Analyses of the onset mechanisms of cardio-stimulatory action by aciclovir.

Cardio-stimulatory actions of aciclovir have been considered to primarily depend on the sympathetically-mediated reflex resulting from its hypotensive effect. To further clarify onset mechanisms of the cardio-stimulatory actions, we initially studied them using isoflurane-anesthetized dogs under thorough ß1-adrenoceptor blockade with atenolol (1 mg/kg, i.v.) (n = 4). Aciclovir (20 mg/kg/10 min, i.v.) decreased mean arterial blood pressure by 10 mmHg, whereas it increased heart rate by 10 bpm and maximum upstroke velocity of ventricular pressure by 928 mmHg/s, and shortened AH interval by 2 ms, indicating that cardio-stimulatory actions were not totally abolished by ß1-adrenoceptor blockade. Then, unknown mechanisms of cardio-stimulatory action were explored. Since aciclovir has a similar chemical structure to theophylline, in silico molecular docking simulation was performed, indicating aciclovir as well as theophylline possesses strong likelihood of interactions with phosphodiesterase 1A, 1C and 3A. Indeed, aciclovir inhibited phosphodiesterase 1A derived from the bovine heart (n = 4), moreover it exerted positive chronotropic action on the atrial tissue preparation of rats along with an increase of tissue cyclic AMP concentration (n = 4). These results indicate that cardio-stimulatory actions of aciclovir could result from not only hypotension-induced, reflex-mediated increase of sympathetic tone but also its inhibitory effects on phosphodiesterase in the heart.

Analysis of antiviral drug properties of thymidine kinase of herpes B virus using recombinant herpes simplex virus 1.

IMPORTANCE: Zoonotic infection of humans with herpes B virus (BV) causes severe neurological diseases. Acyclovir (ACV) and ganciclovir (GCV), most frequently used as anti-herpes drugs, are recommended for prophylaxis and therapy in human BV infection. In this study, we examined the property of BV thymidine kinase (TK) against anti-herpes drugs using a recombinant herpes simplex virus type 1 (HSV-1) carrying BV TK gene. We found that HSV-1 carrying BV TK was similarly sensitive to GCV as HSV-1 carrying varicella zoster virus TK. In addition, we demonstrated that BV TK was not mutated in the GCV- and ACV-resistant HSV-1 carrying BV TK, suggesting that ACV- or GCV-resistant BV might be rare during treatment with these antiviral drugs. These data can provide a new insight into the properties of BV TK in terms of the development of drug resistance.

Clinical evaluation of antiviral combination treatment in cats with feline herpesvirus-1 infection.

Feline herpesvirus-1 (FHV-1) can cause lifelong problems such as rhinotracheitis and ocular disease due to latency and reactivation in affected cats. The particular effects of antiviral drugs have been separately investigated in previous studies for decades and little is known about the combination treatment in active FHV-1 infection. Therefore, we aimed to evaluate the effects of antiviral combination on clinical effectiveness in cats with naturally occurring FHV-1 infection. 28 cats suffering from clinical signs of sneezing, nasal congestion, conjunctivitis, and eye/nose discharge were involved in this study following FHV-1 DNA detection by PCR assay in oculo-oropharyngeal samples. The treatment protocol was as follows: oral famciclovir and L-lysine, ophthalmic acyclovir, and subcutaneous amoxicillin plus clavulanic acid. The symptoms improved each day and total recovery success rate was 80% reduction in clinical scores at the end of the treatment on day 10 (p<0.001). Additionally, PCR was found to be negative for FHV-1 DNA in 82.1% of the samples after the treatment. There were mild decreases in neutrophil and monocyte counts (p>0.05). The arginine to lysine ratio decreased in favour of lysine (p<0.01). As a result, the antiviral combination treatment with famciclovir, L-lysine and ophthalmic acyclovir, and antibacterial drug appears to be clinically effective for the treatment of naturally occurring active FHV-1 infection in cats. In addition, any adverse clinical effect has not been determined associated with the antiviral combination during the study.

Large Subunit of the Human Herpes Simplex Virus Terminase as a Promising Target in Design of Anti-Herpesvirus Agents.

