A Reverse Mutation E143K within the PrM Protein of Zika Virus Asian Lineage Natal RGN Strain Increases Infectivity and Cytopathicity.

Zika virus (ZIKV) is a positive-sense single-stranded RNA virus in the Flaviviridae, which is classified into two different lineages Asian and African. The outbreak of ZIKV Asian lineage isolates in 2015-2016 is associated with the increase in cases with prenatal microcephaly and Guillain-Barré syndrome, and has sparked attention throughout the world. Genome sequence alignment and the analysis of Asian and African lineage isolates indicate that amino acid changes, particular in positively charged amino acid substitutions in the pr region of prM protein might involve a phenotypic change that links with the global outbreak of ZIKV Asian-lineage. The study generated and characterized the virological properties of wild type and mutants of single-round infectious particles (SRIPs) and infectious clones (i.c.s) of ZIKV Asian-lineage Natal RGN strain, and then identified the function of amino acid substitutions at the positions 139 [Asn139→Ser139 (N139S)] and 143 [Glu143→Lys143 (E143K)] in ZIKV polyproteins (located within the pr region of prM protein) in the infectivity and cytopathogenicity. The E143K SRIP and i.c. of Natal RGN strain exhibited relatively higher levels of cytopathic effect, EGFP reporter, viral RNA and protein synthesis, and virus yield in three types of human cell lines, TE617, SF268 and HMC3, compared to wild type (WT), N139S SRIPs and i.c.s, which displayed more efficiency in replication kinetics. Additionally, E143K Natal RGN i.c. had greater activities of virus attachment and entry, yielded higher titers of intracellular and extracellular virions, and assembled the E proteins near to the plasma membrane in infected cells than the other i.c.s. The results indicate that the positively charged amino acid residue Lys143, a conserved residue in the pr region of prM of ZIKV African lineages, plays a crucial role in viral replication kinetics, including viral attachment, entry, assembly and egress. Thus, the negatively charged amino acid residue Glu143 within the pr region of prM leads to an alteration of the phenotypes, in particular, a lower replication efficiency of ZIKV Asian-lineage isolates with the attenuation of infectivity and cytopathicity.

Antidepressant Sertraline Is a Broad-Spectrum Inhibitor of Enteroviruses Targeting Viral Entry through Neutralization of Endolysosomal Acidification.

Enterovirus 71 (EV71) is an etiological agent of hand foot and mouth disease and can also cause neurological complications in young children. However, there are no approved drugs as of yet to treat EV71 infections. In this study, we conducted antiviral drug screening by using a Food and Drug Administration (FDA)-approved drug library. We identified five drugs that showed dose-dependent inhibition of viral replication. Sertraline was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index among the five hits. The antiviral activity of sertraline was noted for other EV serotypes. The drug's antiviral effect is not likely associated with its approved indications as an antidepressant and its mode-of-action as a selective serotonin reuptake inhibitor. The time-of-addition assay revealed that sertraline inhibited an EV71 infection at the entry stage. We also showed that sertraline partitioned into acidic compartments, such as endolysosomes, to neutralize the low pH levels. In agreement with the findings, the antiviral effect of sertraline could be greatly relieved by exposing virus-infected cells to extracellular low-pH culture media. Ultimately, we have identified a use for an FDA-approved antidepressant in broad-spectrum EV inhibition by blocking viral entry through the alkalization of the endolysosomal route.

Toll-Like Receptor 3 Is Involved in Detection of Enterovirus A71 Infection and Targeted by Viral 2A Protease.

