The role of glycoprotein G (gG-2) of herpes simplex virus type 2 (HSV-2) in viral pathogenesis remains poorly understood. gG-2 is cleaved into a secreted form (sgG-2) and a membrane-associated form (mgG-2), but the in vivo function of mgG-2 and the contribution of its glycosylation to immune responses have not been defined. Here, we provide a comprehensive characterization of the N- and O-linked glycosylation profile of mgG-2 and investigate its functional relevance for viral spread and vaccine-induced immunity. Using a mouse genital infection model, we show that an mgG-2-deficient HSV-2 mutant replicates in vaginal epithelial cells but is severely impaired in dissemination to dorsal root ganglia and the central nervous system, identifying mgG-2 as a key determinant of neuronal spread in vivo. In parallel, immunization with recombinant mgG-2 elicited strong humoral and Th1-polarized CD4 + T-cell responses and conferred protection against genital HSV-2 challenge. Importantly, glycosylation of mgG-2 was required for optimal immunogenicity and protection, as deglycosylated variants induced reduced CD4 + T-cell responses and allowed increased viral spread to neuronal tissues. Mechanistically, our findings suggest that glycosylation of mgG-2 modulates antigen recognition and shapes adaptive immune responses that limit viral dissemination after vaccination. Together, these results demonstrate that mgG-2 plays a critical role in HSV-2 pathogenesis and provide a strong rationale for targeting glycosylated mgG-2 in the development of both prophylactic and therapeutic vaccines against HSV-2.

The function of glycoprotein G (gG-2) of herpes simplex virus 2 (HSV-2) during genital infection is unknown. gG-2 is cleaved into a secreted variant (sgG-2) and a membrane associated variant (mgG-2). This work delineates the glycan profile of mgG-2, and demonstrates that mgG-2 is strongly immunogenic, eliciting glycan dependent humoral and Th1-polarized CD4+ T cell responses in a mouse vaccination model. The N- and O-glycosylation of mgG-2 is important to achieve full protection against HSV-2 infection as immunization with deglycosylated mgG-2 resulted in poor CD4+ T cell activation and viral spread to dorsal root ganglia and spinal cord. Furthermore, an mgG-2 negative HSV-2 mutant virus failed to spread to the dorsal root ganglia and the central nervous system of genitally infected mice, despite viral replication in vaginal cells. Our data demonstrate that mgG-2 is important for HSV-2 propagation and that adaptive immune responses targeting the glycosylated protein can prevent neuronal infection, identifying mgG-2 as a promising vaccine candidate.

Background: The knowledge of traditional health practitioners (THPs) on the use of medicinal plant remedies for treatment of human respiratory ailments provides a potential resource for identification of relevant plants and isolation of active ingredients. Purpose: We aimed to identify medicinal plants used by THPs for treatment of respiratory ailments, then screen them in cultured cells for activity against respiratory syncytial virus (RSV), and assess the antiviral potential of identified screening hits. Methods: Forty Rwandese THPs were interviewed to identify medicinal plants used for treatment of respiratory ailments. The indicated plant samples were collected and successively extracted using n-hexane, ethyl acetate, methanol, and water. Extracts were screened for anti-RSV activity and for cytotoxicity in HEp-2 cells. Results: Based on ethnopharmacological survey of THPs, 48 plants used for treating respiratory ailments were identified. Of 203 plant extracts prepared for screening, ten methanolic and four aqueous extracts exhibited anti-RSV activity, manifested by EC50 values ranging from 1.6 to 39.5 µg/ml and selectivity indices (SI, CC50/EC50) of 12–294. Extended studies of an anti-RSV potential of methanolic extract of Senecio maranguensis O. Hoffm. leaves revealed that this preparation targeted the virus fusion (F) protein because extract resistant virus variants carried the double nucleotide substitution at c1104t and a1105g resulting in the T335M amino acid change in the F protein trimer internal cavity. Conclusion: We found that eleven plants, traditionally used against respiratory ailments, exhibited substantial anti-RSV activity in cultured cells, exemplified by the S. maranguensis methanolic extract that affected the activity of RSV F protein.

