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Antiviral Research (v.67, #1)

Prelim II: Editorial Board (pp. ii).

Prelim V: Instructions to Authors (pp. v-vii).

Inside Back Cover: Notes to Authors (pp. co3).

KP-1212/1461, a nucleoside designed for the treatment of HIV by viral mutagenesis by Kevin S. Harris; William Brabant; Sheila Styrchak; Alexander Gall; Richard Daifuku (pp. 1-9).

We report the activities of a novel nucleoside analog against HIV. This nucleoside (KP-1212) is not a chain terminator but exerts its antiviral effects via mutagenesis of the viral genome. Serial passaging of HIV in the presence of KP-1212 causes an increase in the mutation rate of the virus leading to viral ablation. HIV strains resistant to KP-1212 have not yet been isolated. Quite to the contrary, virus treated with KP-1212 exhibited an increased sensitivity not only to KP-1212 but also to another nucleoside reverse transcriptase inhibitor (NRTI), zidovudine. HIV strains resistant to other NRTIs (e.g. zidovudine, lamivudine, stavudine, abacavir, etc.) exhibited no cross-resistance towards KP-1212. Multiple assays confirmed that KP-1212 has a favorable (low) genotoxicity profile when compared to some approved antiviral nucleosides. In addition, KP-1212 is not toxic to mitochondria nor does it exhibit any inhibitory effects on mitochondrial DNA synthesis.

Keywords: HIV; Mutagenesis; Novel nucleoside analog

Mechanism of action of a novel viral mutagenic covert nucleotide: molecular interactions with HIV-1 reverse transcriptase and host cell DNA polymerases by Eisuke Murakami; Aravind Basavapathruni; William D. Bradley; Karen S. Anderson (pp. 10-17).

A novel non-chain terminating nucleoside analog anti-HIV inhibitor, KP-1212 has been designed to form base pairs with multiple bases that may lead to mutagenesis in the HIV-1 viral genome. After multiple replication cycles, the accumulation of mutations surpasses a crucial threshold beyond which the virus can no longer replicate. HIV-1 reverse transcriptase (RT) incorporates the KP-1212 monophosphate into the genome during viral replication after metabolic activation of the KP-1212 nucleoside to the triphosphate. The propensity for forming alternate base pairs with the KP-1212 nucleotide leads to mismatched nucleotides and the subsequent misincorporation is the basis for the inhibitory activity. The results showed that HIV-1 RT and human mitochondrial DNA polymerase (Pol γ) incorporated KP-1212-TP with a significant level of efficiency, whereas mouse DNA polymerase β (Pol β) did not. Misincorporation studies suggest that both HIV-1 RT and Pol γ may cause mutations at significantly high rates. These in vitro data confirm the mechanistic basis of KP-1212 as a viral mutagen but suggest that there may be a potential for toxicity to the mitochondria.

Keywords: KP-1212; Mutagen; HIV-1; Reverse transcriptase; Nucleoside analog

Identification of natural compounds with antiviral activities against SARS-associated coronavirus by Shi-you Li; Cong Chen; Hai-qing Zhang; Hai-yan Guo; Hui Wang; Lin Wang; Xiang Zhang; Shi-neng Hua; Jun Yu; Pei-gen Xiao; Rong-song Li; Xuehai Tan (pp. 18-23).

More than 200 Chinese medicinal herb extracts were screened for antiviral activities against Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay for virus-induced cytopathic effect (CPE). Four of these extracts showed moderate to potent antiviral activities against SARS-CoV with 50% effective concentration (EC₅₀) ranging from 2.4±0.2 to 88.2±7.7μg/ml. Out of the four, Lycoris radiata was most potent. To identify the active component, L. radiata extract was subjected to further fractionation, purification, and CPE/MTS assays. This process led to the identification of a single substance lycorine as an anti-SARS-CoV component with an EC₅₀ value of 15.7±1.2nM. This compound has a CC₅₀ value of 14980.0±912.0nM in cytotoxicity assay and a selective index (SI) greater than 900. The results suggested that four herbal extracts and the compound lycorine are candidates for the development of new anti-SARS-CoV drugs in the treatment of SARS.

Keywords: Severe Acute Respiratory Syndrome coronavirus (SARS-CoV); Drug screening; Natural products; Lycorine; Lycoris radiata; herb

Acetone, ethanol and methanol extracts of Phyllanthus urinaria inhibit HSV-2 infection in vitro by Chien-Min Yang; Hua-Yew Cheng; Ta-Chen Lin; Lien-Chai Chiang; Chun-Ching Lin (pp. 24-30).

Phyllanthus urinaria Linnea (Euphorbiaceae) is one of the traditional medicinal plants that are widely applied by oriental people, especially by Chinese and Indian, to ameliorate various kinds of ailments. Many biological activities, including anti-hepatitis B virus, anti-Epstein–Barr virus and anti-retroviral reverse transcriptase, of P. urinaria have been reported, but not against herpes simplex virus (HSV). In this study, the anti-HSV-1 and HSV-2 activities of different solvents extracted from P. urinaria were investigated in vitro by plaque reduction assay. Results showed that acetone, ethanol and methanol extracts of P. urinaria inhibited HSV-2 but not HSV-1 infection. The 50% inhibitory concentration against HSV-2 infection (IC₅₀) of acetone, ethanol and methanol extracts was 4.3±0.5, 5.0±0.4 and 4.0±0.9mcg/ml, respectively. All three extracts showed no cytotoxic effect against Vero cells at concentrations of 10.0mcg/ml or below. The time-of-addition study demonstrated that these three extracts were only effective when added during the HSV-2 infection which, therefore, suggested that they disturb the initial stage of HSV-2 infection. Furthermore, they can diminish virus infectivity without significantly affecting incubation time and temperature. Therefore, the acetone, ethanol and methanol extracts of P. urinaria were concluded to likely inhibit HSV-2 infection through disturbing the early stage of virus infection and through diminishing the virus infectivity.

