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Antiviral Research (v.84, #2)
Reverse genetics technology for Rift Valley fever virus: Current and future applications for the development of therapeutics and vaccines by Michele Bouloy; Ramon Flick (pp. 101-118).
The advent of reverse genetics technology has revolutionized the study of RNA viruses, making it possible to manipulate their genomes and evaluate the effects of these changes on their biology and pathogenesis. The fundamental insights gleaned from reverse genetics-based studies over the last several years provide a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. This review summarizes the successes and stumbling blocks in the development of reverse genetics technologies for Rift Valley fever virus and their application to the further dissection of its pathogenesis and the design of new therapeutics and safe and effective vaccines.
Keywords: Vaccine; Rift Valley fever virus; Non-structural protein; Bunyavirus; Phlebovirus; Reverse genetics; cDNA clone; Infectious clone; Minigenome rescue; Virus-like particle; Attenuation
Reverse genetics technology for Rift Valley fever virus: Current and future applications for the development of therapeutics and vaccines by Michele Bouloy; Ramon Flick (pp. 101-118).
The advent of reverse genetics technology has revolutionized the study of RNA viruses, making it possible to manipulate their genomes and evaluate the effects of these changes on their biology and pathogenesis. The fundamental insights gleaned from reverse genetics-based studies over the last several years provide a new momentum for the development of designed therapies for the control and prevention of these viral pathogens. This review summarizes the successes and stumbling blocks in the development of reverse genetics technologies for Rift Valley fever virus and their application to the further dissection of its pathogenesis and the design of new therapeutics and safe and effective vaccines.
Keywords: Vaccine; Rift Valley fever virus; Non-structural protein; Bunyavirus; Phlebovirus; Reverse genetics; cDNA clone; Infectious clone; Minigenome rescue; Virus-like particle; Attenuation
Wanted, dead or alive: New viral vaccines by Ian J. Amanna; Mark K. Slifka (pp. 119-130).
Vaccination is one of the most effective methods used for protecting the public against infectious disease. Vaccines can be segregated into two general categories: replicating vaccines (i.e., live, attenuated vaccines) and non-replicating vaccines (e.g., inactivated or subunit vaccines). It has been assumed that live attenuated vaccines are superior to non-replicating vaccines in terms of the quality of the antiviral immune response, the level of protective immunity, and the duration of protective immunity. Although this a prevalent viewpoint within the field, there are several exceptions to the rule. Here, we will explore the historical literature in which some of these conclusions have been based, including “Experiments of Nature” and describe examples of the efficacy of replicating vaccines compared to their non-replicating counterparts. By building a better understanding of how successful vaccines work, we hope to develop better “next-generation” vaccines as well as new vaccines against HIV—a pathogen of global importance for which no licensed vaccine currently exists.
Keywords: Immunological memory; T cell; B cell; Antibody; Vaccine; Virus
Wanted, dead or alive: New viral vaccines by Ian J. Amanna; Mark K. Slifka (pp. 119-130).
Vaccination is one of the most effective methods used for protecting the public against infectious disease. Vaccines can be segregated into two general categories: replicating vaccines (i.e., live, attenuated vaccines) and non-replicating vaccines (e.g., inactivated or subunit vaccines). It has been assumed that live attenuated vaccines are superior to non-replicating vaccines in terms of the quality of the antiviral immune response, the level of protective immunity, and the duration of protective immunity. Although this a prevalent viewpoint within the field, there are several exceptions to the rule. Here, we will explore the historical literature in which some of these conclusions have been based, including “Experiments of Nature” and describe examples of the efficacy of replicating vaccines compared to their non-replicating counterparts. By building a better understanding of how successful vaccines work, we hope to develop better “next-generation” vaccines as well as new vaccines against HIV—a pathogen of global importance for which no licensed vaccine currently exists.
Keywords: Immunological memory; T cell; B cell; Antibody; Vaccine; Virus
Inhibition of HSV-1 ocular infection with morpholino oligomers targeting ICP0 and ICP27 by Megan Moerdyk-Schauwecker; David A. Stein; Kathleen Eide; Robert E. Blouch; Rob Bildfell; Patrick Iversen; Ling Jin (pp. 131-141).