The phytochemical investigation of the stem bark of Morus mesozygia afforded the fourteen known secondary metabolites moracin N (1), moracin S (2), mulberrofuran L (3), moracin C (4), moracin L (5), moracin M (6), moracin D (7), artopithecin A (8), isobavachalcone (9), morachalcone A (10), 2,2՛,4,4՛-tetrahydroxychalcone (11), 3β-acetoxy-urs-12-en-11-one (12), betulinic acid (13), and 4,4'-diphenylmethane-bis(methyl) carbamate (14). Their structures were elucidated by NMR spectroscopic and mass spectrometric analyzes and their cytotoxicity (CC₅₀ for HEKa, IMR-90, and HPrEC), anti-inflammatory (TNF-α, NF-κB, and NO inhibition), antibacterial (MIC), antitumor (IC₅₀), and antiviral (CPE-based and plaque reduction assays) activities were studied. Moracin D (7) showed potent anti-inflammatory activity towards the release of NF-κB (0.57 < IC50 < 1.21 μM). 3β-Acetoxy-urs-12-en-11-one (12) had potent antibacterial activity towards Bacillus subtilis and Micrococcus luteus, with MIC values of 12.71 and 15.59 μM, respectively. Moracin M (6) had the highest antitumor activity (IC50 = 19.80 μM) against SK-MEL-28 human melanoma cells. Isobavachalcone (9) exhibited activity against Human Rhinovirus 2 (HRV-2; IC50 = 7.01 μM) with a selectivity index (SI) = 9.1 as compared to HeLa cells. None of the compounds exhibited significant antiviral activity against respiratory syncytial virus (RSV) or herpes simplex virus type 2 (HSV-2). Out of the isolated compounds, moracin D (7), isobavachalcone (9) and marsformoxide B (12), may be considered to be promising leads for the development of anti-inflammatory (NF-κB), antiviral (HRV-2), and antibacterial (B. subtilis and M. luteus) agents, respectively.

BACKGROUND:Tick-borne encephalitis (TBE) is a neurological disease caused by the tick-borne encephalitis virus (TBEV). Despite available vaccines, breakthrough infections occur, some fatal. OBJECTIVES:As no antiviral therapy for TBE is currently approved, this study evaluated the in vitro activity of already licenced remdesivir (RDV) and sofosbuvir (SOF) for possible drug repurposing against TBEV. METHODS:TBEV was cultured in A549 cells, and the inhibitory effects of RDV (GS-5734), its parent nucleotide GS-441524, and SOF (GS-7977) were assessed. RESULTS:After 78 h, RDV demonstrated significantly lower EC50 values than SOF (0.14 vs. 11 µM) based on TBEV RNA levels measured by RT-qPCR. RDV also had a lower mean EC50 (0.55 µM) compared to GS-441524 and SOF (>8.9 and 13.1 µM, respectively) using crystal violet staining after 5 days. After 11 passages of TBEV in the presence of RDV, emergence of virus with a higher EC50 (1.32 vs. 0.55 µM) was detected with two mutations (L3122F and Y3278F) in NS5, the viral RNA-dependent RNA polymerase (RdRp), and one substitution in envelope (E) protein (E402G). Similarly, SOF resistance appeared after 20 passages, increasing EC50 values (35.5 vs. 10 µM). CONCLUSION:RDV exhibits potent in vitro antiviral activity against TBEV via specific targeting of the viral RdRp as confirmed by the emergence of resistance-associated double NS5 substitutions in vitro in the presence of RDV. While the potential in vivo implications of the observed RDV resistance remain to be determined, these in vitro data support further assessment of RDV for the treatment of TBEV infection.

