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.

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.

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.

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.

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.

Co-circulation of Aedes flavivirus,Culex flavivirus,and Quang Binh virus in Shanghai,China

Background:With increases in global travel and trade,the spread of arboviruses is undoubtedly alarming.Pathogen detection in field-caught mosquitoes can provide the earliest possible warning of transmission.Insect-specific flavivirus(ISFV)has been first detected in 1991 and documented worldwide in the latest ten years.Although infection with ISFVs is apparently limited to insects,an increase in the infection rate of mosquito-borne flaviviruses may be able to induce cytopathic effects in vertebrate cells during co-infection with other human pathogens.However,little is known whether ISFVs persist in most regions of China.Methods:During the mosquito activity season in 2016,a surveillance program was carried out to detect ISFVs in mosquitoes in metropolitan Shanghai,China.The presence of ISFVs was randomly tested in different species of mosquitoes using RT-PCR-based and hemi-nested PCR assays,following by the sequencing of PCR products.Sequences from positive pooled samples were compared with those deposited in GenBank.Thereafter,sequences of representative insect flaviviruses were used for further phylogenetic and molecular evolutionary analyses.Results:Our investigations showed:(1)the presence of Aedes flavivirus(AEFV)in 11/161 pooled samples(nine pools in Songjiang District,one pool in Huangpu District,and one pool in Qingpu District)of Aedes albopictus,(2)the presence of Quang Binh virus(QBV)in 10/195 pooled samples(all in Chongming District)of Culex tritaeniorhynchus;and(3)the presence of Culex flavivirus(CxFV)in 9/228 pooled samples(six pools in Pudong New Area,two pools in Huangpu District,and one pool in Chongming District)of Cx.pipiens.Furthermore,phylogenetic analyses of the gene sequences of envelope proteins indicated that Shanghai CxFV strains belonged to the Asia/USA genotype.The overall maximum likelihood estimation values(and 95%confidence interval)for CxFV,QBV,and AEFV in mosquitoes collected in Shanghai in 2016 were 1.34(0.66-2.45),1.65(0.87-2.85),and 1.51(0.77-2.70)per 1000,respectively.Conclusions:This study reveals the presence and the geographical distribution of ISFVs,and determines the genetic variation and the infection rate of ISFVs in Shanghai,China.At least,three insect flaviviruses including ISFVs,AEFV,CxFV,and QBV,co-circulate in this area.To our knowledge,this is the first report of AEFV in China.

The Role of Host Cytoskeleton in Flavivirus Infection

The family of flaviviruses is one of the most medically important groups of emerging arthropod-borne viruses. Host cell cytoskeletons have been reported to have close contact with flaviviruses during virus entry, intracellular transport, replication, and egress process, although many detailed mechanisms are still unclear. This article provides a brief overview of the function of the most prominent flaviviruses-induced or-hijacked cytoskeletal structures including actin, microtubules and intermediate filaments, mainly focus on infection by dengue virus, Zika virus and West Nile virus. We suggest that virus interaction with host cytoskeleton to be an interesting area of future research.

The Restrictome of Flaviviruses

Flaviviruses are a genus of mostly arthropod-borne RNA viruses that cause a range of pathologies in humans.Basic knowledge on flaviviruses is rapidly expanding,partly due to their status as frequent emerging or re-emerging pathogens.Flaviviruses include the dengue,Zika,West Nile,tick-borne encephalitis and yellow fever viruses(DENV,ZIKV,WNV,TBEV and YFV,respectively).As is the case with other families of viruses,the success of productive infection of human cells by flaviviruses depends in part on the antiviral activity of a heterogeneous group of cellular antiviral proteins called restriction factors.Restriction factors are the effector proteins of the cell-autonomous innate response against viruses,an immune pathway that also includes virus sensors as well as intracellular and extracellular signal mediators such as type I interferons(IFN-I).In this review,I summarize recent progress toward the identification and characterization of flavivirus restriction factors.In particular,I focus on IFI6,Schlafen 11,FMRP,OAS-RNase L,RyDEN,members of the TRIM family of proteins(TRIM5α,TRIM19,TRIM56,TRIM69 and TRIM79α)and a new mechanism of action proposed for viperin.Recent and future studies on this topic will lead to a more complete picture of the flavivirus restrictome,defined as the ensemble of cellular factors with demonstrated anti-flaviviral activity.

