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Antiviral Research (v.75, #3)
Plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle by Els Keyaerts; Leen Vijgen; Christophe Pannecouque; Els Van Damme; Willy Peumans; Herman Egberink; Jan Balzarini; Marc Van Ranst (pp. 179-187).
We describe the antiviral activity of plant lectins with specificity for different glycan structures against the severe acute respiratory syndrome coronavirus (SARS-CoV) and the feline infectious peritonitis virus (FIPV) in vitro. The SARS-CoV emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and FIPV infection causes a chronic and often fatal peritonitis in cats. A unique collection of 33 plant lectins with different specificities were evaluated. The plant lectins possessed marked antiviral properties against both coronaviruses with EC50 values in the lower microgram/ml range (middle nanomolar range), being non-toxic (CC50) at 50–100μg/ml. The strongest anti-coronavirus activity was found predominantly among the mannose-binding lectins. In addition, a number of galactose-, N-acetylgalactosamine-, glucose-, and N-acetylglucosamine-specific plant agglutinines exhibited anti-coronaviral activity. A significant correlation (with an r-value of 0.70) between the EC50 values of the 10 mannose-specific plant lectins effective against the two coronaviruses was found. In contrast, little correlation was seen between the activity of other types of lectins. Two targets of possible antiviral intervention were identified in the replication cycle of SARS-CoV. The first target is located early in the replication cycle, most probably viral attachment, and the second target is located at the end of the infectious virus cycle.
Keywords: Coronavirus; Plant lectins; Antiviral; SARS-CoV; Mannose
Synergistic inhibition of HIV-1 infection by combinations of soluble polyanions with other potential microbicides by Katherine E. Gantlett; Jonathan N. Weber; Quentin J. Sattentau (pp. 188-197).
Several polyanionic compounds with potential for use as topically applied microbicides to prevent HIV-1 sexual transmission, such as PRO 2000, are currently in phase III clinical efficacy trials. Microbicidal formulations may well comprise combinations of inhibitors to increase potency, reduce dose and minimize problems of HIV-1 resistance. We have therefore evaluated in vitro, the anti-HIV-1 activity of two leading polyanionic microbicides combined with other antiretroviral agents with microbicidal potential. Dextran sulfate (DS) and PRO 2000 were combined with the neutralizing antibody IgG1b12, the peptide-based fusion inhibitor T20, the CCR5 antagonist TAK779 and the cyanobacterial protein cyanovirin-N. Anti-HIV-1 activity was assessed in a single cycle replication assay using pseudoviruses carrying a luciferase reporter gene and the envelope glycoproteins from HIV-1 isolates JR-FL (R5) and HxB2 (X4), against both immortalized and primary CD4+ cell targets. The data were analyzed for synergy using Calcusyn™ software. Results indicate that PRO 2000 and DS can act synergistically with most inhibitors tested, although the degree of synergy depends on inhibitor concentration and combination. These data provide a rational basis for testing of microbicide combinations in vivo.
Keywords: HIV-1; Microbicide; Synergy; Antiretroviral; Polyanion
Synthesis and anti-HIV activity of (−)-β-d-(2 R,4 R)-1,3-dioxolane-2,6-diamino purine (DAPD) (amdoxovir) and (−)-β-d-(2 R,4 R)-1,3-dioxolane guanosine (DXG) prodrugs by Janarthanan Narayanasamy; Manik R. Pullagurla; Ashoke Sharon; Jianing Wang; Raymond F. Schinazi; Chung K. Chu (pp. 198-209).
Prodrugs of (−)-β-d-(2 R,4 R)-1,3-dioxolane-2,6-diamino purine (DAPD), organic salts of DAPD, 5′-l-valyl DAPD and N−1 substituted (−)-β-d-(2 R,4 R)-1,3-dioxolane guanosine (DXG) have been synthesized with the objective of finding molecules which might be superior to DAPD and DXG in solubility as well as pharmacologic profiles. Synthesized prodrugs were evaluated for anti-HIV activity against HIV-1LAI in primary human lymphocytes (PBM cells) as well as their cytotoxicity in PBM, CEM and Vero cells. DAPD prodrugs, modified at the C6 position of the purine ring, demonstrated several folds of enhanced anti-HIV activity in comparison to the parent compound DAPD without increasing the toxicity. The presence of alkyl amino groups at the C6 position of the purine ring increased the antiviral potency several folds, and the most potent compound (−)-β-d-(2 R,4 R)-1,3-dioxolane-2-amino-6-aminoethyl purine (8) was 17 times more potent than that of DAPD. 5′-l-Valyl DAPD20 and organic acid salts21–24 also exhibited enhanced anti-HIV activity in comparison to DAPD, while DXG prodrugs16 and17 exhibited lower potency than that of DXG or DAPD.
