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Antiviral Research (v.66, #2-3)
Development of antiviral therapy for severe acute respiratory syndrome by Jindrich Cinatl Jr.; Martin Michaelis; Gerold Hoever; Wolfgang Preiser; Hans Wilhelm Doerr (pp. 81-97).
A new disease, the severe acute respiratory distress syndrome (SARS), caused by the SARS coronavirus (SARS-CoV), emerged at the beginning of 2003 and rapidly spread throughout the world. Although the disease had disappeared in June 2003 its re-emergence cannot be excluded. The development of vaccines against SARS-CoV may take years. Therefore, the availability of effective antiviral drugs against SARS-CoV may be crucial for the control of future SARS outbreaks. In this review, experimental and clinical data about potential anti-SARS drugs is summarised and discussed. Animal model studies will be needed to help to determine which interventions warrant controlled clinical testing.
Keywords: Anti-viral therapy; SARS-CoV; Ribavirin
Interferon alfacon1 is an inhibitor of SARS-corona virus in cell-based models by Jason Paragas; Lawrence M. Blatt; Chris Hartmann; John W. Huggins; Tim P. Endy (pp. 99-102).
Preliminary data examining interferon alfacon1 treatment of SARS-CoV (severe acute respiratory syndrome-corona virus)-infected patients suggests this therapy is well tolerated and of therapeutic benefit. We report herein that interferon alfacon1, has potent in vitro antiviral activity against SARS-CoV. In a cytopathic effect protection (CPE) assay, interferon alfacon1 inhibited the generation of CPE in a dose-dependent manner with an IC50 of 0.001μg/ml, a clinically achievable level. Furthermore, interferon alfacon1 also demonstrated significant antiviral activity in yield reduction and plaque reduction assays. The in vitro antiviral activity of interferon alfacon1 against SARS-CoV suggests continued evaluation of interferon alfacon1 as a therapeutic treatment for patients infected with SARS-CoV.
Keywords: Interferon; Alfacon1; SARS-corona; Antiviral; Therapy
The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell by L.B. Talarico; C.A. Pujol; R.G.M. Zibetti; P.C.S. Faría; M.D. Noseda; M.E.R. Duarte; E.B. Damonte (pp. 103-110).
Two homogeneous sulfated polysaccharides obtained from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata, the kappa/iota/nu carrageenan G3d and thedl-galactan hybrid C2S-3, were assayed for their antiviral properties against the four serotypes of dengue virus (DENV) in different host cell types. Both seaweed derivatives were selective inhibitors of DENV-2 multiplication in Vero cells with inhibitory concentration 50% (IC50) values around 1μg/ml and selectivity indices >1000. The compounds had a lower antiviral effect against DENV-3 (IC50 values in the range 13.9–14.2μg/ml), an even lower effect against DENV-4 (IC50 values in the range 29.3 to >50μg/ml) and were totally inactive against DENV-1. With respect to the host cell, the polysulfates were inhibitors of DENV-2 and DENV-3 in the human hepatoma HepG2 and foreskin PH cells, with similar antiviral effectiveness as in Vero cells, but were totally inactive in mosquito C6/36 HT cells. Mechanistic studies demonstrated that G3d and C2S-3 were active DENV-2 inhibitors only when added together with the virus or early after infection, and both initial processes of virus adsorption and internalization are the main targets of these compounds. Therefore, the variations in antiviral activity of the polysaccharides depending on the viral serotype and the host cell may be ascribed to differences in the virus-cell interaction leading to virus entry.
Keywords: Dengue virus; Flaviviruses; Antiviral activity; Sulfated polysaccharides; Viral entry
Immunogenicity of enterovirus 70 capsid protein VP1 and its non-overlapping N- and C-terminal fragments by Dequan Chen; Chris Duggan; Donald E. Texada; Thomas B. Reden; Lakshmana M. Kooragayala; Marlyn P. Langford (pp. 111-117).