Flavivirus-like particles in the cardiac myocyte of a patient with arrhythmogenic right ventricular cardiomyopathy

Vires infection and subsequent myocardial damage and repairing were regarded as important aspects in the pathogenesis of dilated cardiomyopathy. Enterovirus and its association with myocarditis and dilated cardiomyopathy have been investigated extensively. Arrhythmogenic right ventricular cardiomyopathy （ARVC） is a relatively uncommon kind of cardiomyopathy which could cause malignant arrhythmia and sudden death in human. Although some ARVC patients have family history and some gene defects were found to be associated with ARVC, enterovims genome was shown in endomyocardial samples from the patients with ARVC and it suggested possible association between ARVC and vires infective myocarditis. Here, we present an electron microscopic evidence for a flaviviral infection of myocytes from a case of ARVC.

Flavivirus RNA cap methyltransferase:structure,function,and inhibition

Many flaviviruses are significant human pathogens.The plus-strand RNA genome of a flavivirus contains a 5′terminal cap 1 structure(m7GpppAmG).The flavivirus encodes one methyltransferase(MTase),located at the Nterminal portion of the NS5 RNA-dependent RNA polymerase(RdRp).Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development.The NS5 MTase catalyzes both guanine N7 and ribose 2'-OH methylations during viral cap formation.Representative flavivirus MTases,from dengue,yellow fever,and West Nile virus(WNV),sequentially generate GpppA!m7GpppA!m7GpppAm.Despite the existence of two distinct methylation activities,the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine(SAM),the methyl donor.This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2'-O methyl groups from SAM during the sequential reactions.Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2'-OH on the first transcribed nucleotide.Mutant enzymes with different methylation defects can trans complement one another in vitro,demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro.In the context of the infectious virus,defects in both methylations,or a defect in the N7 methylation alone,are lethal to WNV.However,viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-typeWNV challenge.The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and,thus,methyltransferase represents a novel and promising target for flavivirus therapy.

Powassan virus:A tick borne flavivirus infecting humans

Infection of humans by Powassan virus(POWV)occurs rarely but is potentially life‐threatening.First isolated in Ontario,Canada in 1958,the presence of POWV has been confirmed in three countries:Canada,the USA,and Russia.Although a limited number of human cases has been reported thus far,the infection rate has shown signs of increasing during the 21^(st) century.Interestingly,POWV and a genetically close variant,deer tick virus(DTV),are the only member of the tick‐borne flaviviruses known to be endemic in North America and maintain in respective tick‐host cycles.In this review,we briefly summarize current knowledge involving the epidemiology and etiology,pathogenesis and immunity,molecular evolution,and protein functions of POWV,aiming to increase our understanding of the virus and unlock the potential to control this lethal pathogen.These data may also provide tools to minimize the future threat of other emerging and re‐emerging viruses.

Screening of novel synthetic derivatives of dehydroepiandrosterone for antivirals against flaviviruses infections

Flaviviruses are important arthropod-borne pathogens that represent an immense global health problem.Their unprecedented epidemic rate and unpredictable clinical features underscore an urgent need for antiviral interventions.Dehydroepiandrosterone(DHEA)is a natural occurring adrenal-derived steroid in the human body that has been associated in protection against various infections.In the present study,the plaque assay based primary screening was conducted on 32 synthetic derivatives of DHEA against Japanese encephalitis virus(JEV)to identify potent anti-flaviviral compounds.Based on primary screening,HAAS-AV3026 and HAAS-AV3027 were selected as hits from DHEA derivatives that exhibited strong antiviral activity against JEV(IC_(50)=2.13 and 1.98μmol/L,respectively)and Zika virus(ZIKV)(IC_(50)=3.73 and 3.42μmol/L,respectively).Mechanism study indicates that HAAS-AV3026 and HAAS-AV3027 do not exhibit inhibitory effect on flavivirus binding and entry process,while significantly inhibit flavivirus infection at the replication stage.Moreover,indirect immunofluorescence assay,Western blot analyses,and quantitative reverse transcription-PCR(qRT-PCR)revealed a potent antiviral activity of DHEA derivatives hits against JEV and ZIKV in terms of inhibition of viral infection,protein production,and viral RNA synthesis in Vero cells.Taken together,our results may provide a basis for the development of new antivirals against flaviviruses.

Mosquito salivary protein promotes flavivirus transmission

With the support by the National Natural Science Foundation of China,the research team led by Prof.Cheng Gong (程功)at Tsinghua-Peking Center for Life Sciences,School of Medicine,Tsinghua University,identified a key mosquito salivary protein related to flavivirus transmission,which was published in Nature Communications (2020,11:260).