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Antiviral Research (v.93, #1)
Do viral infections mimic bacterial sepsis? The role of microvascular permeability: A review of mechanisms and methods by B.E. Steinberg; N.M. Goldenberg; W.L. Lee (pp. 2-15).
► Control mechanisms of endothelial permeability. ► Changes in permeability in bacterial sepsis. ► Methods for endothelial barrier investigation. ► Case studies: hantaviruses, human herpesvirus 8, dengue virus.A dysregulated immune response and functional immunosuppression have been considered the major mechanisms of the bacterial sepsis syndrome. More recently, the loss of endothelial barrier function and resultant microvascular leak have been found to be a key determinant of the pathogenesis of bacterial sepsis. Whether a similar paradigm applies to systemic viral syndromes is not known. Answering this question has far-reaching implications for the development of future anti-viral therapeutic strategies. In this review, we provide an overview of the structure and function of the endothelium and how its barrier integrity is compromised in bacterial sepsis. The various in vitro and in vivo methodologies available to investigate vascular leak are reviewed. Emphasis is placed on the advantages and limitations of cell culture techniques, which represent the most commonly used methods. Within this context, we appraise recent studies of three viruses – hantavirus, human herpes virus 8 and dengue virus – that suggest microvascular leak may play a role in the pathogenesis of these viral infections. We conclude with a discussion of how endothelial barrier breakdown may occur in other viral infections such as H5N1 avian influenza virus.
Keywords: Endothelial barrier; Sepsis; Hemorrhagic fever; Dengue virus; Human herpes virus 8; H5N1 avian influenza
Do viral infections mimic bacterial sepsis? The role of microvascular permeability: A review of mechanisms and methods by B.E. Steinberg; N.M. Goldenberg; W.L. Lee (pp. 2-15).
► Control mechanisms of endothelial permeability. ► Changes in permeability in bacterial sepsis. ► Methods for endothelial barrier investigation. ► Case studies: hantaviruses, human herpesvirus 8, dengue virus.A dysregulated immune response and functional immunosuppression have been considered the major mechanisms of the bacterial sepsis syndrome. More recently, the loss of endothelial barrier function and resultant microvascular leak have been found to be a key determinant of the pathogenesis of bacterial sepsis. Whether a similar paradigm applies to systemic viral syndromes is not known. Answering this question has far-reaching implications for the development of future anti-viral therapeutic strategies. In this review, we provide an overview of the structure and function of the endothelium and how its barrier integrity is compromised in bacterial sepsis. The various in vitro and in vivo methodologies available to investigate vascular leak are reviewed. Emphasis is placed on the advantages and limitations of cell culture techniques, which represent the most commonly used methods. Within this context, we appraise recent studies of three viruses – hantavirus, human herpes virus 8 and dengue virus – that suggest microvascular leak may play a role in the pathogenesis of these viral infections. We conclude with a discussion of how endothelial barrier breakdown may occur in other viral infections such as H5N1 avian influenza virus.
Keywords: Endothelial barrier; Sepsis; Hemorrhagic fever; Dengue virus; Human herpes virus 8; H5N1 avian influenza
Inhibition of Junin virus RNA synthesis by an antiviral acridone derivative by Claudia S. Sepúlveda; Cybele C. García; Mirta L. Fascio; Norma B. D’Accorso; Maite L. Docampo Palacios; Rolando F. Pellón; Elsa B. Damonte (pp. 16-22).
► The acridone3f is a potent and selective inhibitor of in vitro Junin virus infection. ► The antiviral activity of acridone was due to inhibition of viral RNA synthesis. ► Addition of exogenous guanosine produced partial reversion of the antiviral activity.There are no specific approved drugs for the treatment of agents of viral hemorrhagic fevers (HF) and antiviral therapies against these viruses are urgently needed. The present study characterizes the potent and selective antiviral activity against the HF causing arenavirus Junin virus (JUNV) of the compound 10-allyl-6-chloro-4-methoxy-9(10 H)-acridone, designated3f. The effectiveness of3f to inhibit JUNV multiplication was not importantly affected by the initial multiplicity of infection, with similar effective concentration 50% (EC50) values in virus yield inhibition assays performed in Vero cells in the range of 0.2–40 plaque forming units (PFU)/cell. Mechanistic studies demonstrated that3f did not affect the initial steps of adsorption and internalization. The subsequent process of viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR in compound-treated cells relative to non-treated cells. The addition of exogenous guanosine rescued the infectivity and RNA synthesis of JUNV in3f-treated cells in a dose-dependent manner, but the reversal was partial, suggesting that the reduction of the GTP pool contributed to the antiviral activity of3f, but it was not the main operative mechanism. The comparison of3f with two other viral RNA inhibitors, ribavirin and mycophenolic acid, showed that ribavirin did not act against JUNV through the cellular enzyme inosine monophosphate dehydrogenase (IMPDH) inhibition whereas the anti-JUNV activity of mycophenolic acid was mainly targeted at this enzyme.
Keywords: Junin virus; Hemorrhagic fever virus; Antiviral; Acridone; RNA synthesis; Inosine monophosphate dehydrogenase (IMPDH)
Inhibition of Junin virus RNA synthesis by an antiviral acridone derivative by Claudia S. Sepúlveda; Cybele C. García; Mirta L. Fascio; Norma B. D’Accorso; Maite L. Docampo Palacios; Rolando F. Pellón; Elsa B. Damonte (pp. 16-22).
► The acridone3f is a potent and selective inhibitor of in vitro Junin virus infection. ► The antiviral activity of acridone was due to inhibition of viral RNA synthesis. ► Addition of exogenous guanosine produced partial reversion of the antiviral activity.There are no specific approved drugs for the treatment of agents of viral hemorrhagic fevers (HF) and antiviral therapies against these viruses are urgently needed. The present study characterizes the potent and selective antiviral activity against the HF causing arenavirus Junin virus (JUNV) of the compound 10-allyl-6-chloro-4-methoxy-9(10 H)-acridone, designated3f. The effectiveness of3f to inhibit JUNV multiplication was not importantly affected by the initial multiplicity of infection, with similar effective concentration 50% (EC50) values in virus yield inhibition assays performed in Vero cells in the range of 0.2–40 plaque forming units (PFU)/cell. Mechanistic studies demonstrated that3f did not affect the initial steps of adsorption and internalization. The subsequent process of viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR in compound-treated cells relative to non-treated cells. The addition of exogenous guanosine rescued the infectivity and RNA synthesis of JUNV in3f-treated cells in a dose-dependent manner, but the reversal was partial, suggesting that the reduction of the GTP pool contributed to the antiviral activity of3f, but it was not the main operative mechanism. The comparison of3f with two other viral RNA inhibitors, ribavirin and mycophenolic acid, showed that ribavirin did not act against JUNV through the cellular enzyme inosine monophosphate dehydrogenase (IMPDH) inhibition whereas the anti-JUNV activity of mycophenolic acid was mainly targeted at this enzyme.
Keywords: Junin virus; Hemorrhagic fever virus; Antiviral; Acridone; RNA synthesis; Inosine monophosphate dehydrogenase (IMPDH)
Identification of an antioxidant small-molecule with broad-spectrum antiviral activity by Rekha G. Panchal; St. Patrick Reid; Julie P. Tran; Alison A. Bergeron; Jay Wells; Krishna P. Kota; Javad Aman; Sina Bavari (pp. 23-29).
► Identified compound with broad-spectrum antiviral activity. ► Identified compound has anti-oxidant properties. ► Inhibition of oxidative stress for developing host-oriented antiviral therapeutics.The highly lethal filoviruses, Ebola and Marburg cause severe hemorrhagic fever in humans and non-human primates. To date there are no licensed vaccines or therapeutics to counter these infections. Identifying novel pathways and host targets that play an essential role during infection will provide potential targets to develop therapeutics. Small molecule chemical screening for Ebola virus inhibitors resulted in identification of a compound NSC 62914. The compound was found to exhibit anti-filovirus activity in cell-based assays and in vivo protected mice following challenge with Ebola or Marburg viruses. Additionally, the compound was found to inhibit Rift Valley fever virus, Lassa virus and Venezuelan equine encephalitis virus in cell-based assays. Investigation of the mechanism of action of the compound revealed that it had antioxidant properties. Specifically, compound NSC 62914 was found to act as a scavenger of reactive oxygen species, and to up-regulate oxidative stress-induced genes. However, four known antioxidant compounds failed to inhibit filovirus infection, thus suggesting that the mechanistic basis of the antiviral function of the antioxidant NSC 62914 may involve modulation of multiple signaling pathways/targets.
Keywords: Abbreviations; EBOV; Ebola virus; MARV; Marburg virus; ROS; reactive oxygen species; RVFV; Rift Valley fever virusBroad-spectrum; Filoviruses; High-content; Antioxidant
Identification of an antioxidant small-molecule with broad-spectrum antiviral activity by Rekha G. Panchal; St. Patrick Reid; Julie P. Tran; Alison A. Bergeron; Jay Wells; Krishna P. Kota; Javad Aman; Sina Bavari (pp. 23-29).
► Identified compound with broad-spectrum antiviral activity. ► Identified compound has anti-oxidant properties. ► Inhibition of oxidative stress for developing host-oriented antiviral therapeutics.The highly lethal filoviruses, Ebola and Marburg cause severe hemorrhagic fever in humans and non-human primates. To date there are no licensed vaccines or therapeutics to counter these infections. Identifying novel pathways and host targets that play an essential role during infection will provide potential targets to develop therapeutics. Small molecule chemical screening for Ebola virus inhibitors resulted in identification of a compound NSC 62914. The compound was found to exhibit anti-filovirus activity in cell-based assays and in vivo protected mice following challenge with Ebola or Marburg viruses. Additionally, the compound was found to inhibit Rift Valley fever virus, Lassa virus and Venezuelan equine encephalitis virus in cell-based assays. Investigation of the mechanism of action of the compound revealed that it had antioxidant properties. Specifically, compound NSC 62914 was found to act as a scavenger of reactive oxygen species, and to up-regulate oxidative stress-induced genes. However, four known antioxidant compounds failed to inhibit filovirus infection, thus suggesting that the mechanistic basis of the antiviral function of the antioxidant NSC 62914 may involve modulation of multiple signaling pathways/targets.
Keywords: Abbreviations; EBOV; Ebola virus; MARV; Marburg virus; ROS; reactive oxygen species; RVFV; Rift Valley fever virusBroad-spectrum; Filoviruses; High-content; Antioxidant
Identification of a novel resistance mutation for benzimidazole inhibitors of the HCV RNA-dependent RNA polymerase by Leen Delang; Mathy Froeyen; Piet Herdewijn; Johan Neyts (pp. 30-38).
► Novel resistance mutation (T389S) observed for HCV benzimidazole inhibitors. ► T389S results in moderate resistance levels to benzimidazole inhibitors. ► A structural hypothesis for the emergence of this mutation is formulated. ► Substitutions to the benzimidazole scaffold can affect the resistance mutations.Non-nucleoside inhibitors of the RNA-dependent RNA polymerase of the hepatitis C virus that are based on a benzimidazole or indole scaffold have been reported to interact with thumb domain 1 of the enzyme. Escape mutants that confer in vitro resistance to these inhibitors map to amino acids P495, P496 or V499. We here report a novel resistance mutation (T389S/A) that was identified following resistance selection with the benzimidazole non-nucleoside polymerase inhibitor JT-16 in HCV Con-1 subgenomic replicon (genotype 1b). This JT-16 resistant replicon retained wild-type susceptibility to protease inhibitors and nucleoside polymerase inhibitors. Replicons that carry mutations T389A and T389S have moderate levels of resistance to JT-16 (7- and 13-fold, respectively). Mutation P495A is associated with high-level (44-fold) resistance. Surprisingly, this previously reported ‘key’ mutation for benzimidazole resistance, P495A, was detected in only 15% of the resistant population. Furthermore, the replication fitness of the T389S mutant was significantly higher than that of the P495A mutant. By means of molecular modeling a structural hypothesis was formulated to explain the emergence of the T389S/A mutation in the JT-16 resistant replicon. Our data demonstrate that low-level resistant, but fit, variants can develop during in vitro resistance selection with the benzimidazole inhibitor JT-16. Moreover, different substitutions to the benzimidazole scaffold can affect the (pattern of) resistance mutations that emerge during resistance selection.
Keywords: Hepatitis C virus; Non-nucleoside polymerase inhibitor; Benzimidazole; Resistance
Identification of a novel resistance mutation for benzimidazole inhibitors of the HCV RNA-dependent RNA polymerase by Leen Delang; Mathy Froeyen; Piet Herdewijn; Johan Neyts (pp. 30-38).
