Viral-host molecular interactions and metabolic modulation:Strategies to inhibit flaviviruses pathogenesis

Flaviviruses,which include globally impactful pathogens,such as West Nile virus,yellow fever virus,Zika virus,Japanese encephalitis virus,and dengue virus,contribute significantly to human infections.Despite the ongoing emergence and resurgence of flavivirus-mediated pathogenesis,the absence of specific therapeutic options remains a challenge in the prevention and treatment of flaviviral infections.Through the intricate processes of fusion,transcription,replication,and maturation,the complex interplay of viral and host metabolic interactions affects pathophysiology.Crucial interactions involve metabolic molecules,such as amino acids,glucose,fatty acids,and nucleotides,each playing a pivotal role in the replication and maturation of flaviviruses.These viral-host metabolic molecular interactions hijack and modulate the molecular mechanisms of host metabolism.A comprehensive understanding of these intricate metabolic pathways offers valuable insights,potentially unveiling novel targets for therapeutic interventions against flaviviral pathogenesis.This review emphasizes promising avenues for the development of therapeutic agents that target specific metabolic molecules,such as amino acids,glucose,fatty acids,and nucleotides,which interact with flavivirus replication and are closely linked to the modulation of host metabolism.The clinical limitations of current drugs have prompted the development of new inhibitory strategies for flaviviruses based on an understanding of the molecular interactions between the virus and the host.

Resveratrol as an epigenetic therapy for flavivirus infection:A narrative review

Flaviviruses are a group of positive-stranded RNA viruses that cause a broad spectrum of severe illnesses in humans worldwide.Clinical manifestations of flavivirus infections range from mild febrile illness to hemorrhage,shock,and neurological manifestations.Flavivirus infections cause a substantial global health impact,with an estimated more than 400 million cases of infections annually.Hence,an understanding of flavivirus-host interaction is urgently needed for new antiviral therapeutic strategies.In recent years,many aspects concerning epigenetic therapy for viral infections have been addressed,including methylation of the genome,acetylation/deacetylation of histone complex and microRNA regulation.In this context,we surveyed and reviewed the literature and summarized the epigenetic effects of resveratrol,a natural polyphenol with potential anti-viral properties,on flavivirus infections.

Development of Double Antibody Sandwich ELISA for Detection of Duck or Goose Flavivirus

In order to establish double antibody sandwich enzyme-linked immunosorbent assay （DAS-ELISA） for detection of duck or goose flavivirus, polyclonal antibody against the flavivirus strain JS804 in geese and monoclonal antibody against the E protein of flavivirus strain JS804 in geese were used as the capture antibody and detection antibody, respectively. The optimal dilution of the capture antibody and detecting antibody capable of detecting the flavivirus strain JS804 in geese were 1：3 200 and 1：160 in the check-board titration, respectively. The reaction time of sample was 1 h, and the optimal working dilution of HRP-labeled goat-anti-mouse IgG was 1：10 000. The positive standard value was 0.247 （OD450.m）. The geese flavivirus could be detected at a minimal concentration of 1.875 μg mL^-1. The ELISA had no cross-reaction with Newcastle disease virus （NDV）, Avian influenza virus （AIV）, Infectious bronchitis virus （IBV）, Infectious bursal disease virus （IBDV）, Duck hepatitis virus （DHV）, and Gosling plague virus （GPV）. Twenty clinical samples were detected by the DAS-ELISA and RT-PCR respectively, with the agreement rate of 75%. The results revealed that the DAS-ELISA possessed favorable specificity and higher sensitivity, indicating a suitable method for rapid detection of the duck or goose flavivirus.

The Flavivirus Protease As a Target for Drug Discovery

Many flaviviruses are significant human pathogens causing considerable disease burdens,including encephalitis and hemorrhagic fever,in the regions in which they are endemic.A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication,such as the viral protease.During viral replication,the flavivirus genome is translated as a single polyprotein precursor,which must be cleaved into individual proteins by a complex of the viral protease,NS3,and its cofactor,NS2B.Because this cleavage is an obligate step of the viral life-cycle,the flavivirus protease is an attractive target for antiviral drug development.In this review,we will survey recent drug development studies targeting the NS3 active site,as well as studies targeting an NS2B/NS3interaction site determined from flavivirus protease crystal structures.