Keywords: Nucleoside; Prodrug; Dioxolane-guanine; Dioxolane-2,6-diaminopurine; DAPD; Amdoxovir; Anti-HIV activity
Molecular basis of antagonism between K70E and K65R tenofovir-associated mutations in HIV-1 reverse transcriptase by R.M. Kagan; T.-S. Lee; L. Ross; R.M. Lloyd Jr.; M.A. Lewinski; S.J. Potts (pp. 210-218).
The K70E mutation in HIV-1 reverse transcriptase was observed in 10% of virologic non-responders of the abacavir/lamivudine/tenofovir arm of ESS30009, alone, or in mixtures with K65R by population sequencing. Clonal analysis of six ESS30009 K70E isolates failed to identify double mutants carrying K65R+K70E. Site-directed K70E mutants had a replication capacity of 97±29%, but only 2.4±0.9% for K65R+K70E and 0.01% for K65R+K70E+M184V mutants. K65R+K70E phenotypic fold changes for abacavir, lamivudine and tenofovir were comparable to reported values for K65R alone. In molecular dynamic simulations, the ɛ-amino group of K65 was positioned 2.7±0.1Å from the γ-phosphate of the dTTP ligand and stabilized the triphosphate. In the R65 mutant, this distance increased to 4.2±0.4Å and the interaction energy with the ligand was less favorable, but the K70 ɛ-amino group was repositioned closer to the γ-phosphate and had a more favorable interaction energy. In the double mutant, E70 could not stabilize the γ-phosphate, resulting in a more severe defect. The net effect of the atomic-level changes in the double mutant may be to destabilize the pyrophosphate leaving group of the ligand, more severely affecting the catalytic rate of the polymerization reaction than the R65 single mutation.
Keywords: HIV-1; Reverse transcriptase; Tenofovir; K65R; K70E; Molecular dynamics
Neuraminidase inhibitor susceptibility of porcine H3N2 influenza A viruses isolated in Germany between 1982 and 1999 by Katja Bauer; Christina Schrader; Jochen Suess; Peter Wutzler; Michaela Schmidtke (pp. 219-226).
As an intermediate host of avian and human influenza A viruses (FLUAV) pigs may play a potential role in interspecies virus transmission and reassortment of viral genes including those conferring antiviral drug resistance. Porcine FLUAV isolated in Germany between 1989 and 2001 contains mutations in the M2 gene inducing amantadine resistance. No data exist on neuraminidase inhibitor (NAI) susceptibility of these porcine FLUAV. We studied the antiviral activity of NAI against seven selected H3N2 FLUAV isolated from pigs in Germany between 1982 and 1999. All isolates were susceptible towards oseltamivir and zanamivir in neuraminidase enzyme-inhibition assays. Both compounds inhibited virus spreading and reduced the virus yields and plaque size at low concentrations. Higher concentrations were necessary to reduce the plaque number. Two isolates that differed in glycosylation pattern of viral hemagglutinin (HA) showed markedly reduced drug susceptibility in cell culture-based assays.
Keywords: Porcine influenza; Neuraminidase inhibitors; Oseltamivir; Zanamivir; Phenotyping; Genotyping; Resistance
In vitro anti-HIV and -HSV activity and safety of sodium rutin sulfate as a microbicide candidate by Jian Tao; Qinxue Hu; Jing Yang; Rurun Li; Xiuyi Li; Chengping Lu; Chaoyin Chen; Ling Wang; Robin Shattock; Kunlong Ben (pp. 227-233).
Sodium rutin sulfate (SRS) is a sulfated rutin modified from the natural flavonol glycoside rutin. Here, we investigated its in vitro anti-HIV and -HSV activities and its cytotoxic profile. Fifty percent inhibitory concentration (IC50) values of SRS against HIV-1 X4 virus IIIB, HIV-1 R5 isolates Ada-M and Ba-L were 2.3±0.2, 4.5±2.0 and 8.5±3.8μM with a selectivity index (SI) of 563, 575 and 329, respectively. Its IC50 against primary R5 HIV-1 isolate from Yunnan province in China was 13.1±5.5μM, with a SI of 197. In contrast, unsulfated rutin had no activity against any of the HIV-1 isolates tested. Further study indicated that SRS blocked viral entry and virus–cell fusion likely through interacting with the HIV-1 envelope glycoprotein. SRS also demonstrated some activity against human herpes simplex virus (HSV) with an IC50 of 88.3±0.1μM and a SI of 30. The 50% cytotoxicity concentration (CC50) of SRS was >3.0mM, as determined in human genital ME180, HeLa and primary human foreskin fibroblast cells. Minimum inhibitory concentration of SRS for vaginal lactobacilli was >3.0mM. These results collectively indicate that SRS represents a novel candidate for anti-HIV-1/HSV microbicide development.