Alternative therapies are needed for HSV-1 infections in patients refractory to treatment with Acyclovir (ACV) and its derivatives. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA analogues that enter cells readily and reduce target gene expression through steric blockage of complementary RNA. When applied before or soon after infection PPMO targeting the translation-start-site regions of HSV-1 ICP0 or ICP27 mRNA reduced HSV-1 plaque formation by 70–98% in vitro. The ICP0 PPMO also reduced ACV-resistant HSV-1 (strain 615.9) plaque formation by 70–90%, while an equivalent dose of ACV produced only 40–50% inhibition when the treatment was applied between 1 and 3hpi. Seven daily topical treatments of 100μg ICP0 PPMO caused no gross or microscopic damage to the corneas of uninfected mice. Topical application of 10μg ICP0 PPMO to the eyes of HSV-1 infected mice reduced the incidence of eye disease by 37.5–50% compared to controls. This study demonstrates that topically applied PPMO holds promise as an antiviral drug candidate against HSV-1 ocular infection.
Keywords: Herpes virus; Antiviral; Antisense; Morpholino oligomer
Inhibition of HSV-1 ocular infection with morpholino oligomers targeting ICP0 and ICP27 by Megan Moerdyk-Schauwecker; David A. Stein; Kathleen Eide; Robert E. Blouch; Rob Bildfell; Patrick Iversen; Ling Jin (pp. 131-141).
Alternative therapies are needed for HSV-1 infections in patients refractory to treatment with Acyclovir (ACV) and its derivatives. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA analogues that enter cells readily and reduce target gene expression through steric blockage of complementary RNA. When applied before or soon after infection PPMO targeting the translation-start-site regions of HSV-1 ICP0 or ICP27 mRNA reduced HSV-1 plaque formation by 70–98% in vitro. The ICP0 PPMO also reduced ACV-resistant HSV-1 (strain 615.9) plaque formation by 70–90%, while an equivalent dose of ACV produced only 40–50% inhibition when the treatment was applied between 1 and 3hpi. Seven daily topical treatments of 100μg ICP0 PPMO caused no gross or microscopic damage to the corneas of uninfected mice. Topical application of 10μg ICP0 PPMO to the eyes of HSV-1 infected mice reduced the incidence of eye disease by 37.5–50% compared to controls. This study demonstrates that topically applied PPMO holds promise as an antiviral drug candidate against HSV-1 ocular infection.
Keywords: Herpes virus; Antiviral; Antisense; Morpholino oligomer
Sustained and specific in vitro inhibition of HIV-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes by Reginald Clayton; Asa Ohagen; Francois Nicol; Alfred M. Del Vecchio; Tim H.M. Jonckers; Olivia Goethals; Marnix Van Loock; Lieve Michiels; John Grigsby; Zheng Xu; Yuan Peng Zhang; Lester L. Gutshall; Mark Cunningham; Haiyan Jiang; Sharon Bola; Robert T. Sarisky; Kurt Hertogs (pp. 142-149).
Selective delivery of antiretrovirals to human immunodeficiency virus (HIV) infected cells may reduce toxicities associated with long-term highly active antiretroviral therapy (HAART), may improve therapeutic compliance and delay the emergence of resistance. We developed sterically stabilized pegylated liposomes coated with targeting ligands derived from the Fab’ fragment of HIV-gp120-directed monoclonal antibody F105, and evaluated these liposomes as vehicles for targeted delivery of a novel HIV-1 protease inhibitor. We demonstrated that the immunoliposomes were selectively taken up by HIV-1-infected cells and localized intracellularly, enabling the establishment of a cytoplasmic reservoir of protease inhibitor. In antiviral experiments, the drug delivered by the immunoliposomes showed greater and longer antiviral activity than comparable concentrations of free drug or drug encapsulated in non-targeted liposomes. In conclusion, by combining a targeting moiety with drug-loaded liposomes, efficient and specific uptake by non-phagocytic HIV-infected cells was facilitated, resulting in drug delivery to infected cells. This approach to targeted delivery of antiretroviral compounds may enable the design of drug regimens for patients that allow increased therapeutic adherence and less toxic treatment of HIV infection.