A Virus-type Specific Serological Diagnosis of Flavivirus Infection Using Virus-like Particles

Many flaviviruses are emerging and reemerging pathogens, such as West Nile virus （WNV）, dengue virus （DENV）, yellow fever virus （YFV）, and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members, plaque reduction neutralization test （PRNT） is the only available assay to determine the infecting flavivirus type. Since PRNT requires culturing raw viruses, it must be performed in biosafety levet-3 or level-4 containment for many flaviviruses, and takes more than ten days to complete. To overcome these problems, we have developed flavivirus viral-like particles （VLPs） that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps： （i） VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h; （ii）the neutralized VLPs are used to infect Vero cells; and （iii） the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen （as quantified by the luciferase activities） is the etiologic agent. As a proof-of-concept, we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the ＂gold standard＂ of the classic PRNT; importantly, it shortens the assay time from 〉10 days to 〈1 day, and can be performed in biosafety level-2 facility.

Regulation of cell survival and death during Flavivirus infections

Flaviviruses, ss(+) RNA viruses, include many of mankind's most important pathogens. Their pathogenicity derives from their ability to infect many types of cells including neurons, to replicate, and eventually to kill the cells. Flaviviruses can activate tumor necrosis factor α and both intrinsic(Bax-mediated) and extrinsic pathways to apoptosis. Thus they can use many approaches for activating these pathways. Infection can lead to necrosis if viral load is extremely high or to other types of cell death if routes to apoptosis are blocked. Dengue and Japanese Encephalitis Virus can also activate autophagy. In this case the autophagy temporarily spares the infected cell, allowing a longer period of reproduction for the virus, and the autophagy further protects the cell against other stresses such as those caused by reactive oxygen species. Several of the viral proteins have been shown to induce apoptosis or autophagy on their own, independent of the presence of other viral proteins. Given the versatility of these viruses to adapt to and manipulate the metabolism, and thus to control the survival of, the infected cells, we need to understand much better how the specific viral proteins affect the pathways to apoptosis and autophagy. Only in this manner will we be able to minimize the pathology that they cause.

Real-time RT-PCR Assay for the Detection of Culex flavivirus

Based on the Culex flavivirus (CxFV) E gene sequences in GenBank, CxFV-specific primers and probes were designed for real-time reverse transcription-polymerase chain reaction (RT-qPCR). The specificity test revealed that CxFV could be detected using RT-qPCR with the specific CxFV primers and probes; other species of arboviruses were not detected. The stability test demonstrated a coefficient of variation of <1.5%. A quantitative standard curve for CxFV RT-qPCR was established. Quantitative standard curve analysis revealed that the lower detection limit of the RT-qPCR system is 100 copies/mu L. Moreover, RT-qPCR was used to detect CxFV viral RNA in mosquito pool samples. In conclusion, we established a real-time RT-PCR assay for CxFV detection, and this assay is more sensitive and efficient than general RT-PCR. This technology may be used to monitor changes in the environmental virus levels.

Development and Application of the Nested PCR Assay for Duck Flavivirus

[ Objective] Aim to establish a kind of efficient detection method for duck flavivirus（DFV）. E Method] The method of nested PCR based on flavivirus universal primers which were designed according to the GeneBank flavivirus gene sequence. [ Result] The degenerate universal prim ers Flav P1 -Flav P4 were designed by genome comparison to target NS5 gene conserved area. The system could only amplify flavivirus purpose gene and the sensitivity was 90 copies/iJL, higher than the ordinary PCR 1 000 times. Homology and evolutionary analysis showed that duck flavivirus belonged to mosquito-born flavivirus, NTAV group, similar with Tembusu and BYD virus. [ Conclusion] Primers of the nested PCR system had good universality and specificity and method had high sensitivity. This system successfully detected flavivirus and clarified the evolution station of DFV.