Herpes simplex virus type 1 (HSV-1) is an extremely widespread pathogen characterized by recurrent infections. HSV-1 most commonly causes painful blisters or sores around the mouth or on the genitals, but it can also cause keratitis or, rarely, encephalitis. First-line and second-line antiviral drugs used to treat HSV infections, acyclovir and related compounds, as well as foscarnet and cidofovir, selectively inhibit herpesvirus DNA polymerase (DNA-pol). It has been previously found that (S)-4-[6-(purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine (compound 1) exhibits selective anti-herpesvirus activity against HSV-1 in cell culture, including acyclovir-resistant mutants, so we consider it as a lead compound. In this work, the selection of HSV-1 clones resistant to the lead compound was carried out. High-throughput sequencing of resistant clones and reference HSV-1/L2 parent strain was performed to identify the genetic determinants of the virus's resistance to the lead compound. We identified a candidate mutation presumably associated with resistance to the virus, namely the T321I mutation in the UL15 gene encoding the large terminase subunit. Molecular modeling was used to evaluate the affinity and dynamics of the lead compound binding to the putative terminase binding site. The results obtained suggest that the lead compound, by binding to pUL15, affects the terminase complex. pUL15, which is directly involved in the processing and packaging of viral DNA, is one of the crucial components of the HSV terminase complex. The loss of its functional activity leads to disruption of the formation of mature virions, so it represents a promising drug target. The discovery of anti-herpesvirus agents that affect biotargets other than DNA polymerase will expand our possibilities of targeting HSV infections, including those resistant to baseline drugs.

An industrial procedure for the intestinal permeability enhancement of acyclovir: in-vitro and histological evidence.

Acyclovir, an antiviral drug, has low bioavailability due to its low permeability. Consequently, high drug doses and frequent administration are required. This study investigates the use of span 60, at different concentrations, as a granulating agent to enhance drug permeability using an industrial procedure on a pilot scale. The micromeritics, drug content, drug crystallinity, drug partition coefficient, and drug release of the produced formulations were examined. The findings revealed an enhanced drug partition coefficient, suggesting drug entrapment in the polar portion of span 60. The drug release profiles exhibited rapid and complete drug release. The improvement of the drug permeability was evaluated using a modified non-everted sac technique. Notably, drug permeability through the rabbit intestine significantly improved, as evidenced by various calculated permeation parameters, providing insights into the drug absorption mechanism. The widening of the paracellular pathway was observed through histological examination of the rabbit intestinal segment, which aligns with the drug absorption mechanism. The utilization of a paracellular pathway enhancer as a granulating agent holds promise as a strategy to enhance the oral bioavailability of class III drugs. Overall, this study presents a novel drug delivery approach to enhance drug permeation and bioavailability, with potential implications for other medications.

Inhibition of herpes simplex virus by wedelolactone via targeting viral envelope and cellular TBK1/IRF3 and SOCS1/STAT3 pathways.

OBJECTIVES: Development of novel antiherpes simplex virus (HSV) agents with active mechanisms different from nucleoside analogues is of high importance. Herein, we investigated the anti-HSV activities and mechanisms of wedelolactone (WDL) both in vitro and in vivo. METHODS: Cytopathic effect (CPE) inhibition assay, plaque assay, and western blot assay were used to evaluate the anti-HSV effects of WDL in vitro. The immunofluorescence assay, RT-PCR assay, plaque reduction assay, sandwich ELISA assay, syncytium formation assay, tanscriptome analysis and western blot assay were used to explore the anti-HSV mechanisms of WDL. The murine encephalitis and vaginal models of HSV infection were performed to evaluate the anti-HSV effects of WDL in vivo. RESULTS: WDL possessed inhibitory effects against both HSV-1 and HSV-2 in different cells with low toxicity, superior to the effects of acyclovir. WDL can directly inactivate the HSV particle via destruction of viral envelope and block HSV replication process after virus adsorption, different from the mechanisms of acyclovir. WDL may influence the host genes and signaling pathways related to HSV infection and immune responses. WDL can mainly interfere with the TBK1/IRF3 and SOCS1/STAT3 pathways to reduce HSV infection and inflammatory responses. Importantly, WDL treatment markedly improved mice survival, attenuated inflammatory symptoms, and reduced the virus titres in both HSV-1 and HSV-2 infected mice. CONCLUSIONS: Thus, the natural compound WDL has the potential to be developed into a novel anti-HSV agent targeting both viral envelope and cellular TBK1/IRF3 and SOCS1/STAT3 pathways.