Currently no practical treatment method or effective virus vaccine is available for acute hemorrhagic conjunctivitis (AHC) caused by enterovirus 70 (EV70). Antibodies to UV-inactivated EV70 (J670/71 epidemic isolate) and to the inclusion bodies of recombinant proteins of full-length EV70 VP1 (GST-VP1m), its non-overlapping terminal fragments N138 (1–138 aa) and C170 (141–310 aa) (or GST-N138m and GST-C170) were developed in rabbits. The anti-EV70 neutralizing activities of the rabbit sera were determined by standard neutralization assays. The antibodies to UV-inactivated EV70, were immuno-reactive with EV70 capsid proteins VP1 and VP3 of four EV70 epidemic isolates (KW/97, T260/74, J670/71 and AE/72) in Western-blot analysis, and immunoprecipitated the capsid proteins VP1 and VP3 from the cell lysates of virus-infected human Chang's conjunctival (HCC) cells. The antibodies to GST-VP1m, GST-N138m and GST-C170, immunoprecipitated only the VP1 proteins of the four EV70 isolates. Anti-EV70 J670/71 antibodies and the antibodies to the three recombinant VP1 proteins were all capable of immunoprecipitating EV70 whole-virus of the four EV70 epidemic isolates grown in HCC cells. The anti-EV70 virion antibodies neutralized EV70 isolates with titers of 6000–10,000units/ml while the antibodies to GST-VP1m, GST-N138m or GST-C170 neutralized EV70 isolates with titers of 20–320units/ml. The results suggest that (a) immunization with bacterially produced recombinant EV70 VP1 and its non-overlapping N- and C-terminal fragments, was capable of eliciting EV70-neutralizing antibodies; (b) the neutralization titers of antibodies to the recombinant VP1 proteins were lower than that of antibodies to the UV-inactivated EV70 virions; and (c) the non-overlapping N138 and C170 fragments of EV70 VP1 both harbor independent anti-EV70 neutralization antigenic sites.
Keywords: Enterovirus 70; VP1; Recombinant protein; Neutralizing antibody; Neutralization antigenic site
Highly potent anti-HIV-1 activity isolated from fermented Polygonum tinctorium Aiton by Yu Zhong; Yoshiyuki Yoshinaka; Tadahiro Takeda; Noriko Shimizu; Sayaka Yoshizaki; Yoshio Inagaki; Shinobu Matsuda; Gisho Honda; Nobutaka Fujii; Naoki Yamamoto (pp. 119-128).
A water-soluble extract of fermented Polygonum tinctorium Aiton (Polygonaceae) called Sukumo, exhibited a potent inhibitory activity against HIV type 1 in vitro. The extract potently suppressed acute HIV-1 (IIIB) infection in MT-4 cells with EC50 values of 0.5μg/ml but exhibited low cytotoxicity to MT-4 cells even at a high concentration (CC50>1000μg/ml). It also inhibited giant cell formation in co-cultures of HIV-infected cells and uninfected Molt-4 cells. Sukumo extract was found to interact with both the viral envelope glycoprotein and cellular receptors, thus blocking virus-cell binding and virus-induced syncytium formation. There was a good correlation between the extract's anti-HIV-1 activity and its inhibitory effects on HIV-1 binding. It also suppressed replication of herpes simplex virus type 1 in Vero cells with an EC50 of 11.56μg/ml. On the other hand, there was no appreciable activity against influenza A virus, poliovirus or SARS corona virus when tested at concentrations ranging from 3.2–400μg/ml as shown by microscopic image analysis for cytopathic effect (CPE). Physico-chemical studies revealed that the anti-HIV activity in the extract was essentially maintained after boiling at 100°C in 1N HCl or 1N NaOH, and after treatment with 100mM NaIO4. The inhibitory activity of the extract was also not reduced after pronase digestion. The active factor in the extract is likely to be a novel compound(s) having a polyanionic substructure and a molecular weight of 10,000–50,000.
Keywords: Polygonum tinctorium; Sukumo; extract; HIV-1; HSV-1; Viral entry
The olive leaf extract exhibits antiviral activity against viral haemorrhagic septicaemia rhabdovirus (VHSV) by Vicente Micol; Nuria Caturla; Laura Pérez-Fons; Vicente Más; Luis Pérez; Amparo Estepa (pp. 129-136).
A commercial plant extract derived from olive tree leaf ( Olea europaea) (LExt) and its major compound, oleuropein (Ole), inhibited the in vitro infectivity of the viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus. Incubation of virus with LExt or Ole before infection reduced the viral infectivity to 10 and 30%, respectively. Furthermore, LExt drastically decreased VHSV titers and viral protein accumulation (virucidal effect) in a dose dependent manner when added to cell monolayers 36h post-infection. On the other hand, both the LExt and Ole were able to inhibit cell-to-cell membrane fusion induced by VHSV in uninfected cells, suggesting interactions with viral envelope. Therefore, we propose that O. europaea could be used as a potential source of promising natural antivirals, which have demonstrated to lack impact on health and environment. In addition, Ole could be used to design other related antiviral agents.
