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The balance of the segmented genome derived from naturally occurring influenza A viruses (IAVs) is delicate and vulnerable to foreign insertions, thus most reporter IAVs up to date are generated using the backbone of the laboratory-adapted strains. In this study, we constructed a reporter influenza A/H3N2 virus (A/NY-HiBiT) which was derived from a clinical isolate, by placing a minimized HiBiT tag to the N-terminus of the viral nuclear-export protein (NEP). Here, we show that this 11-amino acid HiBiT tag did not adversely impact the viral genome balance, and the recombinant A/NY-HiBiT virus maintains its relative stability. Moreover, the replication profile of the HiBiT-tagged virus can be measured by a simple Nano-Glo assay, providing a robust high-throughput screening (THS) platform. We used this platform to evaluate a collection of the pre-purified fractions which were derived from rare Chinese medicinal materials, and we identified three fractions, including wild Trametes robiniophila (50% methanol fraction), Ganoderma (water fraction), and wild Phellinus igniarius (ethyl acetate fraction), as potent anti-IAV actives. Our results demonstrate that this IAV reporter can be used as a powerful HTS platform for antiviral development.

Influenza A virus (IAV) genome comprises eight negative-sense RNA segments, of which the replication is well orchestrated and the delicate balance of multiple segments are dynamically regulated throughout IAV life cycle. However, previous studies seldom discuss these balances except for functional hemagglutinin-neuraminidase balance that is pivotal for both virus entry and release. Therefore, we attempt to revisit IAV life cycle by highlighting the critical role of "genome balance". Moreover, we raise a "balance regression" model of IAV evolution that the virus evolves to rebalance its genome after reassortment or interspecies transmission, and direct a "balance compensation" strategy to rectify the "genome imbalance" as a result of artificial modifications during creation of recombinant IAVs. This review not only improves our understanding of IAV life cycle, but also facilitates both basic and applied research of IAV in future.

Ebola virus (EBOV) is one of the most deadliest agents already known, causing periodic epidemic of a severe hemorrhagic fever disease in Africa. Although two monoclonal antibody (mAb) drugs have recently received approval in the USA, additional therapeutics are still needed to combat potential outbreaks of resistance variants and other closely related ebola viruses. In this chapter, we describe the current understanding of the EBOV entry process and summarize the approaches, strategies, and advances in discovery and development of EBOV entry inhibitors, including therapeutic antibodies, peptides, small molecules, natural products, and other chemical structures.

Influenza A virus (IAV) causes great morbidity and mortality worldwide every year. However, there are only a limited number of drugs clinically available against IAV infection. Further, emergence of drug-resistant strains can render those drugs ineffective. Thus there is an unmet medical need to develop new anti-influenza agents. In this study, we show that punicalagin from plants possesses strong anti-influenza activity with a low micromolar IC50 value in tissue culture. Using a battery of bioassays such as single-cycle replication assay, neuraminidase (NA) inhibition assay, and virus yield reduction assay, we demonstrate that the primary mechanism of action (MOA) of punicalagin is the NA-mediated viral release. Moreover, punicalagin can inhibit replication of different strains of influenza A and B viruses, including oseltamivir-resistant virus (NA/H274Y), indicating that punicalagin is a broad spectrum antiviral against both IAV and IBV. Further, although punicalagin targets NA like oseltamivir, it has a different MOA. These results suggest that punicalagin is an influenza NA inhibitor that may be further developed as a novel antiviral against influenza viruses.