Keywords: Abbreviations; AIDS; acquired immune deficiency syndrome; AZT; 3′-azido-3′-deoxythymidine; CC; 50; 50% cytotoxic concentration; ELISA; enzyme-linked immunosorbent assay; FCS; fetal calf serum; HIV; human immunodeficiency virus; HSV; herpes simplex virus; IC; 50; 50% inhibitory concentration; IL-2; interleukin-2; MIC; minimum inhibitory concentration; NO; nitric oxide; PBMC; peripheral blood mononuclear cells; PHA; phytohemagglutinin; SI; selectivity index; SRS; sodium rutin sulfate; CCID; 50; 50% cell culture infectious doseMicrobicide; HIV-1; HSV; Sodium rutin sulfate; Rutin; Viral entry
Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector by Yongping Jiang; Kangzhen Yu; Hongbo Zhang; Pingjing Zhang; Chenjun Li; Guobin Tian; Yanbing Li; Xijun Wang; Jinying Ge; Zhigao Bu; Hualan Chen (pp. 234-241).
H5N1 influenza viruses have caused significant disease and deaths in various parts of the world in several species, including humans. Vaccination combined with culling can provide an attractive method for outbreak containment. Using synthesized oligos and overlapping extension PCR techniques, we constructed an H5 HA gene, optiHA, containing chicken biased codons based on the HA amino acid sequence of the highly pathogenic H5N1 virus A/goose/Guangdong/1/96 (GS/GD/96). The optiHA and wild-type HA genes were inserted into plasmids pCI or pCAGGS, and designated as pCIoptiHA, pCAGGoptiHA, pCIHA and pCAGGHA, respectively. To evaluate vaccine efficacy, groups of 3-week-old specific pathogen free (SPF) chickens were intramuscularly injected with the four plasmids. Sera were collected on a weekly basis post-vaccination (p.v.) for hemagglutination inhibition (HI) assays and neutralization (NT) antibody detection. All chickens receiving pCAGGoptiHA and pCAGGHA developed high levels of HI and NT antibodies at 3 weeks p.v., and were completely protected from lethal H5 virus challenge, while only partial protection was induced by inoculation with the other two plasmids.A second experiment was conducted to evaluate if a lower dose of the pCAGGoptiHA vaccine could be effective, results indicated that two doses of 10μg of pCAGGoptiHA could induce complete protection in chickens against H5 lethal virus challenge. Based on our results, we conclude that construction optimization could dramatically increase the H5 HA gene DNA vaccine efficacy in chickens, and therefore, greatly decrease the dose necessary for inducing complete protection in chickens.
Keywords: Avian influenza; H5 subtype; DNA vaccine; Codon optimization
Chemical properties, mode of action, and in vivo anti-herpes activities of a lignin–carbohydrate complex from Prunella vulgaris by Yongwen Zhang; Paul Pui-Hay But; Vincent Eng-Choon Ooi; Hong-Xi Xu; Gillian D. Delaney; Spencer H.S. Lee; Song F. Lee (pp. 242-249).
The chemical nature, the mode of action, and the in vitro and in vivo anti-HSV activities of the polysaccharide from Prunella vulgaris were characterized. The polysaccharide was isolated by ethanol precipitation, dialysis, CTAB precipitation, and gel exclusion chromatography. The isolated compound (PPS-2b) was a lignin–carbohydrate complex with a molecular weight of 8500. The carbohydrate moiety was composed of glucose, galactose, mannose, galacturonic acid, rhamnose, xylose, and arabinose with glucose as the major sugar. In plaque reduction assay, PPS-2b showed activities against HSV-1 and HSV-2. The anti-HSV activity could be abolished by periodate oxidation. Mechanism studies showed that PPS-2b inactivated HSV-1 directly, blocked HSV-1 binding to Vero cells, and inhibited HSV-1 penetration into Vero cells. A similar inhibition was observed with a gC-deficient strain of HSV-1. The in vivo activities of a Prunella cream formulated with a semi-purified fraction was assessed in a HSV-1 skin lesion model in guinea pigs and a HSV-2 genital infection model in BALB/c mice. Guinea pigs that received the Prunella cream treatment showed a significant reduction ( P<0.01) in skin lesions. Mice that received the Prunella cream treatment showed a significant reduction ( P<0.01) in mortality. In conclusion, the anti-HSV compound from P. vulgaris is a lignin–polysaccharide complex with potent activity against HSV-1 and HSV-2. Its mode of action appears to be inhibiting viral binding and penetration into host cells.