Enterovirus A71 (EV-A71) has emerged as a major pathogen causing hand, foot, and mouth disease, as well as neurological disorders. The host immune response affects the outcomes of EV-A71 infection, leading to either resolution or disease progression. However, the mechanisms of how the mammalian innate immune system detects EV-A71 infection to elicit antiviral immunity remain elusive. Here, we report that the Toll-like receptor 3 (TLR3) is a key viral RNA sensor for sensing EV-A71 infection to trigger antiviral immunity. Expression of TLR3 in HEK293 cells enabled the cells to sense EV-A71 infection, leading to type I, IFN-mediated antiviral immunity. Viral double-stranded RNA derived from EV-A71 infection was a key ligand for TLR3 detection. Silencing of TLR3 in mouse and human primary immune cells impaired the activation of IFN-ß upon EV-A71 infection, thus reinforcing the importance of the TLR3 pathway in defending against EV-A71 infection. Our results further demonstrated that TLR3 was a target of EV-A71 infection. EV-A71 protease 2A was implicated in the downregulation of TLR3. Together, our results not only demonstrate the importance of the TLR3 pathway in response to EV-A71 infection, but also reveal the involvement of EV-A71 protease 2A in subverting TLR3-mediated antiviral defenses.

Tubacin, an HDAC6 Selective Inhibitor, Reduces the Replication of the Japanese Encephalitis Virus via the Decrease of Viral RNA Synthesis.

Japanese encephalitis virus (JEV), a neurotropic flavivirus, annually causes over 30,000 Japanese Encephalitis (JE) cases in East and Southeast Asia. Histone deacetylases (HDACs) modulate lysine acetylation of histones and non-histone proteins, regulating many processes including inflammation and antiviral immune response. This study investigated antiviral activity of pan- and selective-HDAC inhibitors as host-targeting agents against JEV. Among HDAC inhibitors, selective HDAC6 inhibitors (tubastatin-A (TBSA) and tubacin) concentration-dependently inhibited JEV-induced cytopathic effect and apoptosis, as well as reduced virus yield in human cerebellar medulloblastoma cells. The 50% inhibitory concentration (IC50) values of virus yield was 0.26 µM for tubacin and 1.75 µM for TBSA, respectively. Tubacin (IC50 of 1.52 µM), but not TBSA, meaningfully blocked the production of intracellular infectious virus particles. In time-of-addition assays, the greatest potency of antiviral activity was observed in the mode of pre-treatment with tubacin (IC50 of 1.89 µM) compared to simultaneous (IC50 of 4.88 µM) and post-treatment (IC50 of 2.05 µM) modes. Interestingly, tubacin induced the hyperacetylation of a HDAC6 substrate Hsp90 and reduced the interaction of Hsp90 with JEV NS5 protein. Novobiocin, an Hsp90 inhibitor, diminished the NS5 protein amount and virus replication in JEV-infected cells. Meantime, tubacin suppressed the NS5 expression and antisense RNA genome synthesis in infected cells. Tubacin-induced Hsp90 hyperacetylation was suggested to influence the NS5 activity in JEV replication. Therefore, tubacin had a high potential of a host-targeting agent against JEV, exhibiting preventive and therapeutic activities against JEV infection.

Development of a fluorescence resonance energy transfer-based intracellular assay to identify novel enterovirus 71 antivirals.

Enterovirus 71 (EV71) is considered one of the most virulent pathogens in the family Picornaviridae. However, there have been no effective treatments for the severe complications caused by EV71. Development of new drugs against targets that are essential for viral replication often requires screening large collections of compounds, for which a high-throughput screening platform is needed. In this study, a drug-screening platform was developed based on a genetically engineered cell line that displays fluorescence resonance energy transfer (FRET) and shows a real-time and quantifiable impairment of FRET upon EV71 infection. A library of small molecules consisting of 1280 compounds with defined bioactivities was used for screening drugs with anti-EV71 activity; accurate, rapid, and robust results were obtained from this screening procedure. Ten drugs were identified in the primary screening, and their antiviral activities were indicated by dose-dependent elevation of FRET. Among these, AC-93253, mitoxantrone and N-bromoacetamide had not been reported as enterovirus inhibitors, and it was confirmed that they were able to suppress viral yields in a dose-dependent manner. Taken together, these studies demonstrate the feasibility of this FRET-based platform for efficient screening and identification of novel compounds with activity against EV71 infection.

SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6.

Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLPro) reportedly inhibits the production of type I interferons (IFNs) and pro-inflammatory cytokines in Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I) pathways. The study investigated the inhibitory effect and its antagonistic mechanism of SARS-CoV PLPro on TLR7-mediated cytokine production. TLR7 agonist (imiquimod (IMQ)) concentration-dependently induced activation of ISRE-, NF-κB- and AP-1-luciferase reporters, as well as the production of IFN-α, IFN-ß, TNF-α, IL-6 and IL-8 in human promonocyte cells. However, SARS-CoV PLPro significantly inhibited IMQ-induced cytokine production through suppressing the activation of transcription factors IRF-3, NF-κB and AP-1. Western blot analysis with anti-Lys48 and anti-Lys63 ubiquitin antibodies indicated the SARS-CoV PLPro removed Lys63-linked ubiquitin chains of TRAF3 and TRAF6, but not Lys48-linked ubiquitin chains in un-treated and treated cells. The decrease in the activated state of TRAF3 and TRAF6 correlated with the inactivation of TBK1 in response to IMQ by PLPro. The results revealed that the antagonism of SARS-CoV PLPro on TLR7-mediated innate immunity was associated with the negative regulation of TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals.

Idarubicin is a broad-spectrum enterovirus replication inhibitor that selectively targets the virus internal ribosomal entry site.

Enterovirus 71 (EV71) causes life-threatening diseases with neurological manifestations in young children. However, the treatment of EV71 infections remains an unmet medical need. Idarubicin (IDR) is an anthracycline compound that is used therapeutically for certain types of tumour. In this study, we identified IDR as an EV71 inhibitor, which displayed antiviral potency in the submicromolar range and substantially protected cells from the cytopathic effects and cell death caused by EV71 infections. The antiviral effects extended to several other enterovirus (EV) species, and these effects were independent of cytotoxicity or topoisomerase inhibition. Structure-activity relationship studies indicated the importance of the anthracycline scaffold for anti-EV potency. IDR effectively blocked the synthesis of viral protein and RNA, but not the viral proteolysis processes. Moreover, anthracyclines were demonstrated to suppress EV internal ribosomal entry site (IRES)-mediated translation; conversely, the cellular p53 IRES activity was not sensitive to IDR action. Inhibition of IRES-mediated translation by IDR correlated with the affinity of binding between IDR and the particular IRES. Moreover, IDR impaired binding between the EV71 IRES RNA and hnRNP A1, a known host IRES trans-acting factor. In sum, we have identified a USA FDA-approved anticancer drug with the new indication as a selective EV IRES binder and inhibitor. The finding may also provide leads for the development of novel antiviral therapies directed at the EV IRES RNA.

Antiviral Potential of a Novel Compound CW-33 against Enterovirus A71 via Inhibition of Viral 2A Protease.

Enterovirus A71 (EV-A71) in the Picornaviridae family causes hand-foot-and-mouth disease, aseptic meningitis, severe central nervous system disease, even death. EV-A71 2A protease cleaves Type I interferon (IFN)-α/ß receptor 1 (IFNAR1) to block IFN-induced Jak/STAT signaling. This study investigated anti-EV-A7l activity and synergistic mechanism(s) of a novel furoquinoline alkaloid compound CW-33 alone and in combination with IFN-ß Anti-EV-A71 activities of CW-33 alone and in combination with IFN-ß were evaluated by inhibitory assays of virus-induced apoptosis, plaque formation, and virus yield. CW-33 showed antiviral activities with an IC50 of near 200 µM in EV-A71 plaque reduction and virus yield inhibition assays. While, anti-EV-A71 activities of CW-33 combined with 100 U/mL IFN-ß exhibited a synergistic potency with an IC50 of approximate 1 µM in plaque reduction and virus yield inhibition assays. Molecular docking revealed CW-33 binding to EV-A71 2A protease active sites, correlating with an inhibitory effect of CW33 on in vitro enzymatic activity of recombinant 2A protease IC50 = 53.1 µM). Western blotting demonstrated CW-33 specifically inhibiting 2A protease-mediated cleavage of IFNAR1. CW-33 also recovered Type I IFN-induced Tyk2 and STAT1 phosphorylation as well as 2\',5\'-OAS upregulation in EV-A71 infected cells. The results demonstrated CW-33 inhibiting viral 2A protease activity to reduce Type I IFN antagonism of EV-A71. Therefore, CW-33 combined with a low-dose of Type I IFN could be applied in developing alternative approaches to treat EV-A71 infection.

