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Antiviral Research (v.70, #3)
Intracellular uptake of thymidine and antiherpetic drugs for thymidine kinase-deficient mutants of herpes simplex virus type 1 by Pan Kee Bae; Jee Hyun Kim; Hae Soo Kim; In Kwon Chung; Sang Gi Paik; Chong-Kyo Lee (pp. 93-104).
The influence of the thymidine (Thd) kinase (TK) of herpes simplex virus type 1 (HSV-1) on the intracellular uptake and anabolism of nucleosides has been investigated. To compare the differences between the TK-positive (TK+) and TK-deficient strains, acyclovir (ACV)-resistant strains were cloned from a cell culture and classified into 2 groups, viz. the TK-partial (TKp) and TK-negative (TK−). The cellular uptake of thymidine was highly dependent on the viral TK (vTK) activity. The TK+ strain showed the highest level of intracellular thymidine uptake, the TKp strain a moderate level, which varied from strain to strain, and the TK− and mock strains showed little uptake. The inhibition of viral replication by ACV, ganciclovir (GCV) and penciclovir (PCV) did not decrease the Thd uptake at all. On the contrary, a notable increase found to be induced by ACV. The influence of the vTK on the uptake of GCV or PCV was much greater than that of ACV. The metabolism was generally less dependent on the vTK activity than the influx. The influx and phosphorylation rates of GCV and PCV were dependent on the substrate specificity of the vTK.
Keywords: Herpes simplex virus; Thymidine kinase; TK-deficiency; Drug-resistance; Antiherpetic drug; Nucleoside uptake; Nucleoside anabolism
Inhibition of measles virus and subacute sclerosing panencephalitis virus by RNA interference by Momoko Otaki; Kiyonao Sada; Hiroyasu Kadoya; Motoko Nagano-Fujii; Hak Hotta (pp. 105-111).
Subacute sclerosing panencephalitis (SSPE) is a rare, but fatal outcome of measles virus (MeV) infection. SSPE develops after prolonged persistence of mutated MeV called SSPE virus. Although a combination therapy using interferon and inosiplex or ribavirin appears to prolong survival time to some extent, there is currently no effective treatment to completely cure SSPE and a new treatment strategy is greatly needed. In this study, we adopted RNA interference (RNAi) strategy and examined whether small interfering RNAs (siRNAs) can be used to inhibit replication of MeV and SSPE virus. We report here that siRNAs targeted against L mRNA of MeV, either synthetic siRNAs or those generated by pcPUR+U6i-based expression plasmids, effectively and specifically inhibited replication of both MeV and SSPE virus without exhibiting any cytotoxic effect. The L protein of MeV is a major component of RNA-dependent RNA polymerase that is essential for viral RNA replication, and yet it is least abundant among all the MeV proteins expressed. Therefore, mRNA encoding the L protein would be a good target for RNAi strategy. The present results imply the possibility that our siRNAs against MeV L mRNA are among the potential candidates to be used to treat patients with SSPE.
Keywords: RNA interference; siRNA; Subacute sclerosing panencephalitis; Measles virus; L protein
Yatein from Chamaecyparis obtusa suppresses herpes simplex virus type 1 replication in HeLa cells by interruption the immediate-early gene expression by Yuh-Chi Kuo; Yueh-Hsiung Kuo; Yuang-Lian Lin; Wei-Jern Tsai (pp. 112-120).
Inhibitory effects of methanolic extracts from nine Chinese herbs on herpes simplex virus type 1 (HSV-1) replication were studied. By a bioassay-guided fractionation procedure, yatein (C22H23O7; M.W.399) was isolated from Chamaecyparis obtusa; yatein significantly suppressed HSV-1 multiplication in HeLa cells without apparent cytotoxicity. To further localize the point in the HSV-1 replication cycle where arrest occurred, a set of key regulatory events leading to the viral multiplication was examined, including viral immediate-early (α) and late (γ) gene expression and DNA replication. Results indicated that levels of glycoprotein B (gB) and gC mRNA expression in HeLa cells were impeded by yatein. Data from polymerase chain reaction showed that replication of HSV-1 DNA in HeLa cells was arrested by yatein. Furthermore, yatein decreased ICP0 and ICP4 gene expression in HeLa cells. Results of an electrophoretic mobility shift assay demonstrated that yatein interrupted the formation of α- trans-induction factor/C1/Oct-1/GARAT multiprotein complex. The mechanisms of antiviral action of yatein seem to be mediated, by inhibiting HSV-1 α gene expression, including expression of the ICP0 and ICP4 genes, and by arresting HSV-1 DNA synthesis and structural protein expression in HeLa cells. These results suggest that yatein is an antiviral agent against HSV-1 replication.
Keywords: HSV-1; Yatein; gB; ICP4; α-; Trans; -induction factor
Combination chemotherapy, a potential strategy for reducing the emergence of drug-resistant influenza A variants by Natalia A. Ilyushina; Nicolai V. Bovin; Robert G. Webster; Elena A. Govorkova (pp. 121-131).
