The Topical Novel Formulations of Interferon α-2в Effectively Inhibit HSV-1 Keratitis in the Rabbit Eye Model and HSV-2 Genital Herpes in Mice.

Herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) are widespread human pathogens that establish chronic latent infections leading to recurrent episodes. Current treatments are limited, necessitating the development of novel antiviral strategies. This study aimed to assess the antiviral efficacy of novel topical formulations containing interferon alpha-2b (IFN α-2b) against HSV-1 and HSV-2. The formulations, Oftalmoferon® forte (eye drops) and Interferon Vaginal Tablets, demonstrated potent antiviral effects against HSV-1 and HSV-2 in Vero cells, respectively, with concentration-dependent inhibition of viral replication. Subsequently, their efficacy was tested in animal models: HSV-1 keratitis in the rabbit eye model and HSV-2 genital herpes in mice. Oftalmoferon® forte effectively treated HSV-1 keratitis, reducing clinical symptoms and ulcerations compared to virus control. Interferon Vaginal Tablets showed promising results in controlling HSV-2 genital herpes in mice, improving survival rates, reducing clinical signs, weight loss and viral replication. The novel IFN α-2b formulations exhibited significant antiviral activity against HSV infections in cell culture and animal models. These findings suggest the potential of these formulations as alternative treatments for HSV infections, particularly in cases resistant to current therapies. Further studies are warranted to optimize treatment regimens and assess clinical efficacy in humans.

Dynamic label-free analysis of SARS-CoV-2 infection reveals virus-induced subcellular remodeling.

Assessing the impact of SARS-CoV-2 on organelle dynamics allows a better understanding of the mechanisms of viral replication. We combine label-free holotomographic microscopy with Artificial Intelligence to visualize and quantify the subcellular changes triggered by SARS-CoV-2 infection. We study the dynamics of shape, position and dry mass of nucleoli, nuclei, lipid droplets and mitochondria within hundreds of single cells from early infection to syncytia formation and death. SARS-CoV-2 infection enlarges nucleoli, perturbs lipid droplets, changes mitochondrial shape and dry mass, and separates lipid droplets from mitochondria. We then used Bayesian network modeling on organelle dry mass states to define organelle cross-regulation networks and report modifications of organelle cross-regulation that are triggered by infection and syncytia formation. Our work highlights the subcellular remodeling induced by SARS-CoV-2 infection and provides an Artificial Intelligence-enhanced, label-free methodology to study in real-time the dynamics of cell populations and their content.

Multi-omics analysis reveals the dynamic interplay between Vero host chromatin structure and function during vaccinia virus infection.

The genome folds into complex configurations and structures thought to profoundly impact its function. The intricacies of this dynamic structure-function relationship are not well understood particularly in the context of viral infection. To unravel this interplay, here we provide a comprehensive investigation of simultaneous host chromatin structural (via Hi-C and ATAC-seq) and functional changes (via RNA-seq) in response to vaccinia virus infection. Over time, infection significantly impacts global and local chromatin structure by increasing long-range intra-chromosomal interactions and B compartmentalization and by decreasing chromatin accessibility and inter-chromosomal interactions. Local accessibility changes are independent of broad-scale chromatin compartment exchange (~12% of the genome), underscoring potential independent mechanisms for global and local chromatin reorganization. While infection structurally condenses the host genome, there is nearly equal bidirectional differential gene expression. Despite global weakening of intra-TAD interactions, functional changes including downregulated immunity genes are associated with alterations in local accessibility and loop domain restructuring. Therefore, chromatin accessibility and local structure profiling provide impactful predictions for host responses and may improve development of efficacious anti-viral counter measures including the optimization of vaccine design.

Virological characteristics of a SARS-CoV-2-related bat coronavirus, BANAL-20-236.