Cloning and Sequence Analysis of E Gene from Chicken Flavivirus Isolate CJD05

[ Objective] This experiment aimed to find out the origin and genetic evolution relationship of chicken flavivirus （CFV） CJD05 strain in Fujian Province. [Method] A pair of primers were designed and synthesized according to the sequences of E gene from Duck flavivirus （DFV） iso- late BYD-1. E gene of CFV isolate CJD05 was specially amplified and its sequences were analyzed. [Result] The target bar which was cloned from CFV isolate C, JD05 was I 503 bp. Homology analysis was conducted to compare E gene nucleotide sequence of CFV isolate CJD05 with DFV iso- late BYD-I and goose flavivirus （GFV） isolate JS804. Results indicated that isolate nuclectide homologies were 99.2% and 99.3%, and amino acid homologies were 99.0% and 98.6% respectively. [Conclusion] CFV isolate C, JD05, DFV isolate BYD-1 and GFV isolate JS804 were highly homologous. The homology of CFV isolate CJD05 with Tembusu virus （TMUV） was higher than with other arthropod-borne flaviviruses.

Research Progress on a Novel Duck Flavivirus Disease

A severe egg-drop disease caused by the infection of a novel duck flavivirus outbroke successively in many provinces in southeastern China since 2010.It was identified as a duck Tembusu virus（DTMUV）and named by Chinese Association of Animal Science and Veterinary Medicine in the first symposium on waterfowl disease control,which was presumed to be a mosquito-borne flavivirus of the Ntaya virus subgroup in the genus Flavivirus,family Flaviviridae.Currently,a large number of studies have been conducted on the epidemiology,clinical symptoms and pathological changes,etiology,and rapid diagnoses of the virus.The disease remains a constant threat to the duck industry.In order to provide reference for subsequent in-depth study,in this paper,research progress on the disease was summarized based on previous studies.Furthermore,the potential infection or asymptomatic infection in humans should be evaluated as soon as possible.

A broadly protective antibody that targets the flavivirus NS1 protein

There are no approved flaviviral therapies and the development of vaccines against flaviruses has the potential of being undermined by antibody-dependent enhancement(ADE).The flavivirus nonstructural protein 1(NS1)is a promising vaccine antigen with low ADE risk but has yet to be explored as a broad-spectrum therapeutic antibody target.Here,we provide the structural basis of NS1 antibody cross-reactivity through cocrystallization of the antibody 1G5.3 with NS1 proteins from dengue and Zika viruses.

外泌体在蚊媒黄病毒感染中的调控作用研究

外泌体是一种广泛存在于人体体液中的由真核细胞分泌的一种细胞外囊泡,直径30~100nm。根据其生物发生和结构的特性,外泌体可以携带宿主细胞中的生物信息物质至其他细胞,在细胞间通信、生理病理反应中起着重要作用。受全球气候环境影响,蚊媒传染病逐渐对人类健康造成威胁,其中一种重要的病原体为黄病毒科病毒,包括登革病毒、寨卡病毒和流行性乙型脑炎病毒等。外泌体在黄病毒感染的过程中起着至关重要的作用,可促进病毒传播或激活宿主的免疫反应对抗病毒。本文主要总结了外泌体在蚊媒黄病毒科病毒感染中发挥调控作用的研究进展,以便更深入地了解病毒与宿主细胞的相互作用,从而开发出可作为防治蚊媒传染病的候选细胞靶点。

蚊媒黄病毒传播机制及疫苗与药物研发进展

蚊媒病毒可通过蚊虫叮咬在宿主和蚊虫媒介之间传播循环。由蚊媒传播的病毒达数百种之多,每年可造成数亿人感染,这些病毒感染可引起严重的人类疾病,如出血热、关节炎、脑炎和脑膜炎等,严重者可导致死亡。蚊媒病毒大部分为RNA病毒,其中黄病毒科的蚊媒病毒流行最为广泛,包括登革病毒、寨卡病毒、黄热病毒、乙型脑炎病毒和西尼罗病毒等。目前虽然已有针对少数蚊媒黄病毒的有效疫苗,例如预防黄热病病毒和乙型脑炎病毒的疫苗,但仍然没有针对大多数蚊媒黄病毒的有效预防性疫苗和抗病毒疗法。因此,全面了解蚊媒黄病毒在脊椎动物宿主与蚊虫之间的感染与传播的机制,可能为抗蚊媒黄病毒的疫苗与药物研发提供新的思路与目标,从而使我们能够更有效地预测和控制蚊媒黄病毒在自然界传播和流行,为应对蚊媒黄病毒造成的公共卫生威胁提供新的解决方案。本文首先描述了蚊媒黄病毒的生物学特性和流行病学特性,接下来介绍了蚊媒黄病毒的传播途径与媒介模型,并进一步全面总结了目前对蚊媒黄病毒在宿主和媒介之间传播机制及病毒在蚊虫媒介中的感染机制的研究,同时针对蚊媒黄病毒的新型疫苗研发和药物筛选策略,对未来针对蚊媒黄病毒的机制研究与抗病毒策略开发进行了展望。