Rapid development of resistant influenza variants after amantadine treatment is one of the main drawbacks of M2 blockers. On the other hand, the emergence of variants with low susceptibility to the neuraminidase (NA) inhibitors is limited. In the present study we examined whether combination therapy with two classes of anti-influenza drugs can affect the emergence of resistant variants in vitro. We observed that virus yields of human A/Nanchang/1/99 (H1N1), A/Panama/2007/99 (H3N2), and A/Hong Kong/156/97 (H5N1) viruses in MDCK cells were significantly reduced ( P<0.005) when the cells were treated with the combination of amantadine and low doses of oseltamivir carboxylate (≤1μM). After five sequential passages in MDCK cells, the M2 protein of viruses cultivated with amantadine alone mutated at positions V27A and S31N/I. Viruses cultivated with oseltamivir carboxylate (≥0.001μM) possessed mutations in the hemagglutinin (HA) protein. These variants showed reduced efficiency of binding to sialic acid receptors and decreased sensitivity to NA inhibitor in plaque reduction assay. Importantly, no mutations in the HA, NA, and M2 proteins were detected when the drugs were used in combination. Our results suggest that combination chemotherapy with M2 blocker and NA inhibitor reduced the emergence of drug-resistant influenza variants in vitro. This strategy could be an option for the control of influenza virus infection, and combinations with other novel drugs should be explored.
Keywords: Influenza A virus; Oseltamivir; Amantadine; Drug resistance
The efficacy of combined therapy of arsenic trioxide and alpha interferon in human T-cell leukemia virus type-1-infected squirrel monkeys ( Saimiri sciureus) by Jean Michel Heraud; Frank Mortreux; Fabrice Merien; Hugues Contamin; Renaud Mahieux; Jean Francois Pouliquen; Eric Wattel; Antoine Gessain; Hugues de Thé; Ali Bazarbachi; Olivier Hermine; Mirdad Kazanji (pp. 132-139).
Human T-cell lymphotropic virus type 1 (HTLV-1)-associated adult T-cell leukemia/lymphoma (ATLL) has a poor prognosis owing to its intrinsic resistance to chemotherapy. Although zidovudine (AZT) and alpha interferon (IFN-α) give rise to some response and improve the prognosis of ATLL, alternative therapies are needed. Arsenic trioxide (As2O3) has been shown to synergize with IFN-α in arresting cell growth and inducing apoptosis of ATLL cells in vitro. In this study, we evaluated the toxicity and the efficacy of this combined treatment in HTLV-1-infected squirrel monkeys ( Saimiri sciureus) and HTLV-1 infected cell lines derived therefrom. We first show that treatment with As2O3 and IFN-α can induce growth arrest in HTLV-1-transformed monkey T-cell lines in vitro. We then show that treatment of squirrel monkeys with As2O3 in vivo is highly toxic at 0.9 or 0.3mg/day but not at 0.14mg/day for up to 2 weeks. Although the combination of As2O3 and IFN-α did not affect significantly the HTLV-1 proviral load in infected monkeys, it reduced the absolute numbers of CD3+, CD4+ and CD8+ cells during treatment, with a significant reduction in the total number of circulating HTLV-1 flower cells in the infected monkeys with chronic ATLL-like disease.
Keywords: Arsenic; Interferon-alpha; HTLV-1; Squirrel monkeys; Treatment; Proviral load; Flower cells
Edible bird's nest extract inhibits influenza virus infection by Chao-Tan Guo; Tadanobu Takahashi; Wakoto Bukawa; Noriko Takahashi; Hirokazu Yagi; Koichi Kato; Kazuya I.-P. Jwa Hidari; Daisei Miyamoto; Takashi Suzuki; Yasuo Suzuki (pp. 140-146).
Edible bird's nest (EBN) is the nest of the swift that is made from its saliva. Although EBN has been widely used for enhancing immunocompetence, its antiviral efficacy has not been studied in detail. We found that EBN extract could strongly inhibit infection with influenza viruses in a host range-independent manner when it was hydrolyzed with Pancreatin F. Western blotting assay showed that the EBN extract bound to influenza virus. Furthermore, EBN extract could neutralize the infection of MDCK cells with influenza viruses and inhibit hemagglutination of influenza viruses to erythrocytes, but it could not inhibit the activity of influenza virus sialidase. Fluorometric HPLC indicated that the major molecular species of sialic acid in EBN is N-acetylneuraminic acid. The results suggest that EBN is a safe and valid natural source for the prevention of influenza viruses.
Keywords: Influenza; Bird nest; Sialic acid
Effects of ozone exposure on inactivation of intra- and extracellular enterovirus 71 by Ya-Ching Lin; Sheng-Chi Wu (pp. 147-153).
In this study, the potential of ozone in inactivating enterovirus 71 (EV71) free particles was investigated using either various ozone flow rates of 100, 80 or 60mg/h or a constant flow rate of 80mg/h, given to culture medium or various pH culture media containing EV71, respectively. Results demonstrated that EV71 inactivation by ozone was related to the kinetics of ozone solubility, ∼99% inactivation being obtained in the exponential phase of ozone solubility. However, the inactivation rate was dependent on the ozone input flow rate and positively enhanced at acidic pH. Inactivation of intracellular EV71 was also studied. At a constant ozone supply of 60mg/h, a significant reduction of intracellular virus titer (≥99%, p<0.01) was obtained after 45 or 60min exposure but with low cell viability. Upon 30min exposure, however, 45% cell viability was retained. The results indicate that the inactivating effect of ozone on intracellular EV71 virus is dependent on exposure duration.
Keywords: Enterovirus 71; Ozone; Virus inactivation