► Novel resistance mutation (T389S) observed for HCV benzimidazole inhibitors. ► T389S results in moderate resistance levels to benzimidazole inhibitors. ► A structural hypothesis for the emergence of this mutation is formulated. ► Substitutions to the benzimidazole scaffold can affect the resistance mutations.Non-nucleoside inhibitors of the RNA-dependent RNA polymerase of the hepatitis C virus that are based on a benzimidazole or indole scaffold have been reported to interact with thumb domain 1 of the enzyme. Escape mutants that confer in vitro resistance to these inhibitors map to amino acids P495, P496 or V499. We here report a novel resistance mutation (T389S/A) that was identified following resistance selection with the benzimidazole non-nucleoside polymerase inhibitor JT-16 in HCV Con-1 subgenomic replicon (genotype 1b). This JT-16 resistant replicon retained wild-type susceptibility to protease inhibitors and nucleoside polymerase inhibitors. Replicons that carry mutations T389A and T389S have moderate levels of resistance to JT-16 (7- and 13-fold, respectively). Mutation P495A is associated with high-level (44-fold) resistance. Surprisingly, this previously reported ‘key’ mutation for benzimidazole resistance, P495A, was detected in only 15% of the resistant population. Furthermore, the replication fitness of the T389S mutant was significantly higher than that of the P495A mutant. By means of molecular modeling a structural hypothesis was formulated to explain the emergence of the T389S/A mutation in the JT-16 resistant replicon. Our data demonstrate that low-level resistant, but fit, variants can develop during in vitro resistance selection with the benzimidazole inhibitor JT-16. Moreover, different substitutions to the benzimidazole scaffold can affect the (pattern of) resistance mutations that emerge during resistance selection.
Keywords: Hepatitis C virus; Non-nucleoside polymerase inhibitor; Benzimidazole; Resistance
CpG and poly(I:C) stimulation of dendritic cells and fibroblasts limits herpes simplex virus type 1 infection in an IFNβ-dependent and -independent way by Giel R. Gaajetaan; Tanja H. Geelen; Gert E. Grauls; Cathrien A. Bruggeman; Frank R. Stassen (pp. 39-47).
► CpG stimulation of pDCs results in a strong antiviral effect. ► The observed antiviral effects of TLR ligands are mainly IFNβ-dependent. ► No significant antiviral role for IFNα is observed after TLR stimulation of DCs. ► The strong antiviral effect of poly(I:C) is DC-independent.Viral activation of toll-like receptors (TLRs) on dendritic cells (DCs) leads to production of various cytokines, including antiviral type I interferons (IFNs). Synthetic ligands specific for TLRs are also able to induce the production of type I IFNs (IFNα/β) by DCs, suggesting that these ligands have potential as antiviral drugs. In this in vitro study we extensively investigated the antiviral activity of various TLR ligands. Mouse bone marrow (BM) cells were differentiated into plasmacytoid and conventional DCs (pDCs and cDCs), stimulated with various TLR ligands and tested the antiviral abilities of collected supernatants in an in vitro herpes simplex virus type 1 (HSV-1) infection model. We observed a significant IFNβ-, (but not IFNα-) dependent reduction in HSV-1 infection when a mixed pDC/cDC population was stimulated with the TLR9 ligand CpG. In the absence of pDCs, TLR stimulation resulted in less pronounced antiviral effects. The most pronounced antiviral effect was observed when both DC subsets were stimulated with poly(I:C). A similar noticeable antiviral effect was observed when fibroblasts (L929 cells) were stimulated directly with poly(I:C). These poly(I:C)-mediated antiviral effects were only partially IFNβ-mediated and probably TLR independent. These data demonstrate that TLR ligands are not only able to produce type I IFN but can indeed act as antiviral drugs. In particular poly(I:C), which exerts its antiviral effects even in the absence of DCs, may become a promising drug e.g. to prevent respiratory infections by topical intranasal application.
Keywords: Abbreviations; BM; bone marrow; BM-DC; BM-derived DC; cDC; conventional dendritic cell; CPE; cytopathogenic effect; DC; dendritic cell; FL; Flt-3L; GM; GM-CSF; HSV; herpes simplex virus; IFN; interferon; LPS; lipopolysaccharide; MCMV; mouse cytomegalovirus; MDA5; melanoma-differentiation-associated gene 5; ODN; oligodeoxynucleotides; pDC; plasmacytoid dendritic cell; PRR; pattern recognition receptor; TLR; toll-like receptor; UNG; uracil-N-glycosylaseToll-like receptor; Interferon; Dendritic cell; HSV-1
CpG and poly(I:C) stimulation of dendritic cells and fibroblasts limits herpes simplex virus type 1 infection in an IFNβ-dependent and -independent way by Giel R. Gaajetaan; Tanja H. Geelen; Gert E. Grauls; Cathrien A. Bruggeman; Frank R. Stassen (pp. 39-47).
► CpG stimulation of pDCs results in a strong antiviral effect. ► The observed antiviral effects of TLR ligands are mainly IFNβ-dependent. ► No significant antiviral role for IFNα is observed after TLR stimulation of DCs. ► The strong antiviral effect of poly(I:C) is DC-independent.Viral activation of toll-like receptors (TLRs) on dendritic cells (DCs) leads to production of various cytokines, including antiviral type I interferons (IFNs). Synthetic ligands specific for TLRs are also able to induce the production of type I IFNs (IFNα/β) by DCs, suggesting that these ligands have potential as antiviral drugs. In this in vitro study we extensively investigated the antiviral activity of various TLR ligands. Mouse bone marrow (BM) cells were differentiated into plasmacytoid and conventional DCs (pDCs and cDCs), stimulated with various TLR ligands and tested the antiviral abilities of collected supernatants in an in vitro herpes simplex virus type 1 (HSV-1) infection model. We observed a significant IFNβ-, (but not IFNα-) dependent reduction in HSV-1 infection when a mixed pDC/cDC population was stimulated with the TLR9 ligand CpG. In the absence of pDCs, TLR stimulation resulted in less pronounced antiviral effects. The most pronounced antiviral effect was observed when both DC subsets were stimulated with poly(I:C). A similar noticeable antiviral effect was observed when fibroblasts (L929 cells) were stimulated directly with poly(I:C). These poly(I:C)-mediated antiviral effects were only partially IFNβ-mediated and probably TLR independent. These data demonstrate that TLR ligands are not only able to produce type I IFN but can indeed act as antiviral drugs. In particular poly(I:C), which exerts its antiviral effects even in the absence of DCs, may become a promising drug e.g. to prevent respiratory infections by topical intranasal application.
Keywords: Abbreviations; BM; bone marrow; BM-DC; BM-derived DC; cDC; conventional dendritic cell; CPE; cytopathogenic effect; DC; dendritic cell; FL; Flt-3L; GM; GM-CSF; HSV; herpes simplex virus; IFN; interferon; LPS; lipopolysaccharide; MCMV; mouse cytomegalovirus; MDA5; melanoma-differentiation-associated gene 5; ODN; oligodeoxynucleotides; pDC; plasmacytoid dendritic cell; PRR; pattern recognition receptor; TLR; toll-like receptor; UNG; uracil-N-glycosylaseToll-like receptor; Interferon; Dendritic cell; HSV-1
Identification of novel virus inhibitors by influenza A virus specific reporter cell based screening by Junjie Zhang; Ting Liu; Xiaomei Tong; Gang Li; Jinghua Yan; Xin Ye (pp. 48-54).
► We generated an influenza virus reporter cell line to screen a small compound library. ► Ten compounds were identified to have inhibitory activity against influenza A virus. ► Four of them blocked the activity of vRNP. ► The compound NSC 335506 inhibited HA-mediated membrane fusion. ► The reporter cell is a very useful tool to screen inhibitors against influenza A virus.As influenza viruses have developed resistance towards current drugs, it is urgent to find potential novel antiviral inhibitors. Here we generated an influenza virus reporter cell line in which the luciferase gene was driven by the influenza virus promoter and screened a small compound library (NCI Diversity Set II). Ten compounds were identified to have inhibitory activity against influenza A virus H1N1. Among them, four compounds blocked influenza virus replication through inhibiting the activity of vRNP. The compound NSC 335506 inhibited HA-mediated membrane fusion. It showed the inhibitory activity against H1N1, H9N2 and H5N1 subtype but not H3N2. Our results demonstrated that influenza virus reporter cell is a very useful tool to identify novel inhibitors against influenza A virus.
Keywords: Influenza A virus; Cell line based screening; Antiviral inhibitors; HA; vRNP
Identification of novel virus inhibitors by influenza A virus specific reporter cell based screening by Junjie Zhang; Ting Liu; Xiaomei Tong; Gang Li; Jinghua Yan; Xin Ye (pp. 48-54).
► We generated an influenza virus reporter cell line to screen a small compound library. ► Ten compounds were identified to have inhibitory activity against influenza A virus. ► Four of them blocked the activity of vRNP. ► The compound NSC 335506 inhibited HA-mediated membrane fusion. ► The reporter cell is a very useful tool to screen inhibitors against influenza A virus.As influenza viruses have developed resistance towards current drugs, it is urgent to find potential novel antiviral inhibitors. Here we generated an influenza virus reporter cell line in which the luciferase gene was driven by the influenza virus promoter and screened a small compound library (NCI Diversity Set II). Ten compounds were identified to have inhibitory activity against influenza A virus H1N1. Among them, four compounds blocked influenza virus replication through inhibiting the activity of vRNP. The compound NSC 335506 inhibited HA-mediated membrane fusion. It showed the inhibitory activity against H1N1, H9N2 and H5N1 subtype but not H3N2. Our results demonstrated that influenza virus reporter cell is a very useful tool to identify novel inhibitors against influenza A virus.
Keywords: Influenza A virus; Cell line based screening; Antiviral inhibitors; HA; vRNP
Precore/core promoter mutations and hepatitis B virus genotype in hepatitis B and C dually infected patients treated with interferon-based therapy by Chao-Hung Hung; Chien-Hung Chen; Sheng-Nan Lu; Jing-Houng Wang; Tsung-Hui Hu; Chao-Min Huang; Ming-Chao Tsai; Chuan-Mo Lee (pp. 55-63).
► Prevalence and implications of HBV mutation/genotype in dual BC infection is unknown. ► Dual infection has a higher prevalence of genotype C and less G1896A mutation. ► Genotype C is associated with more A1762T/G1764A mutation, but less G1896A mutation. ► Specific HBV mutations could predict long-term HBV response after IFN therapy.We studied the prevalence and distribution of precore/basal core promoter (BCP) mutations and hepatitis B virus (HBV) genotypes in HBV/hepatitis C virus (HCV) dually-infected patients, and evaluated their impact on long-term HBV response of interferon (IFN)-based therapy. The HBV genotypes and sequences of the precore/BCP regions were determined in 180 HBV/HCV dually-infected patients and were compared with 90 age, sex and hepatitis B e antigen-matched chronic hepatitis B controls. Serum HBV DNA and hepatitis B surface antigen (HBsAg) were assessed every 3–6months after therapy with IFN or pegylated-IFN plus ribavirin in 135 dually-infected patients with active hepatitis C. Dually-infected patients had a higher prevalence of genotype C HBV ( P=0.022) and a lower frequency of G1896A mutation ( P=0.004) as compared with controls. Among dually-infected patients, genotype C was associated with a higher frequency of A1762T/G1764A mutation ( P<0.001), but with lower HBV DNA ( P<0.001) and a lower frequency of A1752T/G ( P=0.008), C1799G ( P<0.001) and G1896A mutation (P <0.001) than genotype B. Based on Cox proportional hazards model, young age (hazard ratio (HR)=0.952, P=0.001), sustained virological response to HCV (HR=4.638, P=0.044), C1766T mutation (HR=5.216, P=0.003) and A1846T mutation (HR=2.332, P=0.031) correlated with HBV DNA reactivation (⩾2000IU/ml) after therapy. Age (HR=1.068, P=0.020), G1896A mutation (HR=0.140, P=0.01) and A1846T mutation (HR=0.086, P=0.018) were associated with HBsAg seroclearance independently. In conclusion, specific mutations in the precore/BCP regions could be useful in predicting long-term HBV response in HBV/HCV dually-infected patients treated with IFN-based therapy.
Keywords: Dual hepatitis B and C; Interferon; Genotype; Precore/core promoter mutation; HBsAg seroclearanceAbbreviations; AST; aspartate aminotransferase; ALT; alanine aminotransferase; BCP; basal core promoter; CI; confidence interval; HR; hazard ratio; HBeAg; hepatitis B e antigen; HBsAg; hepatitis B surface antigen; HBV; hepatitis B virus; HCV; hepatitis C virus; HCC; hepatocellular carcinoma; IFN; interferon; IU; international unit; PCR; polymerase chain reaction; SVR; sustained virological response
Precore/core promoter mutations and hepatitis B virus genotype in hepatitis B and C dually infected patients treated with interferon-based therapy by Chao-Hung Hung; Chien-Hung Chen; Sheng-Nan Lu; Jing-Houng Wang; Tsung-Hui Hu; Chao-Min Huang; Ming-Chao Tsai; Chuan-Mo Lee (pp. 55-63).