Eupafolin and Ethyl Acetate Fraction of Kalanchoe gracilis Stem Extract Show Potent Antiviral Activities against Enterovirus 71 and Coxsackievirus A16.

Enterovirus 71 (EV71) and coxsackievirus A16 (CoxA16) are main pathogens of hand-foot-and-mouth disease, occasionally causing aseptic meningitis and encephalitis in tropical and subtropical regions. Kalanchoe gracilis, Da-Huan-Hun, is a Chinese folk medicine for treating pain and inflammation, exhibiting antioxidant and anti-inflammatory activities. Our prior report (2012) cited K. gracilis leaf extract as moderately active against EV71 and CoxA16. This study further rates antienteroviral potential of K. gracilis stem (KGS) extract to identify potent antiviral fractions and components. The extract moderately inhibits viral cytopathicity and virus yield, as well as in vitro replication of EV71 (IC50 = 75.18 µ g/mL) and CoxA16 (IC50 = 81.41 µ g/mL). Ethyl acetate (EA) fraction of KGS extract showed greater antiviral activity than that of n-butanol or aqueous fraction: IC50 values of 4.21 µ g/mL against EV71 and 9.08 µ g/mL against CoxA16. HPLC analysis, UV-Vis absorption spectroscopy, and plaque reduction assay indicate that eupafolin is a vital component of EA fraction showing potent activity against EV71 (IC50 = 1.39 µ M) and CoxA16 (IC50 = 5.24 µ M). Eupafolin specifically lessened virus-induced upregulation of IL-6 and RANTES by inhibiting virus-induced ERK1/2, AP-1, and STAT3 signals. Anti-enteroviral potency of KGS EA fraction and eupafolin shows the clinical potential against EV71 and CoxA16 infection.

Calcium flux and calpain-mediated activation of the apoptosis-inducing factor contribute to enterovirus 71-induced apoptosis.

Enterovirus 71 (EV71) is a causative agent of an array of childhood diseases with severe neurological manifestations implicated. EV71 infection is known to induce caspase-dependent apoptosis in cell cultures and animal models. However, whether an alternative apoptotic pathway independent of caspase activation can be triggered by EV71 infection has not been explored. In this study, we showed that calcium (Ca²âº)-activated calpains are capable of mediating caspase-independent pathway activation during EV71-induced apoptosis in HeLa cells. Results from subcellular fractionation analysis and confocal imaging indicated that during EV71 infection, apoptosis-inducing factor (AIF), a primary mediator of the caspase-independent pathway, became truncated and translocated from the mitochondrion to nucleus. This was accompanied by the release of cytochrome c, and sharply decreased mitochondrial membrane potential. AIF knockdown data indicated significant protection against apoptotic cell death, with greater protection provided by the addition of a pan-caspase inhibitor. The Ca²âº-dependent, calpain isoforms 1 and 2, but not cathepsins, were proven crucial for the altered AIF behaviour as studied by the pharmacological inhibitor and the knockdown approaches. We then analysed Ca²âº dynamics in the infected cells and found elevated levels of mitochondrial Ca²âº. Treatment with ruthenium red, a mitochondrial Ca²âº influx inhibitor, significantly blocked calpain activations and AIF cleavage. Our conclusion was that calpain activation via Ca²âº flux plays an essential role in eliciting an AIF-mediated, caspase-independent apoptotic pathway in EV71-infected cells. These findings should be useful for understanding the virus-induced cytopathology and the impact of Ca²âº homeostasis on EV71 infection.

