Doxycycline is an antibiotic derived from tetracycline that possesses antimicrobial and anti-inflammatory activities. Antiviral activity of doxycycline against dengue virus has been reported previously; however, its anti-dengue properties need further investigation. This study was conducted to determine the potential activity of doxycycline against dengue virus replication in vitro. Doxycycline inhibited the dengue virus serine protease (DENV2 NS2B-NS3pro) with an IC50 value of 52.3 ± 6.2 μM at 37 °C (normal human temperature) and 26.7 ± 5.3 μM at 40 °C (high fever temperature). The antiviral activity of doxycycline was first tested at different concentrations against DENV2 using a plaque-formation assay. The virus titter decreased significantly after applying doxycycline at levels lower than its 50 % cytotoxic concentration (CC50, 100 μM), showing concentration-dependent inhibition with a 50 % effective concentration (EC50) of approximately 50 μM. Doxycycline significantly inhibited viral entry and post-infection replication of the four dengue serotypes, with serotype-specific inhibition (high activity against DENV2 and DENV4 compared to DENV1 and DENV3). Collectively, these findings underline the need for further experimental and clinical studies on doxycycline, utilizing its anti-dengue and anti-inflammatory activities to attenuate the clinical symptoms of dengue virus infection.
Viruses from the genus Enterovirus (EV) of the Picornaviridae family are known to cause diseases such as hand foot and mouth disease (HFMD), respiratory diseases, encephalitis and myocarditis. The capsid of EV is an attractive target for the development of direct-acting small molecules that can interfere with viral entry. Some of the capsid binders have been evaluated in clinical trials but the majority have failed due to insufficient efficacy or unacceptable off-target effects. Furthermore, most of the capsid binders exhibited a low barrier to resistance. Alternatively, host-targeting inhibitors such as peptides derived from the capsid of EV that can recognize cellular receptors have been identified. However, the majority of these peptides displayed low anti-EV potency (µM range) as compared to the potency of small molecule compounds (nM range). Nonetheless, the development of anti-EV peptides is warranted as they may complement the small-molecules in a drug combination strategy to treat EVs. Lastly, structure-based approach to design antiviral peptides should be utilized to unearth potent anti-EV peptides.
In the search for anti-SARS-CoV-2 drugs, much attention is given to safe and widely available native compounds. The green tea component epigallocatechin 3 gallate (EGCG) is particularly promising because it reportedly inhibits viral replication and viral entry in vitro. However, conclusive evidence for its predominant activity is needed. We tested EGCG effects on the native virus isolated from COVID-19 patients in two independent series of experiments using VERO cells and two different treatment schemes in each series. The results confirmed modest cytotoxicity of EGCG and its substantial antiviral activity. The preincubation scheme aimed at infection prevention has proven particularly beneficial. We complemented that finding with a detailed investigation of EGCG interactions with viral S-protein subunits, including S2, RBD, and the RBD mutant harboring the N501Y mutation. Molecular modeling experiments revealed N501Y-specific stacking interactions in the RBD-ACE2 complex and provided insight into EGCG interference with the complex formation. Together, these findings provide a molecular basis for the observed EGCG effects and reinforce its prospects in COVID-19 prevention therapy.
Despite the importance of DENV as a human pathogen, there is no specific treatment or protective vaccine. Successful entry into the host cells is necessary for establishing the infection. Recently, the virus entry step has become an attractive therapeutic strategy because it represents a barrier to suppress the onset of the infection. Four putative antiviral peptides were designed to target domain III of DENV-2 E protein using BioMoDroid algorithm. Two peptides showed significant inhibition of DENV when simultaneously incubated as shown by plaque formation assay, RT-qPCR, and Western blot analysis. Both DET4 and DET2 showed significant inhibition of virus entry (84.6% and 40.6% respectively) using micromolar concentrations. Furthermore, the TEM images showed that the inhibitory peptides caused structural abnormalities and alteration of the arrangement of the viral E protein, which interferes with virus binding and entry. Inhibition of DENV entry during the initial stages of infection can potentially reduce the viremia in infected humans resulting in prevention of the progression of dengue fever to the severe life-threatening infection, reduce the infected vector numbers, and thus break the transmission cycle. Moreover these peptides though designed against the conserved region in DENV-2 would have the potential to be active against all the serotypes of dengue and might be considered as Hits to begin designing and developing of more potent analogous peptides that could constitute as promising therapeutic agents for attenuating dengue infection.