► Prevalence and implications of HBV mutation/genotype in dual BC infection is unknown. ► Dual infection has a higher prevalence of genotype C and less G1896A mutation. ► Genotype C is associated with more A1762T/G1764A mutation, but less G1896A mutation. ► Specific HBV mutations could predict long-term HBV response after IFN therapy.We studied the prevalence and distribution of precore/basal core promoter (BCP) mutations and hepatitis B virus (HBV) genotypes in HBV/hepatitis C virus (HCV) dually-infected patients, and evaluated their impact on long-term HBV response of interferon (IFN)-based therapy. The HBV genotypes and sequences of the precore/BCP regions were determined in 180 HBV/HCV dually-infected patients and were compared with 90 age, sex and hepatitis B e antigen-matched chronic hepatitis B controls. Serum HBV DNA and hepatitis B surface antigen (HBsAg) were assessed every 3–6months after therapy with IFN or pegylated-IFN plus ribavirin in 135 dually-infected patients with active hepatitis C. Dually-infected patients had a higher prevalence of genotype C HBV ( P=0.022) and a lower frequency of G1896A mutation ( P=0.004) as compared with controls. Among dually-infected patients, genotype C was associated with a higher frequency of A1762T/G1764A mutation ( P<0.001), but with lower HBV DNA ( P<0.001) and a lower frequency of A1752T/G ( P=0.008), C1799G ( P<0.001) and G1896A mutation (P <0.001) than genotype B. Based on Cox proportional hazards model, young age (hazard ratio (HR)=0.952, P=0.001), sustained virological response to HCV (HR=4.638, P=0.044), C1766T mutation (HR=5.216, P=0.003) and A1846T mutation (HR=2.332, P=0.031) correlated with HBV DNA reactivation (⩾2000IU/ml) after therapy. Age (HR=1.068, P=0.020), G1896A mutation (HR=0.140, P=0.01) and A1846T mutation (HR=0.086, P=0.018) were associated with HBsAg seroclearance independently. In conclusion, specific mutations in the precore/BCP regions could be useful in predicting long-term HBV response in HBV/HCV dually-infected patients treated with IFN-based therapy.
Keywords: Dual hepatitis B and C; Interferon; Genotype; Precore/core promoter mutation; HBsAg seroclearanceAbbreviations; AST; aspartate aminotransferase; ALT; alanine aminotransferase; BCP; basal core promoter; CI; confidence interval; HR; hazard ratio; HBeAg; hepatitis B e antigen; HBsAg; hepatitis B surface antigen; HBV; hepatitis B virus; HCV; hepatitis C virus; HCC; hepatocellular carcinoma; IFN; interferon; IU; international unit; PCR; polymerase chain reaction; SVR; sustained virological response
Single nucleotide polymorphism in the promoter region of the CD209 gene is associated with human predisposition to severe forms of tick-borne encephalitis by Andrey V. Barkhash; Andrey A. Perelygin; Vladimir N. Babenko; Margo A. Brinton; Mikhail I. Voevoda (pp. 64-68).
► Genetic predisposition to tick-borne encephalitis virus-induced disease was studied. ► Russian patients with various clinical forms of tick-borne encephalitis were tested. ► Human CD209 gene encoding DC-SIGN (C-type lectin) was selected as a candidate gene. ► Two SNPs in the promoter region of the CD209 gene were selected as genetic markers. ► CD209 gene rs2287886 SNP is associated with the severity of tick-borne encephalitis.Tick-borne encephalitis virus (TBEV) is a neurotropic, positive-sense RNA virus of the genus Flavivirus (family Flaviviridae) which can cause a variety of clinical manifestations in humans. Previously the severity and outcome of dengue fever and hepatitis C (diseases caused by viruses from the family Flaviviridae) were associated with the rs4804803 single nucleotide polymorphism (SNP) located in the promoter region of the human CD209 gene. This gene encodes dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN), a C-type lectin pathogen-recognition receptor expressed on the surface of dendritic cells and some types of macrophages. In the current study, a possible association between two SNPs in the promoter region of the CD209 gene (rs4804803 and rs2287886) and predisposition to severe forms of TBEV-induced disease was investigated. The genotypic, allelic and haplotypic frequencies of these SNPs were analyzed in 136 non-immunized Russian patients with different clinical manifestations of tick-borne encephalitis (TBE) and in a control group. An increase in the frequency of the rs2287886 SNP AA homozygotes and the A allele was detected among patients with severe central nervous system disease compared with the group of patients with meningitis ( P=0.003 and 0.019), or a combined group of patients with mild forms (fever and meningitis) ( P=0.003 and 0.026), or the control group ( P=0.007 and 0.035). Thus, our results suggest that the CD209 gene promoter region rs2287886 SNP is associated with predisposition to severe forms of TBE in the Russian population.
Keywords: Tick-borne encephalitis (TBE); Human genetic predisposition; Dendritic cell-specific intercellular adhesion molecule-3 (ICAM3)-grabbing non-integrin (DC-SIGN); CD209; gene; Single nucleotide polymorphism (SNP)
Single nucleotide polymorphism in the promoter region of the CD209 gene is associated with human predisposition to severe forms of tick-borne encephalitis by Andrey V. Barkhash; Andrey A. Perelygin; Vladimir N. Babenko; Margo A. Brinton; Mikhail I. Voevoda (pp. 64-68).
► Genetic predisposition to tick-borne encephalitis virus-induced disease was studied. ► Russian patients with various clinical forms of tick-borne encephalitis were tested. ► Human CD209 gene encoding DC-SIGN (C-type lectin) was selected as a candidate gene. ► Two SNPs in the promoter region of the CD209 gene were selected as genetic markers. ► CD209 gene rs2287886 SNP is associated with the severity of tick-borne encephalitis.Tick-borne encephalitis virus (TBEV) is a neurotropic, positive-sense RNA virus of the genus Flavivirus (family Flaviviridae) which can cause a variety of clinical manifestations in humans. Previously the severity and outcome of dengue fever and hepatitis C (diseases caused by viruses from the family Flaviviridae) were associated with the rs4804803 single nucleotide polymorphism (SNP) located in the promoter region of the human CD209 gene. This gene encodes dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN), a C-type lectin pathogen-recognition receptor expressed on the surface of dendritic cells and some types of macrophages. In the current study, a possible association between two SNPs in the promoter region of the CD209 gene (rs4804803 and rs2287886) and predisposition to severe forms of TBEV-induced disease was investigated. The genotypic, allelic and haplotypic frequencies of these SNPs were analyzed in 136 non-immunized Russian patients with different clinical manifestations of tick-borne encephalitis (TBE) and in a control group. An increase in the frequency of the rs2287886 SNP AA homozygotes and the A allele was detected among patients with severe central nervous system disease compared with the group of patients with meningitis ( P=0.003 and 0.019), or a combined group of patients with mild forms (fever and meningitis) ( P=0.003 and 0.026), or the control group ( P=0.007 and 0.035). Thus, our results suggest that the CD209 gene promoter region rs2287886 SNP is associated with predisposition to severe forms of TBE in the Russian population.
Keywords: Tick-borne encephalitis (TBE); Human genetic predisposition; Dendritic cell-specific intercellular adhesion molecule-3 (ICAM3)-grabbing non-integrin (DC-SIGN); CD209; gene; Single nucleotide polymorphism (SNP)
SP600125 inhibits Orthopoxviruses replication in a JNK1/2 -independent manner: Implication as a potential antipoxviral by Anna C.T.C. Pereira; Jamária A.P. Soares-Martins; Flávia G.G. Leite; André F.P. Da Cruz; Alice A. Torres; Thais Souto-Padrón; Erna G. Kroon; Paulo C.P. Ferreira; Cláudio A. Bonjardim (pp. 69-77).
The pharmacological inhibitor SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] has been largely employed as a c-JUN N-terminal kinase (JNK1/2) inhibitor. In this study, we evaluated whether pretreatment with SP600125 was able to prevent Orthopoxviruses Vaccinia virus (VACV), Cowpox virus (CPXV) and modified Vaccinia virus Ankara (MVA) replication. We found that incubation with SP600125 not only blocked virus-stimulated JNK phosphorylation, but also, significantly reduced virus production. We observed 1–3 log decline in viral yield depending on the cell line infected (A31, BSC-40 or BHK-21). The reduction in viral yield correlated with a dramatic impact on virus morphogenesis progress, intracellular mature viruses (IMV) were barely detected. Despite the fact that SP600125 can act as an efficient anti-orthopoxviral compound, we also provide evidence that this antiviral effect is not specifically exerted through JNK1/2 inhibition. This conclusion is supported by the fact that viral titers measured after infections of JNK1/2 knockout cells were not altered as compared to those of wild-type cells. In contrast, a decline in viral titers was verified when the infection of KO cells was carried out in the presence of the pharmacological inhibitor. SP600125 has been the focus of recent studies that have evaluated its action on diverse viral infections including DNA viruses. Our data support the notion that SP600125 can be regarded as a potential antipoxviral compound.
Keywords: Poxvirus; SP600125; Antipoxviral; Antiviral; JNK; Morphogenesis
SP600125 inhibits Orthopoxviruses replication in a JNK1/2 -independent manner: Implication as a potential antipoxviral by Anna C.T.C. Pereira; Jamária A.P. Soares-Martins; Flávia G.G. Leite; André F.P. Da Cruz; Alice A. Torres; Thais Souto-Padrón; Erna G. Kroon; Paulo C.P. Ferreira; Cláudio A. Bonjardim (pp. 69-77).
The pharmacological inhibitor SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] has been largely employed as a c-JUN N-terminal kinase (JNK1/2) inhibitor. In this study, we evaluated whether pretreatment with SP600125 was able to prevent Orthopoxviruses Vaccinia virus (VACV), Cowpox virus (CPXV) and modified Vaccinia virus Ankara (MVA) replication. We found that incubation with SP600125 not only blocked virus-stimulated JNK phosphorylation, but also, significantly reduced virus production. We observed 1–3 log decline in viral yield depending on the cell line infected (A31, BSC-40 or BHK-21). The reduction in viral yield correlated with a dramatic impact on virus morphogenesis progress, intracellular mature viruses (IMV) were barely detected. Despite the fact that SP600125 can act as an efficient anti-orthopoxviral compound, we also provide evidence that this antiviral effect is not specifically exerted through JNK1/2 inhibition. This conclusion is supported by the fact that viral titers measured after infections of JNK1/2 knockout cells were not altered as compared to those of wild-type cells. In contrast, a decline in viral titers was verified when the infection of KO cells was carried out in the presence of the pharmacological inhibitor. SP600125 has been the focus of recent studies that have evaluated its action on diverse viral infections including DNA viruses. Our data support the notion that SP600125 can be regarded as a potential antipoxviral compound.
Keywords: Poxvirus; SP600125; Antipoxviral; Antiviral; JNK; Morphogenesis
A conserved matrix epitope based DNA vaccine protects mice against influenza A virus challenge by Prashant Kumar; Madhu Khanna; Binod Kumar; Roopali Rajput; Akhil C. Banerjea (pp. 78-85).
► We construct a plasmid (pDNA) encoding a conserved matrix epitope of influenza A virus. ► We examine the immune response after injecting the pDNA in mice model. ► Immunogenicity of pDNA compared with inactivated virus and complete matrix protein. ► pDNA vaccination cross-protects the mice against lethal infection with the virus.DNA vaccination represents a unique strategy to overcome the limitations of immunization with conventional vaccines which is restricted by the high variability of influenza viruses. We evaluated the protective efficacy of a plasmid DNA (pDNA), encoding an evolutionarily conserved epitope of viral matrix protein, against the influenza A virus infection. It was found that the mice immunized via the intra-muscular route purely elicited cell mediated immune response to the pDNA, with enhanced level of Th1 cytokines viz. IL-12 and IFNγ production in the stimulated splenocyte supernatant. The cytotoxic T lymphocytes in the spleen of immunized mice significantly lysed the virus-infected MDCK cells. A significant decrease in virus replication was also observed in the lungs of immunized mice and 83% of the mice were protected against the lethal challenge of influenza A viruses. These findings suggest that the plasmid DNA expressing a single matrix epitope may serve as a promising vaccine candidate to provide effective immunity in the susceptible (mouse) population.
Keywords: Influenza; Epitope; Vaccine
A conserved matrix epitope based DNA vaccine protects mice against influenza A virus challenge by Prashant Kumar; Madhu Khanna; Binod Kumar; Roopali Rajput; Akhil C. Banerjea (pp. 78-85).