Vaccination is the most prevalent prophylactic means for controlling seasonal influenza infections. However, an effective vaccine usually takes at least 6 months to develop for the circulating strains. Therefore, new therapeutic options are needed for the acute treatment of influenza infections to control this virus and prevent epidemics/pandemics from developing. We have discovered fast-acting, orally bioavailable acylated 4-aminopiperidines with an effective mechanism of action targeting viral hemagglutinin (HA). Our data show that these compounds are potent entry inhibitors of influenza A viruses. We present docking studies that suggest an HA binding site for these inhibitors on H5N1. Compound 16 displayed a significant decrease of viral titer when evaluated in the infectious assays with influenza virus H1N1 (A/Puerto Rico/8/1934) or H5N1 (A/Vietnam/1203/2004) strains and the oseltamivir-resistant strain with the most common H274Y mutation. In addition, compound 16 showed significant synergistic activity with oseltamivir in vitro.

Background: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Rhizoma paridis saponins (RPS), the main bioactive ingredients of Paris polyphylla Smith var. yunnanensis (PPY), have been proved to have remarkable effects on NSCLC cell lines. However, the multi-component synergistic effects and mechanisms of RPS on NSCLC have not been elucidated. Objective: To decipher the multi-RPS synergistic effects and mechanisms against NSCLC based on network pharmacology combined with segmented solid-phase extraction (SPE) and bioactivity screening method. Methods: Firstly, segmented SPE and cytotoxicity assays were performed to screen the RPS-enrichment fraction of PPY, and the steroidal saponins in it were identified by LC-MS/MS. Then, a network pharmacology analysis was performed to predict the potential therapeutic targets of RPS on NSCLC. Finally, viable cell counting tests and RT-qPCR were utilized to verify the synergistic effects and mechanisms of RPS. Results: 48 potentially active compounds were identified from the 30% MeOH/EtOAc fraction of PPY (30% M/E PPY). The results of the network pharmacology analysis indicated that RPS exerted joint effects by regulating six key targets in the PI3K-AKT signaling pathway. In vitro experiments showed that due to the synergistic effects, 30% M/E PPY at 13.90 μg/mL could exert a stronger inhibitory activity on A549 cells by reducing the overexpression of six hub genes compared with the parallel control groups. Conclusion: This research elaborates on the multi-RPS synergy mechanisms against NSCLC and provides a way to develop new combination medicines for NSCLC.

Our previous efforts have proved that modifications targeting the 150-cavity of influenza neuraminidase can achieve more potent and more selective inhibitors. In this work, four subseries of C5-NH

Hemagglutinin (HA) plays a critical role during influenza virus receptor binding and subsequent membrane fusion process, thus HA has become a promising drug target. For the past several decades, we and other researchers have discovered a series of HA inhibitors mainly targeting its fusion machinery. In this review, we summarize the advances in HA-targeted development of small molecule inhibitors. Moreover, we discuss the structural basis and mode of action of these inhibitors, and speculate upon future directions toward more potent inhibitors of membrane fusion and potential anti-influenza drugs.

Background In the urgent campaign to develop therapeutics against SARS-CoV-2, natural products have been an important source of new lead compounds. Results We herein identified two natural products, ginkgolic acid and anacardic acid, as inhibitors using a high-throughput screen targeting the SARS-CoV-2 papain-like protease (PLpro). Moreover, our study demonstrated that the two hit compounds are dual inhibitors targeting the SARS-CoV-2 3-chymotrypsin-like protease (3CLpro) in addition to PLpro. A mechanism of action study using enzyme kinetics further characterized the two compounds as irreversible inhibitors against both 3CLpro and PLpro. Significantly, both identified compounds inhibit SARS-CoV-2 replication in vitro at nontoxic concentrations. Conclusions Our finding provides two novel natural products as promising SARS-CoV-2 antivirals.

Our previous efforts have proved that modifications targeting the 150-cavity of influenza neuraminidase can achieve more potent and more selective inhibitors. In this work, four subseries of C5-NH2 modified oseltamivir derivatives were designed and synthesized to explore every region inside the 150-cavity. Among them, compound 23d was exceptionally potent against the whole panel of Group-1 NAs with IC50 values ranging from 0.26 to 0.73 nM, being 15-53 times better than oseltamivir carboxylate (OSC) and 7-11 times better than zanamivir. In cellular assays, 23d showed more potent or equipotent antiviral activities against corresponding virus strains compared to OSC with no cytotoxicity. Furthermore, 23d exhibited high metabolic stability in human liver microsomes (HLM) and low inhibitory effect on main cytochrome P450 enzymes. Notably, 23d displayed favorable druggability in vivo and potent antiviral efficacy in the embryonated egg model and mice model. Overall, 23d appears to be a promising candidate for the treatment of influenza virus infection.

