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Antiviral Research (v.82, #1)
GSK983: A novel compound with broad-spectrum antiviral activity by Robert Harvey; Kevin Brown; Qin Zhang; Margaret Gartland; Leslie Walton; Christine Talarico; Wendell Lawrence; Dean Selleseth; Neil Coffield; Jeffry Leary; Kelly Moniri; Sara Singer; Jay Strum; Kristjan Gudmundsson; Karen Biron; Karen R. Romines; Phiroze Sethna (pp. 1-11).
GSK983, a novel tetrahydrocarbazole, inhibits the replication of a variety of unrelated viruses in vitro with EC50 values of 5–20nM. Both replication of the adenovirus Ad-5 and the polyoma virus SV-40, and episomal maintenance of human papillomaviruses (HPV) and Epstein-Barr virus (EBV) are susceptible to GSK983. The compound does not inhibit all viruses; herpes simplex virus (HSV-1), human immunodeficiency virus (HIV), and lytic replication of EBV were not susceptible at concentrations below 1μM. GSK983 does inhibit the growth of cell lines immortalized by HTLV-1, EBV, HPV, SV40 and Ad-5, with EC50 values in the range of 10–40nM. Depending on the cell line, the compound induces either apoptosis or cytostasis at concentrations over 20nM. GSK983 also inhibits cell lines immortalized by non-viral mechanisms, but has little effect on primary cells. The CC50 values for keratinocytes, fibroblasts, lymphocytes, endothelial, and bone marrow progenitor cells are all above 10μM. The pattern of inhibition, which includes diverse viruses as well as growth of immortalized cells of varied origins, suggests the target is a host cell protein, rather than a viral protein. Preliminary mechanism studies indicate that GSK983 acts by inducing a subset of interferon-stimulated genes.
Keywords: Novel antiviral; GSK983; Tetrahydrocarbazole; Interferon-stimulated genes
A recombinant, infectious human parainfluenza virus type 3 expressing the enhanced green fluorescent protein for use in high-throughput antiviral assays by Jason P. Roth; Joseph K.-K. Li; Donald F. Smee; John D. Morrey; Dale L. Barnard (pp. 12-21).
The ability to rescue an infectious, recombinant, negative-stranded, RNA virus from a complementary DNA (cDNA) clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this study, the enhanced green fluorescent protein (EGFP) gene was inserted into the human parainfluenza virus type 3 (HPIV-3) antigenome and a recombinant, infectious virus was rescued. Maximum EGFP expression levels, measured by fluorescence, were seen at day 3. Comparison of a 3-day, viral expressed EGFP fluorescence assay to a 7-day, neutral red assay, based on complete cell destruction in virus infected MA-104 cells, yielded Z′-factor values of 0.83 and 0.70, respectively. A 3-day, endpoint EGFP-based antiviral assay and a 7-day, endpoint neutral red based antiviral assay were run in parallel to establish antiviral sensitivity profiles of 23 compounds based on selective index (SI) values. Using an SI threshold of 10, the EGFP-based antiviral assay had a sensitivity of 100% and a specificity of 54%. Thus, the use of an EGFP-based antiviral assay for testing potential antiviral compounds against HPIV-3 in a high-throughput format may be justified.
Keywords: Human parainfluenza virus type 3; Enhanced green fluorescent protein; Recombinant virus; Antiviral assay
Retroviral self-inactivation in the mouse vagina induced by short DNA by Lina Wittmer-Elzaouk; Jiunshan Jung-Shiu; Jochen Heinrich; Karin Moelling (pp. 22-28).
Human immunodeficiency virus (HIV) has been shown to undergo self-destruction upon treatment of cell-free virions with partially double-stranded oligodeoxynucleotides targeting the polypurine tract (PPT) of the viral RNA in the virus particle. The ODN forms a local hybrid with the PPT activating the viral RNase H to prematurely cleave the genomic RNA. Here we are describing the self-destruction of a recombinant lentivirus harboring the PPT of HIV in a mouse vagina model. We showed a decrease in viral RNA levels in cell-free virus particles and a reduction reverse transcribed complementary DNA (cDNA) in virus-infected human and primary murine cells by incubation with ODNs. In the vagina simultaneous, prophylactic or therapeutic ODN treatments led to a significant reduction in viral RNA levels. Our finding may have some relevance for the design of other viral self-destruction approaches. It may lead to a microbicide for reduction of sexual and mother-to-child transmission.
Keywords: Lentiviral vector; HIV; RNase H; Microbicide; Virus suicide; Hairpin-loop DNA
Rapid identification of oseltamivir-resistant influenza A(H1N1) viruses with H274Y mutation by RT-PCR/restriction fragment length polymorphism assay by Lizheng Guo; Rebecca J. Garten; Angie S. Foust; Wendy M. Sessions; Margaret Okomo-Adhiambo; Larisa V. Gubareva; Alexander I. Klimov; Xiyan Xu (pp. 29-33).