BACKGROUND: Although several SARS-CoV-2-related coronaviruses (SC2r-CoVs) were discovered in bats and pangolins, the differences in virological characteristics between SARS-CoV-2 and SC2r-CoVs remain poorly understood. Recently, BANAL-20-236 (B236) was isolated from a rectal swab of Malayan horseshoe bat and was found to lack a furin cleavage site (FCS) in the spike (S) protein. The comparison of its virological characteristics with FCS-deleted SARS-CoV-2 (SC2ΔFCS) has not been conducted yet. METHODS: We prepared human induced pluripotent stem cell (iPSC)-derived airway and lung epithelial cells and colon organoids as human organ-relevant models. B236, SARS-CoV-2, and artificially generated SC2ΔFCS were used for viral experiments. To investigate the pathogenicity of B236 in vivo, we conducted intranasal infection experiments in hamsters. FINDINGS: In human iPSC-derived airway epithelial cells, the growth of B236 was significantly lower than that of the SC2ΔFCS. A fusion assay showed that the B236 and SC2ΔFCS S proteins were less fusogenic than the SARS-CoV-2 S protein. The infection experiment in hamsters showed that B236 was less pathogenic than SARS-CoV-2 and even SC2ΔFCS. Interestingly, in human colon organoids, the growth of B236 was significantly greater than that of SARS-CoV-2. INTERPRETATION: Compared to SARS-CoV-2, we demonstrated that B236 exhibited a tropism toward intestinal cells rather than respiratory cells. Our results are consistent with a previous report showing that B236 is enterotropic in macaques. Altogether, our report strengthens the assumption that SC2r-CoVs in horseshoe bats replicate primarily in the intestinal tissues rather than respiratory tissues. FUNDING: This study was supported in part by AMED ASPIRE (JP23jf0126002, to Keita Matsuno, Kazuo Takayama, and Kei Sato); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers "UTOPIA" (JP223fa627001, to Kei Sato), AMED SCARDA Program on R&D of new generation vaccine including new modality application (JP223fa727002, to Kei Sato); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005h0001, to Takasuke Fukuhara, and Keita Matsuno); AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108493, to Takasuke Fukuhara; JP22fk0108617 to Takasuke Fukuhara; JP22fk0108146, to Kei Sato; JP21fk0108494 to G2P-Japan Consortium, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, and Kei Sato; JP21fk0108425, to Kazuo Takayama and Kei Sato; JP21fk0108432, to Kazuo Takayama, Takasuke Fukuhara and Kei Sato; JP22fk0108534, Terumasa Ikeda, and Kei Sato; JP22fk0108511, to Yuki Yamamoto, Terumasa Ikeda, Keita Matsuno, Shinya Tanaka, Kazuo Takayama, Takasuke Fukuhara, and Kei Sato; JP22fk0108506, to Kazuo Takayama and Kei Sato); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa Ikeda; and JP22fk0410039, to Kei Sato); AMED Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125008 to Keita Matsuno); AMED CREST (JP21gm1610005, to Kazuo Takayama; JP22gm1610008, to Takasuke Fukuhara; JST PRESTO (JPMJPR22R1, to Jumpei Ito); JST CREST (JPMJCR20H4, to Kei Sato); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041, to G2P-Japan Consortium, Keita Matsuno, Takasuke Fukuhara and Kei Sato); JST SPRING (JPMJSP2108 to Shigeru Fujita); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham Nasser; 20K15767, to Jumpei Ito); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to Kei Sato); JSPS Research Fellow DC2 (22J11578, to Keiya Uriu); JSPS Research Fellow DC1 (23KJ0710, to Yusuke Kosugi); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa Ikeda); World-leading Innovative and Smart Education (WISE) Program 1801 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to Naganori Nao); Ministry of Health, Labour and Welfare (MHLW) under grant 23HA2010 (to Naganori Nao and Keita Matsuno); The Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (to Kei Sato); International Joint Research Project of the Institute of Medical Science, the University of Tokyo (to Terumasa Ikeda and Takasuke Fukuhara); The Tokyo Biochemical Research Foundation (to Kei Sato); Takeda Science Foundation (to Terumasa Ikeda and Takasuke Fukuhara); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Foundation (to Terumasa Ikeda); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Hirose Foundation (to Tomokazu Tamura); and Mitsubishi Foundation (to Kei Sato).

The glycosylation deficiency of flavivirus NS1 attenuates virus replication through interfering with the formation of viral replication compartments.