Keywords: Oleuropein; Olive leaf extract; VHSV; Antiviral; Viral membrane fusion
Mesuol, a natural occurring 4-phenylcoumarin, inhibits HIV-1 replication by targeting the NF-κB pathway by Nieves Márquez; Rocío Sancho; Luis M. Bedoya; José Alcamí; José Luis López-Pérez; Arturo San Feliciano; Bernd L. Fiebich; Eduardo Muñoz (pp. 137-145).
Coumarins and structurally related compounds have been recently shown to inhibit replication of human immunodeficiency virus (HIV) and thus, exhibit a therapeutic potential. In this study we report that mesuol and isomesuol, two 4-phenyl coumarins, isolated from the tree Marila pluricostata, suppress HIV-1 replication in Jurkat T cells. These coumarins do not affect the reverse transcription and intregration steps of the viral cycle and their antiviral effect is additive with that of azidothymidine (AZT). In addition, mesuol inhibits TNFα-induced HIV-1-LTR transcriptional activity by targeting the nuclear factor-κB (NF-κB) pathway. While mesuol does not prevent either the binding of NF-κB to DNA or the phosphorylation and degradation of NF-κB inhibitory protein, IκBα, it inhibits the phosphorylation and the transcriptional activity of the NF-κB p65 subunit in TNFα-stimulated cells. These results highlight the potential of the NF-κB transcription factor as a target for anti-HIV-1 compounds such as 4-phenyl coumarins, which could serve as lead compounds for the development of additional therapeutic approaches against AIDS.
Keywords: 4-Phenyl-coumarins; Mesuol; HIV-1; LTR; NF-κB
Putative antiviral activity in hemolymph from adult Pacific oysters, Crassostrea gigas by Cécile Olicard; Tristan Renault; Corinne Torhy; Abdenour Benmansour; Nathalie Bourgougnon (pp. 147-152).
Innate, non-specific resistance mechanisms are important to pathogens, particularly for delaying virus replication at the onset of infection. Innate immunity constitutes the first line of defense in vertebrates and is the only one in invertebrates. Little is known about possible antiviral substances in invertebrates. The present work concerns a study of antiviral substances in hemolymph from adult Crassostrea gigas oysters. Despite the detection of cytotoxicity in fresh filtered hemolymph for both mammalian (CC50: 750μg/ml) and fish cells (CC50: >2000μg/ml for EPC cells and 345μg/ml for RTG-2 cells), an antiviral substance was detected. Fresh filtered hemolymph was capable of inhibiting the replication of herpes simplex virus type 1 in vitro at an EC50 of 425μg/ml (total proteins) and the replication of infectious pancreatic necrosis virus in EPC and RTG-2 cells at 217 and 156μg/ml (total proteins), respectively.
Keywords: Oyster; Crassostrea gigas; Antiviral defense; HSV-1; IPNV; VHSV; Hemolymph
Effects of HIV Q151M-associated multi-drug resistance mutations on the activities of (−)-β-d-1′,3′-dioxolan guanine by Joy Y. Feng; Florence Myrick; Boulbaba Selmi; Jérôme Deval; Bruno Canard; Katyna Borroto-Esoda (pp. 153-158).
The multi-drug resistance HIV-1 genotype A62V/V75I/F77L/F116Y/Q151M is associated with resistance to many nucleoside reverse transcriptase inhibitors including AZT, ddI, ddC, d4T, abacavir, and 3TC. In this study, we evaluated the antiviral activity of (−)-β-d-1′,3′-dioxolan guanine (DXG) towards mutant HIV-1 containing V75I/F77L/F116Y/Q151M (V75Icomplex) and A62V/V75I/F77L/F116Y/Q151M (A62Vcomplex) in MT-2 cells. We further investigated the mechanism of resistance by studying the incorporation of DXG 5′-triphosphate (DXG-TP) during DNA synthesis by mutant enzymes containing single mutations at Q151M or A62V, and the V75Icomplex and A62Vcomplex using pre-steady state kinetic analysis. Our studies showed that mutant virus containing V75Icomplex and A62Vcomplex were both more than 23-fold resistant to DXG, and this correlated with the 68- and 20-fold resistance changes observed in the enzymatic assay. Compared to the wild-type enzyme, DXG-TP was incorporated 39- and 21-fold less efficiently by the mutant enzyme containing V75Icomplex and A62Vcomplex, mainly due to decreases in the rate of incorporation. The A62V mutation significantly increased the rate of incorporation ( kpol) for both dGTP (3-fold) and DXG-TP (7.9-fold), while the binding affinity of A62V HIV-1 RT for DXG-TP was decreased 14-fold. At the enzyme level, the addition of the A62V mutation to V75I/F77L/F116Y/Q151M moderately (3.4-fold) reversed the resistance to DXG-TP.