► We construct a plasmid (pDNA) encoding a conserved matrix epitope of influenza A virus. ► We examine the immune response after injecting the pDNA in mice model. ► Immunogenicity of pDNA compared with inactivated virus and complete matrix protein. ► pDNA vaccination cross-protects the mice against lethal infection with the virus.DNA vaccination represents a unique strategy to overcome the limitations of immunization with conventional vaccines which is restricted by the high variability of influenza viruses. We evaluated the protective efficacy of a plasmid DNA (pDNA), encoding an evolutionarily conserved epitope of viral matrix protein, against the influenza A virus infection. It was found that the mice immunized via the intra-muscular route purely elicited cell mediated immune response to the pDNA, with enhanced level of Th1 cytokines viz. IL-12 and IFNγ production in the stimulated splenocyte supernatant. The cytotoxic T lymphocytes in the spleen of immunized mice significantly lysed the virus-infected MDCK cells. A significant decrease in virus replication was also observed in the lungs of immunized mice and 83% of the mice were protected against the lethal challenge of influenza A viruses. These findings suggest that the plasmid DNA expressing a single matrix epitope may serve as a promising vaccine candidate to provide effective immunity in the susceptible (mouse) population.
Keywords: Influenza; Epitope; Vaccine
Novel HBsAg markers tightly correlate with occult HBV infection and strongly affect HBsAg detection by Valentina Svicher; Valeria Cento; Martina Bernassola; Maria Neumann-Fraune; Formijn Van Hemert; Mengjie Chen; Romina Salpini; Chang Liu; Roberta Longo; Michela Visca; Sara Romano; Valeria Micheli; Ada Bertoli; Caterina Gori; Francesca Ceccherini-Silberstein; Cesare Sarrecchia; Massimo Andreoni; Mario Angelico; Antonella Ursitti; Alberto Spanò; Jing Maria Zhang; Jens Verheyen; Giuseppina Cappiello; Carlo Federico Perno (pp. 86-93).
► HBsAg genetic markers correlated with occult HBV D genotype infection in vivo were studied. ► Twenty HBsAg-mutations significantly correlated for the first time with OBI were found. ► They localized in the major HBV B-cell-epitope, and in HBsAg-capsid interaction region. ► They strongly affect, up to abrogate, HBsAg detection. ► Thus, they can affect full-reliability of diagnostic-assays for HBsAg-detection.Occult HBV infection (OBI) is a threat for the safety of blood-supply, and has been associated with the onset of HBV-related hepatocellular carcinoma and lymphomagenesis. Nevertheless, genetic markers in HBsAg (particularly in D-genotype, the most common in Europe) significantly associated with OBI in vivo are missing. Thus, the goal of this study is to define: (i) prevalence and clinical profile of OBI among blood-donors; (ii) HBsAg-mutations associated with OBI; (iii) their impact on HBsAg-detection. OBI was searched among 422,278 blood-donors screened by Nucleic-Acid-Testing. Following Taormina-OBI-definition, 26 (0.006%) OBI-patients were identified. Despite viremia <50IU/ml, HBsAg-sequences were obtained for 25/26 patients (24/25 genotype-D). OBI-associated mutations were identified by comparing OBI-HBsAg with that of 82 chronically-infected (genotype-D) patients as control. Twenty HBsAg-mutations significantly correlated for the first time with OBI. By structural analysis, they localized in the major HBV B-cell-epitope, and in HBsAg-capsid interaction region. 14/24 OBI-patients (58.8%) carried in median 3 such mutations (IQR:2.0–6.0) against 0 in chronically-infected patients. By co-variation analysis, correlations were observed for R122P+S167L (phi=0.68, P=0.01), T116N+S143L (phi=0.53, P=0.03), and Y100S+S143L (phi=0.67, p<0.001).Mutants (obtained by site-directed mutagenesis) carrying T116N, T116N+S143L, R122P, R122P+Q101R, or R122P+S167L strongly decreased HBsAg-reactivity (54.9±22.6S/CO, 31.2±12.0S/CO, 6.1±2.4S/CO, 3.0±1.0S/CO and 3.9±1.3S/CO, respectively) compared to wild-type (306.8±64.1S/CO). Even more, Y100S and Y100S+S143L supernatants show no detectable-HBsAg (experiments in quadruplicate).In conclusions, unique HBsAg-mutations in genotype-D, different than those described in genotypes B/C (rarely found in western countries), tightly correlate with OBI, and strongly affect HBsAg-detection. By altering HBV-antigenicity and/or viral-particle maturation, they may affect full-reliability of universal diagnostic-assays for HBsAg-detection.
Keywords: HBV; Occult infection; HBsAg structure; HBsAg detection
Novel HBsAg markers tightly correlate with occult HBV infection and strongly affect HBsAg detection by Valentina Svicher; Valeria Cento; Martina Bernassola; Maria Neumann-Fraune; Formijn Van Hemert; Mengjie Chen; Romina Salpini; Chang Liu; Roberta Longo; Michela Visca; Sara Romano; Valeria Micheli; Ada Bertoli; Caterina Gori; Francesca Ceccherini-Silberstein; Cesare Sarrecchia; Massimo Andreoni; Mario Angelico; Antonella Ursitti; Alberto Spanò; Jing Maria Zhang; Jens Verheyen; Giuseppina Cappiello; Carlo Federico Perno (pp. 86-93).
► HBsAg genetic markers correlated with occult HBV D genotype infection in vivo were studied. ► Twenty HBsAg-mutations significantly correlated for the first time with OBI were found. ► They localized in the major HBV B-cell-epitope, and in HBsAg-capsid interaction region. ► They strongly affect, up to abrogate, HBsAg detection. ► Thus, they can affect full-reliability of diagnostic-assays for HBsAg-detection.Occult HBV infection (OBI) is a threat for the safety of blood-supply, and has been associated with the onset of HBV-related hepatocellular carcinoma and lymphomagenesis. Nevertheless, genetic markers in HBsAg (particularly in D-genotype, the most common in Europe) significantly associated with OBI in vivo are missing. Thus, the goal of this study is to define: (i) prevalence and clinical profile of OBI among blood-donors; (ii) HBsAg-mutations associated with OBI; (iii) their impact on HBsAg-detection. OBI was searched among 422,278 blood-donors screened by Nucleic-Acid-Testing. Following Taormina-OBI-definition, 26 (0.006%) OBI-patients were identified. Despite viremia <50IU/ml, HBsAg-sequences were obtained for 25/26 patients (24/25 genotype-D). OBI-associated mutations were identified by comparing OBI-HBsAg with that of 82 chronically-infected (genotype-D) patients as control. Twenty HBsAg-mutations significantly correlated for the first time with OBI. By structural analysis, they localized in the major HBV B-cell-epitope, and in HBsAg-capsid interaction region. 14/24 OBI-patients (58.8%) carried in median 3 such mutations (IQR:2.0–6.0) against 0 in chronically-infected patients. By co-variation analysis, correlations were observed for R122P+S167L (phi=0.68, P=0.01), T116N+S143L (phi=0.53, P=0.03), and Y100S+S143L (phi=0.67, p<0.001).Mutants (obtained by site-directed mutagenesis) carrying T116N, T116N+S143L, R122P, R122P+Q101R, or R122P+S167L strongly decreased HBsAg-reactivity (54.9±22.6S/CO, 31.2±12.0S/CO, 6.1±2.4S/CO, 3.0±1.0S/CO and 3.9±1.3S/CO, respectively) compared to wild-type (306.8±64.1S/CO). Even more, Y100S and Y100S+S143L supernatants show no detectable-HBsAg (experiments in quadruplicate).In conclusions, unique HBsAg-mutations in genotype-D, different than those described in genotypes B/C (rarely found in western countries), tightly correlate with OBI, and strongly affect HBsAg-detection. By altering HBV-antigenicity and/or viral-particle maturation, they may affect full-reliability of universal diagnostic-assays for HBsAg-detection.
Keywords: HBV; Occult infection; HBsAg structure; HBsAg detection
RNA interference inhibits replication of tick-borne encephalitis virus in vitro by Katharina Achazi; Pranav Patel; Ravish Paliwal; Aleksandar Radonić; Matthias Niedrig; Oliver Donoso-Mantke (pp. 94-100).
► We developed small interfering RNAs (siRNA) targeted to the TBE virus genome. ► siRNAs reduce the quantity of TBE virus particles, genome, and protein up to 85%. ► The 50% inhibitory dose of the shRNA plasmid was 0.05 μg/ml. ► RNA interference could become valuable tool for controlling TBE virus infections.Each year, up to 10,000 cases of infections with the flavivirus tick-borne encephalitis (TBE) virus that affect the central nervous system are reported in Europe and Asia. Due to the potentially severe adverse effects of post-exposure prophylaxis with TBE virus hyperimmunoglobulin, TBE can currently only be treated symptomatically. An RNA interference (RNAi) approach to inhibit TBE virus replication was therefore developed. In this study we demonstrate for the first time that small interfering RNAs (siRNAs) targeted at the TBE virus genome reduce the quantity of infectious TBE virus particles, TBE virus genome, and TBE virus protein in vitro by up to 85%. The 50% inhibitory dose (DI50) of the shRNA plasmid was only 0.05μg/ml. As RNAi-based therapeutics for other diseases are already being evaluated in phases II and III clinical trials, it is possible that RNAi could become valuable tool for controlling TBE virus infection.
Keywords: Abbreviations; DAPI; 4′,6-diamidino-2-phenylindole; DI; 50; 50% inhibitory dose; DMEM; Dulbecco’s modified Eagle’s medium; dsRNA; double-stranded RNA; EGFP; enhanced green fluorescent protein; GE; genome equivalent; HIV; human immunodeficiency virus; MOI; multiplicity of infection; PFU; plaque forming units; RISC; RNA-induced silencing complex; RNAi; RNA interference; RT-qPCR; quantitative real-time RT-PCR; shRNA; short hairpin RNA; siRNA; small interfering RNA; TBE; tick-borne encephalitisFlavivirus; Tick-borne encephalitis virus; Tick-borne encephalitis; RNA interference; Small interfering RNA; Antiviral drug
RNA interference inhibits replication of tick-borne encephalitis virus in vitro by Katharina Achazi; Pranav Patel; Ravish Paliwal; Aleksandar Radonić; Matthias Niedrig; Oliver Donoso-Mantke (pp. 94-100).
► We developed small interfering RNAs (siRNA) targeted to the TBE virus genome. ► siRNAs reduce the quantity of TBE virus particles, genome, and protein up to 85%. ► The 50% inhibitory dose of the shRNA plasmid was 0.05 μg/ml. ► RNA interference could become valuable tool for controlling TBE virus infections.Each year, up to 10,000 cases of infections with the flavivirus tick-borne encephalitis (TBE) virus that affect the central nervous system are reported in Europe and Asia. Due to the potentially severe adverse effects of post-exposure prophylaxis with TBE virus hyperimmunoglobulin, TBE can currently only be treated symptomatically. An RNA interference (RNAi) approach to inhibit TBE virus replication was therefore developed. In this study we demonstrate for the first time that small interfering RNAs (siRNAs) targeted at the TBE virus genome reduce the quantity of infectious TBE virus particles, TBE virus genome, and TBE virus protein in vitro by up to 85%. The 50% inhibitory dose (DI50) of the shRNA plasmid was only 0.05μg/ml. As RNAi-based therapeutics for other diseases are already being evaluated in phases II and III clinical trials, it is possible that RNAi could become valuable tool for controlling TBE virus infection.
Keywords: Abbreviations; DAPI; 4′,6-diamidino-2-phenylindole; DI; 50; 50% inhibitory dose; DMEM; Dulbecco’s modified Eagle’s medium; dsRNA; double-stranded RNA; EGFP; enhanced green fluorescent protein; GE; genome equivalent; HIV; human immunodeficiency virus; MOI; multiplicity of infection; PFU; plaque forming units; RISC; RNA-induced silencing complex; RNAi; RNA interference; RT-qPCR; quantitative real-time RT-PCR; shRNA; short hairpin RNA; siRNA; small interfering RNA; TBE; tick-borne encephalitisFlavivirus; Tick-borne encephalitis virus; Tick-borne encephalitis; RNA interference; Small interfering RNA; Antiviral drug
Potent anti-respiratory syncytial virus activity of a cholestanol-sulfated tetrasaccharide conjugate by Anna Lundin; Tomas Bergström; Carla R. Andrighetti-Fröhner; Loubna Bendrioua; Vito Ferro; Edward Trybala (pp. 101-109).
► We modified sulfated oligosaccharides by addition of cholestanol to reducing end. ► The modification improved anti-RS virus potency of native oligosaccharide. ► The compound showed virucidal activity against RS-virus.A number of different viruses including respiratory syncytial virus (RSV) initiate infection of cells by binding to cell surface glycosaminoglycans and sulfated oligo- and polysaccharide mimetics of these receptors exhibit potent antiviral activity in cultured cells. We investigated whether the introduction of different lipophilic groups to the reducing end of sulfated oligosaccharides would modulate their anti-RSV activity. Our results demonstrate that the cholestanol-conjugated tetrasaccharide (PG545) exhibited ∼5- to 16-fold enhanced anti-RSV activity in cultured cells compared with unmodified sulfated oligosaccharides. Furthermore, PG545 displayed virus-inactivating (virucidal) activity, a feature absent in sulfated oligosaccharides. To inhibit RSV infectivity PG545 had to be present during the initial steps of viral infection of cells. The anti-RSV activity of PG545 was due to both partial inhibition of the virus attachment to cells and a more profound interference with some post-attachment steps as PG545 efficiently neutralized infectivity of the cell-adsorbed virus. The anti-RSV activity of PG545 was reduced when tested in the presence of human nasal secretions. Serial passages of RSV in the presence of increasing concentrations of PG545 selected for weakly resistant viral variants that comprised the F168S and the P180S amino acid substitutions in the viral G protein. Altogether we identified a novel and potent inhibitor of RSV, which unlike sulfated oligo- and polysaccharide compounds, could irreversibly inactivate RSV infectivity.
