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Antiviral Research (v.73, #1)
Administration of a dual toll-like receptor 7 and toll-like receptor 8 agonist protects against influenza in rats by David M. Hammerbeck; Gary R. Burleson; Craig J. Schuller; John P. Vasilakos; Mark Tomai; Elaine Egging; Felicia R. Cochran; Susan Woulfe; Richard L. Miller (pp. 1-11).
Toll-like receptors (TLR) detect conserved molecular patterns expressed by pathogens. Detection of the “molecular signature” for RNA viruses including influenza has been attributed to TLR3, TLR7, and TLR8. In the present study, compound 3M-011 was shown to be a synthetic human TLR7/8 agonist and cytokine inducer. 3M-011 was investigated as a stand-alone immune response modifier in a rat model of human influenza. Intranasal (IN) administration of 3M-011 significantly inhibited H3N2 influenza viral replication in the nasal cavity when administered from 72h before IN viral inoculation to 6h after inoculation. Viral inhibition correlated with the ability of the TLR7/8 agonist to stimulate type I interferon (IFN) and other cytokines such as tumor necrosis factor-α, interleukin-12, and IFN-γ from rat peripheral blood mononuclear cells. Prophylactic administration of TLR7/8 agonist also suppressed influenza viral titers in the lung, which corresponded with local IFN production. The activity of the TLR7/8 agonist resulted in greater inhibition of viral titers compared to rat recombinant IFN-α administered in a comparable dosing regimen. These studies indicate that TLR7/8 agonists may have prophylactic and therapeutic benefits in the treatment of respiratory viral infections, such as influenza, when administered prior to or shortly after viral inoculation.
Keywords: Toll-like receptor; Influenza; Innate immunity; Interferon; Cytokine
Administration of a dual toll-like receptor 7 and toll-like receptor 8 agonist protects against influenza in rats by David M. Hammerbeck; Gary R. Burleson; Craig J. Schuller; John P. Vasilakos; Mark Tomai; Elaine Egging; Felicia R. Cochran; Susan Woulfe; Richard L. Miller (pp. 1-11).
Toll-like receptors (TLR) detect conserved molecular patterns expressed by pathogens. Detection of the “molecular signature” for RNA viruses including influenza has been attributed to TLR3, TLR7, and TLR8. In the present study, compound 3M-011 was shown to be a synthetic human TLR7/8 agonist and cytokine inducer. 3M-011 was investigated as a stand-alone immune response modifier in a rat model of human influenza. Intranasal (IN) administration of 3M-011 significantly inhibited H3N2 influenza viral replication in the nasal cavity when administered from 72h before IN viral inoculation to 6h after inoculation. Viral inhibition correlated with the ability of the TLR7/8 agonist to stimulate type I interferon (IFN) and other cytokines such as tumor necrosis factor-α, interleukin-12, and IFN-γ from rat peripheral blood mononuclear cells. Prophylactic administration of TLR7/8 agonist also suppressed influenza viral titers in the lung, which corresponded with local IFN production. The activity of the TLR7/8 agonist resulted in greater inhibition of viral titers compared to rat recombinant IFN-α administered in a comparable dosing regimen. These studies indicate that TLR7/8 agonists may have prophylactic and therapeutic benefits in the treatment of respiratory viral infections, such as influenza, when administered prior to or shortly after viral inoculation.
Keywords: Toll-like receptor; Influenza; Innate immunity; Interferon; Cytokine
Inhibition of replication and transcription activator and latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus by morpholino oligomers by Yan-Jin Zhang; Kai-Yu Wang; David A. Stein; Deendayal Patel; Rheba Watkins; Hong M. Moulton; Patrick L. Iversen; David O. Matson (pp. 12-23).
Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistance to nucleases. In this study, RTA and LANA mRNA in PEL cells were targeted by antisense peptide-conjugated PMO (P-PMO) in an effort to suppress KSHV replication. Highly efficient P-PMO uptake by PEL cells was observed. Treatment of PEL cells with a RTA P-PMO (RP1) reduced RTA expression in a dose-dependent and sequence-specific manner, and also caused a significant decrease in several KSHV early and late gene products, including vIL-6, vIRF-1, and ORF-K8.1A. KSHV viral DNA levels were reduced both in cells and culture supernatants of RP1 P-PMO-treated cells, indicating that KSHV lytic replication was suppressed. Treatment of BCBL-1 cells with P-PMO against LANA resulted in a reduction of LANA expression. Cell viability assays detected no cytotoxicity from P-PMO alone, within the concentration range used for the experiments in this study. These results suggest that RP1 P-PMO can specifically block KSHV replication, and further study is warranted.