BACKGROUND: Flavivirus is a challenge all over the world. The replication of flavivirus takes place within membranous replication compartments (RCs) derived from endoplasmic reticulum (ER). Flavivirus NS1 proteins have been proven essential for the formation of viral RCs by remodeling the ER. The glycosylation of flavivirus NS1 proteins is important for viral replication, yet the underlying mechanism remains unclear. METHODS: HeLa cells were used to visualize the ER remodeling effects induced by NS1 expression. ZIKV replicon luciferase assay was performed with BHK-21 cells. rZIKV was generated from BHK-21 cells and the plaque assay was done with Vero Cells. Liposome co-floating assay was performed with purified NS1 proteins from 293T cells. RESULTS: We found that the glycosylation of flavivirus NS1 contributes to its ER remodeling activity. Glycosylation deficiency of NS1, either through N-glycosylation sites mutations or tunicamycin treatment, compromises its ER remodeling activity and interferes with viral RCs formation. Disruption of NS1 glycosylation results in abnormal aggregation of NS1, rather than reducing its membrane-binding activity. Consequently, deficiency in NS1 glycosylation impairs virus replication. CONCLUSIONS: In summary, our results highlight the significance of NS1 glycosylation in flavivirus replication and elucidate the underlying mechanism. This provides a new strategy for combating flavivirus infections.

Novel benzimidazole angiotensin receptor blockers with anti-SARS-CoV-2 activity equipotent to that of nirmatrelvir: computational and enzymatic studies.

BACKGROUND: Hypertension worsens outcomes in SARS-CoV-2 patients. Sartans, a type of antihypertensive angiotensin receptor blocker-(ARB), reduce COVID-19 morbidity and mortality by targeting angiotensin-converting enzyme-2 (ACE2). This study aimed to evaluate the antiviral and antihypertensive effects of nirmatrelvir, commercial sartans (candesartan, losartan, and losartan carboxylic (Exp3174)), and newly synthesized sartans (benzimidazole-N-biphenyl carboxyl (ACC519C) and benzimidazole-N-biphenyl tetrazole (ACC519T)), compared to nirmatrelvir, the antiviral component of Paxlovid. RESEARCH DESIGN AND METHODS: Surface plasmon resonance (SPR) and enzymatic studies assessed drug effects on ACE2. Antiviral abilities were tested with SARS-CoV-2-infected Vero E6 cells, and antihypertensive effects were evaluated using angiotensin II-contracted rabbit iliac arteries. RESULTS: Benzimidazole-based candesartan and ACC519C showed antiviral activity comparable to nirmatrelvir (95% inhibition). Imidazole-based losartan, Exp3174, and ACC519T were less potent (75%-80% and 50%, respectively), with Exp3174 being the least effective. SPR analysis indicated high sartans-ACE2 binding affinity. Candesartan and nirmatrelvir combined had greater inhibitory and cytopathic effects (3.96%) than individually (6.10% and 5.08%). ACE2 enzymatic assays showed varying effects of novel sartans on ACE2. ACC519T significantly reduced angiotensin II-mediated contraction, unlike nirmatrelvir and ACC519T(2). CONCLUSION: This study reports the discovery of a new class of benzimidazole-based sartans that significantly inhibit SARS-CoV-2, likely due to their interaction with ACE2.

Cleavage of the syncytial protein of J paramyxovirus is required for its ability to promote cell-cell fusion.

Cell-cell fusion mediated by most paramyxovirus requires fusion protein (F) and attachment protein (H, HN, or G). The F protein is proteolytic cleaved to be fusogenically active. J paramyxovirus (JPV) has a unique feature in the family Paramyxoviridae: It encodes an integral membrane protein, syncytial protein (SP, formerly known as transmembrane protein, TM), which is essential in JPV-promoted cell-cell fusion (i.e., syncytial). In this study, we report that cleavage of SP is essential for its syncytial-promoting activity. We have identified the cleavage site of SP at amino acid residues 172 to 175, LKTG, and deletion of the "LKTG" residues abolished SP protein cleavage and its ability to promote cell-cell fusion. Replacing the cleavage site LKTG with a factor Xa protease cleavage site allows cleavage of the SP with factor Xa protease and restores its ability to promote cell-cell fusion. Furthermore, results from a hemifusion assay indicate that cleavage of SP plays an important role in the progression from the intermediate hemifusion state to a complete fusion. This work indicates that SP has many characteristics of a fusion protein. We propose that SP is likely a cell-cell fusion-promoting protein.