Keywords: Respiratory syncytial virus; PG545; Sulfated oligosaccharides; Virucidal activity
Potent anti-respiratory syncytial virus activity of a cholestanol-sulfated tetrasaccharide conjugate by Anna Lundin; Tomas Bergström; Carla R. Andrighetti-Fröhner; Loubna Bendrioua; Vito Ferro; Edward Trybala (pp. 101-109).
► We modified sulfated oligosaccharides by addition of cholestanol to reducing end. ► The modification improved anti-RS virus potency of native oligosaccharide. ► The compound showed virucidal activity against RS-virus.A number of different viruses including respiratory syncytial virus (RSV) initiate infection of cells by binding to cell surface glycosaminoglycans and sulfated oligo- and polysaccharide mimetics of these receptors exhibit potent antiviral activity in cultured cells. We investigated whether the introduction of different lipophilic groups to the reducing end of sulfated oligosaccharides would modulate their anti-RSV activity. Our results demonstrate that the cholestanol-conjugated tetrasaccharide (PG545) exhibited ∼5- to 16-fold enhanced anti-RSV activity in cultured cells compared with unmodified sulfated oligosaccharides. Furthermore, PG545 displayed virus-inactivating (virucidal) activity, a feature absent in sulfated oligosaccharides. To inhibit RSV infectivity PG545 had to be present during the initial steps of viral infection of cells. The anti-RSV activity of PG545 was due to both partial inhibition of the virus attachment to cells and a more profound interference with some post-attachment steps as PG545 efficiently neutralized infectivity of the cell-adsorbed virus. The anti-RSV activity of PG545 was reduced when tested in the presence of human nasal secretions. Serial passages of RSV in the presence of increasing concentrations of PG545 selected for weakly resistant viral variants that comprised the F168S and the P180S amino acid substitutions in the viral G protein. Altogether we identified a novel and potent inhibitor of RSV, which unlike sulfated oligo- and polysaccharide compounds, could irreversibly inactivate RSV infectivity.
Keywords: Respiratory syncytial virus; PG545; Sulfated oligosaccharides; Virucidal activity
MEK1–ERKs signal cascade is required for the replication of Enterovirus 71 (EV71) by Bo Wang; Hao Zhang; Meng Zhu; Zhijun Luo; Yihong Peng (pp. 110-117).
► We observed in vitro infection with EV71 induced a biphasic activation of ERK. ► Inhibition of ERK activation and knockdown of MEK1 clearly impaired virus production. ► By contrast, silencing MEK2 expression did not such effect. ► Both MEK isoforms were activated and translocated to nucleus upon EV71 infection. ► We conclude that only MEK1–ERKs activation is necessary for the virus production.The role of the MEK1–ERK signaling cascade in the replication cycle of Enterovirus 71 (EV71), the primary cause of hand, foot and mouth disease (HFMD), has been analyzed. In vitro infection with EV71 induced a biphasic activation of ERK. The two phases of activation appeared to be triggered by different mechanisms, with the first phase being activated by the binding of viral particles to the membrane receptor of host cells and the second probably being in response to the production of new virus particles. Inhibition of ERK activation by U0126 was found to severely impair virus production. A similar reduction in EV71 replication was also observed when MEK1 expression was subject to knockdown using specific siRNAs. By contrast knockdown of MEK2 expression showed that it was dispensable for virus replication cycle, despite both MEK isoforms being activated and translocated to the nucleus equally well in response to virus infection. Overall, this study suggests distinct functions of the two isoforms of MEK in EV71 replication cycle, with an essential role for MEK1 in stimulating the ERK signaling cascade to promote virus replication. Taken together with our previous work on herpes simplex virus type 2 (HSV2) this study highlights MEK1 as a potential broad antiviral molecular target.
Keywords: EV71; Viral replication cycle; MEK1; MEK2; ERK; siRNAs
MEK1–ERKs signal cascade is required for the replication of Enterovirus 71 (EV71) by Bo Wang; Hao Zhang; Meng Zhu; Zhijun Luo; Yihong Peng (pp. 110-117).
► We observed in vitro infection with EV71 induced a biphasic activation of ERK. ► Inhibition of ERK activation and knockdown of MEK1 clearly impaired virus production. ► By contrast, silencing MEK2 expression did not such effect. ► Both MEK isoforms were activated and translocated to nucleus upon EV71 infection. ► We conclude that only MEK1–ERKs activation is necessary for the virus production.The role of the MEK1–ERK signaling cascade in the replication cycle of Enterovirus 71 (EV71), the primary cause of hand, foot and mouth disease (HFMD), has been analyzed. In vitro infection with EV71 induced a biphasic activation of ERK. The two phases of activation appeared to be triggered by different mechanisms, with the first phase being activated by the binding of viral particles to the membrane receptor of host cells and the second probably being in response to the production of new virus particles. Inhibition of ERK activation by U0126 was found to severely impair virus production. A similar reduction in EV71 replication was also observed when MEK1 expression was subject to knockdown using specific siRNAs. By contrast knockdown of MEK2 expression showed that it was dispensable for virus replication cycle, despite both MEK isoforms being activated and translocated to the nucleus equally well in response to virus infection. Overall, this study suggests distinct functions of the two isoforms of MEK in EV71 replication cycle, with an essential role for MEK1 in stimulating the ERK signaling cascade to promote virus replication. Taken together with our previous work on herpes simplex virus type 2 (HSV2) this study highlights MEK1 as a potential broad antiviral molecular target.
Keywords: EV71; Viral replication cycle; MEK1; MEK2; ERK; siRNAs
In vitro anti-hepatitis B and SARS virus activities of a titanium-substituted-heteropolytungstate by Yan-fei Qi; Hong Zhang; Juan Wang; Yanfang Jiang; Jinhua Li; Ye Yuan; Shiyao Zhang; Kun Xu; Yangguang Li; Juan Li; Junqi Niu; Enbo Wang (pp. 118-125).
► A structural determined heteropolytungstate has been synthesized. ► The heteropolytungstate can efficiently inhibit HBV replication in vitro. ► The heteropolytungstate also shows high anti-SARS virus activity in vitro.A structural determined heteropolytungstate, [K4(H2O)8Cl][K4(H2O)4PTi2W10O40]·NH2OH1, has been synthesized and evaluated for in vitro antiviral activities against hepatitis B (HBV) and SARS virus. The identity and high purity of compound1 were confirmed by elemental analysis, NMR, IR analysis and single-crystal X-ray diffraction. The compound1, evaluated in HepG 2.2.15 cells expressing permanently HBV, significantly reduced the levels of HBV antigens and HBV DNA in a dose-dependent and time-dependent manner. EC50 values were determined to be 54μM for HBeAg, 61μM for HBsAg and 2.66μM for supernatant HBV DNA, as compared to 1671, 1570, 169μM, respectively, for the commercially-available hepatitis B drug adefovir dipivoxil (ADV). Intracellular cccDNA, pgRNA and HBcAg were also found to be decreased by compound1 in a concentration-dependent manner. Cytotoxicity results showed that compound1 has low toxicity in HepG 2 cells with CC50 value of 515.20μM. The results indicate that compound1 can efficiently inhibit HBV replication in HepG 2.2.15 cells line in vitro. Additionally, compound1 also shows high anti-SARS activity at an EC50 of 7.08μM and toxicity with a CC50 of 118.6μM against MDCK cells.
Keywords: Abbreviations; HBV; hepatitis B virus; INF-α; interferon alpha; 3TC; nucleoside analog lamivudine; POMs; Polyoxometalates; SARS; severe acute respiratory syndrome; DMEM; Dulbecco’s Modified Eagle’s Medium; FBS; fetal bovine serum; Vero-E; 6; African green monkey kidney cells; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ADV; adefovir dipivoxil; CC; 50; 50% cytotoxic dose; TI; therapeutic index; EC; 50; 50% effective concentration; CC; 0; maximal noncytotoxic concentration; EC; 90; 90% effective concentration; MDCK; Madin–Darby canine kidneyHeteropolytungstate; Antiviral activity; Hepatitis B virus; SARS virus; In vitro
In vitro anti-hepatitis B and SARS virus activities of a titanium-substituted-heteropolytungstate by Yan-fei Qi; Hong Zhang; Juan Wang; Yanfang Jiang; Jinhua Li; Ye Yuan; Shiyao Zhang; Kun Xu; Yangguang Li; Juan Li; Junqi Niu; Enbo Wang (pp. 118-125).
► A structural determined heteropolytungstate has been synthesized. ► The heteropolytungstate can efficiently inhibit HBV replication in vitro. ► The heteropolytungstate also shows high anti-SARS virus activity in vitro.A structural determined heteropolytungstate, [K4(H2O)8Cl][K4(H2O)4PTi2W10O40]·NH2OH1, has been synthesized and evaluated for in vitro antiviral activities against hepatitis B (HBV) and SARS virus. The identity and high purity of compound1 were confirmed by elemental analysis, NMR, IR analysis and single-crystal X-ray diffraction. The compound1, evaluated in HepG 2.2.15 cells expressing permanently HBV, significantly reduced the levels of HBV antigens and HBV DNA in a dose-dependent and time-dependent manner. EC50 values were determined to be 54μM for HBeAg, 61μM for HBsAg and 2.66μM for supernatant HBV DNA, as compared to 1671, 1570, 169μM, respectively, for the commercially-available hepatitis B drug adefovir dipivoxil (ADV). Intracellular cccDNA, pgRNA and HBcAg were also found to be decreased by compound1 in a concentration-dependent manner. Cytotoxicity results showed that compound1 has low toxicity in HepG 2 cells with CC50 value of 515.20μM. The results indicate that compound1 can efficiently inhibit HBV replication in HepG 2.2.15 cells line in vitro. Additionally, compound1 also shows high anti-SARS activity at an EC50 of 7.08μM and toxicity with a CC50 of 118.6μM against MDCK cells.
Keywords: Abbreviations; HBV; hepatitis B virus; INF-α; interferon alpha; 3TC; nucleoside analog lamivudine; POMs; Polyoxometalates; SARS; severe acute respiratory syndrome; DMEM; Dulbecco’s Modified Eagle’s Medium; FBS; fetal bovine serum; Vero-E; 6; African green monkey kidney cells; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ADV; adefovir dipivoxil; CC; 50; 50% cytotoxic dose; TI; therapeutic index; EC; 50; 50% effective concentration; CC; 0; maximal noncytotoxic concentration; EC; 90; 90% effective concentration; MDCK; Madin–Darby canine kidneyHeteropolytungstate; Antiviral activity; Hepatitis B virus; SARS virus; In vitro
Molecular modeling study on the resistance mechanism of HCV NS3/4A serine protease mutants R155K, A156V and D168A to TMC435 by Weiwei Xue; Dabo Pan; Ying Yang; Huanxiang Liu; Xiaojun Yao (pp. 126-137).
Display Omitted► Resistance mechanism of three main HCV NS3/4A mutants to TMC435 were investigated. ► R155K and D168A mutations break the intermolecular salt bridges network. ► A156V mutation disrupts the two substrate-like intermolecular hydrogen bonds. ► The three key mutations make NS3A change the induced-fit binding conformation.Hepatitis C virus (HCV) NS3/4A protease represents an attractive drug target for antiviral therapy. However, drug resistance often occurs, making many protease inhibitors ineffective and allowing viral replication to occur. Herein, based on the recently determined structure of NS3/4A–TMC435 complex, atomic-level models of the key residue mutated (R155K, A156V and D168A) NS3/4A–TMC435 complexes were constructed. Subsequently, by using molecular dynamics simulations, binding free energy calculation and substrate envelope analysis, the structural and energetic changes responsible for drug resistance were investigated. The values of the calculated binding free energy follow consistently the order of the experimental activities. More importantly, the computational results demonstrate that R155K and D168A mutations break the intermolecular salt bridges network at the extended S2 subsite and affect the TMC435 binding, while A156V mutation leads to a significant steric clash with TMC435 and further disrupts the two canonical substrate-like intermolecular hydrogen bond interactions (TMC435(N1–H46)⋯Arg155(O) and Ala157(N–H)⋯TMC435(O2)). In addition, by structural analysis, all the three key residue mutations occur outside the substrate envelope and selectively weaken TMC435’s binding affinity without effect on its natural substrate peptide (4B5A). These findings could provide some insights into the resistance mechanism of NS3/4A protease mutants to TMC435 and would be critical for the development of novel inhibitors that are less susceptible to drug resistance.