In the beginning of 2007–2008 Northern Hemisphere influenza season, the frequency of influenza A(H1N1) viruses bearing a previously defined oseltamivir resistance conferring amino acid change of Histidine to Tyrosine at position 274 (H274Y) of the neuraminidase (NA) increased dramatically. In order to rapidly detect such resistant viruses, an RT-PCR/restriction fragment length polymorphism (RT-PCR/RFLP) assay targeting amino acid 274 of the N1 NA molecule was developed to investigate the presence or absence of the H274Y mutation. The reverse primer was engineered to produce a BspHI site in the amplicon for oseltamivir-sensitive viruses with Histidine at position 274 (274H). A total of 50 influenza A(H1N1) specimens including 30 oseltamivir-sensitive and 20 oseltamivir-resistant ones submitted to the Centers for Disease Control and Prevention (CDC) during the 2007–2008 influenza season were successfully characterized by this assay. The assay was specific for grown A(H1N1) viruses and original clinical specimens, with a lower limit of detection of approximately 10 RNA transcript copies per reaction. Our RT-PCR/RFLP assay provides a simple, rapid and sensitive tool to monitor the emergence and spread of H274Y oseltamivir-resistant influenza A(H1N1) viruses.
Keywords: Oseltamivir resistance; Influenza A(H1N1) virus; H274Y; RT-PCR/RFLP
Different neuraminidase inhibitor susceptibilities of human H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany from 2001 to 2005/2006 by Katja Bauer; Martina Richter; Peter Wutzler; Michaela Schmidtke (pp. 34-41).
In the flu season 2005/2006 amantadine-resistant human influenza A viruses (FLUAV) of subtype H3N2 circulated in Germany. This raises questions on the neuraminidase inhibitor (NAI) susceptibility of FLUAV. To get an answer, chemiluminescence-based neuraminidase inhibition assays were performed with 51 H1N1, H1N2, and H3N2 FLUAV isolated in Germany from 2001 to 2005/2006. According to the mean IC50 values (0.38–0.91nM for oseltamivir and 0.76–1.13nM for zanamivir) most H1N1 and H3N2 FLUAV were NAI-susceptible. But, about four times higher zanamivir concentrations were necessary to inhibit neuraminidase activity of H1N2 viruses. Two H1N1 isolates were less susceptible to both drugs in NA inhibition as well as virus yield reduction assays. Results from sequence analysis of viral hemagglutinin and neuraminidase genes and evolutionary analysis of N2 gene revealed (i) different subclades for N2 in H1N2 and H3N2 FLUAV that could explain the differences in zanamivir susceptibility among these viruses and (ii) specific amino acid substitutions in the neuraminidase segment of the two less NAI-susceptible H1N1 isolates. One H3N2 was isolate proved to be a mixture of a NA deletion mutant and full-length NA viruses.
Keywords: Influenza A virus; Neuraminidase inhibitors; Oseltamivir; Zanamivir; Resistance; Evolution
Replicons from genotype 1b HCV-positive sera exhibit diverse sensitivities to anti-HCV reagents by Go Nishimura; Masanori Ikeda; Kyoko Mori; Takahide Nakazawa; Yasuo Ariumi; Hiromichi Dansako; Nobuyuki Kato (pp. 42-50).
Half of the population of genotype 1 HCV is resistant to current pegylated-interferon-α (PEG-IFN-α) and ribavirin therapy. The resistance to IFN therapy is an urgent problem, especially in patients with genotype 1 HCV infection. However, sensitivities among HCV strains to anti-HCV reagents including IFNs have not been thoroughly addressed. Here, we established three different subgenomic replicons (1B-4, 1B-5, and KAH5 strains) in addition to our previously established replicon (O strain). We comparatively examined the sensitivities of four replicons to IFN-α, IFN-γ, IFN-λ, cyclosporine A, and fluvastatin. Among the replicons, the 1B-4 and KAH5 replicons were the most sensitive and resistant, respectively to IFN-λ (EC50: 1.50ng/ml vs. 8.50ng/ml) and fluvastatin (EC50: 2.82μM vs. 7.87μM), although these replicons possessed similar features in terms of genetic distance from the O strain, HCV RNA expression levels, and sensitivity to IFN-α (EC50: 1.44IU/ml vs. 1.37IU/ml) and cyclosporine A (EC50: 0.71μg/ml vs. 0.96μg/ml). These replicons are thus useful tools for examining the mechanism of anti-HCV activity, especially in IFN-λ and statins.
