Antiviral Rotenoids and Isoflavones Isolated from Millettia oblata ssp. teitensis.

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 µg/mL, respectively. Compounds 6, 8, 10, 11, and 14 showed anti-RSV activity with IC50 values of 0.4-10 µM, while compound 3 exhibited anti-HRV-2 (human rhinovirus 2) activity with an IC50 of 4.2 µM. 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.

Identification of epidermal growth factor receptor-tyrosine kinase inhibitor targeting the VP1 pocket of human rhinovirus.

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 EC50 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.

Structure and Role of O-Linked Glycans in Viral Envelope Proteins.

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.

Crotofolane Diterpenoids and Other Constituents Isolated from Croton kilwae.

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-3ß,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.

Modified ent-Abietane Diterpenoids from the Leaves of Suregada zanzibariensis.

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 ß-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.

Cellular Chondroitin Sulfate and the Mucin-like Domain of Viral Glycoprotein C Promote Diffusion of Herpes Simplex Virus 1 While Heparan Sulfate Restricts Mobility.

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.

Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike-ACE2 Interaction.

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.

Anti-respiratory syncytial virus and anti-herpes simplex virus activity of six Tanzanian medicinal plants with extended studies of Erythrina abyssinica stem bark.

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.

Antiviral iridoid glycosides from Clerodendrum myricoides.

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 and 2 Infections during Differentiation of Human Cortical Neurons.

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.

Herpes Simplex Virus Type 2 Mucin-Like Glycoprotein mgG Promotes Virus Release from the Surface of Infected Cells.

The contribution of virus components to liberation of herpes simplex virus type 2 (HSV-2) progeny virions from the surface of infected cells is poorly understood. We report that the HSV-2 mutant deficient in the expression of a mucin-like membrane-associated glycoprotein G (mgG) exhibited defect in the release of progeny virions from infected cells manifested by ~2 orders of magnitude decreased amount of infectious virus in a culture medium as compared to native HSV-2. Electron microscopy revealed that the mgG deficient virions were produced in infected cells and present at the cell surface. These virions could be forcibly liberated to a nearly native HSV-2 level by the treatment of cells with glycosaminoglycan (GAG)-mimicking oligosaccharides. Comparative assessment of the interaction of mutant and native virions with surface-immobilized chondroitin sulfate GAG chains revealed that while the mutant virions associated with GAGs ~fourfold more extensively, the lateral mobility of bound virions was much poorer than that of native virions. These data indicate that the mgG of HSV-2 balances the virus interaction with GAG chains, a feature critical to prevent trapping of the progeny virions at the surface of infected cells.

Surveillance of wastewater revealed peaks of SARS-CoV-2 preceding those of hospitalized patients with COVID-19.

SARS-CoV-2 was discovered among humans in Wuhan, China in late 2019, and then spread rapidly, causing a global pandemic. The virus was found to be transmitted mainly by respiratory droplets from infected persons or by direct contact. It was also shown to be excreted in feces, why we investigated whether the virus could be detected in wastewater and if so, to which extent its levels reflects its spread in society. Samples of wastewater from the city of Gothenburg, and surrounding municipalities in Sweden were collected daily from mid-February until June 2020 at the Rya wastewater treatment plant. Flow proportional samples of wastewater were collected to ensure that comparable amounts were obtained for analysis. Daily samples were pooled into weekly samples. Virus was concentrated on a filter and analyzed by RT-qPCR. The amount of SARS-CoV-2 varied with peaks approximately every four week, preceding variations in number of newly hospitalized patients by 19-21 days. At that time virus testing for COVID-19 was limited to patients with severe symptoms. Local differences in viral spread was shown by analyzing weekly composite samples of wastewater from five sampling sites for four weeks. The highest amount of virus was found from the central, eastern, and northern parts of the city. SARS-CoV-2 was also found in the treated effluent wastewater from the WWTP discharged into the recipient, the Göta River, although with a reduction of 4-log10. The viral peaks with regular temporal intervals indicated that SARS-CoV-2 may have a cluster spread, probably reflecting that the majority of infected persons only spread the disease during a few days. Our results are important for both the planning of hospital care and to rapidly identify and intervene against local spread of the virus.