Keywords: HCV NS3/4A protease; TMC435; Drug resistance; Substrate envelope; Molecular dynamics simulations
Molecular modeling study on the resistance mechanism of HCV NS3/4A serine protease mutants R155K, A156V and D168A to TMC435 by Weiwei Xue; Dabo Pan; Ying Yang; Huanxiang Liu; Xiaojun Yao (pp. 126-137).
Display Omitted► Resistance mechanism of three main HCV NS3/4A mutants to TMC435 were investigated. ► R155K and D168A mutations break the intermolecular salt bridges network. ► A156V mutation disrupts the two substrate-like intermolecular hydrogen bonds. ► The three key mutations make NS3A change the induced-fit binding conformation.Hepatitis C virus (HCV) NS3/4A protease represents an attractive drug target for antiviral therapy. However, drug resistance often occurs, making many protease inhibitors ineffective and allowing viral replication to occur. Herein, based on the recently determined structure of NS3/4A–TMC435 complex, atomic-level models of the key residue mutated (R155K, A156V and D168A) NS3/4A–TMC435 complexes were constructed. Subsequently, by using molecular dynamics simulations, binding free energy calculation and substrate envelope analysis, the structural and energetic changes responsible for drug resistance were investigated. The values of the calculated binding free energy follow consistently the order of the experimental activities. More importantly, the computational results demonstrate that R155K and D168A mutations break the intermolecular salt bridges network at the extended S2 subsite and affect the TMC435 binding, while A156V mutation leads to a significant steric clash with TMC435 and further disrupts the two canonical substrate-like intermolecular hydrogen bond interactions (TMC435(N1–H46)⋯Arg155(O) and Ala157(N–H)⋯TMC435(O2)). In addition, by structural analysis, all the three key residue mutations occur outside the substrate envelope and selectively weaken TMC435’s binding affinity without effect on its natural substrate peptide (4B5A). These findings could provide some insights into the resistance mechanism of NS3/4A protease mutants to TMC435 and would be critical for the development of novel inhibitors that are less susceptible to drug resistance.
Keywords: HCV NS3/4A protease; TMC435; Drug resistance; Substrate envelope; Molecular dynamics simulations
Intranasal administration of live Lactobacillus species facilitates protection against influenza virus infection in mice by Ha-Na Youn; Dong-Hun Lee; Yu-Na Lee; Jae-Keun Park; Seong-Su Yuk; Si-Yong Yang; Hyun-Jeong Lee; Seo-Hyung Woo; Hyoung-Moon Kim; Joong-Bok Lee; Seung-Yong Park; In-Soo Choi; Chang-Seon Song (pp. 138-143).
► Lactobacillus species facilitates protection against influenza virus infection in mice. ► Intranasal administration bestowed higher protective efficacy than the oral route. ► Live bacteria conferred higher protection against influenza than dead bacteria. ► There were huge differences in protective effects of various Lactobacillus strains on influenza virus infection.Influenza virus infections continue to be a significant public health problem. For improved therapies and preventive measures against influenza, there has been an increased tendency in modern medicine involving the use of probiotics. In this study, we compared the protective efficacy of various live and dead Lactobacillus species against challenge with influenza virus in mice according to the administration route and dose. In addition, to understand the underlying mechanism behind this clinical protective effect, we performed immunologic assays including examination of IgA levels and cytokine profiles in the lung. The survival rate of mice receiving intranasal administration of Lactobacillus was higher than after oral administration, and administration of live bacteria was more protective than of dead bacteria. The lung levels of interleukin (IL)-12 and IgA were significantly increased ( P<0.05). Conversely, the levels of the pro-inflammatory cytokines tumor necrosis factor-alpha and IL-6 were decreased. Interestingly, there were huge differences in protective effects of various Lactobacillus strains on influenza virus infection. Therefore, for clinical applications, selection of effective strains could be critical and individually optimized application regimens of the selected strains are required.
Keywords: Influenza virus; Mice; Lactobacillus; Probiotics; Immunomodulation
Intranasal administration of live Lactobacillus species facilitates protection against influenza virus infection in mice by Ha-Na Youn; Dong-Hun Lee; Yu-Na Lee; Jae-Keun Park; Seong-Su Yuk; Si-Yong Yang; Hyun-Jeong Lee; Seo-Hyung Woo; Hyoung-Moon Kim; Joong-Bok Lee; Seung-Yong Park; In-Soo Choi; Chang-Seon Song (pp. 138-143).
► Lactobacillus species facilitates protection against influenza virus infection in mice. ► Intranasal administration bestowed higher protective efficacy than the oral route. ► Live bacteria conferred higher protection against influenza than dead bacteria. ► There were huge differences in protective effects of various Lactobacillus strains on influenza virus infection.Influenza virus infections continue to be a significant public health problem. For improved therapies and preventive measures against influenza, there has been an increased tendency in modern medicine involving the use of probiotics. In this study, we compared the protective efficacy of various live and dead Lactobacillus species against challenge with influenza virus in mice according to the administration route and dose. In addition, to understand the underlying mechanism behind this clinical protective effect, we performed immunologic assays including examination of IgA levels and cytokine profiles in the lung. The survival rate of mice receiving intranasal administration of Lactobacillus was higher than after oral administration, and administration of live bacteria was more protective than of dead bacteria. The lung levels of interleukin (IL)-12 and IgA were significantly increased ( P<0.05). Conversely, the levels of the pro-inflammatory cytokines tumor necrosis factor-alpha and IL-6 were decreased. Interestingly, there were huge differences in protective effects of various Lactobacillus strains on influenza virus infection. Therefore, for clinical applications, selection of effective strains could be critical and individually optimized application regimens of the selected strains are required.
Keywords: Influenza virus; Mice; Lactobacillus; Probiotics; Immunomodulation
The 2008–2009 H1N1 influenza virus exhibits reduced susceptibility to antibody inhibition: Implications for the prevalence of oseltamivir resistant variant viruses by Wai Lan Wu; Siu-Ying Lau; Yixin Chen; Genyan Wang; Bobo Wing-Yee Mok; Xi Wen; Pui Wang; Wenjun Song; Tianwei Lin; Kwok-Hung Chan; Kwok-Yung Yuen; Honglin Chen (pp. 144-153).
A naturally-occurring H275Y oseltamivir resistant variant of influenza A (H1N1) virus emerged in 2007, subsequently becoming prevalent worldwide, via an undetermined mechanism. To understand the antigenic properties of the H275Y variant, oseltamivir resistant and susceptible strains of H1N1 viruses were analyzed by hemagglutination inhibition (HI) and microneutralization assays. HI analysis with H1-positive sera obtained from seasonal flu vaccine immunized and non-immunized individuals, and H1-specific monoclonal antibodies, revealed that resistant strains exhibited a reduced reactivity to these antisera and antibodies in the HI assay, as compared to susceptible strains. Neutralization assay testing demonstrated that oseltamivir resistant H1N1 strains are also less susceptible to antibody inhibition during infection. Mice inoculated with a resistant clinical isolate exhibit 4-fold lower virus-specific antibody titers than mice infected with a susceptible strain under the same conditions. Resistant and sensitive variants of 2009 pandemic H1N1 virus did not exhibit such differences. While HA1 and NA phylogenetic trees show that both oseltamivir resistant and susceptible strains belong to clade 2B, NA D354G and HA A189T substitutions were found exclusively, and universally, in oseltamivir resistant variants. Our results suggest that the reduced susceptibility to antibody inhibition and lesser in vivo immunogenicity of the oseltamivir resistant 2008–2009 H1N1 influenza A virus is conferred by coupled NA and HA mutations, and may contribute to the prevalence of this H1N1 variant.
Keywords: Influenza; H1N1; Oseltamivir; Resistance; Neuraminidase; H275Y
The 2008–2009 H1N1 influenza virus exhibits reduced susceptibility to antibody inhibition: Implications for the prevalence of oseltamivir resistant variant viruses by Wai Lan Wu; Siu-Ying Lau; Yixin Chen; Genyan Wang; Bobo Wing-Yee Mok; Xi Wen; Pui Wang; Wenjun Song; Tianwei Lin; Kwok-Hung Chan; Kwok-Yung Yuen; Honglin Chen (pp. 144-153).
A naturally-occurring H275Y oseltamivir resistant variant of influenza A (H1N1) virus emerged in 2007, subsequently becoming prevalent worldwide, via an undetermined mechanism. To understand the antigenic properties of the H275Y variant, oseltamivir resistant and susceptible strains of H1N1 viruses were analyzed by hemagglutination inhibition (HI) and microneutralization assays. HI analysis with H1-positive sera obtained from seasonal flu vaccine immunized and non-immunized individuals, and H1-specific monoclonal antibodies, revealed that resistant strains exhibited a reduced reactivity to these antisera and antibodies in the HI assay, as compared to susceptible strains. Neutralization assay testing demonstrated that oseltamivir resistant H1N1 strains are also less susceptible to antibody inhibition during infection. Mice inoculated with a resistant clinical isolate exhibit 4-fold lower virus-specific antibody titers than mice infected with a susceptible strain under the same conditions. Resistant and sensitive variants of 2009 pandemic H1N1 virus did not exhibit such differences. While HA1 and NA phylogenetic trees show that both oseltamivir resistant and susceptible strains belong to clade 2B, NA D354G and HA A189T substitutions were found exclusively, and universally, in oseltamivir resistant variants. Our results suggest that the reduced susceptibility to antibody inhibition and lesser in vivo immunogenicity of the oseltamivir resistant 2008–2009 H1N1 influenza A virus is conferred by coupled NA and HA mutations, and may contribute to the prevalence of this H1N1 variant.
Keywords: Influenza; H1N1; Oseltamivir; Resistance; Neuraminidase; H275Y
Preparation and characterization of egg yolk immunoglobulin Y specific to influenza B virus by Junlin Wen; Suqing Zhao; Daigui He; Yuane Yang; Yueming Li; Sisi Zhu (pp. 154-159).
► Egg yolk antibody (IgY) based immunotherapy is promising in combating influenza. ► Immunized hen producing numerous inexpensive IgY antibody. ► Western blot demonstrates virus membrane protein-bonding specificity. ► Plaque reduction assay showed in vitro antiviral ability. ► Intranasal treatment resulted in significant virus clearance in challenged mice.The aim of this study was to prepare egg yolk immunoglobulin (IgY) for use in the prevention and treatment of influenza B viral infections. Laying hens were immunized with inactivated influenza B virus (IBV), and IgY was isolated from the egg yolk by multiple polyethylene glycol (PEG) 6000 extraction and ammonium sulfate purification steps. The titers and specificity of the purified antibodies were assessed. The specific IgY titer increased beginning the second week after the first immunization, with the titer peaking at the fifth week. The yield of IgY was 76.5mg per yolk, and the purity was 98.2%. The use of western blotting and the hemagglutination inhibition (HI) test demonstrated that IBV-specific IgY binds specifically to influenza B virus proteins, and a plaque reduction assay revealed the neutralization efficacy of IBV-specific IgY at reducing influenza infection in MDCK cells. Furthermore, when mice were treated intranasally prior to or after influenza B virus infection, IBV-specific IgY protected the mice from influenza infection or reduced viral replication in their lungs, respectively. These findings indicate that IgY is an easily prepared and rich source of antibodies that offers a potential alternative strategy for preventing and treating influenza B infections.
Keywords: Antiviral; Influenza B virus; Egg yolk immunoglobulin (IgY); Positive immunization; Plaque reduction assay
Preparation and characterization of egg yolk immunoglobulin Y specific to influenza B virus by Junlin Wen; Suqing Zhao; Daigui He; Yuane Yang; Yueming Li; Sisi Zhu (pp. 154-159).
► Egg yolk antibody (IgY) based immunotherapy is promising in combating influenza. ► Immunized hen producing numerous inexpensive IgY antibody. ► Western blot demonstrates virus membrane protein-bonding specificity. ► Plaque reduction assay showed in vitro antiviral ability. ► Intranasal treatment resulted in significant virus clearance in challenged mice.The aim of this study was to prepare egg yolk immunoglobulin (IgY) for use in the prevention and treatment of influenza B viral infections. Laying hens were immunized with inactivated influenza B virus (IBV), and IgY was isolated from the egg yolk by multiple polyethylene glycol (PEG) 6000 extraction and ammonium sulfate purification steps. The titers and specificity of the purified antibodies were assessed. The specific IgY titer increased beginning the second week after the first immunization, with the titer peaking at the fifth week. The yield of IgY was 76.5mg per yolk, and the purity was 98.2%. The use of western blotting and the hemagglutination inhibition (HI) test demonstrated that IBV-specific IgY binds specifically to influenza B virus proteins, and a plaque reduction assay revealed the neutralization efficacy of IBV-specific IgY at reducing influenza infection in MDCK cells. Furthermore, when mice were treated intranasally prior to or after influenza B virus infection, IBV-specific IgY protected the mice from influenza infection or reduced viral replication in their lungs, respectively. These findings indicate that IgY is an easily prepared and rich source of antibodies that offers a potential alternative strategy for preventing and treating influenza B infections.