一种从鸭新分离的黄病毒研究初报

从以产蛋下降为主的樱桃谷种鸭以及出现神经症状的雏鸭各分离出1株病毒,分别命名为BZ株和LC株。该2株病毒对SPF鸡胚和健康鸭胚均能产生相同的病变,分离病毒不能凝集鸡、鸭、鹅、鸽等的红细胞,在鸭胚成纤维细胞(DEF)能够产生典型的细胞病变(CPE),电镜下观察到约50nm的病毒粒子。病理组织学研究表明,二者在临床上均可导致脑组织危害,表现为脑膜水肿、血管充血和皮质层神经胶质细胞增生等。血清学检测表明,分离病毒与禽流感病毒(AIV)、鸭瘟病毒(DEV)、新城疫病毒(NDV)等病原无交叉。生物学特性鉴定该病原为有囊膜单股RNA病毒。利用不同禽病的特异性引物分别进行PCR或RT-PCR,均未扩增出特异条带。设计随机引物进行RT-PCR,扩增出基因片段,利用GenBank进行Blast同源比较,结果发现,分离病毒与以色列火鸡脑膜脑炎病毒(Israel turkey meningo-encephalitis virus,TMEV)和在马来西亚发现的Tembumu病毒至少在2段基因上具有较高的同源性,属于黄病毒属。测序表明,分离病毒与Tembumu病毒的非结构蛋白(NS5基因)和囊膜蛋白(E基因)的核苷酸同源性为86.7%～90.2%和87.0%～91.8%,与TMEV的NS5基因和E基因的同源性为72.4%～73.2%和72.7%～72.8%。2分离株之间E基因和NS5基因的核苷酸同源性均为99.5%。血清中和试验表明,BZ株阳性血清可以中和LC病毒,因此证实二者可能是同一种病毒。综合以上研究,建议将该病命名为"鸭病毒性脑炎"(Duck viral encephalitis disease)。

鹅源黄病毒的分离和初步鉴定

本试验从广东地区临床表现为体温升高、采食量减少、拉稀粪、站立不稳、运动障碍的幼龄病鹅群,无菌采集病料,进行病原的分离传代。研究结果表明,该病毒对番鸭胚的半数致死量(ELD50)为10-2.68/0.2mL,在pH 7.2的条件下不能凝集鸭、鸡、鹅、鸽的红细胞,人工感染15日龄健康幼鹅能复制出与自然病例相同的临床症状和病变。用BYD病毒E基因片段的特异性引物,对试验获得毒株进行RT-PCR扩增和序列分析,证明获得的序列与GenBank中收录的发现于中国引起鸭产蛋下降综合征的新型黄病毒—BYD病毒(登录号为JF312912)和中国江苏发现的鹅黄病毒JS804株(登录号为JF895923)的同源性最高(有2个碱基的差异),均达到了99%以上,对试验获得的毒株暂定为鹅源BYD病毒广东株1(简称为BYD-GD1)。

鸭出血性卵巢炎病毒RT-PCR检测方法的建立

参照GenBank上登录的黄病毒科黄病毒属NS5基因片段序列设计引物,建立能检测引起鸭出血性卵巢炎病毒的RT-PCR方法。该方法能从鸭出血性卵巢炎病毒扩增出1条约470 bp的特异性片段,从H9亚型禽流感病毒、鸭呼肠孤病毒、鸭瘟病毒、鸭肝炎病毒、鸭源禽Ⅰ型副粘病毒均不能扩增出目的片段;敏感性试验显示建立的RT-PCR方法最低可检出20 pg病毒核酸。以上结果表明建立的RT-PCR方法具有较好的特异性、敏感性和准确性,可用于该病的临床诊断和流行病学调查。

