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.

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。

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.

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.

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.