Variant Analysis of the Thymidine Kinase and DNA Polymerase Genes of Herpes Simplex Virus in Korea: Frequency of Acyclovir Resistance Mutations.

The thymidine kinase (TK) and DNA polymerase (pol) genes of the herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) are two important genes involved in antiviral resistance. We investigated the genetic polymorphisms of the HSV-TK and pol genes in clinical isolates from Korean HSV-infected patients using next-generation sequencing (NGS) for the first time in Korea. A total of 81 HSV-1 and 47 HSV-2 isolates were examined. NGS was used to amplify and sequence the TK and pol genes. Among the 81 HSV-1 isolates, 12 and 17 natural polymorphisms and 9 and 23 polymorphisms of unknown significance in TK and pol were found, respectively. Two HSV-1 isolates (2.5%) exhibited the E257K amino acid substitution in TK, associated with antiviral resistance. Out of 47 HSV-2 isolates, 8 natural polymorphisms were identified in TK, and 9 in pol, with 13 polymorphisms of unknown significance in TK and 10 in pol. No known resistance-related mutations were observed in HSV-2. These findings contribute to our understanding of the genetic variants associated with antiviral resistance in HSV-1 and HSV-2 in Korea, with frequencies of known antiviral resistance-related mutations of 2.5% and 0% in HSV-1 and HSV-2, respectively.

Genotypic testing improves detection of antiviral resistance in human herpes simplex virus.

BACKGROUND: Antiviral resistance in human herpes simplex viruses (HSV) remains a significant clinical challenge in immunocompromised populations. Although molecular tests have largely replaced viral culture for HSV diagnosis and molecular antiviral resistance testing is available for many viruses, HSV resistance testing continues to rely on phenotypic, viral culture-based methods, requiring weeks for results. Consequently, treatment of suspected HSV resistance remains largely empiric. METHODS: We used HSV whole genome sequencing and a database of previously characterized HSV acyclovir and foscarnet resistance mutations to evaluate the performance of genotypic antiviral resistance testing among 19 control strains compared to in-house plaque reduction assay (PRA) and 25 clinical isolates sent for reference lab PRA antiviral resistance testing. RESULTS: Among control strains, 23/29 (79.3%) results were concordant, 5 (17.2%) were indeterminate, and 1 (3.4%) was discordant. Indeterminate results were caused by variants of uncertain significance (VUS), including mutations without published phenotypes and mutations with contradictory results. Among clinical isolates, 14/40 (35%) results were concordant, 17 (42.5%) were indeterminate, and 9 (22.5%) were discordant. All discordant results were in reportedly phenotypically-susceptible HSV-1 strains yet possessed resistance mutations. Three contained resistant subpopulations. 6/8 (75%) discordant phenotypes were concordant with resistant genotypes upon repeat PRA. CONCLUSIONS: These data support the combination of genotypic and phenotypic testing to diagnose HSV resistance more accurately and likely more rapidly than phenotypic testing alone. Genotypic context of resistance mutations and the ability of viral strains to form plaques in culture may affect phenotypic resistance results, highlighting the limitations of PRA alone as a gold standard method.

Acyclovir-resistant HSV-1 isolates among immunocompromised patients in southern Taiwan: Low prevalence and novel mutations.

Herpes simplex virus type 1 (HSV-1) can establish latency in humans and easily relapse in immunocompromised patients, with significant mortality. Treatment with acyclovir (ACV) can result in the emergence of HSV resistance. A total of 440 frozen HSV-1 isolates collected from 318 patients from January 2014 to July 2019 were obtained from National Cheng Kung University Hospital in southern Taiwan. These 440 isolates were subjected to phenotypic studies for ACV-resistance by initial screening with the plaque reduction assay (PRA) and further validation by the DNA reduction assay (DRA). The ACV-resistant strains were further investigated by Sanger sequencing for the full-length UL23 and UL30 genes, which encode thymidine kinase and DNA polymerase, respectively. Hematological malignancies or hematopoietic stem-cell transplantation patients accounted for 56.9% (124/218) among the immunocompromised patients (218/318) in this study. Repeated sampling for HSV testing was 50% (109/218) in immunocompromised patients. Only 1.38% (3/218) of immunocompromised patients and 0.9% (3/318) of all patients developed ACV-resistant HSV-1 as measured by phenotypic screening assays. It is noteworthy that a novel Y248D mutation in the UL23 gene from an immunocompromised patient was found by both PRA and DRA. In 3D protein predicting analysis, uncharged Y248 was located at an alpha-helix and substituted by negative-charged D248, which may alter the function of viral thymidine kinase. Besides, three unreported mutations related to natural polymorphism were found in virus isolates from two immunocompetent patients, including 683-688 deletion, R227H, and A351D in the UL30 gene. These data show that the prevalence of ACV-resistant HSV-1 among immunocompromised patients in southern Taiwan is low. These results will be helpful for the clinical management and treatment of HSV infections.