Antiviral Ability of Kalanchoe gracilis Leaf Extract against Enterovirus 71 and Coxsackievirus A16.

Pandemic infection or reemergence of Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) occurs in tropical and subtropical regions, being associated with hand-foot-and-mouth disease, herpangina, aseptic meningitis, brain stem encephalitis, pulmonary edema, and paralysis. However, effective therapeutic drugs against EV71 and CVA16 are rare. Kalanchoe gracilis (L.) DC is used for the treatment of injuries, pain, and inflammation. This study investigated antiviral effects of K. gracilis leaf extract on EV71 and CVA16 replications. HPLC analysis with a C-18 reverse phase column showed fingerprint profiles of K. gracilis leaf extract had 15 chromatographic peaks. UV/vis absorption spectra revealed peaks 5, 12, and 15 as ferulic acid, quercetin, and kaempferol, respectively. K. gracilis leaf extract showed little cytotoxicity, but exhibited concentration-dependent antiviral activities including cytopathic effect, plaque, and virus yield reductions. K. gracilis leaf extract was shown to be more potent in antiviral activity than ferulic acid, quercetin, and kaempferol, significantly inhibiting in vitro replication of EV71 (IC(50) = 35.88 µg/mL) and CVA16 (IC(50) = 42.91 µg/mL). Moreover, K. gracilis leaf extract is a safe antienteroviral agent with the inactivation of viral 2A protease and reduction of IL-6 and RANTES expressions.

Fisetin and rutin as 3C protease inhibitors of enterovirus A71.

Enterovirus A71 (EV-A71) causes severe complications: encephalitis, pulmonary edema, and death. No effective drug has been approved for clinical use. This study investigated the antiviral effects of flavonoids against EV-A71. An in vitro inhibitor screening assay using recombinant EV-A71 3C protease (3Cpro) demonstrated fisetin and rutin inhibiting 3Cpro enzymatic activity in a dose-dependent manner. Cell-based fluorescence resonance energy transfer (FRET) assay with an EV-A71 3Cpro cleavage motif probe also confirmed that fisetin and rutin inhibited the replication of EV-A71 in cells. A virus replication assay indicated that fisetin and rutin reduced significantly the EV-A71-induced cytopathic effect and viral plaque titers in RD cells culture. The IC(50) values of plaque reduction against EV-A71 were 85 µM for fisetin and 110 µM for rutin. Therapeutic indices (CC50/IC50 of plaque reduction assays) of fisetin and rutin exceeded 10. The study suggests that fisetin and rutin inhibit the replication of EV-A71.

A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus.

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities.

Severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferon-induced responses through downregulation of extracellular signal-regulated kinase 1-mediated signalling pathways.

Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a deubiquitinating enzyme, reportedly blocks poly I : C-induced activation of interferon regulatory factor 3 and nuclear factor kappa B, reducing interferon (IFN) induction. This study investigated type I IFN antagonist mechanism of PLpro in human promonocytes. PLpro antagonized IFN-α-induced responses such as interferon-stimulated response element- and AP-1-driven promoter activation, protein kinase R, 2'-5'-oligoadenylate synthetase (OAS), interleukin (IL)-6 and IL-8 expression, and signal transducers and activators of transcription (STAT) 1 (Tyr701), STAT1 (Ser727) and c-Jun phosphorylation. A proteomics approach demonstrated downregulation of extracellular signal-regulated kinase (ERK) 1 and upregulation of ubiquitin-conjugating enzyme (UBC) E2-25k as inhibitory mechanism of PLpro on IFN-α-induced responses. IFN-α treatment significantly induced mRNA expression of UBC E2-25k, but not ERK1, causing time-dependent decrease of ERK1, but not ERK2, in PLpro-expressing cells. Poly-ubiquitination of ERK1 showed a relationship between ERK1 and ubiquitin proteasome signalling pathways associated with IFN antagonism by PLpro. Combination treatment of IFN-α and the proteasome inhibitor MG-132 showed a time-dependent restoration of ERK1 protein levels and significant increase of ERK1, STAT1 and c-Jun phosphorylation in PLpro-expressing cells. Importantly, PD098059 (an ERK1/2 inhibitor) treatment significantly reduced IFN-α-induced ERK1 and STAT1 phosphorylation, inhibiting IFN-α-induced expression of 2'-5'-OAS in vector control cells and PLpro-expressing cells. Overall results proved downregulation of ERK1 by ubiquitin proteasomes and suppression of interaction between ERK1 and STAT1 as type I IFN antagonist function of SARS-CoV PLpro.