Keywords: KSHV; RTA; LANA; Morpholino; Antiviral; Antisense
Inhibition of replication and transcription activator and latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus by morpholino oligomers by Yan-Jin Zhang; Kai-Yu Wang; David A. Stein; Deendayal Patel; Rheba Watkins; Hong M. Moulton; Patrick L. Iversen; David O. Matson (pp. 12-23).
Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The KSHV replication and transcription activator (RTA) and latency-associated nuclear antigen (LANA) play key roles in activating KSHV lytic replication and maintaining KSHV latency, respectively. Phosphorodiamidate morpholino oligomers (PMO) are similar to short single-stranded DNA oligomers, but possess a modified backbone that confers highly specific binding and resistance to nucleases. In this study, RTA and LANA mRNA in PEL cells were targeted by antisense peptide-conjugated PMO (P-PMO) in an effort to suppress KSHV replication. Highly efficient P-PMO uptake by PEL cells was observed. Treatment of PEL cells with a RTA P-PMO (RP1) reduced RTA expression in a dose-dependent and sequence-specific manner, and also caused a significant decrease in several KSHV early and late gene products, including vIL-6, vIRF-1, and ORF-K8.1A. KSHV viral DNA levels were reduced both in cells and culture supernatants of RP1 P-PMO-treated cells, indicating that KSHV lytic replication was suppressed. Treatment of BCBL-1 cells with P-PMO against LANA resulted in a reduction of LANA expression. Cell viability assays detected no cytotoxicity from P-PMO alone, within the concentration range used for the experiments in this study. These results suggest that RP1 P-PMO can specifically block KSHV replication, and further study is warranted.
Keywords: KSHV; RTA; LANA; Morpholino; Antiviral; Antisense
Hepatitis B virus is inhibited by RNA interference in cell culture and in mice by Ruo-Su Ying; Cai Zhu; Xue-Gong Fan; Ning Li; Xue-Fei Tian; Hong-Bo Liu; Bao-Xin Zhang (pp. 24-30).
For chronic hepatitis B virus (HBV) infection the effects of current therapies are limited. Recently, RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we studied the effects of HBV-specific 21-bp short hairpin RNAs (shRNAs) targeted to the surface antigen (HBsAg) region and the core antigen (HBcAg) region both in a cell culture system and in a mouse model for HBV replication.HBsAg and hepatitis B e antigen (HBeAg) in the media of the cells and in the sera of the mice were analyzed by time-resolved immunofluorometric assay, intracellular HBcAg by immunofluorescence assay, HBsAg and HBcAg in the livers of the mice by immunohistochemical assay, HBV DNA by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and HBV mRNA by semi-quantitative reverse transcriptase PCR (RT-PCR).Transfection with the shRNAs induced an RNAi response. Secreted HBsAg was reduced by >80% in cell culture and >90% in mouse serum, and HBeAg was also significantly inhibited. Immunofluorescence detection of intracellular HBcAg revealed 76% reduction. In the liver tissues by immunohistochemical detection, there were no HBsAg-positive cells and >70% reduction of HBcAg-positive cells for shRNA-1. And for shRNA-2 the detection of HBsAg and HBcAg also revealed substantial reduction. The shRNAs caused a significant inhibition in the levels of viral mRNA relative to the controls. HBV DNA was reduced by >40% for shRNA-1 and >60% for shRNA-2.RNAi is capable of inhibiting HBV replication and expression in vitro and in vivo and thus may constitute a new therapeutic strategy for HBV infection.
Keywords: HBV; RNAi; Hydrodynamics; HepG2.2.15; Animal model
Hepatitis B virus is inhibited by RNA interference in cell culture and in mice by Ruo-Su Ying; Cai Zhu; Xue-Gong Fan; Ning Li; Xue-Fei Tian; Hong-Bo Liu; Bao-Xin Zhang (pp. 24-30).