Virus neutralization assays for human respiratory syncytial virus using airway organoids.

Neutralizing antibodies are considered a correlate of protection against severe human respiratory syncytial virus (HRSV) disease. Currently, HRSV neutralization assays are performed on immortalized cell lines like Vero or A549 cells. It is known that assays on these cell lines exclusively detect neutralizing antibodies (nAbs) directed to the fusion (F) protein. For the detection of nAbs directed to the glycoprotein (G), ciliated epithelial cells expressing the cellular receptor CX3CR1 are required, but generation of primary cell cultures is expensive and labor-intensive. Here, we developed a high-throughput neutralization assay based on the interaction between clinically relevant HRSV grown on primary cells with ciliated epithelial cells, and validated this assay using a panel of infant sera. To develop the high-throughput neutralization assay, we established a culture of differentiated apical-out airway organoids (Ap-O AO). CX3CR1 expression was confirmed, and both F- and G-specific monoclonal antibodies neutralized HRSV in the Ap-O AO. In a side-by-side neutralization assay on Vero cells and Ap-O AO, neutralizing antibody levels in sera from 125 infants correlated well, although titers on Ap-O AO were consistently lower. We speculate that these lower titers might be an actual reflection of the neutralizing antibody capacity in vivo. The organoid-based neutralization assay described here holds promise for further characterization of correlates of protection against HRSV disease.

Anti-SARS-CoV-2 Viral Activity of Sweet Potato Trypsin Inhibitor via Downregulation of TMPRSS2 Activity and ACE2 Expression In Vitro and In Vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever recorded in human history, causing significant losses to both life and economies globally. However, the available evidence is currently insufficient to establish the effectiveness and safety of antiviral drugs or vaccines. The entry of the virus into host cells involves binding to angiotensin-converting enzyme 2 (ACE2), a cell surface receptor, via its spike protein. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a host surface protease, cleaves and activates the virus's S protein, thus promoting viral infection. Plant protease inhibitors play a crucial role in protecting plants against insects and/or microorganisms. The major storage proteins in sweet potato roots include sweet potato trypsin inhibitor (SWTI), which accounts for approximately 60% of the total water-soluble protein and has been found to possess a variety of health-promoting properties, including antioxidant, anti-inflammatory, ACE-inhibitory, and anticancer functions. Our study found that SWTI caused a significant reduction in the expression of the ACE2 and TMPRSS2 proteins, without any adverse effects on cells. Therefore, our findings suggest that the ACE2 and TMPRSS2 axis can be targeted via SWTI to potentially inhibit SARS-CoV-2 infection.

Magnolol against enterovirus 71 by targeting Nrf2-SLC7A11-GSH pathway.

Enterovirus 71 (EV71), a prominent pathogen associated with hand, foot, and mouth disease (HFMD), has been reported worldwide. To date, the advancement of effective drugs targeting EV71 remains in the preliminary experimental stage. In this study, magnolol demonstrated a significant dose-dependent inhibition of EV71 replication in vitro. It upregulated the overall expression level of nuclear factor erythroid 2 - related factor 2 (Nrf2) and facilitated its nucleus translocation, resulting in the increased expression of various ferroptosis inhibitory genes. This process led to a reduction in reactive oxygen species (ROS) accumulation induced by viral infection. Additionally, magnolol exhibited a broad-spectrum antiviral effect against enteroviruses. Notably, treatment with magnolol substantially enhanced the survival rate of EV71-infected mice, attenuated viral load in heart, liver, brain, and limb tissues, and mitigated tissue inflammation. Taken together, magnolol emerges as a promising candidate for the development of anti-EV71 drugs.

Metal-Organic Frameworks: Unconventional Nanoweapons against COVID.