Keywords: Antiviral; Influenza B virus; Egg yolk immunoglobulin (IgY); Positive immunization; Plaque reduction assay
Deletion of the vaccinia virus F13L gene results in a highly attenuated virus that mounts a protective immune response against subsequent vaccinia virus challenge by Inge Vliegen; Guang Yang; Dennis Hruby; Robert Jordan; Johan Neyts (pp. 160-166).
► Deletion of the F13L gene from vaccinia virus results in a highly attenuated virus in mice. ► F13L deleted vaccinia virus induces a protective immune response against a subsequent infection. ► F13L deleted vaccinia virus as novel smallpox vaccine strategy.Vaccinia virus F13L encodes the envelope protein p37, which is the target of the anti-pox virus drug ST-246 () and that is required for production of extracellular vaccinia virus. The F13L (p37)-deleted (and ST-246 resistant) vaccinia virus recombinant (Vac-ΔF13L) produced smaller plaques than the wild-type vaccinia (Western Reserve vaccinia). In addition, Vac-ΔF13L proved, when inoculated either intravenously or intracutaneously in both immunocompetent and immunodeficient (athymic nude or SCID) mice, to be severely attenuated. Intravenous or intracutaneous inoculation of immunocompetent mice with the ΔF13L virus efficiently protected against a subsequent intravenous, intracutaneous or intranasal challenge with vaccinia WR (Western Reserve). This was corroborated by the observation that Vac-ΔF13L induced a humoral immune response against vaccinia following either intravenous or intracutaneous challenge. In conclusion, F13L-deleted vaccinia virus may have the potential to be developed as a smallpox vaccine.
Keywords: Smallpox; Vaccinia; Vaccination; F13L; Mouse
Deletion of the vaccinia virus F13L gene results in a highly attenuated virus that mounts a protective immune response against subsequent vaccinia virus challenge by Inge Vliegen; Guang Yang; Dennis Hruby; Robert Jordan; Johan Neyts (pp. 160-166).
► Deletion of the F13L gene from vaccinia virus results in a highly attenuated virus in mice. ► F13L deleted vaccinia virus induces a protective immune response against a subsequent infection. ► F13L deleted vaccinia virus as novel smallpox vaccine strategy.Vaccinia virus F13L encodes the envelope protein p37, which is the target of the anti-pox virus drug ST-246 () and that is required for production of extracellular vaccinia virus. The F13L (p37)-deleted (and ST-246 resistant) vaccinia virus recombinant (Vac-ΔF13L) produced smaller plaques than the wild-type vaccinia (Western Reserve vaccinia). In addition, Vac-ΔF13L proved, when inoculated either intravenously or intracutaneously in both immunocompetent and immunodeficient (athymic nude or SCID) mice, to be severely attenuated. Intravenous or intracutaneous inoculation of immunocompetent mice with the ΔF13L virus efficiently protected against a subsequent intravenous, intracutaneous or intranasal challenge with vaccinia WR (Western Reserve). This was corroborated by the observation that Vac-ΔF13L induced a humoral immune response against vaccinia following either intravenous or intracutaneous challenge. In conclusion, F13L-deleted vaccinia virus may have the potential to be developed as a smallpox vaccine.
Keywords: Smallpox; Vaccinia; Vaccination; F13L; Mouse
E17A mutation in HIV-1 Vpr confers resistance to didanosine in association with thymidine analog mutations by Slim Fourati; Isabelle Malet; Carolin A. Guenzel; Cathia Soulie; Priscilla Maidou-Peindara; Laurence Morand-Joubert; Marc Wirden; Sophie Sayon; Gilles Peytavin; Anne Simon; Christine Katlama; Serge Benichou; Vincent Calvez; Anne-Geneviève Marcelin (pp. 167-174).
► Vpr E17A frequent in antiretroviral experienced patients failing HAART. ► Vpr E17A associated with TAMs and the use of didanosine in patients treatment histories. ► Decrease in didanosine susceptibility of HIV-1 harboring Vpr E17A with TAMs in phenotypic assays.HIV-1 accessory Vpr protein is involved in the reverse transcription process and has been shown to modulate the virus mutation rate. This process may play a role in the kinetics of appearance of drug resistance mutations under antiretroviral treatment.Vpr sequences were analyzed from plasma viruses derived from 97 HIV-1-infected individuals failing antiretroviral treatment and 63 antiretroviral-naïve patients. Vpr genetic variability was analyzed for association with specific drug treatment and drug resistance mutations. Biological and virological experiments were employed to characterize a mutation in Vpr found to be associated with virological failure.E17A mutation located in the first α-helix of Vpr was more prevalent in HAART-treated individuals compared to untreated individuals. E17A was associated with thymidine analog mutations (TAMs) in reverse transcriptase M41L, L210W and T215Y and with the use of didanosine in the patients’ treatment histories. E17A had no impact on the biochemical and functional properties of Vpr, and did not affect kinetics of replication of wild-type or TAMs-containing viruses. However, its association with TAMs and the use of didanosine was consistent with phenotypic susceptibility assays showing a significant 3-fold decrease in didanosine susceptibility of viruses harboring Vpr E17A combined with TAMs compared to viruses harboring TAMs alone.These findings highlight a novel role of Vpr in HIV-1 drug resistance. Vpr E17A confers resistance to didanosine when associated with TAMs. Whether Vpr E17A facilitates excision of didanosine is still to be determined.
Keywords: HIV-1; Vpr; Drug resistance; Didanosine
E17A mutation in HIV-1 Vpr confers resistance to didanosine in association with thymidine analog mutations by Slim Fourati; Isabelle Malet; Carolin A. Guenzel; Cathia Soulie; Priscilla Maidou-Peindara; Laurence Morand-Joubert; Marc Wirden; Sophie Sayon; Gilles Peytavin; Anne Simon; Christine Katlama; Serge Benichou; Vincent Calvez; Anne-Geneviève Marcelin (pp. 167-174).
► Vpr E17A frequent in antiretroviral experienced patients failing HAART. ► Vpr E17A associated with TAMs and the use of didanosine in patients treatment histories. ► Decrease in didanosine susceptibility of HIV-1 harboring Vpr E17A with TAMs in phenotypic assays.HIV-1 accessory Vpr protein is involved in the reverse transcription process and has been shown to modulate the virus mutation rate. This process may play a role in the kinetics of appearance of drug resistance mutations under antiretroviral treatment.Vpr sequences were analyzed from plasma viruses derived from 97 HIV-1-infected individuals failing antiretroviral treatment and 63 antiretroviral-naïve patients. Vpr genetic variability was analyzed for association with specific drug treatment and drug resistance mutations. Biological and virological experiments were employed to characterize a mutation in Vpr found to be associated with virological failure.E17A mutation located in the first α-helix of Vpr was more prevalent in HAART-treated individuals compared to untreated individuals. E17A was associated with thymidine analog mutations (TAMs) in reverse transcriptase M41L, L210W and T215Y and with the use of didanosine in the patients’ treatment histories. E17A had no impact on the biochemical and functional properties of Vpr, and did not affect kinetics of replication of wild-type or TAMs-containing viruses. However, its association with TAMs and the use of didanosine was consistent with phenotypic susceptibility assays showing a significant 3-fold decrease in didanosine susceptibility of viruses harboring Vpr E17A combined with TAMs compared to viruses harboring TAMs alone.These findings highlight a novel role of Vpr in HIV-1 drug resistance. Vpr E17A confers resistance to didanosine when associated with TAMs. Whether Vpr E17A facilitates excision of didanosine is still to be determined.
Keywords: HIV-1; Vpr; Drug resistance; Didanosine
Inhibition or deficiency of cathepsin B leads defects in HIV-1 Gag pseudoparticle release in macrophages and HEK293T cells by Soon-Duck Ha; Sangwook Park; Clayton James Hattlmann; Stephen Dominic Barr; Sung Ouk Kim (pp. 175-184).
► The cathepsin B inhibitor CA-074Me prevents HIV-1 Gag particle release. ► Macrophages deficient in cathepsin B are defective in releasing HIV-1 Gag particles. ► CA-074Me also prevents the production of enveloped viruses such as HSV and influenza A virus. ► Cathepsin B can be a new therapeutic target for treating HIV-1 and other enveloped viral infections.Human immunodeficiency virus type 1 (HIV-1) egresses from infected cells through utilizing the host membrane budding mechanisms. Assembly of HIV-1 Gag particles occurs on membranes where the Gag multimers subsequently bud off and form enveloped viral particles. In certain cell types such as macrophages, HIV-1 Gag particles have shown to be released into intracellular virus containing compartments (VCC) such as late endosomes, multivesicular bodies (MVBs) or invaginated plasma membrane pockets. Here, we showed that macrophages or HEK293T cells treated with the cathepsin B (CTSB)-specific inhibitor CA-074Me or cells deficient in CTSB failed to release HIV-1 Gag pseudoparticles into the extracellular environment. Based on immunofluorescence and electron microscopy, these cells retained the pseudoparticles in heterogeneous intracellular VCC. CA-074Me was also able to inhibit propagation of two enveloped viruses, herpes simplex virus and influenza A virus, but not non-enveloped enterovirus. These results suggest that CTSB is required for the efficient release of HIV-1 Gag pseudoparticles and targeting CTSB can be a new therapeutic strategy for inhibiting egress of HIV-1 and other enveloped viruses.
Keywords: HIV; Cathepsin B; Trafficking; Egress; Enveloped virus
Inhibition or deficiency of cathepsin B leads defects in HIV-1 Gag pseudoparticle release in macrophages and HEK293T cells by Soon-Duck Ha; Sangwook Park; Clayton James Hattlmann; Stephen Dominic Barr; Sung Ouk Kim (pp. 175-184).
► The cathepsin B inhibitor CA-074Me prevents HIV-1 Gag particle release. ► Macrophages deficient in cathepsin B are defective in releasing HIV-1 Gag particles. ► CA-074Me also prevents the production of enveloped viruses such as HSV and influenza A virus. ► Cathepsin B can be a new therapeutic target for treating HIV-1 and other enveloped viral infections.Human immunodeficiency virus type 1 (HIV-1) egresses from infected cells through utilizing the host membrane budding mechanisms. Assembly of HIV-1 Gag particles occurs on membranes where the Gag multimers subsequently bud off and form enveloped viral particles. In certain cell types such as macrophages, HIV-1 Gag particles have shown to be released into intracellular virus containing compartments (VCC) such as late endosomes, multivesicular bodies (MVBs) or invaginated plasma membrane pockets. Here, we showed that macrophages or HEK293T cells treated with the cathepsin B (CTSB)-specific inhibitor CA-074Me or cells deficient in CTSB failed to release HIV-1 Gag pseudoparticles into the extracellular environment. Based on immunofluorescence and electron microscopy, these cells retained the pseudoparticles in heterogeneous intracellular VCC. CA-074Me was also able to inhibit propagation of two enveloped viruses, herpes simplex virus and influenza A virus, but not non-enveloped enterovirus. These results suggest that CTSB is required for the efficient release of HIV-1 Gag pseudoparticles and targeting CTSB can be a new therapeutic strategy for inhibiting egress of HIV-1 and other enveloped viruses.
Keywords: HIV; Cathepsin B; Trafficking; Egress; Enveloped virus
Prevalence, virology and antiviral drugs susceptibility of hepatitis B virus rtN238H polymerase mutation from 1865 Chinese patients with chronic hepatitis B by Yanwei Zhong; Jiyun Lv; Jin Li; Xiaoyan Xing; Hua Zhu; Heling Su; Li Chen; Xianzhi Zhou (pp. 185-190).
► The prevalence and susceptibility to LAM and ADV of the hepatitis B virus rtN238H mutation. ► Frequently occur in genotype B-infected patients, even without exogenous selection pressures. ► Do neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro.Amino acid substitutions at positions rtN238T/D of the hepatitis B virus (HBV) polymerase have been reported as potential mutations associated with adefovir (ADV) resistance. In this study, we characterized the prevalence of the rtN238H mutation and determined the susceptibility to LAM and ADV using phenotypic analyzes in vitro. One thousand eight hundred and sixty-five HBsAg-positive patients with chronic HBV (CHB) infection were included in this study. HBV genotypes and reverse transcriptase (RT) mutations were determined by direct sequencing. Replication-competent HBV constructs containing the naturally occurring rtN238H mutation were generated and replication capacity and susceptibility to LAM and ADV in transiently transfected hepatoma cell lines were determined. Among 1865 enrolled HBV infected patients, 8.8% (165/1865) showed mutations in the rtN238 locus (143 males/22 females, 91 treatment-naive, 42 ADV-treated, 16 LAM-treated and 16 ADV+LAM-treated), namely 86% rtN238H (142/165), 5.5% rtN238S (9/165), 5.5% rtN238T (9/165) and 3% rtN238D (5/165). Among the rtN238H mutant strains, there were no significant differences between ADV- or/and LAM- treated patients and treated-naive patients (42% vs. 58%). Compared with wild-type HBV, this mutant displayed an equivalent susceptibility to LAM or ADV in phenotypic assays. Importantly, we found that the incidence rate of rtN238H was higher in HBV genotype B infected patients than HBV genotype C subsets (80.3% vs. 19.7%), even without exogenous selection pressures. As rtN238H did neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtN238H likely represents background polymorphisms rather than resistance mutations with clinical implications. The incidence of rtN238H may be associated with HBV genotype.