Japanese encephalitis (JE), a mosquito-borne viral disease, is endemic to the entire east and southeast Asia, and some other parts of the world. Currently, there is no effective therapeutic available for JE; therefore, finding the effective antiviral agent against JEV replication is crucial. In the present study, the in vitro antiviral activity of baicalein and quercetin, two purportedly antiviral bioflavonoids, was evaluated against Japanese encephalitis virus (JEV) replication in Vero cells. Anti-JEV activities of these compounds were examined on different stages of JEV replication cycle. The effects of the compounds on virus replication were determined by foci forming unit reduction assay (FFURA) and quantitative RT-PCR. Baicalein showed potent antiviral activity with IC(50) = 14.28 µg/mL when it was introduced to the Vero cells after adsorption of JEV. Quercetin exhibited weak anti-JEV effects with IC(50) = 212.1 µg/mL when the JEV infected cells were treated with the compound after virus adsorption. However, baicalein exhibited significant effect against JEV adsorption with IC(50) = 7.27 µg/mL while quercetin did not show any anti-adsorption activity. Baicalein also exhibited direct extracellular virucidal activity on JEV with IC(50) = 3.44 µg/mL. However, results of quantitative RT-PCR experiments confirmed the findings from FFURA. This study demonstrated that baicalein should be considered as an appropriate candidate for further investigations, such as the study of molecular and cellular mechanism(s) of action and in vivo evaluation for the development of an effective antiviral compound against Japanese encephalitis virus.
Dengue is an arthropod-borne viral disease that has become endemic and a global threat in many countries with no effective antiviral drug available currently. This study showed that flavonoids: silymarin and baicalein could inhibit the dengue virus in vitro and were well tolerated in Vero cells with a half-maximum cytotoxic concentration (CC50) of 749.70 µg/mL and 271.03 µg/mL, respectively. Silymarin and baicalein exerted virucidal effects against DENV-3, with a selective index (SI) of 10.87 and 21.34, respectively. Baicalein showed a better inhibition of intracellular DENV-3 progeny with a SI of 7.82 compared to silymarin. Baicalein effectively blocked DENV-3 attachment (95.59%) to the Vero cells, while silymarin prevented the viral entry (72.46%) into the cells, thus reducing viral infectivity. Both flavonoids showed promising antiviral activity against all four dengue serotypes. The in silico molecular docking showed that silymarin could bind to the viral envelope (E) protein with a binding affinity of - 8.5 kcal/mol and form hydrogen bonds with the amino acids GLN120, TRP229, ASN89, and THR223 of the E protein. Overall, this study showed that silymarin and baicalein exhibited potential anti-DENV activity and could serve as promising antiviral agents for further development against dengue infection.
Influenza A virus is a particularly problematic virus because of its ability to cause high levels of morbidity on a global scale within a remarkably short period of time. It also has the potential to kill very large numbers of people as occurred in the Spanish influenza pandemic in 1918. Options for antiviral therapy are limited because of the paucity of available drugs and the rapid mutation rate of the virus leading to the emergence of drug-resistant strains. The current H1N1 pandemic and potential threats posed by other strains highlight the need to develop novel therapeutic and prophylactic strategies. Here, we summarize the current state and recent developments of peptide-based inhibitors of influenza A virus.