Three new (1-3) and six known rotenoids (5-10), along with three known isoflavones (11-13), were isolated from the leaves of Millettia oblata ssp. teitensis. A new glycosylated isoflavone (4), four known isoflavones (14-18), and one known chalcone (19) were isolated from the root wood extract of the same plant. The structures were elucidated by NMR and mass spectrometric analyses. The absolute configuration of the chiral compounds was established by a comparison of experimental ECD and VCD data with those calculated for the possible stereoisomers. This is the first report on the use of VCD to assign the absolute configuration of rotenoids. The crude leaves and root wood extracts displayed anti-RSV (human respiratory syncytial virus) activity with IC50 values of 0.7 and 3.4 mu g/mL, respectively. Compounds 6, 8, 10, 11, and 14 showed anti-RSV activity with IC50 values of 0.4-10 mu M, while compound 3 exhibited anti-HRV-2 (human rhinovirus 2) activity with an IC50 of 4.2 mu M. Most of the compounds showed low cytotoxicity for laryngeal carcinoma (HEp-2) cells; however compounds 3, 11, and 14 exhibited low cytotoxicity also in primary lung fibroblasts. This is the first report on rotenoids showing antiviral activity against RSV and HRV viruses.

The phytochemical investigation of the leaves and the roots of Suregada procera afforded the new ent-abietane diterpenoid sureproceriolide A (1) along with the known secondary metabolites 8,14(3:11,12a-diepoxy-13(15)abietane-16,12-olid (2), jolkinolide A (3), jolkinolide E (4), ent-pimara-8(14),15-dien-19-oic acid (5), sitosterol (6), oleana-9(11):12-dien-3 beta-ol (7), and oleic acid (8). Their structures were elucidated by NMR spectroscopic and mass spectrometric analyses, and the structure of jolkinolide A (3) was confirmed by single-crystal X-ray diffraction analysis. Sureproceriolide A (1) showed modest activity against the Gram-positive bacterium Staphylococcus lugdunensis (MIC = 31.44 mu M), and sitosterol (6) against the Gram-negative bacterium Porphyromonas gingivalis (IC50 = 45.37 mu M). Jolkinolide A (3) and E (4) as well as sitosterol (6) inhibited the release of NOS (IMR-90 cells), TNF-alpha (HaCaT cells) and NF-xB (HaCaT cells), with IC50 values of 0.43, 3.21, and 10.32 mu M, respectively. Compound 6 showed antitumoral activity against SK-MEL-28 (IC50 = 20.66 mu M) and CCD-13Lu (IC50 = 24.70 mu M) cell lines, with no cytotoxic effect against the prostate cells PrEC (CC50 > 300 mu M).

ABSTRACT Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC 50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.

Six new crotofolane diterpenoids (1–6) and 13 known compounds (7–19) were isolated from the MeOH–CH2Cl2 (1:1, v/v) extracts of the leaves and stem bark of Croton kilwae. The structures of the new compounds were elucidated by extensive analysis of spectroscopic and mass spectrometric data. The structure of crotokilwaepoxide A (1) was confirmed by single-crystal X-ray diffraction, allowing for the determination of its absolute configuration. The crude extracts and the isolated compounds were investigated for antiviral activity against respiratory syncytial virus (RSV) and human rhinovirus type-2 (HRV-2) in HEp-2 and HeLa cells, respectively, for antibacterial activity against the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli, and for antimalarial activity against the Plasmodium falciparum Dd2 strain. ent-3β,19-Dihydroxykaur-16-ene (7) and ayanin (16) displayed anti-RSV activities with IC50 values of 10.2 and 6.1 μM, respectively, while exhibiting only modest cytotoxic effects on HEp-2 cells that resulted in selectivity indices of 4.9 and 16.4. Compounds 2 and 5 exhibited modest anti-HRV-2 activity (IC50 of 44.6 μM for both compounds), while compound 16 inhibited HRV-2 with an IC50 value of 1.8 μM. Compounds 1–3 showed promising antiplasmodial activities (80–100% inhibition) at a 50 μM concentration.