新型黄病毒在种鹅中垂直传播的研究

【目的】证实新型黄病毒在种鹅中是否存在垂直传播,为控制该病提供可靠的流行病学依据。【方法】采用巢式RT-PCR和病毒分离技术,对患病种鹅病料、不同孵化阶段死胚、出雏蛋壳内部冲洗液、弱雏样品进行黄病毒分离及基因检测。【结果】在患病种鹅、孵化过程中死亡鹅胚尿囊液、出雏蛋壳内壁洗液和雏鹅中均能检测和分离到黄病毒,其中患病种鹅病料的黄病毒阳性率为100.0%,孵化死亡鹅胚黄病毒阳性率为39.6%,出雏蛋壳内部冲洗液黄病毒阳性率为45.0%;1日龄和15日龄弱雏黄病毒阳性率为80.0%。【结论】黄病毒可以通过鹅胚传到下一代,即新型黄病毒在种鹅中存在垂直传播的现象。

新型鹅黄病毒JS804毒株的分离与鉴定

2010年4月至11月,江苏等地鸭、鹅发生了一种以产蛋率、采食量显著下降,出现脑炎样神经症状为特征的传染病。对发病鹅进行剖检观察,鸭胚尿囊腔途径接种发病鹅病料分离病原,电镜观察病毒粒子。对健康鹅接种分离病毒进行动物回归试验。在病毒核酸背景未知的情况下,应用非序列依赖性单引物扩增法结合DNA酶处理（DNase-sequence independent single primer amplification,DNase-SISPA）对病原基因进行扩增,并在此基础上设计了1对特异性引物对病毒基因进行PCR扩增。剖检结果发现病鹅的脑膜、肺脏、肝脏、心脏、卵巢等多器官出血,脾脏肿大坏死。含毒鸭胚尿囊膜超薄切片电镜观察显示：病毒粒子直径为50~60 nm。动物回归试验成功复制出该病并分离到病毒。应用DNase-SISPA方法发现了3个病毒相关基因片段,经序列比对分析,与黄病毒属（Flavi-virus）坦布苏病毒（Tembusu virus）基因序列有很高的同源性,分别为96%、88%、93%。据此设计特异性引物扩增出一段985 bp基因片段,该片段与黄病毒属坦布苏病毒E基因的核苷酸同源性为91%,氨基酸同源性为97%。将分离的鹅黄病毒毒株命名为Goose/Jiangsu/804/2010（简称JS804）。研究结果表明：此次鸭、鹅新发疫病的病原为一种新的黄病毒。

海南省虫媒病毒分离物的初步鉴定

目的：鉴定海南省虫媒病毒分离物。方法：现场捕蚊，分离与鉴定虫媒病毒，主要方法为逆转录聚合酶链反应等。结果：１９９５年从海南省琼中县大丰农场、三亚市立才农场、万宁县兴隆农场、乐东县尖峰林场及西沙捕获的４种蚊虫中分离到１４株虫媒病毒，均可在Ｃ６／３６细胞、ＶｅｒｏＥ６细胞组织培养中增殖，并产生明显的病变；对酸、乙醚敏感，抵抗５－碘脱氧尿苷，属ＲＮＡ病毒。结论：经血清学和逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）初步鉴定，有８株为黄病毒科病毒。

一种新的黄病毒导致蛋鸭产蛋下降及死亡

为探求2010年在上海、浙江和江苏等地鸭群出现产蛋下降甚至停止的发病原因,对浙江省4个发病鸭场发病鸭进行病理剖检以及病毒的PCR鉴定。解剖病鸭发现其脾脏肿大明显,肝脏有针尖状白色点状坏死。进一步的病理学变化分析发现病鸭的肝脏呈现程度不一的脂肪变性和空泡变性,肝细胞呈现局灶性溶解,脾细胞坏死。采集临床发病鸭的脾脏,提取总RNA,用随机引物进行反转录,然后用多种病毒的特异性引物进行PCR鉴定。结果显示,4个鸭场的病料呈鸭黄病毒PCR阳性,其他病毒呈阴性,说明这些鸭场均感染了鸭黄病毒,此病毒在浙江省已广泛流行。