Novel UL23 and UL30 substitutions in HSV1 and HSV2 viruses related to polymorphism or drug resistance.

Data on herpes simplex virus (HSV) polymorphism as well as acyclovir (ACV) and foscarnet (FOS) resistance mutations are not exhaustive and may hinder accurate diagnosis by next-generation sequencing (NGS). Here, we report novel UL23 and UL30 substitutions for HSV1 and HSV2 identified in immunocompromised patients treated for hematological malignancies during the last 6 years of HSV resistance surveillance at the University Hospital of Lyon. For HSV1, 35 novel UL23 substitutions and 52 novel UL30 substitutions were identified. For HSV2, 2 novel UL23 substitutions and 12 novel UL30 substitutions were identified. These results allow to complete the database of HSV1 and HSV2 substitutions, related either to polymorphism or to ACV and FOS resistance.

Inhibition Effects and Mechanisms of Marine Polysaccharide PSSD against Herpes Simplex Virus Type 2.

Genital herpes is a common sexually transmitted disease mainly caused by herpes simplex virus type 2 (HSV-2), which can increase the risk of HIV transmission and is a major health problem in the world. Thus, it is of great significance to develop new anti-HSV-2 drugs with high efficiency and low toxicity. In this study, the anti-HSV-2 activities of PSSD, a marine sulfated polysaccharide, was deeply explored both in vitro and in vivo. The results showed that PSSD had marked anti-HSV-2 activities in vitro with low cytotoxicity. PSSD can directly interact with virus particles to inhibit the adsorption of virus to the cell surface. PSSD may also interact with virus surface glycoproteins to block virus-induced membrane fusion. Importantly, PSSD can significantly attenuate the symptoms of genital herpes and weight loss in mice after gel smear treatment, as well as reducing the titer of virus shedding in the reproductive tract of mice, superior to the effect of acyclovir. In summary, the marine polysaccharide PSSD possesses anti-HSV-2 effects both in vitro and in vivo, and has potential to be developed into a novel anti-genital herpes agent in the future.

Topical formulations containing Trichilia catigua extract as therapeutic options for a genital and an acyclovir-resistant strain of herpes recurrent infection.

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infect, respectively, 67% and 13% of the world population, most commonly causing mild symptoms, such as blisters/ulcers. However, severe conditions such as keratitis, encephalitis, and systemic infections may occur, generally associated with the patient's immunological condition. Although Acyclovir® (ACV) and its analogs are the reference drugs for herpetic infections, the number of ACV-resistant HSV infections is growing exponentially. Therefore, new natural products' bioactive compounds have been studied to develop novel effective anti-herpetics. Trichilia catigua is a plant widely used in traditional medicine, including the treatment of skin diseases and sexual infections. In our study, 16 extracts from the bark of T. catigua, obtained with different solvents and their combinations, were evaluated against HSV-1 AR and HSV-2, respectively, ACV resistance and genital strains in vitro. The extracts with the highest selectivity index were used to prepare new topical anti-herpetic formulations and confirmed in vivo. Two new topical formulations were suggested to treat cutaneous and genital herpetic recurrent lesions. The cytotoxicity and antiviral activity were tested using the MTT method. The cytotoxic (CC50) and inhibitory (IC50) concentrations of 50% and the selectivity index (SI: CC50/IC50) were determined. Tc12, Tc13, and Tc16 were added to the formulations. Infected BALB/c mice were treated for 8 days, and the severity of the herpetic lesions was analyzed daily. All CEs showed a CC50 value ranging from 143 to 400 µg/mL, except for Tc3 and Tc10. Tc12, Tc13, and Tc16 showed the best SI in the 0 h, virucidal, and adsorption inhibition assays. In the in vivo test against HSV-1 AR, the infected animals treated with creams were statistically different from the infected non-treated animals and similar to ACV-treated mice. In HSV-2-infected genitalia, similar effects were found for Tc13 and Tc16 gels. The present study demonstrated that extracts from the bark of T. catigua, traditionally used in folk medicine, are a valuable source of active compounds with anti-herpetic activity. The extracts showed a virucidal mechanism of action and prevented the initial stages of viral replication. The cutaneous and genital infections were strongly inhibited by the Tc12, Tc13, and Tc16 extracts. New topical therapeutic alternatives using Trichilia catigua extracts are suggested for patients infected with ACV-resistant strains of HSV.

Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids.

Herpes simplex encephalitis is a life-threatening disease of the central nervous system caused by herpes simplex viruses (HSVs). Following standard of care with antiviral acyclovir treatment, most patients still experience various neurological sequelae. Here we characterize HSV-1 infection of human brain organoids by combining single-cell RNA sequencing, electrophysiology and immunostaining. We observed strong perturbations of tissue integrity, neuronal function and cellular transcriptomes. Under acyclovir treatment viral replication was stopped, but did not prevent HSV-1-driven defects such as damage of neuronal processes and neuroepithelium. Unbiased analysis of pathways deregulated upon infection revealed tumour necrosis factor activation as a potential causal factor. Combination of anti-inflammatory drugs such as necrostatin-1 or bardoxolone methyl with antiviral treatment prevented the damages caused by infection, indicating that tuning the inflammatory response in acute infection may improve current therapeutic strategies.

Reducing lipid peroxidation attenuates stress-induced susceptibility to herpes simplex virus type 1.

Psychological stress increases the susceptibility to herpes simplex virus type 1 (HSV-1) infection. There is no effective intervention due to the unknown pathogenesis mechanisms. In this study we explored the molecular mechanisms underlying stress-induced HSV-1 susceptibility and the antiviral effect of a natural compound rosmarinic acid (RA) in vivo and in vitro. Mice were administered RA (11.7, 23.4 mg·kg-1·d-1, i.g.) or acyclovir (ACV, 206 mg·kg-1·d-1, i.g.) for 23 days. The mice were subjected to restraint stress for 7 days followed by intranasal infection with HSV-1 on D7. At the end of RA or ACV treatment, mouse plasma samples and brain tissues were collected for analysis. We showed that both RA and ACV treatment significantly decreased stress-augmented mortality and alleviated eye swelling and neurological symptoms in HSV-1-infected mice. In SH-SY5Y cells and PC12 cells exposed to the stress hormone corticosterone (CORT) plus HSV-1, RA (100 µM) significantly increased the cell viability, and inhibited CORT-induced elevation in the expression of viral proteins and genes. We demonstrated that CORT (50 µM) triggered lipoxygenase 15 (ALOX15)-mediated redox imbalance in the neuronal cells, increasing the level of 4-HNE-conjugated STING, which impaired STING translocation from the endoplasmic reticulum to Golgi; the abnormality of STING-mediated innate immunity led to HSV-1 susceptibility. We revealed that RA was an inhibitor of lipid peroxidation by directly targeting ALOX15, thus RA could rescue stress-weakened neuronal innate immune response, thereby reducing HSV-1 susceptibility in vivo and in vitro. This study illustrates the critical role of lipid peroxidation in stress-induced HSV-1 susceptibility and reveals the potential for developing RA as an effective intervention in anti-HSV-1 therapy.

A Herpes Simplex Virus 1 DNA Polymerase Multidrug Resistance Mutation Identified in a Patient With Immunodeficiency and Confirmed by Gene Editing.