Keywords: HIV-1; Pegylated liposome; Immunoliposome; F105; Protease inhibitor; Targeted therapy
Sustained and specific in vitro inhibition of HIV-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes by Reginald Clayton; Asa Ohagen; Francois Nicol; Alfred M. Del Vecchio; Tim H.M. Jonckers; Olivia Goethals; Marnix Van Loock; Lieve Michiels; John Grigsby; Zheng Xu; Yuan Peng Zhang; Lester L. Gutshall; Mark Cunningham; Haiyan Jiang; Sharon Bola; Robert T. Sarisky; Kurt Hertogs (pp. 142-149).
Selective delivery of antiretrovirals to human immunodeficiency virus (HIV) infected cells may reduce toxicities associated with long-term highly active antiretroviral therapy (HAART), may improve therapeutic compliance and delay the emergence of resistance. We developed sterically stabilized pegylated liposomes coated with targeting ligands derived from the Fab’ fragment of HIV-gp120-directed monoclonal antibody F105, and evaluated these liposomes as vehicles for targeted delivery of a novel HIV-1 protease inhibitor. We demonstrated that the immunoliposomes were selectively taken up by HIV-1-infected cells and localized intracellularly, enabling the establishment of a cytoplasmic reservoir of protease inhibitor. In antiviral experiments, the drug delivered by the immunoliposomes showed greater and longer antiviral activity than comparable concentrations of free drug or drug encapsulated in non-targeted liposomes. In conclusion, by combining a targeting moiety with drug-loaded liposomes, efficient and specific uptake by non-phagocytic HIV-infected cells was facilitated, resulting in drug delivery to infected cells. This approach to targeted delivery of antiretroviral compounds may enable the design of drug regimens for patients that allow increased therapeutic adherence and less toxic treatment of HIV infection.
Keywords: HIV-1; Pegylated liposome; Immunoliposome; F105; Protease inhibitor; Targeted therapy
Phyllaemblicin B inhibits Coxsackie virus B3 induced apoptosis and myocarditis by Wang Ya-Feng; Wang Xiao-Yan; Ren Zhe; Qian Chui-Wen; Li Yi-Cheng; Kaio Kitazato; Wang Qing-Duan; Zhang Yan; Zheng Li-Yun; Jiang Jin-Hua; Yang Chong-Ren; Liu Qing; Zhang Ying-Jun; Wang Yi-Fei (pp. 150-158).
Coxsackie virus B3 (CVB3) is believed to be a major contributor to viral myocarditis since virus-associated apoptosis plays a role in the pathogenesis of experimental myocarditis. In this study, we investigated the in vitro and in vivo antiviral activities of Phyllaemblicin B, the main ellagitannin compound isolated from Phyllanthus emblica, a Chinese herb medicine, against CVB3. Herein we report that Phyllaemblicin B inhibited CVB3-mediated cytopathic effects on HeLa cells with an IC50 value of 7.75±0.15μg/mL. In an in vivo assay, treatment with 12mgkg−1d−1 Phyllaemblicin B reduced cardiac CVB3 titers, decreased the activities of LDH and CK in murine serum, and alleviated pathological damages of cardiac muscle in myocarditic mice. Moreover, Phyllaemblicin B clearly inhibited CVB3-associated apoptosis effects both in vitro and in vivo. These results show that Phyllaemblicin B exerts significant antiviral activities against CVB3. Therefore, Phyllaemblicin B may represent a potential therapeutic agent for viral myocarditis.
Keywords: Coxsackie virus B3; Viral myocarditis; Phyllaemblicin B; Antiviral effect; Apoptosis
Phyllaemblicin B inhibits Coxsackie virus B3 induced apoptosis and myocarditis by Wang Ya-Feng; Wang Xiao-Yan; Ren Zhe; Qian Chui-Wen; Li Yi-Cheng; Kaio Kitazato; Wang Qing-Duan; Zhang Yan; Zheng Li-Yun; Jiang Jin-Hua; Yang Chong-Ren; Liu Qing; Zhang Ying-Jun; Wang Yi-Fei (pp. 150-158).