For chronic hepatitis B virus (HBV) infection the effects of current therapies are limited. Recently, RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we studied the effects of HBV-specific 21-bp short hairpin RNAs (shRNAs) targeted to the surface antigen (HBsAg) region and the core antigen (HBcAg) region both in a cell culture system and in a mouse model for HBV replication.HBsAg and hepatitis B e antigen (HBeAg) in the media of the cells and in the sera of the mice were analyzed by time-resolved immunofluorometric assay, intracellular HBcAg by immunofluorescence assay, HBsAg and HBcAg in the livers of the mice by immunohistochemical assay, HBV DNA by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and HBV mRNA by semi-quantitative reverse transcriptase PCR (RT-PCR).Transfection with the shRNAs induced an RNAi response. Secreted HBsAg was reduced by >80% in cell culture and >90% in mouse serum, and HBeAg was also significantly inhibited. Immunofluorescence detection of intracellular HBcAg revealed 76% reduction. In the liver tissues by immunohistochemical detection, there were no HBsAg-positive cells and >70% reduction of HBcAg-positive cells for shRNA-1. And for shRNA-2 the detection of HBsAg and HBcAg also revealed substantial reduction. The shRNAs caused a significant inhibition in the levels of viral mRNA relative to the controls. HBV DNA was reduced by >40% for shRNA-1 and >60% for shRNA-2.RNAi is capable of inhibiting HBV replication and expression in vitro and in vivo and thus may constitute a new therapeutic strategy for HBV infection.
Keywords: HBV; RNAi; Hydrodynamics; HepG2.2.15; Animal model
Inactivation of vesicular stomatitis virus through inhibition of membrane fusion by chemical modification of the viral glycoprotein by Fausto Stauffer; Joari De Miranda; Marcos C. Schechter; Fabiana A. Carneiro; Leonardo T. Salgado; Gisele F. Machado; Andrea T. Da Poian (pp. 31-39).
Membrane fusion is an essential step in the entry of enveloped viruses into their host cells triggered by conformational changes in viral glycoproteins. We have demonstrated previously that modification of vesicular stomatitis virus (VSV) with diethylpyrocarbonate (DEPC) abolished conformational changes on VSV glycoprotein and the fusion reaction catalyzed by the virus. In the present study, we evaluated whether treatment with DEPC was able to inactivate the virus. Infectivity and viral replication were abolished by viral treatment with 0.5mM DEPC. Mortality profile and inflammatory response in the central nervous system indicated that G protein modification with DEPC eliminates the ability of the virus to cause disease. In addition, DEPC treatment did not alter the conformational integrity of surface proteins of inactivated VSV as demonstrated by transmission electron microscopy and competitive ELISA. Taken together, our results suggest a potential use of histidine (His) modification to the development of a new process of viral inactivation based on fusion inhibition.
Keywords: Viral inactivation; Vesicular stomatitis virus; Membrane fusion; Diethylpyrocarbonate
Inactivation of vesicular stomatitis virus through inhibition of membrane fusion by chemical modification of the viral glycoprotein by Fausto Stauffer; Joari De Miranda; Marcos C. Schechter; Fabiana A. Carneiro; Leonardo T. Salgado; Gisele F. Machado; Andrea T. Da Poian (pp. 31-39).
Membrane fusion is an essential step in the entry of enveloped viruses into their host cells triggered by conformational changes in viral glycoproteins. We have demonstrated previously that modification of vesicular stomatitis virus (VSV) with diethylpyrocarbonate (DEPC) abolished conformational changes on VSV glycoprotein and the fusion reaction catalyzed by the virus. In the present study, we evaluated whether treatment with DEPC was able to inactivate the virus. Infectivity and viral replication were abolished by viral treatment with 0.5mM DEPC. Mortality profile and inflammatory response in the central nervous system indicated that G protein modification with DEPC eliminates the ability of the virus to cause disease. In addition, DEPC treatment did not alter the conformational integrity of surface proteins of inactivated VSV as demonstrated by transmission electron microscopy and competitive ELISA. Taken together, our results suggest a potential use of histidine (His) modification to the development of a new process of viral inactivation based on fusion inhibition.
