Phylogenetic Analysis of the Flavivirus-associated Encephalitis Syndromes, Development and Application of a New Molecular Marker for the Rapid Characterization of Flaviviruses

The aim of this study is to explore the phylogenetic relationship of flaviviruses with the known viruses and to establish a rapid PCR-based method for the characterization of the flaviviruses. To this end, phylogenetic analysis of 25 different strains of flaviviruses was carried out on the basis of the full length genomic sequences as well as the sequences of the individual genes and their untranslated regions (UTRs). From this analysis and the extensive sequence alignment studies, a generic primer pair was identified for amplification of a highly conserved region in the NS5 gene, a molecular marker designed as NS5MM in this study, which was different from NS5 region used previously by other groups. This generic primer pair had been validated by using 22 different strains of flaviviruses in the present study. Furthermore, we have successfully applied this new approach to the rapid identification and characterization of 3 new strains of flaviviruses recently isolated in China.

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.

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.

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.

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.

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.

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.

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.

逆转录聚合酶链反应与常规法检测蚊体内黄病毒的比较研究

目的：检测比较蚊体内的黄病毒。方法：采用RT－PCR技术及常规的C6／36细胞培养结合免疫荧光法。结果：无论实验感染的登革热病毒蚊虫标本，还是从野外捕捉的标本，均表明RT－PCR检出黄病毒的阳性率比常规法高，且快速、敏捷，操作简便；常规法检出周期长（2～3周），操作复杂、繁琐和反复多次，检出率低。但常规法能通过细胞病变，观察病毒的毒力，这是RT－PCR所不及的。结论：以RT－PCR结合常规法进行虫媒病毒的研究，将更有利于探索蚊虫与虫媒病毒的关系和内在规律。

鸭坦布苏病毒E蛋白抗原表位的鉴定及其表位交叉反应性分析

为鉴定鸭坦布苏病毒(DTMUV)E蛋白的抗原表位,本研究以纯化的抗DTMUV E蛋白单克隆抗体(MAb)3B6为固相筛选分子,对噬菌体随机12肽库进行淘选,通过对阳性噬菌体克隆测序分析,确定DTMUV E蛋白的模拟表位氨基酸序列,将模拟表位序列与GST蛋白融合表达并进行western blot验证。利用DNAStar对表位序列进行同源性分析,并通过dot blotting方法分析抗原表位在黄病毒阳性血清间的交叉反应性。结果鉴定了DTMUV E蛋白的一个线性抗原表位EVEPPFG,并且该表位在DTMUV与其它黄病毒中均具有高度的保守性。Dot blotting结果显示抗原表位EVEPPFG在黄病毒阳性血清间具有交叉反应性。因此,EVEPPFG为黄病毒共享型的抗原表位,这对于黄病毒E蛋白的抗原结构功能的研究以及研制表位疫苗和诊断抗原等具有重要意义。

黄病毒的分子生物学研究进展

黄病毒属为一类带有包膜的正链ＲＮＡ病毒，可以引起许多种人类疾病。目前，这类病毒的分子生物学研究已经进入了一个突破性的阶段。本文综述总结了我们了解的该病毒科病毒生命周期内的分子生物学。

黑龙江与吉林的部分地区蜱传黄病毒的检测与鉴定

为检测黑龙江、吉林部分地区蜱传黄病毒的多样性,本研究采用布旗法、动物体表摘取法于2018年~2019年对黑龙江、吉林9个采集点进行蜱采集,应用形态学、16S rRNA鉴定蜱种类,并建立RT-PCR方法对蜱携带的黄病毒进行检测,对获得的病毒基因序列进行遗传进化分析。结果显示:采集了2402只蜱,分属于硬蜱科革蜱属、血蜱属和硬蜱属,其中森林革蜱占60.3%(1449/2402)、日本血蜱占14.1%(339/2402)、嗜群血蜱占8.0%(192/2402)和全沟硬蜱占17.6%(422/2402)。从黑龙江全沟硬蜱中检测到阿龙山病毒(ALSV),扩增到1531 nt的NS3-like基因;吉林日本血蜱中检测到恩格耶病毒(NGOV),扩增到916 nt的NS5基因;吉林省森林革蜱中检测到蜱传脑炎病毒(TBEV),获得11051 nt该全长病毒基因组。遗传进化分析显示,ALSV与黑龙江ALSV H3株属于同一进化分支,NGOV是一种新发现的蜱传黄病毒,所获得的TBEV属远东型,与疫苗株同源性为93.86%。本研究初步揭示了该地区的优势蜱种分布,并首次发现了蜱传新型黄病毒NGOV,为该地区蜱传黄病毒的流行情况提供了重要的基础数据。

Recent progress in dengue vaccine development

Dengue virus(DENV) has four distinct serotypes. DENV infection can result in classic dengue fever and life-threatening dengue hemorrhagic fever/dengue shock syndrome. In recent decades, DENV infection has become an important public health concern in epidemic-prone areas. Vaccination is the most effective measure to prevent and control viral infections. However, several challenges impede the development of effective DENV vaccines, such as the lack of suitable animal models and the antibody-dependent enhancement phenomenon. Although no licensed DENV vaccine is available, significant progress has been made. This review summarizes candidate DENV vaccines from recent investigations.