Coxsackie virus B3 (CVB3) is believed to be a major contributor to viral myocarditis since virus-associated apoptosis plays a role in the pathogenesis of experimental myocarditis. In this study, we investigated the in vitro and in vivo antiviral activities of Phyllaemblicin B, the main ellagitannin compound isolated from Phyllanthus emblica, a Chinese herb medicine, against CVB3. Herein we report that Phyllaemblicin B inhibited CVB3-mediated cytopathic effects on HeLa cells with an IC50 value of 7.75±0.15μg/mL. In an in vivo assay, treatment with 12mgkg−1d−1 Phyllaemblicin B reduced cardiac CVB3 titers, decreased the activities of LDH and CK in murine serum, and alleviated pathological damages of cardiac muscle in myocarditic mice. Moreover, Phyllaemblicin B clearly inhibited CVB3-associated apoptosis effects both in vitro and in vivo. These results show that Phyllaemblicin B exerts significant antiviral activities against CVB3. Therefore, Phyllaemblicin B may represent a potential therapeutic agent for viral myocarditis.
Keywords: Coxsackie virus B3; Viral myocarditis; Phyllaemblicin B; Antiviral effect; Apoptosis
Human H1 promoter expressed short hairpin RNAs (shRNAs) suppress avian influenza virus replication in chicken CH-SAH and canine MDCK cells by Arusyak Abrahamyan; Éva Nagy; Serguei P. Golovan (pp. 159-167).
Influenza A virus represents a significant health and economic threat to both humans and animals worldwide. The polymerase III human H1 promoter was used to express nine short hairpin RNAs (shRNAs) targeting nucleocapsid protein (NP) or the acidic component of polymerase complex (PA) genes of avian influenza virus. Tested shRNAs led to a decrease of up to 400 fold in infective titre and up to 80% in the level of viral NP and PA mRNAs in chicken CH-SAH cells ( p<0.05). In mammalian MDCK cells a decrease of up to 106-fold in infective titre, and up to 90% in the level of viral mRNAs ( p<0.05) was detected. While RNAi silencing played a major role in viral inhibition, transfection of any plasmid induced an antiviral interferon response in MDCK cells. In CH-SAH cells, no induction of IFN response was observed by constructs, and the IFN response was suppressed by influenza virus infection. Simultaneous induction of RNAi and IFN responses in CH-SAH cells resulted in 18,500-fold decrease in infective titre ( p<0.05). We also identified novel efficient and conserved RNAi target site in the NP gene which can be used in antiviral cocktails of shRNAs to block the escape of influenza virus.
Keywords: Abbreviations; IFN; interferon; NP; nucleocapsid protein; PA; acidic component of polymerase complex; Pol; polymerase; RNAi; RNA interference; siRNA; short interfering RNA; shRNA; short hairpin RNAChicken; Avian influenza; H1 promoter; MDCK; RNA interference; shRNA
Human H1 promoter expressed short hairpin RNAs (shRNAs) suppress avian influenza virus replication in chicken CH-SAH and canine MDCK cells by Arusyak Abrahamyan; Éva Nagy; Serguei P. Golovan (pp. 159-167).
Influenza A virus represents a significant health and economic threat to both humans and animals worldwide. The polymerase III human H1 promoter was used to express nine short hairpin RNAs (shRNAs) targeting nucleocapsid protein (NP) or the acidic component of polymerase complex (PA) genes of avian influenza virus. Tested shRNAs led to a decrease of up to 400 fold in infective titre and up to 80% in the level of viral NP and PA mRNAs in chicken CH-SAH cells ( p<0.05). In mammalian MDCK cells a decrease of up to 106-fold in infective titre, and up to 90% in the level of viral mRNAs ( p<0.05) was detected. While RNAi silencing played a major role in viral inhibition, transfection of any plasmid induced an antiviral interferon response in MDCK cells. In CH-SAH cells, no induction of IFN response was observed by constructs, and the IFN response was suppressed by influenza virus infection. Simultaneous induction of RNAi and IFN responses in CH-SAH cells resulted in 18,500-fold decrease in infective titre ( p<0.05). We also identified novel efficient and conserved RNAi target site in the NP gene which can be used in antiviral cocktails of shRNAs to block the escape of influenza virus.