Keywords: Viral inactivation; Vesicular stomatitis virus; Membrane fusion; Diethylpyrocarbonate
Replication efficiency of chimeric replicon containing NS5A–5B genes derived from HCV-infected patient sera by Rakesh L. Tripathi; Preethi Krishnan; Yupeng He; Tim Middleton; Tami Pilot-Matias; Chih-Ming Chen; Daryl T.Y. Lau; Stanley M. Lemon; Hongmei Mo; Warren Kati; Akhteruzzaman Molla (pp. 40-49).
A transient subgenomic replicon-based shuttle vector system has been developed to investigate how genetic heterogeneity affects HCV replication efficiency. Individual NS5A or NS5B genes or cassettes containing both NS5A and NS5B genes were amplified from “quasispecies” pools derived from HCV genotype 1a or 1b patient sera using RT-PCR and cloned into their respective shuttle vectors. All shuttle vectors containing NS5A or NS5A–5B genes were constructed with the S2204I “adaptive” mutation because replicons lacking the S2204I mutation replicated poorly. Gene sequences of the quasispecies pools within either genotype 1a or 1b patient samples ranged from 94 to 95% in identity. The replication capacity of 1b shuttle vectors containing patient-derived NS5A or NS5B genes averaged 67 and 75%, respectively, relative to the laboratory-optimized 1b replicon. In contrast, the replication efficiencies of both 1a and 1b shuttle vectors containing patient-derived NS5A–5B gene cassettes averaged around 2% relative to the respective laboratory-optimized replicon. All patient-derived replicons were tested in a transient assay for their sensitivity to either interferon-α (IFN-α) or to the polymerase inhibitor A-782759. Despite the differences in replication efficiency, IC50 values measured for most of the patient-derived replicons were equivalent to the respective values measured in the control laboratory strain replicons. These results demonstrate that patient sequence heterogeneity affects replication efficiency whenever patient-derived NS5A–5B genes are inserted into the laboratory-optimized replicon. The findings also demonstrate the utility of the shuttle vector system to test patient-derived gene sequences for sensitivity to IFN-α and to small molecule inhibitors.
Keywords: HCV; NS5A; NS5B; Sub-genomic replicon; Quasispecies pools; Transient assay
Replication efficiency of chimeric replicon containing NS5A–5B genes derived from HCV-infected patient sera by Rakesh L. Tripathi; Preethi Krishnan; Yupeng He; Tim Middleton; Tami Pilot-Matias; Chih-Ming Chen; Daryl T.Y. Lau; Stanley M. Lemon; Hongmei Mo; Warren Kati; Akhteruzzaman Molla (pp. 40-49).
A transient subgenomic replicon-based shuttle vector system has been developed to investigate how genetic heterogeneity affects HCV replication efficiency. Individual NS5A or NS5B genes or cassettes containing both NS5A and NS5B genes were amplified from “quasispecies” pools derived from HCV genotype 1a or 1b patient sera using RT-PCR and cloned into their respective shuttle vectors. All shuttle vectors containing NS5A or NS5A–5B genes were constructed with the S2204I “adaptive” mutation because replicons lacking the S2204I mutation replicated poorly. Gene sequences of the quasispecies pools within either genotype 1a or 1b patient samples ranged from 94 to 95% in identity. The replication capacity of 1b shuttle vectors containing patient-derived NS5A or NS5B genes averaged 67 and 75%, respectively, relative to the laboratory-optimized 1b replicon. In contrast, the replication efficiencies of both 1a and 1b shuttle vectors containing patient-derived NS5A–5B gene cassettes averaged around 2% relative to the respective laboratory-optimized replicon. All patient-derived replicons were tested in a transient assay for their sensitivity to either interferon-α (IFN-α) or to the polymerase inhibitor A-782759. Despite the differences in replication efficiency, IC50 values measured for most of the patient-derived replicons were equivalent to the respective values measured in the control laboratory strain replicons. These results demonstrate that patient sequence heterogeneity affects replication efficiency whenever patient-derived NS5A–5B genes are inserted into the laboratory-optimized replicon. The findings also demonstrate the utility of the shuttle vector system to test patient-derived gene sequences for sensitivity to IFN-α and to small molecule inhibitors.