Keywords: Hepatitis C virus; Interferon-α; Interferon-γ; Interferon-λ; Cyclosporine A; Statin
Design of oseltamivir analogs inhibiting neuraminidase of avian influenza virus H5N1 by Thanyada Rungrotmongkol; Vladimir Frecer; Wanchai De-Eknamkul; Supot Hannongbua; Stanislav Miertus (pp. 51-58).
Neuraminidase is an important target for design of antiviral agents in the prophylaxis and treatment of avian influenza virus infections. We have shown the applicability of computer-assisted combinatorial techniques in the design, focusing and in silico screening of a virtual library of analogs of oseltamivir (Tamiflu) with the goal to find potent inhibitors of influenza A neuraminidase N1 that fill the cavity found adjacent to the active site. Crystal structure of oseltamivir–N1 complex was used in the structure-based focusing and virtual screening of the designed library. A target-specific Piecewise Linear Potential type 1 scoring function fitted for a training set of 14 carbocyclic inhibitors and validated for three other inhibitors was used to select virtual hits with predicted inhibitory activities in the subnanomolar range. The results of this computational study are useful as a rational guide for synthetic and medicinal chemists who are developing new drugs against the avian influenza virus H5N1.
Keywords: Avian influenza virus subtype H5N1; Neuraminidase inhibitors; Oseltamivir analogs; Computer-assisted combinatorial library design; In silico; screening
Enhanced inhibition of porcine reproductive and respiratory syndrome virus replication by combination of morpholino oligomers by Xue Han; Sumin Fan; Deendayal Patel; Yan-Jin Zhang (pp. 59-66).
Porcine reproductive and respiratory syndrome (PRRS) has caused heavy economic losses in the swine industry worldwide and current strategies to control PRRS are inadequate. Previous studies have shown that peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) can be an effective antiviral against the PRRS virus (PRRSV). PPMO is structurally similar to DNA with modified backbone and is resistant to nuclease. This study was designed to examine increasing inhibitory effect of PPMO combination. Two pairs of PPMOs were identified to have enhanced suppression of PRRSV replication in cell culture, while individual constituents did not work under the same testing conditions. PPMO 5UP1 that is complementary to 5′ terminus of PRRSV genome was paired with 4P1 or 7P1 that are complementary to sequence in the translation initiation regions of ORFs 4 and 7, respectively. The PPMO combination also inhibited replication of heterologous strains in the North American PRRSV genotype. Treatment of the cells with the combinations reduced PRRSV RNA and protein levels. In cell-free or cell-based luciferase reporter assays, the PPMO combination suppressed target mRNA translation more effectively than individual constituents, indicating that the suppression was due to their antisense effect. These results suggest potential application of these PPMO combinations for PRRS control.
Keywords: Porcine reproductive and respiratory syndrome virus; PRRSV; Morpholino; PPMO; Antisense; Antiviral
Synonymous mutations in stem-loop III of Rev responsive elements enhance HIV-1 replication impaired by primary mutations for resistance to enfuvirtide by Mariko Ueno; Eiichi N. Kodama; Kazuya Shimura; Yasuteru Sakurai; Keiko Kajiwara; Yasuko Sakagami; Shinya Oishi; Nobutaka Fujii; Masao Matsuoka (pp. 67-72).
Primary mutations in HIV-1 that are directly involved in the resistance to enfuvirtide have been well documented. However, secondary mutations that are associated with primary mutations and contribute little to the resistance still remain to be elucidated. This study reveals that synonymous mutations at gp41 Q41 (CAG to CAA) or L44 (UUG to CUG) act as secondary mutations. Complementary mutations in the nucleotide level are located in the Rev responsive element (RRE) of the HIV-1 RNA-genome and maintain the replication kinetics of HIV-1 through increasing the structural stability of stem-loop III in the RRE. Therefore, synonymous mutations in the gp41/RRE sequence improve the viral replication impaired by the primary mutations and play a key role as secondary (complementary) mutations.
Keywords: Fusion; gp41; Rev responsive element; Secondary mutation; HIV-1; Replication
Procyanidins and butanol extract of Cinnamomi Cortex inhibit SARS-CoV infection by Min Zhuang; Hong Jiang; Yasuhiro Suzuki; Xiaoguang Li; Peng Xiao; Takashi Tanaka; Hong Ling; Baofeng Yang; Hiroki Saitoh; Lianfeng Zhang; Chuan Qin; Kazuo Sugamura; Toshio Hattori (pp. 73-81).