The global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), named coronavirus disease 2019, has infected more than 8.9 million people worldwide. This calls for urgent effective therapeutic measures. RNA-dependent RNA polymerase (RdRp) activity in viral transcription and replication has been recognized as an attractive target to design novel antiviral strategies. Although SARS-CoV-2 shares less genetic similarity with SARS-CoV (~79%) and Middle East respiratory syndrome coronavirus (~50%), the respective RdRps of the three coronaviruses are highly conserved, suggesting that RdRp is a good broad-spectrum antiviral target for coronaviruses. In this review, we discuss the antiviral potential of RdRp inhibitors (mainly nucleoside analogs) with an aim to provide a comprehensive account of drug discovery on SARS-CoV-2.

Conventional influenza vaccines are based on predicting the circulating viruses year by year, conferring limited effectiveness since the antigenicity of vaccine strains does not always match the circulating viruses. This necessitates development of universal influenza vaccines that provide broader and lasting protection against pan-influenza viruses. The discovery of the highly conserved immunogens (epitopes) of influenza viruses provides attractive targets for universal vaccine design. Here we review the current understanding with broadly protective immunogens (epitopes) and discuss several important considerations to achieve the goal of universal influenza vaccines.

Traditional Chinese medicines (TCMs) have proven to possess advantages in counteracting virus infections according to clinical practices. It’s therefore of great value to discover novel antivirals from TCMs. In this paper, One hundred medicinal plants which have been included in TCM prescriptions for antiviral treatment were selected and prefractionated into 5 fractions each by sequentially using cyclohexane, dichloromethane, ethyl acetate, n-butanol, and water. 500 TCM-simplified extracts were then subjected to a phenotypic screening using a recombinant IAV expressing Gaussia luciferase. Ten TCM fractions were identified to possess antiviral activities against influenza virus. The IC50’s of the hit fractions range from 1.08 to 6.45 μg/mL, while the SIs, from 7.52 to 98.40. Furthermore, all the ten hit fractions inhibited the propagation of progeny influenza virus significantly at 20 μg/mL. The hit TCM fractions deserve further isolation for responsible constituents leading towards anti-influenza drugs. Moreover, a library consisting of 500 simplified TCM extracts was established, facilitating antiviral screening in quick response to emerging and re-emerging viruses such as Ebola virus and current SARS-CoV-2 pandemic.

Lignans are widely produced by various plant species; they are a class of natural products that share structural similarity. They usually contain a core scaffold that is formed by two or more phenylpropanoid units. Lignans possess diverse pharmacological properties, including their antiviral activities that have been reported in recent years. This review discusses the distribution of lignans in nature according to their structural classification, and it provides a comprehensive summary of their antiviral activities. Among them, two types of antiviral lignans—podophyllotoxin and bicyclol, which are used to treat venereal warts and chronic hepatitis B (CHB) in clinical, serve as examples of using lignans for antivirals—are discussed in some detail. Prospects of lignans in antiviral drug discovery are also discussed.

The emerging SARS-CoV-2 infection is the cause of the global COVID-19 pandemic. To date, there are limited therapeutic options available to fight this disease. Here we examined the inhibitory abilities of two broad-spectrum antiviral natural products chebulagic acid (CHLA) and punicalagin (PUG) against SARS-CoV-2 viral replication. Both CHLA and PUG reduced virus-induced plaque formation in Vero-E6 monolayer at noncytotoxic concentrations, by targeting the enzymatic activity of viral 3-chymotrypsin-like cysteine protease (3CLpro) as allosteric regulators. Our study demonstrates the potential use of CHLA and PUG as novel COVID-19 therapies.