The SARS-CoV-2 (COVID-19) pandemic outbreak led to enormous social and economic repercussions worldwide, felt even to this date, making the design of new therapies to combat fast-spreading viruses an imperative task. In the face of this, diverse cutting-edge nanotechnologies have risen as promising tools to treat infectious diseases such as COVID-19, as well as challenging illnesses such as cancer and diabetes. Aside from these applications, nanoscale metal-organic frameworks (nanoMOFs) have attracted much attention as novel efficient drug delivery systems for diverse pathologies. However, their potential as anti-COVID-19 therapeutic agents has not been investigated. Herein, we propose a pioneering anti-COVID MOF approach by studying their potential as safe and intrinsically antiviral agents through screening various nanoMOF. The iron(III)-trimesate MIL-100 showed a noteworthy antiviral effect against SARS-CoV-2 at the micromolar range, ensuring a high biocompatibility profile (90% of viability) in a real infected human cellular scenario. This research effectively paves the way toward novel antiviral therapies based on nanoMOFs, not only against SARS-CoV-2 but also against other challenging infectious and/or pulmonary diseases.

TLR4 signalling: the key to controlling EV71 replication and inflammatory response.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enterovirus 71 (EV71) that frequently affects children, leading to severe infections in some cases. In general, when infection occurs, the body upregulates inflammatory responses to eliminate pathogenic microorganisms to protect the host from infection. However, EV71 may inhibit host's innate immunity to promote virus infection. At present, it is not fully understood how EV71 hijack the host cells for its own replication. Toll-like receptor 4 (TLR4), a natural immune receptor, historically associated with bacterial endotoxin-induced inflammatory responses. However, it is still unclear whether and how TLR4 is altered during EV71 infection. In this study, we observed a reduction in both TLR4 protein and gene transcript levels in RD, GES-1, and Vero cells following EV71 infection, as detected by RT-qPCR, immunofluorescence staining and western blot. Furthermore, we observed that the TLR4 downstream molecules of MYD88, p-NF-κB p65, p-TBK1 and related inflammatory cytokines were also reduced, suggesting that antiviral innate immune and inflammatory response were suppressed. To determine the impact of TLR4 changes on EV71 infection, we interfered EV71-infected RD cells with TLR4 agonist or inhibitor and the results showed that activation of TLR4 inhibited EV71 replication, while inhibition of TLR4 promote EV71 replication. Besides, EV71 replication was also promoted in TLR4 siRNA-transfected and EV71-infected RD cells. This suggests that down-regulation the expression of TLR4 by EV71 can inhibit host immune defense to promote EV71 self-replication. This novel mechanism may be a strategy for EV71 to evade host immunity.

Uncovering the Chemical Composition and Biological Potentials of Bupleurum lancifolium Hornem. from Jordan.

The current study was designed to uncover the chemistry and bioactivity potentials of Bupleurum lancifolium growing wild in Jordan. In this context, the fresh aerial parts obtained from the plant material were subjected to hydrodistillation followed by GC/MS analysis. The main components of the HDEO were γ-patchoulene (23.79%), ß-dihydro agarofuran (23.50%), α-guaiene (14.11%), and valencene (13.28%). Moreover, the crude thanolic extract was partitioned to afford two main major fractions, the aqueous methanol (BLM) and butanol (BLB). Phytochemical investigation of both fractions, using conventional chromatographic techniques followed by careful inspection of the spectral data for the isolated compounds (NMR, IR, and UV-Vis), resulted in the characterization of five known compounds, including α-spinasteryl (M1), ethyl arachidate (M2), ethyl myristate (M3), quercetin-3-O-ß-d-glucopyranosyl-(1-4")-α-L-rhamnopyranosyl (B1), and isorhamnetin-3-O-ß-d-glucopyranosyl-(1-4")-α-L-rhamnopyranosyl (B2). The TPC, TFC, and antioxidant activity testing of both fractions and HDEO revealed an interesting ABTS scavenging potential of the BLB fraction compared to the employed positive controls, which is in total agreement with its high TP and TF contents. Cytotoxic evaluation tests revealed that BLM had interesting cytotoxic effects on the normal breast cell line MDA-MB-231 (ATCC-HTB-26) and the normal dermal fibroblast (ATCC® PCS-201-012) and normal African green monkey kidney Vero (ATCC-CCL-81) cell lines. Despite both the BLB and BLM fractions showing interesting AChE inhibition activities (IC50 = 217.9 ± 5.3 µg/mL and 139.1 ± 5.6 µg/mL, respectively), the HDEO revealed an interestingly high AChE inhibition power (43.8 ± 2.7 µg/mL) that far exceeds the one observed for galanthamine (91.4 ± 5.2 µg/mL). The HDEO, BLM, and BLB exhbitied no interesting antimicrobial activity against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa.