Keywords: Abbreviations; IFN; interferon; NP; nucleocapsid protein; PA; acidic component of polymerase complex; Pol; polymerase; RNAi; RNA interference; siRNA; short interfering RNA; shRNA; short hairpin RNAChicken; Avian influenza; H1 promoter; MDCK; RNA interference; shRNA
Efficient induction of cytotoxic T lymphocytes specific for severe acute respiratory syndrome (SARS)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein 1a by Shunsuke Kohyama; Satoshi Ohno; Tatsuya Suda; Maiko Taneichi; Shoichi Yokoyama; Masahito Mori; Akiharu Kobayashi; Hidenori Hayashi; Tetsuya Uchida; Masanori Matsui (pp. 168-177).
Spike and nucleocapsid are structural proteins of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and major targets for cytotoxic T lymphocytes (CTLs). In contrast, non-structural proteins encoded by two-thirds of viral genome are poorly characterized for cell-mediated immunity. We previously demonstrated that nucleocapsid-derived peptides chemically coupled to the surface of liposomes effectively elicited SARS-CoV-specific CTLs in mice. Here, we attempted to identify HLA-A*0201-restricted CTL epitopes derived from a non-structural polyprotein 1a (pp1a) of SARS-CoV, and investigated whether liposomal peptides derived from pp1a were effective for CTL induction. Out of 30 peptides predicted on computational algorithms, nine peptides could significantly induce interferon gamma (IFN-γ)-producing CD8+ T cells in mice. These peptides were coupled to the surface of liposomes, and inoculated into mice. Six liposomal peptides effectively induced IFN-γ-producing CD8+ T cells and seven liposomal peptides including the six peptides primed CTLs showing in vivo killing activities. Further, CTLs induced by the seven liposomal peptides lysed an HLA-A*0201 positive cell line expressing naturally processed, pp1a-derived peptides. Of note, one of the liposomal peptides induced high numbers of long-lasting memory CTLs. These data suggest that surface-linked liposomal peptides derived from pp1a might offer an efficient CTL-based vaccine against SARS.
Keywords: SARS coronavirus; Cytotoxic T lymphocyte; Peptide; Polyprotein 1a; Liposome; Vaccine
Efficient induction of cytotoxic T lymphocytes specific for severe acute respiratory syndrome (SARS)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein 1a by Shunsuke Kohyama; Satoshi Ohno; Tatsuya Suda; Maiko Taneichi; Shoichi Yokoyama; Masahito Mori; Akiharu Kobayashi; Hidenori Hayashi; Tetsuya Uchida; Masanori Matsui (pp. 168-177).
Spike and nucleocapsid are structural proteins of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and major targets for cytotoxic T lymphocytes (CTLs). In contrast, non-structural proteins encoded by two-thirds of viral genome are poorly characterized for cell-mediated immunity. We previously demonstrated that nucleocapsid-derived peptides chemically coupled to the surface of liposomes effectively elicited SARS-CoV-specific CTLs in mice. Here, we attempted to identify HLA-A*0201-restricted CTL epitopes derived from a non-structural polyprotein 1a (pp1a) of SARS-CoV, and investigated whether liposomal peptides derived from pp1a were effective for CTL induction. Out of 30 peptides predicted on computational algorithms, nine peptides could significantly induce interferon gamma (IFN-γ)-producing CD8+ T cells in mice. These peptides were coupled to the surface of liposomes, and inoculated into mice. Six liposomal peptides effectively induced IFN-γ-producing CD8+ T cells and seven liposomal peptides including the six peptides primed CTLs showing in vivo killing activities. Further, CTLs induced by the seven liposomal peptides lysed an HLA-A*0201 positive cell line expressing naturally processed, pp1a-derived peptides. Of note, one of the liposomal peptides induced high numbers of long-lasting memory CTLs. These data suggest that surface-linked liposomal peptides derived from pp1a might offer an efficient CTL-based vaccine against SARS.