The worldwide prevalence of herpes simplex virus (HSV) and the shortage of efficient vaccines and novel therapeutic strategies against HSV are widely global concerns. The abundance on the virion and the major stimulus for the virus-neutralizing antibodies makes gD a predominant candidate for cure of HSV infection. In this study, we generated a monoclonal antibody (mAb), termed m27f, targeting to glycoprotein D (gD) of HSV-2, which also has cross-reactivity against HSV-1 gD. It has a high level of neutralizing activity against both HSV-1 and HSV-2, and binds to a highly conserved region (residues 292–297) within the pro-fusion domain of gD. It can effectively block HSV cell-to-cell spread in vitro. The pre- or post-attachment neutralization assay and syncytium formation inhibition assay revealed that m27f neutralizes HSV at the post-binding stage. Moreover, therapeutic administration of m27f completely prevented infection-related mortality of mice challenged with a lethal dose of HSV-2. Our newly identified epitope for the neutralizing antibody would facilitate studies of gD-based HSV entry or vaccine design, and m27f itself demonstrated a high potential for adaptation as a protective or therapeutic drug against HSV., Highlights • We generated a novel neutralizing mAb m27f whose epitope located in the pro-fusion domain of gD. • The protective efficiency of m27f was evaluated in vitro and in vivo. • m27f neutralizes HSV at the post-binding stage.

The influenza A virus (IAV) causes seasonal epidemics and occasional but devastating pandemics, which are of a major public health concern. Although several antiviral drugs are currently available, there is an urgent need to develop novel antiviral therapies with different mechanisms of action due to emergence of drug resistance. In this study, two related compounds, chebulagic acid (CHLA) and chebulinic acid (CHLI), were identified as novel inhibitors against IAV replication. A reporter virus-based infection assay demonstrated that CHLA and CHLI exhibit no inhibitory effect on IAV entry or RNA replication during the virus replication cycle. Results of viral release inhibition assay and neuraminidase (NA) inhibition assay indicated that CHLA and CHLI exert their inhibitory effect on the NA-mediated viral release. Moreover, oseltamivir-resistance mutation NA/H274Y of NA is susceptible to CHLA or CHLI, suggesting a different mechanism of action for CHLA and CHLI. In summary, CHLA and CHLI are promising new NA inhibitors that may be further developed as novel antivirals against IAVs.

Kidney-yang deficiency syndrome (KYDS) infected with the influenza virus is a suitable model to imitate a population at high-risk to influenza infection with a high rate of morbidity and mortality. However, the specific molecular mechanisms underlying this disease remain unclear. A stable reference gene is essential as an internal control for molecular biology research of this condition. Reverse-transcription-quantitative PCR (RT-qPCR) is considered an extremely sensitive technique used for absolute and relative quantification of target genes transcript levels. To accurately estimate the relative expression of genes in cells from mice with KYDS in response to infection with influenza A virus subtype H1N1 (A/H1N1) virus using RT-qPCR, it is necessary to identify suitable reference genes. In the present study, analysis of 10 reference genes (Act-β, β2m, GAPDH, Gusβ, Tubα, Grcc10, Eif4h, Rnf187, Nedd8 and Ywhae) was performed across a set of 4 tissue types: Lung; heart; liver; and kidney. KYDS mice were inoculated with A/H1N1 virus or a mock control. For analysis, geNorm, BestKeeper, NormFinder, and Bio-Rad Maestro™ statistical programs were used for the estimation of the stability of the reference genes. The results were authenticated through extended experimental settings using a group of 10 samples, parallel to 3 additional innate immune system-associated genes of the host, TLR3, TLR7 and RIG-I, which were also analyzed using the same algorithms. From the 4 algorithms, taking into account the joint analyses of the ranking order outputs, the 2 genes Ywhae and Nedd8 were identified to be the most stable for mice with KYDS following infection with A/H1N1 virus. In contrast, the least stable genes in all 4 tissues were GAPDH and β2m. These results may affect the choice of reference genes in future studies that use RT-qPCR analysis of target genes in experimental conditions, such as mice with KYDS infected with influenza A virus.