Keywords: HCV; NS5A; NS5B; Sub-genomic replicon; Quasispecies pools; Transient assay
A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals by James W. Noah; William Severson; Diana L. Noah; Lynn Rasmussen; E. Lucile White; Colleen B. Jonsson (pp. 50-59).
The spread of highly pathogenic avian influenza across geographical and species barriers underscores the increasing need for novel antivirals to compliment vaccination and existing antiviral therapies. Identification of new antiviral lead compounds depends on robust primary assays for high-throughput screening (HTS) of large compound libraries. We have developed a cell-based screen for potential influenza antivirals that measures the cytopathic effect (CPE) induced by influenza virus (A/Udorn/72, H3N2) infection in Madin Darby canine kidney (MDCK) cells using the luminescent-based CellTiter Glo system. This 72h assay is validated for HTS in 384-well plates and performs more consistently and reliably than methods using neutral red, with Z values>0.8, signal-to-background>30 and signal-to-noise>10. In a blinded pilot screen ( n=10,781) at 10μM concentration, four compounds (with previously demonstrated efficacy against influenza) inhibited viral-induced CPE by >50%, with EC50/CC50 values comparable to those determined by other cell-based assays, thereby validating this assay accuracy and ability to simultaneously evaluate compound cellular availability and/or toxicity. This assay is translatable for screening against other influenza strains, such as avian flu, and may facilitate identification of antivirals for other viruses that induce CPE, such as West Nile or Dengue.
Keywords: Influenza; Antiviral; HTS; High-throughput screening; Cytopathic effect; CellTiter Glo
A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals by James W. Noah; William Severson; Diana L. Noah; Lynn Rasmussen; E. Lucile White; Colleen B. Jonsson (pp. 50-59).
The spread of highly pathogenic avian influenza across geographical and species barriers underscores the increasing need for novel antivirals to compliment vaccination and existing antiviral therapies. Identification of new antiviral lead compounds depends on robust primary assays for high-throughput screening (HTS) of large compound libraries. We have developed a cell-based screen for potential influenza antivirals that measures the cytopathic effect (CPE) induced by influenza virus (A/Udorn/72, H3N2) infection in Madin Darby canine kidney (MDCK) cells using the luminescent-based CellTiter Glo system. This 72h assay is validated for HTS in 384-well plates and performs more consistently and reliably than methods using neutral red, with Z values>0.8, signal-to-background>30 and signal-to-noise>10. In a blinded pilot screen ( n=10,781) at 10μM concentration, four compounds (with previously demonstrated efficacy against influenza) inhibited viral-induced CPE by >50%, with EC50/CC50 values comparable to those determined by other cell-based assays, thereby validating this assay accuracy and ability to simultaneously evaluate compound cellular availability and/or toxicity. This assay is translatable for screening against other influenza strains, such as avian flu, and may facilitate identification of antivirals for other viruses that induce CPE, such as West Nile or Dengue.
Keywords: Influenza; Antiviral; HTS; High-throughput screening; Cytopathic effect; CellTiter Glo
Parameters of inhibition of HIV-1 infection by small anionic microbicides by Andrei N. Vzorov; Jadranka Bozja; Dabney W. Dixon; Luigi G. Marzilli; Richard W. Compans (pp. 60-68).
Sulfonated porphyrins and phthalocyanines have been shown to have anti-HIV activity and are under consideration as microbicides. Both categories of compounds are small negatively charged molecules and both were previously shown to inhibit cell fusion induced by the HIV Env protein and to block binding of gp120 to the CD4 receptor. In the present study we show that these compounds inhibit transmission of cell-associated HIV, inactivate a broad range of HIV-1 primary isolates and are active against DS polyanion-resistant virus. The compounds tested are active over a range of pH values, and possess no detectable activity against normal bacterial flora. These results support the conclusion that anionic tetrapyrroles are promising candidates as microbicides for HIV prevention.
Keywords: HIV; Topical microbicide; Normal vaginal flora
Parameters of inhibition of HIV-1 infection by small anionic microbicides by Andrei N. Vzorov; Jadranka Bozja; Dabney W. Dixon; Luigi G. Marzilli; Richard W. Compans (pp. 60-68).