Dengue is an important global threat caused by dengue virus (DENV) that records an estimated 390 million infections annually. Despite the availability of CYD-TDV as a commercial vaccine, its long-term efficacy against all four dengue virus serotypes remains unsatisfactory. There is therefore an urgent need for the development of antiviral drugs for the treatment of dengue. Peptide was once a neglected choice of medical treatment but it has lately regained interest from the pharmaceutical industry following pioneering advancements in technology. In this review, the design of peptide drugs, antiviral activities and mechanisms of peptides and peptidomimetics (modified peptides) action against dengue virus are discussed. The development of peptides as inhibitors for viral entry, replication and translation is also described, with a focus on the three main targets, namely, the host cell receptors, viral structural proteins and viral non-structural proteins. The antiviral peptides designed based on these approaches may lead to the discovery of novel anti-DENV therapeutics that can treat dengue patients.
Zika virus (ZIKV) infection is a serious public health concern. ZIKV infection has been associated with increased occurrences of microcephaly among newborns and incidences of Guillain-Barré syndrome among adults. No specific therapeutics or vaccines are currently available to treat and protect against ZIKV infection. Here, a plant-secreted phytoalexin, resveratrol (RES), was investigated for its ability to inhibit ZIKV replication in vitro. Several RES treatment regimens were used. The ZIKV titers of mock- and RES-treated infected cell cultures were determined using the focus-forming assay and the Zika mRNA copy number as determined using qRT-PCR. Our results suggested that RES treatment reduced ZIKV titers in a dose-dependent manner. A reduction of >90% of virus titer and ZIKV mRNA copy number was achieved when infected cells were treated with 80 µM of RES post-infection. Pre-incubation of the virus with 80 µM RES showed >30% reduction in ZIKV titers and ZIKV mRNA copy number, implying potential direct virucidal effects of RES against the virus. The RES treatment reduced >70% virus titer in the anti-adsorption assay, suggesting the possibility that RES also interferes with ZIKV binding. However, there was no significant decrease in ZIKV titer when a short-period of RES treatment was applied to cells before ZIKV infection (pre-infection) and after the virus bound to the cells (virus internalization inhibition), implying that RES acts through its continuous presence in the cell cultures after virus infection. Overall, our results suggested that RES exhibited direct virucidal activity against ZIKV and possessed anti-ZIKV replication properties, highlighting the need for further exploration of RES as a potential antiviral molecule against ZIKV infection.
Dengue is an arthropod-borne viral disease that has become endemic and a global threat in over 100 countries. The increase in prevalence would require a long-term measure to control outbreaks. Sanofi Pasteur has licensed the tetravalent dengue vaccine (Dengvaxia) in certain dengue endemic countries. However, the efficacy of the vaccine is limited against certain dengue serotypes and can only be used for individuals from the age from 9 to 45 years old. Over the years, there has been intense research conducted on the development of antivirals against dengue virus (DENV) through either inhibiting the virus replication or targeting the host cell mechanism to block the virus entry. However, no approved antiviral drug against dengue is yet available. In this chapter, we describe the dengue antiviral development workflow including (i) prophylactic, (ii) virucidal, and (iii) postinfection assays that are employed in the antiviral drug screening process against DENV. Further, we demonstrate different methods that can be used to enumerate the reduction in virus foci number including foci-forming unit reduction assay (FFURA), estimation of viral RNA copy number through quantitative real-time PCR, and a high-throughput enzyme linked immunosorbent assay (ELISA)-based quantification of virus particles.
The coronavirus disease 2019 (COVID-19) pandemic with high infectivity and mortality has caused severe social and economic impacts worldwide. Growing reports of COVID-19 patients with multi-organ damage indicated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) may also disturb the cardiovascular system. Herein, we used human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) as the in vitro platform to examine the consequence of SARS-CoV2 infection on iCMs. Differentiated iCMs expressed the primary SARS-CoV2 receptor angiotensin-converting enzyme-II (ACE2) and the transmembrane protease serine type 2 (TMPRSS2) receptor suggesting the susceptibility of iCMs to SARS-CoV2. Following the infection of iCMs with SARS-CoV2, the viral nucleocapsid (N) protein was detected in the host cells, demonstrating the successful infection. Bioinformatics analysis revealed that the SARS-CoV2 infection upregulates several inflammation-related genes, including the proinflammatory cytokine tumor necrosis factor-α (TNF-α). The pretreatment of iCMs with TNF-α for 24 h, significantly increased the expression of ACE2 and TMPRSS2, SASR-CoV2 entry receptors. The TNF-α pretreatment enhanced the entry of GFP-expressing SARS-CoV2 pseudovirus into iCMs, and the neutralization of TNF-α ameliorated the TNF-α-enhanced viral entry. Collectively, SARS-CoV2 elevated TNF-α expression, which in turn enhanced the SARS-CoV2 viral entry. Our findings suggest that, TNF-α may participate in the cytokine storm and aggravate the myocardial damage in COVID-19 patients.