The hemagglutinin (HA) and neuraminidase (NA) of influenza A virus possess antagonistic activities on interaction with sialic acid (SA), which is the receptor for virus attachment. HA binds SA through its receptor-binding sites, while NA is a receptor-destroying enzyme by removing SAs. The function of HA during virus entry has been extensively investigated, however, examination of NA has long been focused to its role in the exit of progeny virus from infected cells, and the role of NA in the entry process is still under-appreciated. This review summarizes the current understanding of the roles of HA and NA in relation to each other during virus entry.

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanums) is a kind of traditional folk medicinal plant which has been used widely in China for its antivirus, antitumor, and other clinical effects. In this study, ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive/MS) was utilized to analyze the chemical constituents of T. hemsleyanums. Fifty-one constituents were clarified, including flavonoids, anthraquinones, esters, fatty acids, phenols, and catechins. In the subsequent quantitative analysis, the contents of ten compounds of rutin, kaempferol, astragalin, quercitrin, quercetin, vitexin-rhamnoside, isorhamnetin, vitexin, emodin-8-O-β-D-glucoside, and isoquercetin in 18 batches of T. hemsleyanums collected from different places of cultivation were determined. Meanwhile, anti-influenza virus bioactivity in vitro of the above samples was detected with Gaussia Luciferase viral titer assay. It was found that the antiviral bioactivity varied from batches to batches in accordance with content difference of the chemical constituents in T. hemsleyanums. Correlation analysis was performed with SPSS software for the association between LC-MS chemometrics and bioactivity of influenza virus inhibition, and 8 constituents of flavonoids showed positive correlation coefficient, which may provide a valuable clue for searching potential antiviral components in T. hemsleyanums.

A new polysaccharide named GRP (Glehniae radix polysaccharide) was isolated and purified from Glehniae radix by hot water extraction, ethanol precipitation, anion-exchange and gel-filtration chromatography. GRP was homogeneous, with a molecular weight of 1.33×104Da, as determined by high-performance size-exclusion chromatography-refractive index detector analysis. Its structural characteristics were investigated and elucidated by methylation analysis, gas chromatography mass spectrometry, Fourier transform infrared and nuclear magnetic resonance spectroscopy. Based on obtained data, GRP was found to be α‑d‑glucan containing (1→6)-linked and (1→3)-linked backbone with a branch of one (1→6)-linked and terminal glucoses submitting at the C-4 position every fourteen residues. The biological activities of GRP upon proliferation of splenic lymphocyte, RAW264.7 cells and A549 cell, and production of nitric oxide (NO) were investigated in vitro. The results showed that GRP exhibited inhibition against A549 cells proliferation and NO production in RAW264.7 cells, and displayed promotion for proliferation of mouse spleen lymphocytes and RAW264.7 cells, which suggested that GRP may have potential immunoregulation, anti-inflammatory and anti-tumor activity.

Influenza A virus (IAV) causes seasonal epidemics and occasional but devastating pandemics, which are major public health concerns. The putative antiviral therapeutics are useful for the treatment of influenza, however, the emerging resistant strains necessitate a constant search for new drug candidates. Here we report the discovery of a novel antiviral agent, compound CBS1194, which was identified by a parallel high-throughput screening (HTS) campaign using two retroviral pseudotypes bearing H7 or H5 hemagglutinins (HAs). Subsequent analyses demonstrated that CBS1194 is specific to IAVs of group 2, while it has no effect against those of group 1. In a time-of-addition assay, CBS1194 showed a significant inhibitory effect during the early phase of viral infection. In addition, HA-mediated hemolysis can be inhibited by CBS1194 treatment, indicating that this compound may target the HA stalk region, which is responsible for membrane fusion. Escape mutant analyses and in silico docking further revealed that CBS1194 fits into a pocket near the fusion peptide, causing steric hindrance that blocks the low-pH induced rearrangement of HA. In summary, our study identifies a novel fusion inhibitor of group 2 IAVs, which has the potential as lead compound for further development.