Regulatory Mechanisms of the Mucin-Like Region on Herpes Simplex Virus during Cellular Attachment.

Mucin-like regions, characterized by a local high density of O-linked glycosylation, are found on the viral envelope glycoproteins of many viruses. Herpes simplex virus type 1 (HSV-1), for example, exhibits a mucin-like region on its glycoprotein gC, a viral protein involved in initial recruitment of the virus to the cell surface via interaction with sulfated glycosaminoglycans. So far, this mucin-like region has been proposed to play a key role in modulating the interactions with cellular glycosaminoglycans, and in particular to promote release of HSV-1 virions from infected cells. However, the molecular mechanisms and the role as a pathogenicity factor remains unclear. Using single virus particle tracking, we show that the mobility of chondroitin sulfate-bound HSV-1 virions is decreased in absence of the mucin-like region. This decrease in mobility correlates with an increase in HSV-1-chondroitin sulfate binding forces as observed using atomic force microscopy-based force spectroscopy. Our data suggest that the mucin-like region modulates virus-glycosaminoglycan interactions by regulating the affinity, type, and number of glycoproteins involved in the virus-glycosaminoglycan interaction. This study therefore presents new evidence for a role of the mucin-like region in balancing the interaction of HSV-1 with glycosaminoglycans and provides further insights into the molecular mechanisms used by the virus to ensure both successful cell entry and release from the infected cell.

Cell Membrane Derived Platform To Study Virus Binding Kinetics and Diffusion with Single Particle Sensitivity.

Discovery and development of new antiviral therapies essentially rely on two key factors: an in-depth understanding of the mechanisms involved in viral infection and the development of fast and versatile drug screening platforms. To meet those demands, we present a biosensing platform to probe virus-cell membrane interactions on a single particle level. Our method is based on the formation of supported lipid bilayers from cell membrane material. Using total internal reflection fluorescence microscopy, we report the contribution of viral and cellular components to the interaction kinetics of herpes simplex virus type 1 with the cell membrane. Deletion of glycoprotein C (gC), the main viral attachment glycoprotein, or deletion of heparan sulfate, an attachment factor on the cell membrane, leads to an overall decrease in association of virions to the membrane and faster dissociation from the membrane. In addition to this, we perform binding inhibition studies using the antiviral compound heparin to estimate its IC50 value. Finally, single particle tracking is used to characterize the diffusive behavior of the virus particles on the supported lipid bilayers. Altogether, our results promote this platform as a complement to existing bioanalytical assays, being at the interface between simplified artificial membrane models and live cell experiments.

Binding Kinetics and Lateral Mobility of HSV-1 on End-Grafted Sulfated Glycosaminoglycans.

Many viruses, including herpes simplex (HSV), are recruited to their host cells via interaction between their envelope glycoproteins and cell-surface glycosaminoglycans (GAGs). This initial attachment is of a multivalent nature, i.e., it requires the establishment of multiple bonds between amino acids of viral glycoproteins and sulfated saccharides on the GAG chain. To gain understanding of how this binding process is modulated, we performed binding kinetics and mobility studies using end-grafted GAG chains that mimic the end attachment of these chains to proteoglycans. Total internal reflection fluorescence microscopy was used to probe binding and release, as well as the diffusion of single HSV-1 particles. To verify the hypothesis that the degree of sulfation, but also the arrangement of sulfate groups along the GAG chain, plays a key role in HSV binding, we tested two native GAGs (chondroitin sulfate and heparan sulfate) and compared our results to chemically sulfated hyaluronan. HSV-1 recognized all sulfated GAGs, but not the nonsulfated hyaluronan, indicating that binding is specific to the presence of sulfate groups. Furthermore we observed that a notable fraction of GAG-bound virions exhibit lateral mobility, although the multivalent binding to the immobilized GAG brushes ensures firm virus attachment to the interface. Diffusion was faster on the two native GAGs, one of which, chondroitin sulfate, was also characterized by the highest association rate per GAG chain. This highlights the complexity of multivalent virus-GAG interactions and suggests that the spatial arrangement of sulfates along native GAG chains may play a role in modulating the characteristics of the HSV-GAG interaction. Altogether, these results, obtained with a minimal and well-controlled model of the cell membrane, provide, to our knowledge, new insights into the dynamics of the HSV-GAG interaction.