Sulfonated porphyrins and phthalocyanines have been shown to have anti-HIV activity and are under consideration as microbicides. Both categories of compounds are small negatively charged molecules and both were previously shown to inhibit cell fusion induced by the HIV Env protein and to block binding of gp120 to the CD4 receptor. In the present study we show that these compounds inhibit transmission of cell-associated HIV, inactivate a broad range of HIV-1 primary isolates and are active against DS polyanion-resistant virus. The compounds tested are active over a range of pH values, and possess no detectable activity against normal bacterial flora. These results support the conclusion that anionic tetrapyrroles are promising candidates as microbicides for HIV prevention.
Keywords: HIV; Topical microbicide; Normal vaginal flora
Cell line dependency for antiviral activity and in vivo efficacy of N-methanocarbathymidine against orthopoxvirus infections in mice by Donald F. Smee; Miles K. Wandersee; Kevin W. Bailey; Min-Hui Wong; Chung K. Chu; Srinivas Gadthula; Robert W. Sidwell (pp. 69-77).
A novel carbocyclic thymidine analog, N-methanocarbathymidine [( N)-MCT], was evaluated for inhibition of orthopoxvirus infections. Efficacy in vitro was assessed by plaque reduction assays against wild-type and cidofovir-resistant strains of cowpox and vaccinia viruses in nine different cell lines. Minimal differences were seen in antiviral activity against wild-type and cidofovir-resistant viruses. ( N)-MCT's efficacy was affected by the cell line used for assay, with 50% poxvirus-inhibitory concentrations in cells as follows: mouse=0.6–2.2μM, rabbit=52–90μM, monkey=87 to >1000μM, and human=39–220μM. Limited studies performed with carbocyclic thymidine indicated a similar cell line dependency for antiviral activity. ( N)-MCT did not inhibit actively dividing uninfected cells at 1000μM. The potency of ( N)-MCT against an S-variant thymidine kinase-deficient vaccinia virus was similar to that seen against S-variant and wild-type viruses in mouse, monkey, and human cells, implicating a cellular enzyme in the phosphorylation of the compound. Mice were intranasally infected with cowpox and vaccinia viruses followed 24h later by intraperitoneal treatment with ( N)-MCT (twice a day for 7 days) or cidofovir (once a day for 2 days). ( N)-MCT treatment at 100 and 30mg/kg/day resulted in 90 and 20% survival from cowpox virus infection, respectively, compared to 0% survival in the placebo group. Statistically significant reductions in lung virus titers on day 5 occurred in 10, 30, and 100mg/kg/day treated mice. These same doses were also active against a lethal vaccinia virus (WR strain) challenge, and protection was seen down to 10mg/kg/day against a lethal vaccinia virus (IHD strain) infection. Cidofovir (100mg/kg/day) protected animals from death in all three infections.
Keywords: Vaccinia virus; Cowpox virus; (; N; )-MCT; Carbocyclic thymidine; Cidofovir; Antiviral; Thymidine kinase
Cell line dependency for antiviral activity and in vivo efficacy of N-methanocarbathymidine against orthopoxvirus infections in mice by Donald F. Smee; Miles K. Wandersee; Kevin W. Bailey; Min-Hui Wong; Chung K. Chu; Srinivas Gadthula; Robert W. Sidwell (pp. 69-77).