Ebola virus is the causative agent of Ebola virus disease in humans. The lethality of Ebola virus infection is about 50%, supporting the urgent need to develop anti-Ebola drugs. Glycoprotein (GP) is the only surface protein of the Ebola virus, which is functionally critical for the virus to attach and enter the host cells, and is a promising target for anti-Ebola virus drug development. In this study, using the recombinant HIV-1/Ebola pseudovirus platform we previously established, we evaluated a small molecule library containing various quinoline compounds for anti-Ebola virus entry inhibitors. Some of the quinoline compounds specifically inhibited the entry of the Ebola virus. Among them, compound SYL1712 was the most potent Ebola virus entry inhibitor with an IC50 of ~1 &mu, M. The binding of SYL1712 to the vial glycoprotein was computationally modeled and was predicted to interact with specific residues of GP. We used the time of the addition assay to show that compound SYL1712 blocks Ebola GP-mediated entry. Finally, consistent with being an Ebola virus entry inhibitor, compound SYL1712 inhibited infectious Ebola virus replication in tissue culture under biosafety level 4 containment, with an IC50 of 2 &mu, M. In conclusion, we identified several related molecules with a diaryl-quinoline scaffold as potential anti-EBOV entry inhibitors, which can be further optimized for anti-Ebola drug development.

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanums) is a kind of traditional folk medicinal plant which has been used widely in China for its antivirus, antitumor, and other clinical effects. In this study, ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive/MS) was utilized to analyze the chemical constituents of T. hemsleyanums. Fifty-one constituents were clarified, including flavonoids, anthraquinones, esters, fatty acids, phenols, and catechins. In the subsequent quantitative analysis, the contents of ten compounds of rutin, kaempferol, astragalin, quercitrin, quercetin, vitexin-rhamnoside, isorhamnetin, vitexin, emodin-8-O-β-D-glucoside, and isoquercetin in 18 batches of T. hemsleyanums collected from different places of cultivation were determined. Meanwhile, anti-influenza virus bioactivity in vitro of the above samples was detected with Gaussia Luciferase viral titer assay. It was found that the antiviral bioactivity varied from batches to batches in accordance with content difference of the chemical constituents in T. hemsleyanums. Correlation analysis was performed with SPSS software for the association between LC-MS chemometrics and bioactivity of influenza virus inhibition, and 8 constituents of flavonoids showed positive correlation coefficient, which may provide a valuable clue for searching potential antiviral components in T. hemsleyanums.

Influenza A virus (IAV) causes seasonal epidemics and occasional but devastating pandemics, which are major public health concerns. Because the effectiveness of seasonal vaccines is highly variable and the currently available drugs are limited in their efficacy because of the emergence of drug resistance, there is an urgent need to develop novel antivirals. In this study, we characterized a recombinant IAV-carrying Gaussia luciferase (Gluc) gene and determined its potential as a tool for evaluating therapeutics. We demonstrated that this recombinant IAV is replication-competent in tissue culture and pathogenic in mice, although it is slightly attenuated compared to the parental virus. Luciferase expression correlated well with virus propagation both in vitro and in vivo, providing a simple measure for viral replication in tissue culture and in mouse lungs. To demonstrate the utility of this virus, ribavirin and oseltamivir phosphate were used to treat the IAV-infected cells and mice, and we observed the dose-dependent inhibition of viral replication by a luciferase assay. Moreover, the decreased luciferase expression in the infected lungs could predict the protective efficacy of antiviral interventions as early as day 2 post virus challenge. In summary, this study provides a new and quantitative approach to evaluate antivirals against IAV.