Keywords: Hepatitis B virus; Lamivudine; Adefovir; Drug resistance; Polymorphism
Prevalence, virology and antiviral drugs susceptibility of hepatitis B virus rtN238H polymerase mutation from 1865 Chinese patients with chronic hepatitis B by Yanwei Zhong; Jiyun Lv; Jin Li; Xiaoyan Xing; Hua Zhu; Heling Su; Li Chen; Xianzhi Zhou (pp. 185-190).
► The prevalence and susceptibility to LAM and ADV of the hepatitis B virus rtN238H mutation. ► Frequently occur in genotype B-infected patients, even without exogenous selection pressures. ► Do neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro.Amino acid substitutions at positions rtN238T/D of the hepatitis B virus (HBV) polymerase have been reported as potential mutations associated with adefovir (ADV) resistance. In this study, we characterized the prevalence of the rtN238H mutation and determined the susceptibility to LAM and ADV using phenotypic analyzes in vitro. One thousand eight hundred and sixty-five HBsAg-positive patients with chronic HBV (CHB) infection were included in this study. HBV genotypes and reverse transcriptase (RT) mutations were determined by direct sequencing. Replication-competent HBV constructs containing the naturally occurring rtN238H mutation were generated and replication capacity and susceptibility to LAM and ADV in transiently transfected hepatoma cell lines were determined. Among 1865 enrolled HBV infected patients, 8.8% (165/1865) showed mutations in the rtN238 locus (143 males/22 females, 91 treatment-naive, 42 ADV-treated, 16 LAM-treated and 16 ADV+LAM-treated), namely 86% rtN238H (142/165), 5.5% rtN238S (9/165), 5.5% rtN238T (9/165) and 3% rtN238D (5/165). Among the rtN238H mutant strains, there were no significant differences between ADV- or/and LAM- treated patients and treated-naive patients (42% vs. 58%). Compared with wild-type HBV, this mutant displayed an equivalent susceptibility to LAM or ADV in phenotypic assays. Importantly, we found that the incidence rate of rtN238H was higher in HBV genotype B infected patients than HBV genotype C subsets (80.3% vs. 19.7%), even without exogenous selection pressures. As rtN238H did neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtN238H likely represents background polymorphisms rather than resistance mutations with clinical implications. The incidence of rtN238H may be associated with HBV genotype.
Keywords: Hepatitis B virus; Lamivudine; Adefovir; Drug resistance; Polymorphism
U18666A, an intra-cellular cholesterol transport inhibitor, inhibits dengue virus entry and replication by Mee Kian Poh; Guanghou Shui; Xuping Xie; Pei-Yong Shi; Markus R. Wenk; Feng Gu (pp. 191-198).
The level of cholesterol in host cells has been shown to affect viral infection. However, it is still not understood why this level of regulation is important for successful infection. We have shown in this study that dengue virus infection was affected when the cholesterol intake in infected cells was disrupted using a cholesterol transport inhibitor, U18666A. The antiviral effect was found to result from two events: retarded viral trafficking in the cholesterol-loaded late endosomes/lysosomes and suppressed de novo sterol biosynthesis in treated infected cells. We also observed an additive antiviral effect of U18666A with C75, a fatty acid synthase inhibitor, suggesting dengue virus relies on both the host cholesterol and fatty acid biosynthesis for successful replication.
Keywords: Dengue virus; Cholesterol; Antiviral; Fatty acids; U18666A
U18666A, an intra-cellular cholesterol transport inhibitor, inhibits dengue virus entry and replication by Mee Kian Poh; Guanghou Shui; Xuping Xie; Pei-Yong Shi; Markus R. Wenk; Feng Gu (pp. 191-198).
The level of cholesterol in host cells has been shown to affect viral infection. However, it is still not understood why this level of regulation is important for successful infection. We have shown in this study that dengue virus infection was affected when the cholesterol intake in infected cells was disrupted using a cholesterol transport inhibitor, U18666A. The antiviral effect was found to result from two events: retarded viral trafficking in the cholesterol-loaded late endosomes/lysosomes and suppressed de novo sterol biosynthesis in treated infected cells. We also observed an additive antiviral effect of U18666A with C75, a fatty acid synthase inhibitor, suggesting dengue virus relies on both the host cholesterol and fatty acid biosynthesis for successful replication.
Keywords: Dengue virus; Cholesterol; Antiviral; Fatty acids; U18666A
Genotypic characterization of herpes simplex virus DNA polymerase UL42 processivity factor by Sonia Burrel; Zaïna Aït-Arkoub; Henri Agut; David Boutolleau (pp. 199-203).
► Involvement of HSV UL42 processivity factor in antiviral resistance field. ► UL42 processivity factor is highly conserved with a weaker variability for HSV-2. ► Lack of evidence for an association with HSV resistance to currently used antivirals. ► Useful data regarding novel anti-HSV drugs targeting UL42/UL30 protein interaction.The herpes simplex virus (HSV) DNA polymerase is composed of the UL30 catalytic subunit and the UL42 processivity factor. The UL42 subunit increases the processivity of the polymerase along the DNA template during replication. The molecular mechanisms of HSV resistance to drugs interfering with viral DNA synthesis reported so far mainly rely on modifications of the viral thymidine kinase and DNA polymerase. We aimed to extensively describe the genetic variations of HSV UL42 processivity factor and to evaluate its potential involvement in resistance to antivirals. The full-length UL42 gene sequence of HSV was investigated among two laboratory strains (KOS and gHSV-2), 94 drug-sensitive clinical isolates and 25 phenotypically ACV-resistant clinical isolates. This work provided extensive data about natural variability of UL42 processivity factor among both HSV-1 and HSV-2 strains and showed that this viral protein is highly conserved among HSV strains, with a weaker variability for HSV-2. The analysis of 25 HSV clinical isolates exhibiting ACV-resistance documented most of the previously reported mutations related to UL42 natural polymorphism in addition to some unpreviously described polymorphisms. Surprisingly, a single-base deletion in UL42 gene sequence leading to a frameshift in the C-terminal region was identified among 3 HSV clinical isolates. From this preliminary study, UL42 processivity factor did not seem to be likely involved in HSV resistance to antivirals.
Keywords: Herpes simplex virus; UL42 processivity factor; UL30 DNA polymerase; Natural polymorphism; Antiviral resistance
Genotypic characterization of herpes simplex virus DNA polymerase UL42 processivity factor by Sonia Burrel; Zaïna Aït-Arkoub; Henri Agut; David Boutolleau (pp. 199-203).
► Involvement of HSV UL42 processivity factor in antiviral resistance field. ► UL42 processivity factor is highly conserved with a weaker variability for HSV-2. ► Lack of evidence for an association with HSV resistance to currently used antivirals. ► Useful data regarding novel anti-HSV drugs targeting UL42/UL30 protein interaction.The herpes simplex virus (HSV) DNA polymerase is composed of the UL30 catalytic subunit and the UL42 processivity factor. The UL42 subunit increases the processivity of the polymerase along the DNA template during replication. The molecular mechanisms of HSV resistance to drugs interfering with viral DNA synthesis reported so far mainly rely on modifications of the viral thymidine kinase and DNA polymerase. We aimed to extensively describe the genetic variations of HSV UL42 processivity factor and to evaluate its potential involvement in resistance to antivirals. The full-length UL42 gene sequence of HSV was investigated among two laboratory strains (KOS and gHSV-2), 94 drug-sensitive clinical isolates and 25 phenotypically ACV-resistant clinical isolates. This work provided extensive data about natural variability of UL42 processivity factor among both HSV-1 and HSV-2 strains and showed that this viral protein is highly conserved among HSV strains, with a weaker variability for HSV-2. The analysis of 25 HSV clinical isolates exhibiting ACV-resistance documented most of the previously reported mutations related to UL42 natural polymorphism in addition to some unpreviously described polymorphisms. Surprisingly, a single-base deletion in UL42 gene sequence leading to a frameshift in the C-terminal region was identified among 3 HSV clinical isolates. From this preliminary study, UL42 processivity factor did not seem to be likely involved in HSV resistance to antivirals.
Keywords: Herpes simplex virus; UL42 processivity factor; UL30 DNA polymerase; Natural polymorphism; Antiviral resistance
Evaluation of inhaled cidofovir as postexposure prophylactic in an aerosol rabbitpox model by Daniel Verreault; Satheesh K. Sivasubramani; James D. Talton; Lara A. Doyle; Joseph D. Reddy; Stephanie Z. Killeen; Peter J. Didier; Preston A. Marx; Chad J. Roy (pp. 204-208).
► An aerosolized form of cidofovir protects rabbits from aerosol rabbitpox challenge. ► Aerosolized cidofovir provided comparable protection at a fraction of the IV therapeutic dosage. ► Aerosolized treatment significantly reduces lung viral load and corresponding pathology associated with RPX disease.Smallpox is considered a biological threat based upon the possibility of deliberate reintroduction into the population, creating an urgent need for effective antivirals. The antiviral drug cidofovir (Cr) has shown to be effective against poxviruses, although route-specific nephrotoxicity has hampered its development for emergency post-exposure prophylaxis (PEP). In this study, we use a micronized dry powder formulation of pharmaceutical-grade Cr (NanoFOVIRTM; Nf) to treat rabbits exposed to aerosolized rabbitpox virus (RPXV) to further evaluate the effectiveness of direct drug delivery to the lung. Naïve rabbits were infected with RPXV by aerosol; three subsets received aerosolized Nf at 0.5, 1.0 or 1.75mg/kg daily for 3days post-exposure, positive and negative control groups received intravenous (IV) Cr treatments and no treatment, respectively. Nf groups showed an antiviral-dose associated survival of 50% (0.5mg/kg), 80% (1.0mg/kg) and 100% (1.75mg/kg). All animals (100%) from the IV-Cr treatment group and none (0%) from the untreated controls survived. Nf (1.75) protected rabbits from RPX at approximately 10% of the equivalent IV-Cr dose. A dose-related effect was observed in clinical development of RPX disease in Nf groups. Significant reduction of RPX-induced pathological changes was observed in Nf (1.75) and IV-Cr groups. Results suggest that Nf may be a viable antiviral for emergency post-exposure prophylaxis and should be evaluated in other models of poxviral disease.
Keywords: Poxvirus; Rabbitpox; Smallpox treatment; Aerosol; Cidofovir
Evaluation of inhaled cidofovir as postexposure prophylactic in an aerosol rabbitpox model by Daniel Verreault; Satheesh K. Sivasubramani; James D. Talton; Lara A. Doyle; Joseph D. Reddy; Stephanie Z. Killeen; Peter J. Didier; Preston A. Marx; Chad J. Roy (pp. 204-208).
► An aerosolized form of cidofovir protects rabbits from aerosol rabbitpox challenge. ► Aerosolized cidofovir provided comparable protection at a fraction of the IV therapeutic dosage. ► Aerosolized treatment significantly reduces lung viral load and corresponding pathology associated with RPX disease.Smallpox is considered a biological threat based upon the possibility of deliberate reintroduction into the population, creating an urgent need for effective antivirals. The antiviral drug cidofovir (Cr) has shown to be effective against poxviruses, although route-specific nephrotoxicity has hampered its development for emergency post-exposure prophylaxis (PEP). In this study, we use a micronized dry powder formulation of pharmaceutical-grade Cr (NanoFOVIRTM; Nf) to treat rabbits exposed to aerosolized rabbitpox virus (RPXV) to further evaluate the effectiveness of direct drug delivery to the lung. Naïve rabbits were infected with RPXV by aerosol; three subsets received aerosolized Nf at 0.5, 1.0 or 1.75mg/kg daily for 3days post-exposure, positive and negative control groups received intravenous (IV) Cr treatments and no treatment, respectively. Nf groups showed an antiviral-dose associated survival of 50% (0.5mg/kg), 80% (1.0mg/kg) and 100% (1.75mg/kg). All animals (100%) from the IV-Cr treatment group and none (0%) from the untreated controls survived. Nf (1.75) protected rabbits from RPX at approximately 10% of the equivalent IV-Cr dose. A dose-related effect was observed in clinical development of RPX disease in Nf groups. Significant reduction of RPX-induced pathological changes was observed in Nf (1.75) and IV-Cr groups. Results suggest that Nf may be a viable antiviral for emergency post-exposure prophylaxis and should be evaluated in other models of poxviral disease.
Keywords: Poxvirus; Rabbitpox; Smallpox treatment; Aerosol; Cidofovir