N- and O-glycans are both important constituents of viral envelope glycoproteins. O-linked glycosylation can be initiated by any of 20 different human polypeptide O-acetylgalactosaminyl transferases, resulting in an important functional O-glycan heterogeneity. O-glycans are organized as solitary glycans or in clusters of multiple glycans forming mucin-like domains. They are functional both in the viral life cycle and in viral colonization of their host. Negatively charged O-glycans are crucial for the interactions between glycosaminoglycan-binding viruses and their host. A novel mechanism, based on controlled electrostatic repulsion, explains how such viruses solve the conflict between optimized viral attachment to target cells and efficient egress of progeny virus. Conserved solitary O-glycans appear important for viral uptake in target cells by contributing to viral envelope fusion. Dual roles of viral O-glycans in the host B cell immune response, either epitope blocking or epitope promoting, may be exploitable for vaccine development. Finally, specific virus-induced O-glycans may be involved in viremic spread.

The diffusion of viruses at the cell membrane is essential to reach a suitable entry site and initiate subsequent internalization. Although many viruses take advantage of glycosaminoglycans (GAG) to bind to the cell surface, little is known about the dynamics of the virus–GAG interactions. Here, single-particle tracking of the initial interaction of individual herpes simplex virus 1 (HSV-1) virions reveals a heterogeneous diffusive behavior, regulated by cell-surface GAGs with two main diffusion types: confined and normal free. This study reports that different GAGs can have competing influences in mediating diffusion on the cells used here: chondroitin sulfate (CS) enhances free diffusion but hinders virus attachment to cell surfaces, while heparan sulfate (HS) promotes virus confinement and increases entry efficiency. In addition, the role that the viral mucin-like domains (MLD) of the HSV-1 glycoprotein C plays in facilitating the diffusion of the virus and accelerating virus penetration into cells is demonstrated. Together, our results shed new light on the mechanisms of GAG-regulated virus diffusion at the cell surface for optimal internalization. These findings may be extendable to other GAG-binding viruses.

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

The leaf extract of Suregada zanzibariensis gave two new modified ent-abietane diterpenoids, zanzibariolides A (1) and B (2), and two known triterpenoids, simiarenol (3) and β-amyrin (4). The structures of the isolated compounds were elucidated based on NMR and MS data analysis. Single-crystal X-ray diffraction was used to establish the absolute configurations of compounds 1 and 2. The crude leaf extract inhibited the infectivity of herpes simplex virus 2 (HSV-2, IC50 11.5 μg/mL) and showed toxicity on African green monkey kidney (GMK AH1) cells at CC50 52 μg/mL. The isolated compounds 1-3 showed no anti-HSV-2 activity and exhibited insignificant toxicity against GMK AH1 cells at ≥100 μM.