The Cholestanol-Conjugated Sulfated Oligosaccharide PG545 Disrupts the Lipid Envelope of Herpes Simplex Virus Particles.

Herpes simplex virus (HSV) and many other viruses, including HIV, initiate infection of host cells by binding to glycosaminoglycan (GAG) chains of cell surface proteoglycans. Although GAG mimetics, such as sulfated oligo- and polysaccharides, exhibit potent antiviral activities in cultured cells, the prophylactic application of these inhibitors as vaginal microbicides failed to protect women upon their exposure to HIV. A possible explanation for this failure is that sulfated oligo- and polysaccharides exhibit no typical virucidal activity, as their interaction with viral particles is largely electrostatic and reversible and thereby vulnerable to competition with GAG-binding proteins of the genital tract. Here we report that the cholestanol-conjugated sulfated oligosaccharide PG545, but not several other sulfated oligosaccharides lacking this modification, exhibited virucidal activity manifested as disruption of the lipid envelope of HSV-2 particles. The significance of the virus particle-disrupting activity of PG545 was also demonstrated in experimental animals, as this compound, in contrast to unmodified sulfated oligosaccharide, protected mice against genital infection with HSV-2. Thus, PG545 offers a novel prophylaxis option against infections caused by GAG-binding viruses.

Mucin-like Region of Herpes Simplex Virus Type 1 Attachment Protein Glycoprotein C (gC) Modulates the Virus-Glycosaminoglycan Interaction.

Glycoprotein C (gC) mediates the attachment of HSV-1 to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying a HSV-1 mutant lacking the mucin-like domain in gC and the corresponding purified mutant protein (gCΔmuc) in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared with native HSV-1 (i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells and reduced release of viral particles from the surface of infected cells). Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared with native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.

The anterior commissure is a pathway for contralateral spread of herpes simplex virus type 1 after olfactory tract infection.

Herpes simplex encephalitis (HSE), targeting the limbic system, is the most common cause of viral encephalitis in the Western world. Two pathways for viral entry to the central nervous system (CNS) in HSE have been suggested: either via the trigeminal nerve or via the olfactory tract. This question remains unsettled, and studies of viral spread between the two brain hemispheres are scarce. Here, we investigated the olfactory infection as a model of infection and tropism of herpes simplex virus 1 (HSV-1), the causative agent of HSE, in the CNS of rats. Rats were instilled with HSV-1 in the right nostril and sacrificed 1-6 days post-infection, and tissues were analysed for viral spread using immunohistochemistry and quantitative PCR (qPCR). After nasal instillation, HSV-1 infected mitral cells of the olfactory bulb (OB) on the right side only, followed by limbic encephalitis. As a novel finding, the anterior commissure (AC) conveyed a rapid transmission of virus between the right and the left OB, acting as a shortcut also between the olfactory cortices. The neuronal cell population that conveyed the viral infection via the AC was positive for the water channel protein aquaporin 9 (AQP9) by immunohistochemistry. Quantification of AQP9 in cerebrospinal fluid samples of HSE patients showed increment as compared to controls. We conclude that the olfactory route and the AC are important for the spread of HSV-1 within the olfactory/limbic system of rats and furthermore, we suggest that AQP9 is involved in viral tropism and pathogenesis of HSE.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Screening and evaluation of anti-respiratory syncytial virus compounds in cultured cells.

Respiratory syncytial virus (RSV) is a highly contagious pathogen that infects mainly ciliated cells of respiratory epithelium and type 1 pneumocytes in the alveoli frequently causing serious respiratory disease in infants, elderly, and immunocompromised patients. At present, prevention/treatment of RSV infection is limited to the use of specific anti-RSV antibody or an aerosol formulation of ribavirin, a drug of suboptimal efficacy and low safety profile. There is an urgent need for development of novel anti-RSV drugs and virucides. Here we describe the cell culture-based methods used in our laboratory in identification of novel inhibitors of RSV including the P13 fusion inhibitor, and the PG545 virucide. Protocols for antiviral screening, evaluation of anti-RSV potency, and elucidation of mode of antiviral activity of test compounds are described.