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.

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.

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.

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.

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.

Persistent delayed auditory memory and executive function deficits 5 years after West Nile Virus Encephalitis: A neuropsychological and neuroimaging single case study

There are presently no in-depth published neuropsychological studies of West Nile Virus (WNV) encephalitis patients that have been well-correlated with high resolution structural MRI. In this study a middleaged male who developed West Nile Virus encephalitis five years previously was examined three times over a two year period. We examined him with the Wechsler Adult Intelligence Scales—Fourth Edition and the Wechsler Memory Scale—Fourth Edition (WAIS-IV/WMS-IV) and Advanced Clinical Solutions battery supplemented by tests of attention, executive, motor and sensory functions. Neuroradiological imaging revealed hypodensities within the left hippocampus in the axial and coronal planes with T2-FLAIR MRI. The man was previously high functioning and although he had prior history of well-controlled epilepsy it seems unlikely that the epilepsy could fully account for the neuropathological changes. The patient had previously completed a demanding six year double science degree program before he became ill with WNV and he had been a successful manager and director of a research company. Delayed auditory memory scores were at least two standard deviation units below age expected levels and semantic fluency and Booklet Category Tests of executive function were also in the impaired range. Moreover the illness onset profile of muscle weakness, extreme fatigue, memory complaints as well as inability to carry out research projects involving planning on the job were highly consistent with WNV encephalitis. If the memory and executive function deficits had been premorbid manifestations of epilepsy it is unlikely he would have attained the levels he did educationally and occupationally. This left hippocampal lesion is characteristic of other encephalitic viral infections such as herpes simplex virus. To our knowledge this is the first lateralized WNV encephalitis medial temporal lobe patient in the published literature.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Mathematical Model for the Transmission Dynamics of Zika Virus Infection with Combined Vaccination and Treatment Interventions

In this paper, we studied the transmission dynamics of ZIKV in the presence of a vector under the combined effects of treatment and vaccination in a hypothetical population. The disease-free εo and endemic ε1 equilibria were established with local stability on εo. We established the basic reproduction number Ro which served as a threshold for measuring the spread of the infection in the population using the next-generation matrix and computed its numerical value to be Ro = 0.0185903201 using the parameter values. It was established that the disease-free equilibrium εo is locally asymptotically stable since Ro < 1;meaning ZIKV infection would be eradicated from the population. The computational results of the study revealed that combining the two interventions of vaccination and treatment concomitantly proffers an optimal control strategy in taming the transmission of the virus than a single intervention strategy.

Neurological manifestations of Zika virus infection

Zika virus(ZIKV) is a flavivirus(Flaviviridae family) transmitted mainly by Aedes mosquitoes. The virus was restricted to the African continent until its spread to south-east Asia in the 1980's, the Micronesia in 2007, the French Polynesia in 2013 and, more recently in the Americas in 2015, where, up to date, the World Health Organization(WHO) has estimated about 3-4 million total cases of ZIKV infection. During outbreaks in the French Polynesia and Brazil in 2013 and 2015, respectively, national health authorities reported potential neurological complications of ZIKV disease, chiefly an upsurge in Guillain-Barré syndrome, which coincided with ZIKV outbreaks. On the other hand, the emergence of ZIKV in Brazil has been associated with a striking increase in the number of reported cases of microcephaly in fetus and newborns, twenty times higher than in that reported in previous years. While investigations are currently assessing whether there is an actual association between neurological complications and ZIKV infections, the evidence was enough worrisome for WHO to declare a public health emergency of international concern. Here we present an updated review addressing what is currently known about the possible association between ZIKV infection and the development of severe neurological disorders.

Type Ⅰ interferon receptor knockout mice as models for infection of highly pathogenic viruses with outbreak potential

Due to their inability to generate a complete immune response, mice knockout for type I interferon （IFN） receptors （Ifnar-/-） are more susceptible to viral infections, and are thus commonly used for pathogenesis studies. This mouse model has been used to study many diseases caused by highly pathogenic viruses from many families, including the Flaviviridae, Filoviridae, Arenaviridae, Bunyaviridae, Henipaviridae, and Togaviridae. In this review, we summarize the findings from these animal studies, and discuss the pros and cons of using this model versus other known methods for studying pathogenesis in animals.