BACKGROUND: Herpes simplex virus 1 can cause severe infections in individuals who are immunocompromised. In these patients, emergence of drug resistance mutations causes difficulties in infection management. METHODS: Seventeen herpes simplex virus 1 isolates were obtained from orofacial/anogenital lesions in a patient with leaky severe combined immunodeficiency over 7 years, before and after stem cell transplantation. Spatial/temporal evolution of drug resistance was characterized genotypically-with Sanger and next-generation sequencing of viral thymidine kinase (TK) and DNA polymerase (DP)-and phenotypically. CRISPR/Cas9 was used to introduce the novel DP Q727R mutation, and dual infection-competition assays were performed to assess viral fitness. RESULTS: Isolates had identical genetic backgrounds, suggesting that orofacial/anogenital infections derived from the same virus lineage. Eleven isolates proved heterogeneous TK virus populations by next-generation sequencing, undetectable by Sanger sequencing. Thirteen isolates were acyclovir resistant due to TK mutations, and the Q727R isolate additionally exhibited foscarnet/adefovir resistance. Recombinant Q727R mutant virus showed multidrug resistance and increased fitness under antiviral pressure. CONCLUSIONS: Long-term follow-up of a patient with severe combined immunodeficiency revealed virus evolution and frequent reactivation of wild-type and TK mutant strains, mostly as heterogeneous populations. The DP Q727R resistance phenotype was confirmed with CRISPR/Cas9, a useful tool to validate novel drug resistance mutations.

Novel inhibitors of HSV-1 protease effective in vitro and in vivo.

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen known to cause infections of diverse severity, ranging from mild ulceration of mucosal and dermal tissues to life-threatening viral encephalitis. In most cases, standard treatment with acyclovir is sufficient to manage the disease progression. However, the emergence of ACV-resistant strains drives the need for new therapeutics and molecular targets. HSV-1 VP24 is a protease indispensable for the assembly of mature virions and, as such, constitutes an interesting target for the therapy. In this study, we present novel compounds, KI207M and EWDI/39/55BF, that block the activity of VP24 protease and consequently inhibit HSV-1 infection in vitro and in vivo. The inhibitors were shown to prevent the egress of viral capsids from the cell nucleus and suppress the cell-to-cell spread of the infection. They were also proven effective against ACV-resistant HSV-1 strains. Considering their low toxicity and high antiviral potency, the novel VP24 inhibitors could provide an alternative for treating ACV-resistant infections or a drug to be used in combined, highly effective therapy.

A potential anti-HIV-1 compound, Q308, inhibits HSV-2 infection and replication in vitro and in vivo.

HSV-2 is a common human pathogen worldwide that causes genital herpes. Due to the lack of an effective HSV-2 vaccine in the foreseeable future, there is an urgent need to develop effective, safe and affordable anti-HSV-2 agents. Our previous studies confirmed that a small-molecule compound, Q308, effectively inhibits the reactivation of latent HIV and might be developed as an anti-HIV-1 agent. Patients infected with HSV-2 are generally more susceptible to HIV-1 infection than normal humans. In this study, we found that Q308 treatment had strong inhibitory activity against both HSV-2 and acyclovir-resistant HSV-2 strains in vitro and reduced the viral titers in tissue. And this treatment effectively ameliorated the cytokine storm and pathohistological changes caused by HSV-2 infection in HSV-2-infected mice. Unlike nucleoside analogs such as acyclovir, Q308 inhibited post-viral entry events by attenuating the synthesis of viral proteins. Furthermore, Q308 treatment blocked HSV-2-induced PI3K/AKT phosphorylation due to its inhibition on viral infection and replication. Overall, Q308 treatment exhibits potent anti-HSV-2 activity by inhibiting viral replication both in vitro and in vivo. Q308 is a promising lead compound for the development of new anti-HSV-2/HIV-1 therapies, particularly against acyclovir-resistant HSV-2 strains.

A bivalent ß-carboline derivative inhibits macropinocytosis-dependent entry of pseudorabies virus by targeting the kinase DYRK1A.