A novel carbocyclic thymidine analog, N-methanocarbathymidine [( N)-MCT], was evaluated for inhibition of orthopoxvirus infections. Efficacy in vitro was assessed by plaque reduction assays against wild-type and cidofovir-resistant strains of cowpox and vaccinia viruses in nine different cell lines. Minimal differences were seen in antiviral activity against wild-type and cidofovir-resistant viruses. ( N)-MCT's efficacy was affected by the cell line used for assay, with 50% poxvirus-inhibitory concentrations in cells as follows: mouse=0.6–2.2μM, rabbit=52–90μM, monkey=87 to >1000μM, and human=39–220μM. Limited studies performed with carbocyclic thymidine indicated a similar cell line dependency for antiviral activity. ( N)-MCT did not inhibit actively dividing uninfected cells at 1000μM. The potency of ( N)-MCT against an S-variant thymidine kinase-deficient vaccinia virus was similar to that seen against S-variant and wild-type viruses in mouse, monkey, and human cells, implicating a cellular enzyme in the phosphorylation of the compound. Mice were intranasally infected with cowpox and vaccinia viruses followed 24h later by intraperitoneal treatment with ( N)-MCT (twice a day for 7 days) or cidofovir (once a day for 2 days). ( N)-MCT treatment at 100 and 30mg/kg/day resulted in 90 and 20% survival from cowpox virus infection, respectively, compared to 0% survival in the placebo group. Statistically significant reductions in lung virus titers on day 5 occurred in 10, 30, and 100mg/kg/day treated mice. These same doses were also active against a lethal vaccinia virus (WR strain) challenge, and protection was seen down to 10mg/kg/day against a lethal vaccinia virus (IHD strain) infection. Cidofovir (100mg/kg/day) protected animals from death in all three infections.
Keywords: Vaccinia virus; Cowpox virus; (; N; )-MCT; Carbocyclic thymidine; Cidofovir; Antiviral; Thymidine kinase
Antiviral interactions of an HCV polymerase inhibitor with an HCV protease inhibitor or interferon in vitro by Gennadiy Koev; Tatyana Dekhtyar; Lixin Han; Ping Yan; Teresa I. Ng; C. Thomas Lin; Hongmei Mo; Akhteruzzaman Molla (pp. 78-83).
The combinations of Abbott Hepatitis C virus (HCV) polymerase A-782759 with either Boehringer Ingelheim HCV NS3 protease inhibitor BILN-2061 or interferon (IFN) displayed additive to synergistic relationships over a range of concentrations of two-drug combination. Treatment of HCV replicon with A-782759, IFN or BILN-2061 for about 16 days resulted in dramatic reductions in HCV RNA (5.1, 3.0 and 3.9log10 RNA copies, respectively). However, none of the compounds tested alone lead to replicon RNA reduction to undetectable levels. Ongoing replication in the presence of A-782759 or BILN-2061 was associated with the appearance of resistant mutations M414T in NS5B and D168V in NS3, respectively. In contrast, a combination of A-782759 with BILN-2061 resulted in greater than 7logs RNA reduction leading to undetectable replicon RNA after 16 days of treatment. Our findings suggest that a monotherapy with either drug alone is likely to result in development of resistant mutants. However, a combination therapy with polymerase inhibitor has the potential to improve the efficacy of IFN or a protease inhibitor alone in vivo, due to the lower likelihood of resistance development.
Keywords: HCV replicon; Polymerase inhibitor; Protease inhibitor; Drug combination; Synergy
Antiviral interactions of an HCV polymerase inhibitor with an HCV protease inhibitor or interferon in vitro by Gennadiy Koev; Tatyana Dekhtyar; Lixin Han; Ping Yan; Teresa I. Ng; C. Thomas Lin; Hongmei Mo; Akhteruzzaman Molla (pp. 78-83).
The combinations of Abbott Hepatitis C virus (HCV) polymerase A-782759 with either Boehringer Ingelheim HCV NS3 protease inhibitor BILN-2061 or interferon (IFN) displayed additive to synergistic relationships over a range of concentrations of two-drug combination. Treatment of HCV replicon with A-782759, IFN or BILN-2061 for about 16 days resulted in dramatic reductions in HCV RNA (5.1, 3.0 and 3.9log10 RNA copies, respectively). However, none of the compounds tested alone lead to replicon RNA reduction to undetectable levels. Ongoing replication in the presence of A-782759 or BILN-2061 was associated with the appearance of resistant mutations M414T in NS5B and D168V in NS3, respectively. In contrast, a combination of A-782759 with BILN-2061 resulted in greater than 7logs RNA reduction leading to undetectable replicon RNA after 16 days of treatment. Our findings suggest that a monotherapy with either drug alone is likely to result in development of resistant mutants. However, a combination therapy with polymerase inhibitor has the potential to improve the efficacy of IFN or a protease inhibitor alone in vivo, due to the lower likelihood of resistance development.
Keywords: HCV replicon; Polymerase inhibitor; Protease inhibitor; Drug combination; Synergy