Medicine, from January 2016 to December 2019, in the morning of Thursdays. The patient was observed in the Baseline, first and second return, and drugs were introduced according to the stage of the disease with subsequent reassessment. The Visual Analogue Scale (VAS) was applied to all evaluation moments. Results: 101 patients were evaluated, and arthralgia was the predominant symptom in the three evaluated moments. According to the VAS, moderate baseline pain was observed in 58.1% and 58.6% of subacute and chronic cases, respectively. On the first return, moderate pain still predominated in 46.2% in subacute cases and 43% in chronic cases. In the second visit, all patients were in the chronic phase of the disease, 43.8% had VAS with no pain. Regarding the number of compromised joints in the Baseline, polyarticular involvement predominated in both subacute (79%) and chronic (74.1%) cases, in the first return, oligoarticular involvement predominated in 53.8% of subacute cases and 54.7% in chronic cases and, the second return, 40.6% of the patients had oligoarticular involvement and 43.8% had no joint involvement. As for the use of medications in the Baseline, 33.4% of subacute cases used antiinflammatory drugs, and 40% of chronic cases used corticosteroids. At the first visit, 25% of chronic patients were already using combined corticosteroids and methotrexate and 15% were using only methotrexate. In the second return, 35.1% used combined methotrexate and corticosteroids, and 64.9% used only methotrexate. Safety in the use of methotrexate was observed in the context of CHIKV treatment, as the number of adverse reactions was minimal (three patients) and the medication was well tolerated. Conclusion: It was observed that with the adjustment of the medications, there was a reduction in joint impairment, VAS showed mild pain indexes and in some cases with no pain, showing the benefit of using therapy in subacute and chronic cases and improving quality of life of these users.

甲病毒载体的研究进展

甲病毒属于披膜病毒科,是一类有包膜的单股正链RNA病毒。近些年来,国内外有关甲病毒载体的研究非常活跃,其已被用于疫苗研究、基因治疗以及分子生物学等研究领域。基于甲病毒载体的构造原理,将其分为三类,分别为复制完全型载体、复制缺陷型载体和DNA/RNA载体。就这三类载体的改造过程进行了探讨,并对这三类载体的特点及其应用研究进展等方面进行了阐述,以期为开发出更安全、更有效的甲病毒载体提供参考。

Structural basis of Semliki Forest virus entry using the very-low-density lipoprotein receptor

Alphaviruses are a group of important viruses that cause significant diseases in humans.Among them,Semliki Forest vi-rus(SFV)not only causes symptoms such as joint pain but also infects neuron cells and induces encephalitis in rodents.Recently,the very-low-density lipoprotein receptor(VLDLR)was identified as the cellular receptor for SFV entry.In this study,we present the cryo-electron microscopy structure of SFV bound to human VLDLR.VLDLR targets E1-DIII region of SFV using its membrane-distal LDLR class A(LA)repeats.Structural and functional analyses emphasize the synergistic role of multiple VLDLR repeats in the SFV entry.Remarkably,VLDLR’s binding mode to SFV closely mirrors that of minor group human rhinoviruses but differs significantly from other alphaviruses’interactions with receptors in the canyon re-gion of the E protein.We also assessed SFV binding to VLDLR or apolipoprotein E receptor 2(ApoER2)proteins in horses and mosquitoes and revealed their use of multiple but different LA repeats for binding.Our findings illuminate SFV’s cross-species infectivity,offering insights into potential antiviral strategies against alphavirus infections.

从海南岛蚊和蜱分离出28株甲病毒属病毒

１９８３～１９８８年在海南岛从自然界捕蚊和蜱分离出非登革病毒和非日本脑炎病毒的２８株病毒，其中２７株是从不同种的蚊分离出的，１株是从蜱分离出的。９０年代初期在美国疾病控制中心的媒介传播的病毒性疾病分部，用全套国际标准化的抗节肢动物携带的病毒的抗体，对这２８株病毒进行了病毒科的鉴别和病毒属的鉴定，结果证明这２８株病毒都属于披膜病毒科的甲病毒属。

甲病毒检测细胞株的构建及其功能鉴定

目的 构建和鉴定可用于甲病毒快速检测的工程细胞株.方法 以辛德毕斯病毒缺陷型复制子载体系统pVaXJ-EGFP-△NSP4为基础,利用慢病毒表达载体pCDH系统,获得含有绿色荧光蛋白（Enhanced green fluorescent protein,EGFP）报告基因的慢病毒表达质粒pCDH-XJ-EGFP.使用脂质体转染法将质粒pCDH-XJ-EGFP和包装质粒pPACKHl Mix共转染HEK293T细胞,48 h、72 h收集慢病毒颗粒用于感染BHK-21细胞,经嘌呤霉素筛选获得用于蚊媒甲病毒检测的细胞系BHK-Alphavirus.结果 BHK-Alphavirus感染辛德毕斯病毒48 h后,可检测到EGFP的表达,表明BHK-Alphavirus能够用于外源甲病毒的检测.BHK-Alphavirus细胞感染甲病毒属中的辛德毕斯病毒（XJ-160和YN87448）、基孔肯雅病毒（SD08Pan）及盖塔病毒（HB0234）均可以检测到特异的绿色荧光,而被黄病毒属的乙脑病毒（P3）、4型登革病毒（P4）和布尼亚病毒属的塔希纳病毒（XJ0625）等其他正链RNA病毒感染后则检测不到绿色荧光.结论 BHK-Alphavirus细胞可用于蚊媒甲病毒的快速检测,且该检测方法特异性良好.

抗甲病毒化合物高通量筛选模型的建立与应用

建立抗甲病毒化合物高通量筛选模型可为筛选和分析抗甲病毒药靶提供技术基础。本研究以含有分泌型荧光素酶(Gaussialuciferase,GLUC)报告基因标记的重组辛德毕斯病毒XJ160-GLUC为分子基础,建立了抗甲病毒化合物的高通量筛选模型,并对感染复数、检测时间等参数进行优化。经过优化,筛选模型的Z’因子值可达到0.71,表明我们所建立的筛选模型具有较好的稳定性。应用该筛选模型对8080个五合一样品进行筛选,最终获得19个具有抗甲病毒活性的阳性化合物。本研究所建立的抗甲病毒化合物高通量筛选模型及抗甲病毒阳性化合物的发现为抗甲病毒靶标相关的研究提供了新的思路和信息。

从烟台地区蚊虫分离到披膜甲属虫媒病毒

目的 从烟台地区自然界捕捉的蚊虫中分离虫媒病毒。方法 对分离的 15株虫媒病毒中的 3株进行理化特性分析 ,电镜检查以及特异性免疫反应观察。结果 多次实验均显示该病毒对乙醚敏感 ,抵抗 5′ 碘脱氧尿苷 (5′ Idu) ,能在白蚊伊蚊C6 / 36传代细胞上生长繁殖 ,并能引起典型细胞病变 ,乳鼠脑内接种不引起规律发病和死亡。电镜观察 ,可见大小相同的球形病毒颗粒 ,毒粒直径约 (5 5± 2 3)nm。该病毒与乙脑病毒和布尼亚病毒科的组特异性免疫腹水均不反应 ,而与披膜病毒科甲属特异性免疫腹水有交叉反应。结论 本次分离到的病毒为披膜甲属虫媒病毒。