Coxsackievirus A7 and Enterovirus A71 Significantly Reduce SARS-CoV-2 Infection in Cell and Animal Models.

In this study, we investigated the features of co-infection with SARS-CoV-2 and the enterovirus vaccine strain LEV8 of coxsackievirus A7 or enterovirus A71 for Vero E6 cells and Syrian hamsters. The investigation of co-infection with SARS-CoV-2 and LEV-8 or EV-A71 in the cell model showed that a competitive inhibitory effect for these viruses was especially significant against SARS-CoV-2. Pre-infection with enteroviruses in the animals caused more than a 100-fold decrease in the levels of SARS-CoV-2 virus replication in the respiratory tract and more rapid clearance of infectious SARS-CoV-2 from the lower respiratory tract. Co-infection with SARS-CoV-2 and LEV-8 or EV-A71 also reduced the severity of clinical manifestations of the SARS-CoV-2 infection in the animals. Additionally, the histological data illustrated that co-infection with strain LEV8 of coxsackievirus A7 decreased the level of pathological changes induced by SARS-CoV-2 in the lungs. Research into the chemokine/cytokine profile demonstrated that the studied enteroviruses efficiently triggered this part of the antiviral immune response, which is associated with the significant inhibition of SARS-CoV-2 infection. These results demonstrate that there is significant viral interference between the studied strain LEV-8 of coxsackievirus A7 or enterovirus A71 and SARS-CoV-2 in vitro and in vivo.

Development of Robust Freeze-Drying Process for Long-Term Stability of rVSV-SARS-CoV-2 Vaccine.

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3-6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2-8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.

Antiviral Activities of Mastoparan-L-Derived Peptides against Human Alphaherpesvirus 1.

Human alphaherpesvirus 1 (HSV-1) is a significantly widespread viral pathogen causing recurrent infections that are currently incurable despite available treatment protocols. Studies have highlighted the potential of antimicrobial peptides sourced from Vespula lewisii venom, particularly those belonging to the mastoparan family, as effective against HSV-1. This study aimed to demonstrate the antiviral properties of mastoparans, including mastoparan-L [I5, R8], mastoparan-MO, and [I5, R8] mastoparan, against HSV-1. Initially, Vero cell viability was assessed in the presence of these peptides, followed by the determination of antiviral activity, mechanism of action, and dose-response curves through plaque assays. Structural analyses via circular dichroism and nuclear magnetic resonance were conducted, along with evaluating membrane fluidity changes induced by [I5, R8] mastoparan using fluorescence-labeled lipid vesicles. Cytotoxic assays revealed high cell viability (>80%) at concentrations of 200 µg/mL for mastoparan-L and mastoparan-MO and 50 µg/mL for [I5, R8] mastoparan. Mastoparan-MO and [I5, R8] mastoparan exhibited over 80% HSV-1 inhibition, with up to 99% viral replication inhibition, particularly in the early infection stages. Structural analysis indicated an α-helical structure for [I5, R8] mastoparan, suggesting effective viral particle disruption before cell attachment. Mastoparans present promising prospects for HSV-1 infection control, although further investigation into their mechanisms is warranted.

Antiviral Activity of Ag5IO6, a Unique Silver Compound.

Pentasilver hexaoxoiodate (Ag5IO6) has broad-spectrum antimicrobial efficacy, including the long-term prevention of microbial adherence, the rapid killing of planktonic microorganisms, and the elimination of mature biofilms. This study's goal was to determine whether it may also have antiviral activity against structurally distinct viruses. Ag5IO6 was tested following ASTM E1052-20, Standard Practice to Assess the Activity of Microbicides Against Viruses in Suspension, against adenovirus type 5, murine norovirus, poliovirus type 1, SARS-CoV-2 (original), and SARS-CoV-2 (omicron) (host cells: H1HeLa, RAW 264.7, LLC-MK2, Vero E6, and Vero E6, respectively). A 0.1 g/mL Ag5IO6 suspension was prepared and the viruses were exposed for 30 min, 4 h, or 24 h. Exposure to Ag5IO6 resulted in complete kill of SARS-CoV-2 (omicron) within 30 min, as well as complete kill of both SARS-CoV-2 (original) and the murine norovirus within 4 h. Ag5IO6 showed increasing activity over time against the adenovirus, but did not achieve a 3-log reduction within 24 h, and showed no antiviral activity against the poliovirus. These results demonstrate that Ag5IO6 has antiviral activity against medically important viruses, in addition to its well-characterized antimicrobial activity, suggesting that it may be valuable in situations where the prevention or simultaneous treatment of microbes and viruses are necessary.