Visualizing and quantifying the differential cleavages of the eukaryotic translation initiation factors eIF4GI and eIF4GII in the enterovirus-infected cell.

Enterovirus (EV) infection has been shown to cause a marked shutoff of host protein synthesis, an event mainly achieved through the cleavages of eukaryotic translation initiation factors eIF4GI and eIF4GII that are mediated by viral 2A protease (2A(pro)). Using fluorescence resonance energy transfer (FRET), we developed genetically encoded and FRET-based biosensors to visualize and quantify the specific proteolytic process in intact cells. This was accomplished by stable expression of a fusion substrate construct composed of the green fluorescent protein 2 (GFP(2)) and red fluorescent protein 2 (DsRed2), with a cleavage motif on eIF4GI or eIF4GII connected in between. The FRET biosensor showed a real-time and quantifiable impairment of FRET upon EV infection. Levels of the reduced FRET closely correlated with the cleavage kinetics of the endogenous eIF4Gs isoforms. The FRET impairments were solely attributed to 2A(pro) catalytic activity, irrespective of other viral-encoded protease, the activated caspases or general inhibition of protein synthesis in the EV-infected cells. The FRET biosensors appeared to be a universal platform for several related EVs. The spatiotemporal and quantitative imaging enabled by FRET can shed light on the protease-substrate behaviors in their normal milieu, permitting investigation into the molecular mechanism underlying virus-induced host translation inhibition.

Real-time monitoring of human enterovirus (HEV)-infected cells and anti-HEV 3C protease potency by fluorescence resonance energy transfer.

A real-time assay system that allows monitoring of intracellular human enterovirus (HEV) protease activity was established using the principle of fluorescence resonance energy transfer (FRET). It was accomplished by engineering cells to constitutively express a genetically encoded FRET probe. The FRET-based probe was designed to contain an enterovirus 71 3C protease (3C(pro)) cleavage motif flanked by the FRET pair composed of green fluorescent protein 2 and red fluorescent protein 2 (DsRed2). Efficient FRET from the stable line was detected in a real-time manner by fluorescence microscopy, and the disruption of FRET was readily monitored upon HEV infection. The level of the repressed FRET was proportional to the input virus titer and the infection duration as measured by the fluorometric method. The FRET biosensor cell line was also responsive to other related HEV serotypes, but not to the phylogenetically distant herpes simplex virus, which was confirmed by Western blot analysis. The FRET biosensor was then utilized to develop a format for the determination of antiviral susceptibility, as the reduced FRET appeared to reflect viral replication. Evaluations of the FRET biosensor system with representative HEV serotypes demonstrated that their susceptibilities to a 3C(pro) inhibitor, rupintrivir, were all accurately determined. In summary, this novel FRET-based system is a means for rapid detection, quantification, and drug susceptibility testing for HEVs, with potential for the development of a high-throughput screening assay.

Application of fluorescence resonance energy transfer resolved by fluorescence lifetime imaging microscopy for the detection of enterovirus 71 infection in cells.