Keywords: Anti-herpes; Prunella; Animal models
Isolation, identification and function of a novel anti-HSV-1 protein from Grifola frondosa by Chang-Qing Gu; Jun-Wen Li; Fuhuan Chao; Min Jin; Xin-Wei Wang; Zhi-Qiang Shen (pp. 250-257).
A novel antiviral protein was purified from an extract of Grifola frondosa fruiting bodies using a procedure that included 40% ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography, and designated GFAHP. This protein inhibited herpes simplex virus type 1 (HSV-1) replication in vitro with an IC50 value of 4.1μg/ml and a therapeutic index >29.3. Higher concentrations of GFAHP (125 and 500μg/ml) also significantly reduced the severity of HSV-1 induced blepharitis, neovascularization, and stromal keratitis in a murine model. Topical administration of GFAHP to the mouse cornea resulted in a significant decrease in virus production (mean virus yields: 3.4log10PFU in the treated group and 4.19log10PFU in the control group). We proved that GFAHP directly inactivates HSV-1 while simultaneously inhibiting HSV-1 penetration into Vero cells. Gel electrophoresis showed that GFAHP had a molecular weight of 29.5kDa. GFAHP was tryptic digested and analyzed from the PMF of matrix assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and nanoelectrospray ionization tandem mass spectrometry. The N-terminal sequence of GFAHP consisted of an 11 amino acid peptide, NH2-REQDNAPCGLN-COOH that did not match any known amino acid sequences, indicating that GFAHP is likely to be a novel antivirus protein. To our knowledge, this is the first report that characterizes an anti-HSV protein from G. frondosa.
Keywords: Grifola frondosa; Protein; Antiviral activity; Identification; HSV-1
The anti-papillomavirus activity of human and bovine lactoferricin by Nitesh Mistry; Peter Drobni; Jonas Näslund; Vivekananda Gupta Sunkari; Håvard Jenssen; Magnus Evander (pp. 258-265).
Human papillomavirus (HPV) cause common warts, laryngeal papilloma and genital condylomata and is necessary for the development of cervical cancer. We have previously found that lactoferrin has antiviral activity against HPV-16 and others have demonstrated that lactoferricin, an N-terminal fragment of lactoferrin, has inhibitory activities against several viruses. Two cell lines and two virus types, HPV-5 and HPV-16, were used to study if lactoferrin and lactoferricin could inhibit HPV pseudovirus (PsV) infection. We demonstrated that bovine lactoferrin (bLf) and human lactoferrin (hLf) were both potent inhibitors of HPV-5 and -16 PsV infections. Among the four lactoferricin derivatives we analyzed, a 15 amino acid peptide from bovine lactoferricin (bLfcin) 17–31 was the most potent inhibitor of both HPV-5 and HPV-16 PsV infection. Among the other derivatives, the human lactoferricin (hLfcin) 1–49 showed some antiviral activity against HPV PsV infection while bLfcin 17–42 inhibited only HPV-5 PsV infection in one of the cell lines. When we studied initial attachment of HPV-16, only bLfcin 17–42 and hLfcin 1–49 had an antiviral effect. This is the first time that lactoferricin was demonstrated to have an inhibitory effect on HPV infection and the antiviral activity differed depending on size, charge and structures of the lactoferricin.
Keywords: Lactoferrin; Lactoferricin; Human papillomavirus; Antiviral activity
Interactions of macrophages with probiotic bacteria lead to increased antiviral response against vesicular stomatitis virus by Martin Ivec; Tanja Botić; Srečko Koren; Mogens Jakobsen; Hana Weingartl; Avrelija Cencič (pp. 266-274).
Macrophages are an important cellular component of the innate immune system and are normally rapidly recruited and/or activated at the site of virus infection. They can participate in the antiviral response by killing infected cells, by producing antiviral cytokines such as nitric oxide and by producing chemokines and immunoregulatory cytokines that enable the adaptive immune response to recognize infected cells and perform antiviral effector functions. Probiotics, as a part of the normal gut intestinal flora, are important in supporting a functional yet balanced immune system. Improving our understanding of their role in the activation of macrophages and their stimulation of proinflammatory cytokine production in early viral infection was the main goal of this study. Our in vitro model study showed that probiotic bacteria, either from the species Lactobacillus or Bifidobacteria have the ability to decrease viral infection by establishing the antiviral state in macrophages, by production of NO and inflammatory cytokines such as interleukin 6 and interferon-gamma. These effects correlated with the mitochondrial activity of infected macrophages, therefore, the measurements of mitochondrial dehydrogenases activity could be implied as the first indicator of potential inhibitory effects of the probiotics on virus replication. The interactions between probiotic bacteria, macrophages and vesicular stomatitis virus (VSV), markedly depended on the bacterial strain studied.
Keywords: Macrophages; Lactobacillus; Bifidobacteria; Antiviral activity; NO; Inflammatory cytokines