A Cullin 5-based complex serves as an essential modulator of ORF9b stability in SARS-CoV-2 replication.

The ORF9b protein, derived from the nucleocapsid's open-reading frame in both SARS-CoV and SARS-CoV-2, serves as an accessory protein crucial for viral immune evasion by inhibiting the innate immune response. Despite its significance, the precise regulatory mechanisms underlying its function remain elusive. In the present study, we unveil that the ORF9b protein of SARS-CoV-2, including emerging mutant strains like Delta and Omicron, can undergo ubiquitination at the K67 site and subsequent degradation via the proteasome pathway, despite certain mutations present among these strains. Moreover, our investigation further uncovers the pivotal role of the translocase of the outer mitochondrial membrane 70 (TOM70) as a substrate receptor, bridging ORF9b with heat shock protein 90 alpha (HSP90α) and Cullin 5 (CUL5) to form a complex. Within this complex, CUL5 triggers the ubiquitination and degradation of ORF9b, acting as a host antiviral factor, while HSP90α functions to stabilize it. Notably, treatment with HSP90 inhibitors such as GA or 17-AAG accelerates the degradation of ORF9b, leading to a pronounced inhibition of SARS-CoV-2 replication. Single-cell sequencing data revealed an up-regulation of HSP90α in lung epithelial cells from COVID-19 patients, suggesting a potential mechanism by which SARS-CoV-2 may exploit HSP90α to evade the host immunity. Our study identifies the CUL5-TOM70-HSP90α complex as a critical regulator of ORF9b protein stability, shedding light on the intricate host-virus immune response dynamics and offering promising avenues for drug development against SARS-CoV-2 in clinical settings.

Median Tissue Culture Infectious Dose 50 (TCID50) Assay to Determine Infectivity of Cytopathic Viruses.

The emergence of zoonotic viruses like severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 have significantly impacted global health and economy. The discovery of other viruses in wildlife reservoir species present a threat for future emergence in humans and animals. Therefore, assays that are less reliant on virus-specific information, such as neutralization assays, are crucial to rapidly develop diagnostics, understand virus replication and pathogenicity, and assess the efficacy of therapeutics against newly emerging viruses. Here, we describe the discontinuous median tissue culture infectious dose 50 (TCID50) assay to quantitatively determine the titer of any virus that can produce a visible cytopathic effect in infected cells.

Antiviral activity of pimecrolimus against dengue virus type 2 infection in vitro and in vivo.

Dengue virus (DENV) infection is a public health concern in several countries and is associated with severe diseases, such as dengue hemorrhagic fever and dengue shock syndrome. DENVs are transmitted to humans via the bites of infected Aedes mosquitoes, and no antiviral therapeutics are currently available. In this work, we aimed to identify antiviral drugs against DENV type 2 (DENV2) infections and selected pimecrolimus as a potential antiviral drug candidate. Pimecrolimus significantly inhibited DENV2-mediated cell death and replication in vitro. We also confirmed a decrease in the number of plaques formed as well as in the envelope protein levels of DENV2. The time-of-addition and course experiments revealed that pimecrolimus inhibited DENV2 infection during the early stages of the virus replication cycle. In an experimental mouse model, orally administered pimecrolimus alleviated body weight loss and lethality caused by DENV2 infection, which we used as readouts of the drug's antiviral potency. Furthermore, pimecrolimus significantly inhibited the DENV2 load and ameliorated focal necrosis in the liver and spleen. Taken together, our in vitro and in vivo findings suggest that pimecrolimus is a promising antiviral drug candidate for the treatment of DENV2 infection.