Keywords: Abbreviations; AZT; 3′-azido-3′-deoxythymidine; AZT; R; AZT-resistant; ddC; 2′,3′-dideoxycytidine; ddI; 2′,3′-dideoxyinosine; d4T; 2′,3′-didehydro-3′-deoxythymidine; DAPD; (−)-β-; d; -2,6-diaminopurine dioxolane; DXG; (−)-β-; d; -1′,3′-dioxolane guanosine; D30/D45; DNA/DNA primer/template 30/45-mer; MP; 5′-monophosphate; MDR; multi-drug resistance; dNTP; 2′-deoxynucleoside 5′-triphosphate, a general term for the natural nucleoside 5′-triphosphates; ddNTP; 2′,3′-dideoxynucleoside 5′-triphosphate; a general term for the analog nucleoside 5′-triphosphates; RT; reverse transcriptase; 3TC; (−)β-; l; -2′,3′-dideoxy-3′-thiacytidine; TP; 5′-triphosphate; wt; wild-typeDAPD; Amdoxovir; DXG; Pre-steady state; HIV-1 reverse transcriptase; Multi-drug resistance; Q151M
Inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (SARS)-associated coronavirus by Masayuki Saijo; Shigeru Morikawa; Shuetsu Fukushi; Tetsuya Mizutani; Hideki Hasegawa; Noriyo Nagata; Naoko Iwata; Ichiro Kurane (pp. 159-163).
The activity of inosine-5′-monophosphate dehydrogenase (IMPDH) inhibitors, mizoribine and ribavirin, against severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) was determined by plaque reduction and yield reduction assays. Mizoribine and ribavirin selectively inhibited replication of SARS-CoV. The 50% inhibitory concentration (IC50) of mizoribine for SARS-CoV Frankfurt-1 and SARS-CoV HKU39849, as determined by plaque reduction was 3.5μg/ml and 16μg/ml, respectively, and the IC50 of ribavirin for SARS-CoV Frankfurt-1 and SARS-CoV HKU39849 was 20μg/ml and 80μg/ml, while the 50% cytotoxic concentration of mizoribine and ribavirin for Vero E6 cells exceeded 200μg/ml. In a yield reduction assay, mizoribine (10μg/ml) and ribavirin (40μg/ml) inhibited the replication of SARS-CoV and reduced the infectious SARS-CoV titers to one-tenth or less. Mizoribine inhibited replication of SARS-CoV more strongly than ribavirin. However, neither drug could completely inhibit replication of SARS-CoV even at concentrations up to 100μg/ml.
Keywords: SARS; Coronavirus; Ribavirin; Mizoribine
Investigation into the ability of GB virus B to replicate in various immortalized cell lines by Victor E. Buckwold; Barbara Collins; Priscilla Hogan; Sherry Rippeon; Jiayi Wei (pp. 165-168).
GB virus B (GBV-B) is the most closely related virus to the hepatitis C virus (HCV) and is an attractive surrogate model system for HCV drug development efforts. Unfortunately, GBV-B can only be grown in the primary hepatocytes of certain non-human primates. We grew GBV-B in tamarins and marmosets and then used this virus in the absence and presence of lipofection reagents to try to infect 20 different cell lines including human primary hepatocytes and marmoset primary hepatocytes. GBV-B only replicated in marmoset primary hepatocytes. We isolated primary hepatocytes from GBV-B-positive and negative tamarins and marmosets and tried to immortalize the cells using SV40 large T-antigen or cell fusion. GBV-B stable cell lines were constructed in Huh7 and HepG2 cell lines, but there was no evidence for viral replication or a response to antiviral agents in these lines. Infectious full-length GBV-B RNA could be transfected into Vero, Huh7 and HepG2 at high efficiency, however there was no evidence for GBV-B replication or a response to antiviral agents. None of these approaches were successful and an in vitro model of GBV-B replication using immortalized cell lines was not produced. We hypothesize that these immortalized cell lines lack liver-specific factors that are required for GBV-B replication.
Keywords: GB virus B; Surrogate model system; Immortalization