Timely and effective virus infection detection is critical for the clinical management and prevention of the disease spread in communities during an outbreak. A range of methods have been developed for this purpose, of which classical serological and viral nucleic acids detection are the most popular. We describe an alternative, imaging-based approach that utilizes fluorescence resonance energy transfer (FRET) resolved by fluorescence lifetime imaging microscopy (FLIM) and demonstrate it on the example of enterovirus 71 (EV71) infection detection. A plasmid construct is developed with the sequence for GFP2 and DsRed2 fluorescent proteins, linked by a 12-amino-acid-long cleavage recognition site for the 2A protease (2A(pro)), encoded by the EV71 genome and specific for the members of Picornaviridae family. In the construct expressed in HeLa cells, the linker binds the fluorophores within the Forster distance and creates a condition for FRET to occur, thus resulting in shortening of the GFP2 fluorescence lifetime. On cells infection with EV71, viral 2A(pro) released to the cytoplasm cleaves the recognition site, causing disruption of FRET through separation of the fluorophores. Thus, increased GFP2 lifetime to the native values, manifested by the time-correlated single-photon counting, serves as an efficient and specific indicator of the EV71 virus infection.

In vivo dynamics of enterovirus protease revealed by fluorescence resonance emission transfer (FRET) based on a novel FRET pair.

An in vivo protease assay suitable for analysis by fluorescence resonance energy transfer (FRET) was developed on the basis of a novel FRET pair. The specifically designed fusion substrate consists of green fluorescent protein 2 (GFP2)-peptide-red fluorescent protein 2 (DsRed2), with a cleavage motif for the enterovirus 2A protease (2Apro) embedded within the peptide region. FRET can be readily visualized in real-time from cells expressing the fusion substrate until a proteolytic cleavage by 2Apro from the input virus. The level of FRET decay is a function of the amount and infection duration of the inoculated virus as measured by a fluorometer assay. The FRET biosensor also responded well to other related enteroviruses but not to a phylogenetically distant virus. Western blot analysis confirmed the physical cleavage of the fusion substrate upon the infections. The study provides proof of principle for applying the FRET technology to diagnostics, screening procedures, and cell biological research.

Reporter cell lines for the detection of herpes simplex viruses.

Virus culture has played significant roles in basic and clinical virology, with a number of advantages that cannot be attainable by modern molecular techniques. However, virus culture is generally a slower process, as it inevitably takes the period of a full replication cycle of a given virus. A genetically modified cell culture with a virus-inducible marker is described here, using a frequently isolated DNA virus (herpes simplex virus) as a model. The assay system relies on expression of the reporter gene driven by a specific viral promoter that is triggered early in the course of viral infection. The reporter gene employed was green fluorescent protein (GFP) or secreted alkaline phosphatase (SEAP), whose assays offer real-time detection or quantification, respectively. This cell-based assay is simple, rapid, sensitive, specific, and quantitative and serves as a phenotypic method for determination of antiviral susceptibilities.

Selective inhibition of enterovirus 71 replication by short hairpin RNAs.

RNA interference (RNAi) is a sequence-specific, post-transcriptional process of mRNA degradation induced by small interfering RNA molecules. In this report, RNAi strategy was exploited to treat the infection of enterovirus 71 (EV71), considered as one of the most virulent pathogens that can cause severe complications in the family of Picornaviridae. We developed short hairpin RNA (shRNA) expression plasmids that significantly inhibited viral protein expression in a sequence-specific and dose-dependent fashion after transient transfection in cell cultures. Stable expression of shRNAs in cultured cells exhibited marked viral resistance in every step assessed in the viral replication. Using cytotoxicity of shRNA-expressing cells as a surrogate marker, it was shown that replication of EV71 was specifically attenuated by these plasmid-derived shRNAs, while replications of other related enteroviruses examined were not. These proof-of-concept studies demonstrated the feasibility of this approach for the therapy of EV71-associated diseases.