Pseudorabies virus (PRV) has become a "new life-threatening zoonosis" since the human-originated PRV strain was first isolated in 2020. To identify novel anti-PRV agents, we screened a total of 107 ß-carboline derivatives and found 20 compounds displaying antiviral activity against PRV. Among them, 14 compounds showed better antiviral activity than acyclovir. We found that compound 45 exhibited the strongest anti-PRV activity with an IC50 value of less than 40 nM. Our in vivo studies showed that treatment with 45 significantly reduced the viral loads and protected mice challenged with PRV. To clarify the mode of action of 45, we conducted a time of addition assay, an adsorption assay, and an entry assay. Our results indicated that 45 neither had a virucidal effect nor affected viral adsorption while significantly inhibiting PRV entry. Using the FITC-dextran uptake assay, we determined that 45 inhibits macropinocytosis. The actin-dependent plasma membrane protrusion, which is important for macropinocytosis, was also suppressed by 45. Furthermore, the kinase DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) was predicted to be a potential target for 45. The binding of 45 to DYRK1A was confirmed by drug affinity responsive target stability and cellular thermal shift assay. Further analysis revealed that knockdown of DYRK1A by siRNA suppressed PRV macropinocytosis and the tumor necrosis factor alpha-TNF-induced formation of protrusions. These results suggested that 45 could restrain PRV macropinocytosis by targeting DYRK1A. Together, these findings reveal a unique mechanism through which ß-carboline derivatives restrain PRV infection, pointing to their potential value in the development of anti-PRV agents.

Evaluation of Histone Demethylase Inhibitor ML324 and Acyclovir against Cyprinid herpesvirus 3 Infection.

Cyprinid herpesvirus 3 (CyHV-3) can cause severe disease in koi and common carp (Cyprinus carpio). Currently, no effective treatment is available against CyHV-3 infection in koi. Both LSD1 and JMJD2 are histone demethylases (HD) and are critical for immediate-early (IE) gene activation essential for lytic herpesvirus replication. OG-L002 and ML324 are newly discovered specific inhibitors of LSD1 and JMJD2, respectively. Here, HD inhibitors were compared with acyclovir (ACV) against CyHV-3 infection in vitro and in vivo. ML324, at 20-50 µM, can completely block ~1 × 103 PFU CyHV-3 replication in vitro, while OG-L002 at 20 µM and 50 µM can produce 96% and 98% inhibition, respectively. Only about 94% inhibition of ~1 × 103 PFU CyHV-3 replication was observed in cells treated with ACV at 50 µM. As expected, CyHV-3 IE gene transcription of ORF139 and ORF155 was blocked within 72 h post-infection (hpi) in the presence of 20 µM ML324. No detectable cytotoxicity was observed in KF-1 or CCB cells treated for 24 h with 1 to 50 µM ML324. A significant reduction of CyHV-3 replication was observed in ~6-month-old infected koi treated with 20 µM ML324 in an immersion bath for 3-4 h at 1-, 3-, and 5-days post-infection compared to the control and ACV treatments. Under heat stress, 50-70% of 3-4-month-old koi survived CyHV-3 infection when they were treated daily with 20 µM ML324 in an immersion bath for 3-4 h within the first 5 d post-infection (dpi), compared to 11-19% and 22-27% of koi in the control and ACV treatments, respectively. Our study demonstrates that ML324 has the potential to be used against CyHV-3 infection in koi.

Ethanol extract from Artemisia argyi leaves inhibits HSV-1 infection by destroying the viral envelope.

Herpes simplex virus type 1 (HSV-1) is a widely disseminated virus that establishes latency in the brain and causes occasional but fatal herpes simplex encephalitis. Currently, acyclovir (ACV) is the main clinical drug used in the treatment of HSV-1 infection, and the failure of therapy in immunocompromised patients caused by ACV-resistant HSV-1 strains necessitates the requirement to develop novel anti-HSV-1 drugs. Artemisia argyi, a Traditional Chinese Medicine, has been historically used to treat inflammation, bacterial infection, and cancer. In this study, we demonstrated the antiviral effect and mechanism of ethanol extract of A. argyi leaves (hereafter referred to as 'AEE'). We showed that AEE at 10 µg/ml exhibits potent antiviral effects on both normal and ACV-resistant HSV-1 strains. AEE also inhibited the infection of HSV-2, rotavirus, and influenza virus. Transmission electron microscopy revealed that AEE destroys the membrane integrity of HSV-1 viral particles, resulting in impaired viral attachment and penetration. Furthermore, mass spectrometry assay identified 12 major components of AEE, among which two new flavones, deoxysappanone B 7,3'-dimethyl ether, and 3,7-dihydroxy-3',4'-dimethoxyflavone, exhibited the highest binding affinity to HSV-1 glycoprotein gB at the surface site critical for gB-gH-gL interaction and gB-mediated membrane fusion, suggesting their involvement in inactivating virions. Therefore, A. argyi is an important source of antiviral drugs, and the AEE may be a potential novel antiviral agent against HSV-1 infection.