ETHNOPHARMACOLOGICAL RELEVANCE:Except for few highly pathogenic viruses, no antiviral drug has been approved for treatment of viral infections in humans. Plant extracts, selected based on their ethno-medical use, represent an important source of compounds for the development of novel candidate antiviral drugs. This especially concerns plants with ethnomedical records on their use in treatment of viral infections. AIM OF THE STUDY:To identify and document medicinal plants used by traditional health practitioners (THPs) for treatment of respiratory infections and muco-cutaneous lesions in order to study their antiviral activity including identification of active components and elucidation of mode of antiviral activity. MATERIALS AND METHODS:The ethno-medical survey was performed in the Kagera region of Tanzania. The THPs were asked for plants used for treatment of signs and symptoms of respiratory infections and watery muco-cutaneous blisters in oral and genital regions. The plants identified were successively extracted with n-hexane, ethyl acetate and water, and the extracts assayed for anti-respiratory syncytial virus (RSV), anti-herpes simplex virus 2 (HSV-2), and anti-human parainfluenza virus 2 (HPIV-2) activity in cultured cells. Antiviral components were separated by ethanol precipitation and CL-6B chromatography, and the mode of antiviral activity elucidated by the time-of-addition assay and selection for the virus variants resistant to antiviral plant extract. RESULTS:THPs identified fifteen plants used for treatment of respiratory infections and muco-cutaneous blisters. The water extract, but not n-hexane or ethyl acetate extracts, of six of these plants including Erythrina abyssinica stem bark, inhibited infectivity of two glycosaminoglycan-binding viruses i.e., RSV and HSV-2 but not the sialic acid binding HPIV-2. An activity-guided separation revealed that antiviral component(s) of water extract of E. abyssinica could be precipitated with ethanol. This sample potently and selectively inhibited RSV and HSV-2 infectivity in cultured cells with IC50 values of 2.1 μg/ml (selectivity index >476) and 0.14 μg/ml (selectivity index >7143) respectively. The sample exhibited inhibitory effect on the virus attachment to and entry into the cells by directly targeting the viral particles. Indeed, 10 consecutive virus passages in HEp-2 cells in the presence of this extract selected for a resistant RSV variant lacking the attachment, viral membrane-associated, G protein due to a stop codon at amino acid residue 33 (Leu33stop). Fractionation of the E. abyssinica extract on a CL-6B column revealed that anti-RSV and HSV-2 activity correlated with carbohydrate content. The most pronounced antiviral activity was associated with a carbohydrate containing ingredient of molecular mass of <5 kDa, which may polymerize to antiviral composites of up to 410 kDa. CONCLUSIONS:Altogether, the water extract of six medicinal plants showed anti-RSV and anti-HSV-2 activities. Extended studies of the stem bark of E. abyssinica identified antiviral components that potently and selectively inhibited infectivity of free RSV and HSV-2 particles, a feature of importance in topical treatment of these infections. This observation confirms ethno-medical information concerning the use of E. abyssinica extract for treatment of respiratory infections and herpetic lesions.

The methanol root extract of Clerodendrum myricoides (Hochst.) Vatke afforded two new (1, 2) and two known (3, 4) iridoid glycosides. The structures of the isolated compounds were established based on NMR, IR, UV and MS data analyses. The crude extract and the isolated constituents were assayed for antiviral activity against the human respiratory syncytial virus (RSV) in human laryngeal epidermoid carcinoma (HEp-2) cells. The crude extract inhibited RSV infectivity at EC50 = 0.21 μg/ml, while it showed cytotoxicity against HEp-2 cells with CC50 = 9 μg/ml. Compound 2 showed 43.2% virus inhibition at 100 μM, while compounds 1 as well as 3 and 4 had only weak antiviral and cytotoxic activities.

Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) can infect the central nervous system (CNS) with dire consequences; in children and adults, HSV-1 may cause focal encephalitis, while HSV-2 causes meningitis. In neonates, both viruses can cause severe, disseminated CNS infections with high mortality rates. Here, we differentiated human induced pluripotent stem cells (iPSCs) towards cortical neurons for infection with clinical CNS strains of HSV-1 or HSV-2. Progenies from both viruses were produced at equal quantities in iPSCs, neuroprogenitors and cortical neurons. HSV-1 and HSV-2 decreased viability of neuroprogenitors by 36.0% and 57.6% (p < 0.0001), respectively, 48 h post-infection, while cortical neurons were resilient to infection by both viruses. However, in these functional neurons, both HSV-1 and HSV-2 decreased gene expression of two markers of synaptic activity, CAMK2B and ARC, and affected synaptic activity negatively in multielectrode array experiments. However, unaltered secretion levels of the neurodegeneration markers tau and NfL suggested intact axonal integrity. Viral replication of both viruses was found after six days, coinciding with 6-fold and 22-fold increase in gene expression of cellular RNA polymerase II by HSV-1 and HSV-2, respectively. Our results suggest a resilience of human cortical neurons relative to the replication of HSV-1 and HSV-2.