We found that the butanol fraction of Cinnamomi Cortex (CC/Fr.2) showed moderate inhibitory activity in wild-type severe acute respiratory syndrome coronavirus (wtSARS-CoV) and HIV/SARS-CoV S pseudovirus infections. The inhibition on pseudovirus was also seen in cells pretreated with the CC and CC/Fr.2 (IC50S, 283.4±16.3 and 149.5±13.5μg/ml, respectively), however the highest activities on wtSARS-CoV were observed when the viruses were treated by the extracts before challenging (IC50S, 43.1±2.8 and 7.8±0.3μg/ml; SIs, 8.4 and 23.1, respectively). Among the compounds fractionated from CC, procyanidin A2 and procyanidin B1 showed moderate anti-wtSARS-CoV activity (IC50S, 29.9±3.3 and 41.3±3.4μM; SIs, 37.35 and 15.69, respectively). We also sought to determine whether they could interfere with the clathrin-dependent endocytosis pathway using transferrin receptor (TfR) as an indicator. CC/Fr.2 inhibited the internalization of TfR but the procyanidins did not. Taken together, CC/Fr.2 contains unknown substances, that could inhibit the infection, probably by interfering with endocytosis, and it also contains procyanidins that did not inhibit the internalization but inhibited the infection. Therefore, CC extracts contain anti-virus activities that act through distinct mechanisms according to differences in the compounds or mixtures.
Keywords: Abbreviations; SARS-CoV; severe acute respiratory syndrome coronavirus; HIV; human immunodeficiency virus; VSV; vesicular stomatitis virus; Wild-type SARS-CoV; wtSARS-CoV; ACE2; angiotensin converting enzyme 2; IC; 50; 50% inhibitory concentration; CC; 50; 50% growth reduction of the cells; SI; selective index, CC; 50; /IC; 50; TCID; 50; 50% tissue culture infectious dose; PBS; phosphate-buffered saline; BSA; bovine serum albumin; DMEM; Dulbecco's modified Eagle's medium; FITC; fluorescein isothiocyanate; PE; phycoerythrin; CHX; cycloheximide; MFI; mean fluorescence intensitySARS-CoV; Medicinal herbs; Cinnamomi Cortex; Pseudovirus; Transferrin receptor
Development of a cell-based assay for high-throughput screening of inhibitors against HCV genotypes 1a and 1b in a single well by Rubina Mondal; Gennadiy Koev; Tami Pilot-Matias; Yupeng He; Teresa Ng; Warren Kati; Akhteruzzaman Molla (pp. 82-88).
The Hepatitis C (HCV) replicon system is a useful tool for the high-volume screening of inhibitors of HCV replication. In this report, a cell-based assay has been described, which monitors the inhibition of HCV genotypes 1a and 1b as well as cytotoxicity, from a single well of a 96-well plate. A mixture of two stable replicon cell lines was used: one containing a 1a-H77 replicon expressing a firefly luciferase reporter, and the other one containing a 1b-N replicon with a secreted alkaline phosphatase reporter, thus allowing us to monitor replication of two HCV genotypes in the same well. Cytotoxicity was measured using the Resazurin cytotoxicity assay. The assay was validated with known HCV inhibitors and showed that the antiviral activity and cytotoxicity of compounds were reproducibly measured under screening conditions. It was also showed that the assay's signal-to-noise ratio and Z′ coefficient were suitable for high-throughput screening. A panel of HCV inhibitors showed a good correlation between EC50 and TD50 values for 1a and 1b replicon activity and cytotoxicity measured using either a single replicon format or mixed replicon format. Thus, the use of this mixed replicon format provides an economical method for simultaneous measurement of compound activity against two HCV genotypes as well as cytotoxicity, thereby reducing cost of reagents and labor as well as improving throughput.
Keywords: HTS; HCV; Replicon; Cell-based assay; SEAP; Luciferase; Resazurin cytotoxicity assay
Anti-influenza virus activity and structure–activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains by Lieve Naesens; Evelien Vanderlinden; Erzsébet Rőth; József Jekő; Graciela Andrei; Robert Snoeck; Christophe Pannecouque; Eszter Illyés; Gyula Batta; Pál Herczegh; Ferenc Sztaricskai (pp. 89-94).
Previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. We here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. The lead compound8e displayed an antivirally effective concentration of 0.4μM, which was consistent amongst influenza A/H1N1, A/H3N2 and B viruses, and a selectivity index ≥50. Structural analogues derived from aglycovancomycin were found to be inactive. The hydrophobic side chain was shown to be an important determinant of activity. The narrow structure–activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. Compound8e proved to be inactive against several unrelated RNA and DNA viruses, except for varicella-zoster virus, against which a favorable activity was noted.
Keywords: Glycopeptide antibiotic; Ristocetin; Influenza; Antiviral