The past decade has seen the re-emergence of Chikungunya virus (CHIKV) as a major global health threat, affecting millions around the world. Although fatal infections are rare among infected patients, the occurrence of long-lasting polyarthralgia has a significant impact on patients' quality of lives and ability to work. These issues were the stimuli for this study to determine the potential of baicalin, a bioflavonoid, as the novel antiviral compound against CHIKV. It was found that baicalin was well tolerated by Vero, BHK-21 and HEK 293T cells with maximal nontoxic doses >600 μM, ≈ 350 μM and ≈110 μM, respectively. Antiviral assays indicated that baicalin was the most effective inhibitor when tested for its direct virucidal activity with EC50 ≈ 7 μM, followed by inhibition of virus entry into the host cell, attachment of virus particle to cellular receptors and finally intracellular replication of viral RNA genome. In silico analysis using molecular docking demonstrated close interactions between baicalin and CHIKV envelope protein with considerably strong binding affinity of -9.7 kcal/mol. qRT-PCR analysis revealed that baicalin had the greatest effect on the synthesis of viral negative stand RNA with EC50 ≈ 0.4 μM followed by the inhibition of synthesis of positive-strand genomic (EC50 ≈ 13 μM) and subgenomic RNAs (EC50 ≈ 14 μM). These readings indicate that the compound efficiently inhibits replicase complexes formation but is a less potent inhibitor of existing replicase complexes. Coherent with this hypothesis, the use of recombinant CHIKV replicons harboring Renilla luciferase marker showed that replication of corresponding replicon RNAs was only slightly downregulated at higher doses of baicalin, with EC50 > 100 μM. Immunofluorescence and western blotting experiments demonstrated dose-dependent inhibition of expression of different viral proteins. It was also observed that levels of important protein markers for cellular autophagy (LC3) and apoptosis (Bax) were reduced in baicalin treatment groups as compared with untreated virus infected controls. In summary, given its low toxicity and high efficacy against CHIKV, baicalin has great potential to be developed as the novel antiviral compound for CHIKV. In vivo studies to evaluate its activity in a more complexed system represent a necessary step for future analysis.
Zika virus (ZIKV) is an arthropod-borne flavivirus that causes newborn microcephaly and Guillian-Barré syndrome in adults. No therapeutics are available to treat ZIKV infection or other flaviviruses. In this study, we explored the inhibitory effect of glycosaminoglycans and analogues against ZIKV infection. Highly sulfated heparin, dextran sulfate and suramin significantly inhibited ZIKV infection in Vero cells. De-sulfated heparin analogues lose inhibitory effect, implying that sulfonate groups are critical for viral inhibition. Suramin, an FDA-approved anti-parasitic drug, inhibits ZIKV infection with 3-5 log10 PFU viral reduction with IC50value of ∼2.5-5 μg/ml (1.93 μM-3.85 μM). A time-of-drug-addition study revealed that suramin remains potent even when administrated at 1-24 hpi. Suramin inhibits ZIKV infection by preventing viral adsorption, entry and replication. Molecular dynamics simulation revealed stronger interaction of suramin with ZIKV NS3 helicase than with the envelope protein. Suramin warrants further investigation as a potential antiviral candidate for ZIKV infection. Heparan sulfate (HS) is a cellular attachment receptor for multiple flaviviruses. However, no direct ZIKV-heparin interaction was observed in heparin-binding analysis, and downregulate or removal of cellular HS with sodium chlorate or heparinase I/III did not inhibit ZIKV infection. This indicates that cell surface HS is not utilized by ZIKV as an attachment receptor.