Keywords: SARS coronavirus; Cytotoxic T lymphocyte; Peptide; Polyprotein 1a; Liposome; Vaccine
A novel HCV NS3 protease mutation selected by combination treatment of the protease inhibitor boceprevir and NS5B polymerase inhibitors by Robert Chase; Angela Skelton; Ellen Xia; Stephanie Curry; Shaotang Liu; Patricia McMonagle; H.-C. Huang; Xiao Tong (pp. 178-184).
Boceprevir (SCH 503034) is an orally active novel inhibitor of the hepatitis C virus (HCV) NS3 protease currently in clinical development for the treatment of hepatitis C. In this in vitro study, we demonstrate that combination of boceprevir with a nucleoside analog or a non-nucleoside HCV NS5B polymerase inhibitor was superior to treatment by single agents in inhibiting viral RNA replication in replicon cells. In the presence of boceprevir (at 5×EC90), the addition of 2′-C-methyl-adenosine or an indole- N-acetamide targeting the polymerase finger-loop site (at 1×EC90) significantly reduced the emergence of resistant replicon colonies. A higher dose (5×EC90) of either of the polymerase inhibitors in combination with boceprevir suppressed replicon resistance further to below detectable levels. Sequencing analysis of replicon cells selected by the combination treatment revealed known resistance mutations to the two polymerase inhibitors but no previously reported resistance mutations to boceprevir. Interestingly, a novel mutation (M175L) in the protease domain was identified. The dually resistant replicon cells were monitored for over 30 passages and sensitivity to polymerase inhibitors was found to decrease over time in a manner that correlated with the increasing prevalence of specific resistance mutations. Importantly, these cells remained sensitive to interferon-α and different classes of polymerase inhibitors. These findings support the rationale for clinical evaluation of combination treatment of HCV protease and polymerase inhibitors.
Keywords: HCV protease inhibitor; HCV polymerase inhibitor; Resistance; Combination therapy
A novel HCV NS3 protease mutation selected by combination treatment of the protease inhibitor boceprevir and NS5B polymerase inhibitors by Robert Chase; Angela Skelton; Ellen Xia; Stephanie Curry; Shaotang Liu; Patricia McMonagle; H.-C. Huang; Xiao Tong (pp. 178-184).
Boceprevir (SCH 503034) is an orally active novel inhibitor of the hepatitis C virus (HCV) NS3 protease currently in clinical development for the treatment of hepatitis C. In this in vitro study, we demonstrate that combination of boceprevir with a nucleoside analog or a non-nucleoside HCV NS5B polymerase inhibitor was superior to treatment by single agents in inhibiting viral RNA replication in replicon cells. In the presence of boceprevir (at 5×EC90), the addition of 2′-C-methyl-adenosine or an indole- N-acetamide targeting the polymerase finger-loop site (at 1×EC90) significantly reduced the emergence of resistant replicon colonies. A higher dose (5×EC90) of either of the polymerase inhibitors in combination with boceprevir suppressed replicon resistance further to below detectable levels. Sequencing analysis of replicon cells selected by the combination treatment revealed known resistance mutations to the two polymerase inhibitors but no previously reported resistance mutations to boceprevir. Interestingly, a novel mutation (M175L) in the protease domain was identified. The dually resistant replicon cells were monitored for over 30 passages and sensitivity to polymerase inhibitors was found to decrease over time in a manner that correlated with the increasing prevalence of specific resistance mutations. Importantly, these cells remained sensitive to interferon-α and different classes of polymerase inhibitors. These findings support the rationale for clinical evaluation of combination treatment of HCV protease and polymerase inhibitors.