The prM glycoprotein is thought to be a chaperone for the proper folding, membrane association and assembly of the envelope protein (E) of flaviviruses. The prM-E and E proteins of the Japanese encephalitis virus (JEV) were expressed in insect cells using both the baculovirus-expression system and the transient expression method. Protein expression was analysed by Western blotting and the cytopathic effect was observed by microscopy. In the baculovirus-expression system the E protein, with or without the prM protein, induced syncytial formation in Sf9 cells. Transient expression of prM-E also induced syncytia in Sf9 cells. Immunofluorescence revealed that in presence of prM, E proteins were endoplasmic reticulum-like in distribution, while in the absence of prM, E proteins were located on the cell surface. Sucrose gradient sedimentation and Western blot analysis indicated that the E protein expressed with or without the prM protein was secreted into the culture medium in particulate form. The formation of virus-like particles (VLPs) in the medium was confirmed by electron microscopy and immunoelectron microscopy. The results suggest that the E protein of JEV in the absence of prM, retained its fusion ability, by either cell surface expression or formation of VLPs. Moreover, based on the observation that co-expression of prM-E in Sf9 cells induced considerable syncytial formation, a novel, safe and simple antiviral screening approach is proposed for studying inhibitory antibodies, peptides or small molecules targeting the JEV E protein.

Influenza A virus infections cause significant morbidity and mortality, and novel antivirals are urgently needed. Influenza RNA-dependent RNA polymerase (RdRp) activity has been acknowledged as a promising target for novel antivirals. In this study, a phenotypic versus target-based screening strategy was established to identify the influenza A virus inhibitors targeting the virus RNA transcription/replication steps by sequentially using an RdRp-targeted screen and a replication-competent reporter virus-based approach using the same compounds. To demonstrate the utility of this approach, a pilot screen of a library of 891 compounds derived from natural products was carried out. Quality control analysis indicates that the primary screen was robust for identification of influenza A virus inhibitors targeting RdRp activity. Finally, two hit candidates were identified, and one was validated as a putative RdRp inhibitor. This strategy can greatly reduce the number of false positives and improve the accuracy and efficacy of primary screening, thereby providing a powerful tool for antiviral discovery.

Ebola virus has caused 26 outbreaks in 10 different countries since its identification in 1976, making it one of the deadliest emerging viral pathogens. The most recent outbreak in West Africa from 2014-16 was the deadliest yet and culminated in 11,310 deaths out of 28,616 confirmed cases. Currently, there are no FDA-approved therapeutics or vaccines to treat Ebola virus infections. The slow development of effective vaccines combined with the severity of past outbreaks emphasizes the need to accelerate research into understanding the virus lifecycle and the development of therapeutics for post exposure treatment. Here we present a summary of the major findings on the Ebola virus replication cycle and the therapeutic approaches explored to treat this devastating disease. The major focus of this review is on small molecule inhibitors.

The cDNA fragment of JEV prME gene was cloned into the baculovirus shuttle vector (bacmid) to construct a recombinant baculovirus vector, defined as AcBac-prME. Then the recombinant baculovirus Ac-prME was obtained by transfecting Sf9 cells with AcBac-prME. Western blot analysis and immunofluorescence results indicated that both prM and E proteins were efficiently expressed in Sf9 cells. Electron microscopy suggested that prME was assembled into JEV-VLPs. To further evaluate the potential of JEV-VLPs as vaccine, the mice were immunized with JEV-VLPs and then challenged with lethal JEV. The results of mice survival and pathological changes demonstrated that the JEV-VLPs performed complete protection against JEV-P3 strain and relieved pathological changes in the mice brain significant. This study suggest that JEV-VLPs would be a potential vaccine for Japanese encephalitis virus.