未分类黄病毒研究进展

对未分类黄病毒的发现过程、结构特征、与其他黄病毒关系等方面做了介绍,并重点对未分类黄病毒的研究进展进行了综述。

Clinical implications and treatment of dengue

Dengue is a common pathogenic disease often proving fatal,more commonly affecting the tropics.Aedes mosquito is the vector for this disease,and outbreaks of dengue often cause mass damage to life.The current review is an effort to present an insight into the causes,etiology,symptoms,transmission,diagnosis,major organs affected,mitigation and line of treatment of this disease with special emphasis on dings of natural origin.The disease has a potential to spread as an endemic,often claiming several lives and thus requires concerted efforts to work out better treatment options.Traditional medicine offers an alternative solution and could be explored as a safer treatment option.Development of a successful vaccine and immunization technique largely remains a challenge and a better antiviral approach needs to be worked out to complement the supportive therapy.No single synthetic molecule has found to be wholly effective enough to offer curative control and the line of treatment mostly utilizes a combination of fluid replacement and antipyretics-analgesics like molecules to provide symptomatic relief.

An immunoglobulin Y that specifically binds to an in silico-predicted unique epitope of Zika virus non-structural 1 antigen

Objective:To identify unique immunogenic epitopes of Zika virus non-structural 1(NS1)antigen and produce immunoglobulin Y(IgY)for potential use in he diagnosis of of Zika virus infection.Methods:Immunogenic epitopes were identified using in silico B-cell epitope prediction.A synthetic peptide analog of the predicted epitope was used to induce antipeptide IgY production in hens which was purified using affinity chromatography.Presence of purified IgY and its binding specificity were performed by gel electrophoresis and ELISA,respectively.Results:Out of the nine continuous epitopes identified,the sequence at position 193-208(LKVREDYSLECDPAVI)was selected and used to produce anti-peptide IgY.The produced IgY was found to bind to the synthetic analog of the Zika virus NS1 immunogenic epitope but not to other flaviviruses and random peptides from other pathogens.Conclusions:In this study,we identified an immunogenic epitope unique to Zika virus that can be used to develop a serodiagnostic tool that specifically detect Zika virus infection.

Construction of an insecticidal baculovirus expressing insect-specific neurotoxin AaIT

Considering the factors which affect gene transcription, translation and the stability of mRNA, without changing the amino acid composition of the encoded polypeptide, AaIT gene encoding insect-specific neurotoxin was designed and synthesized according to bias in codon choice, overall G+C content and G + C content of bases at the third position in codons of polyhedrin genes of baculovirus and of plant genes as well. AaIT gene was fused behind a synthetic gp67 signal sequence and then recombined into the genome of Trichoplusia ni nuclear polyhedrosis virus (TnNPV) by transfer vector pSXIV VI+X3. The recombinant virus TnNPV-AalT (occ+-gal-) was screened. The results of Southern blotting and SDS-PAGE demonstrated that AaIT gene had integrated into the genome of virus and expressed. Bioassays on the 3rd-instar Trichoplusia ni larvae showed that recombinant viruses TnNPV-AalT could shorten the time of killing insect and improve the efficiency of killing agronomically important insects.

Biological and historical overview of Zika virus

The recent outbreak of the Zika virus attracts worldwide attention probably because the most recently affected country(Brazil) will host the 2016 Olympic Game. Zika virus infected cases are now spreading to many other countries and its infection might be linked to some severe medical sequelae. Since its first isolation from the infected monkey in 1947 in Uganda, only a few studies had been taken until recent outbreak. According to the history of referenced publications, there is a 19-year gap from 1989 to 2007. This might be because only mild diseases were diagnosed from Zika virus infected populations. Obviously, the recent reports that Zika virus infection is probably associated with microcephaly of the neonates makes us reevaluate the medical significance of the viral pathogen. It can be transmitted sexually or by mosquito biting. Sexual transmission of the Zika virus distinguishes it from other members of the Genus Flavivirus. Detailed information of the Zika virus is needed through a thorough investigation covering basic, epidemical, subclinical and clinical studies. Here, we reviewed the published information of Zika virus.

Contribution of phylogenetics to understanding the evolution and epidemiology of dengue virus

Dengue virus(DENV)is one of the most important arboviral pathogens in the tropics and subtropics,and nearly one-third of the world's population is at risk of infection.The transmission of DENV involves a sylvatic cycle between nonhuman primates(NHP)and Aedes genus mosquitoes,and an endemic cycle between human hosts and predominantly Aedes aegypti.DENV belongs to the genus Flavivirus of the family Flaviviridae and consists of four antigenically distinct serotypes(DENV-1-4).Phylogenetic analyses of DENV have revealed its origin,epidemiology,and the drivers that determine its molecular evolution in nature.This review discusses how phyloge-netic research has improved our understanding of DENV evolution and how it affects viral ecology and improved our ability to analyze and predict future DENV emergence.