Keywords: Anti-HSV activity; P. urinaria; Plant; Early stage of infection; Virus infectivity

Lactoferrin inhibits enterovirus 71 infection by binding to VP1 protein and host cells by Tsui-Ying Weng; Lien-Cheng Chen; Huey-Wen Shyu; Shun-Hua Chen; Jen-Ren Wang; Chun-Keung Yu; Huan-Yao Lei; Trai-Ming Yeh (pp. 31-37).

The antiviral activities of bovine lactoferrin (LF) against enterovirus 71 (EV71) were studied both in vitro and in vivo. LF protected both human rhabdomyosarcoma and neuroblastoma SK-N-SH cell lines from EV71 infection when it was added at the same time, before, or within 30min after EV71 infection. Using enzyme-linked immunosorbent assay-based binding assay and indirect fluorescent stain, we found that LF could bind to the target cells. Furthermore, it was found that LF could bind to the VP1 protein of EV71, which was blocked in the presence of anti-VP1 antibody. In addition, LF could induce IFN-α expression of SK-N-SH cells and inhibit EV71-induced IL-6 production. Finally, LF protected mice against lethal EV71 challenge. Taken together, these results suggest that LF can inhibit EV71 infection by interacting with both EV71 and host cells.

Keywords: Enterovirus; Lactoferrin

Error-prone replication of West Nile virus caused by ribavirin by Craig W. Day; Donald F. Smee; Justin G. Julander; Vladimir F. Yamshchikov; Robert W. Sidwell; John D. Morrey (pp. 38-45).

Ribavirin has been reported to cause error-prone replication and viral extinction in RNA viruses. The antiviral activity of ribavirin against West Nile virus (WNV) was evaluated in various cell lines to select a model in which mutagenic effects could be studied. The antiviral activity was greatest in HeLa cells as compared to CV-1, L929, Vero, or MA-104 cells. WNV was also passaged sequentially in cell monolayers treated with ribavirin to determine whether cumulative mutations could lead to viral extinction in these cell lines. The virus was abrogated in HeLa cells after 4 passages, while high viral titers persisted after many passages in other cells. A molecular clone of WNV was propagated in HeLa cells treated with 15μg/mL ribavirin, and sequencing of viral genome segments revealed significant increases in transition mutations, demonstrating that ribavirin induced error-prone replication. The relative infectivity of viral RNA synthesized in the presence of ribavirin was shown to be reduced compared with untreated controls. These data support the hypothesis that error catastrophe is one of the modes of action for ribavirin against WNV.

Keywords: West Nile virus; Error catastrophe; Ribavirin; Antiviral; Mutagenesis

Helper T cell cytokine response to ribavirin priming before combined treatment with interferon alpha and ribavirin for patients with chronic hepatitis C by Norihiro Furusyo; Norihiko Kubo; Kazuhiro Toyoda; Hiroaki Takeoka; Shigeki Nabeshima; Masayuki Murata; Makoto Nakamuta; Jun Hayashi (pp. 46-54).

The viral genotype and serum viral level influence the response to interferon (IFN) treatment in patients with chronic hepatitis C virus (HCV) viremia. The aim of this study was to investigate a possible relationship between early virological response and helper T (Th) cell cytokine expansion by 4 weeks of ribavirin (RIB) alone followed by IFN and RIB combined in patients with genotype 1b and a high HCV RNA level, patients reported not to respond well to IFN treatment. Eighty-one patients with genotype 1b and a high HCV RNA level, over 100 international unit per milliliter (KIU/mL) (by Amplicor HCV Monitor), were assigned to two groups: Group A ( N=40) with a 4-week RIB administration followed by a 24-week combination treatment, and Group B ( N=41) with a 24-week combination treatment only. Blood was obtained from each patient on the following schedule: at Baseline (4 weeks before day 0), on day 0 (initiation day of the RIB and IFN combination treatment), weeks 4 (4 weeks after the start of the combination treatment), and at the end of the combination treatment. Flow cytometry was used to investigate sequential changes of IFN-γ producing (Th1) and interleukin-4 producing (Th2) cells from whole blood samples after stimulation with PMA and ionomycin. Serum HCV RNA clearances were 32.5% at week 4, 43.2% at week 8, 85.7% at the end of the combination treatment, and 22.9% within the 24-week follow-up in Group A; and 17.1%, 27.0%, 66.7% and 19.4% in Group B, respectively. The mean Th1/Th2 ratio significantly increased from 15.9 at baseline to 17.6 at day 0 with a decrease of Th2 cells, and then significantly decreased from 17.6 at day 0 to 15.5 at week 4 in Group A, while there was no significant change in Group B between baseline and day 0. In Group A, 13 patients with HCV RNA clearance within 4 weeks had a significantly increased Th1/Th2 ratio, from 14.0 at baseline to 22.1 at day 0, and then a significantly decreased ratio, from 22.1 at day 0 to 15.0 at week 4, while the others had no significant change in the ratio. RIB administration preceding combined treatment of RIB with IFN was more effective in Th2 cell expansion than the usual combined treatment of IFN with RIB and led to a relatively early virological clearance in chronic hepatitis C patients with genotype 1b and a high HCV RNA level.

Keywords: Interferon-α (IFN-α); Ribavirin (RIB); Hepatitis C virus (HCV); Interferon-γ (IFN-γ); Interleukin-4 (Il-4)

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Antiviral Research (v.67, #1)