Keywords: HCV protease inhibitor; HCV polymerase inhibitor; Resistance; Combination therapy
Utility of RNAi-mediated prnp gene silencing in neuroblastoma cells permanently infected by prions: Potentials and limitations by Younghwan Kim; Boram Han; William Titlow; Charles E. Mays; Moosik Kwon; Chongsuk Ryou (pp. 185-193).
Prion diseases are incurable, transmissible neurodegenerative disorders in humans and animals. Because the disease-associated isoform of prion protein, PrPSc, is conformationally converted from cellular prion protein, PrPC, knockdown of PrPC expression by RNA interference (RNAi) implicates therapy for prion diseases. In this study, introduction of small interfering (si) and small hairpin (sh) RNAs targeting the prion protein gene ( prnp) transcripts triggered specific gene silencing and reduced the PrPC level in both prion-free and -infected neuroblastoma cell lines. Furthermore, this approach suppressed PrPSc formation and ultimately eliminated PrPSc from prion-infected cell lines. However, prolonged culture of cured cells resulted in reappearance of PrPSc in the cell population, presumably by de novo PrPSc formation from residual PrPC uncontrolled by RNAi and PrPSc remained under the detection limit. Protein misfolding cyclic amplification assays further confirmed that lysate of cured cells was sufficient to support PrPSc propagation. Our data not only suggest a potential treatment option but also implicate a caveat for using an RNAi approach for prion diseases. These findings provide critical information required to advance RNAi-based prevention and therapy for prion diseases of humans and animals.
Keywords: Prion; PrP; C; PrP; Sc; RNAi; siRNA; shRNA
Utility of RNAi-mediated prnp gene silencing in neuroblastoma cells permanently infected by prions: Potentials and limitations by Younghwan Kim; Boram Han; William Titlow; Charles E. Mays; Moosik Kwon; Chongsuk Ryou (pp. 185-193).
Prion diseases are incurable, transmissible neurodegenerative disorders in humans and animals. Because the disease-associated isoform of prion protein, PrPSc, is conformationally converted from cellular prion protein, PrPC, knockdown of PrPC expression by RNA interference (RNAi) implicates therapy for prion diseases. In this study, introduction of small interfering (si) and small hairpin (sh) RNAs targeting the prion protein gene ( prnp) transcripts triggered specific gene silencing and reduced the PrPC level in both prion-free and -infected neuroblastoma cell lines. Furthermore, this approach suppressed PrPSc formation and ultimately eliminated PrPSc from prion-infected cell lines. However, prolonged culture of cured cells resulted in reappearance of PrPSc in the cell population, presumably by de novo PrPSc formation from residual PrPC uncontrolled by RNAi and PrPSc remained under the detection limit. Protein misfolding cyclic amplification assays further confirmed that lysate of cured cells was sufficient to support PrPSc propagation. Our data not only suggest a potential treatment option but also implicate a caveat for using an RNAi approach for prion diseases. These findings provide critical information required to advance RNAi-based prevention and therapy for prion diseases of humans and animals.
Keywords: Prion; PrP; C; PrP; Sc; RNAi; siRNA; shRNA
Single and multiple mutations in the human cytomegalovirus UL97 gene and their relationship to the enzymatic activity of UL97 kinase for ganciclovir phosphorylation by Silvia I. Sanchez Puch; Verónica L. Mathet; Margarita Porta; María L. Cuestas; José R. Oubiña; Cristina M. Videla; Horacio E. Salomón (pp. 194-198).
In this study we determined that the double mutant M460V/D605E in the UL97 gene of an HCMV isolate from an immunocompromised patient (MMT isolate) is related to resistance to ganciclovir (GCV) therapy. Our results suggest that the aspartic acid-to-glutamic acid substitution at codon 605 may be associated with a natural polymorphism of the UL97 gene, and not with positive selection pressure exerted by the antiviral drug. We also determined that GCV resistance due to the M460V mutation in the HCMV UL97 gene is not offset by a second mutation (D605E) at codon 605. Furthermore, we showed that when the two mutations related to GCV resistance were simultaneously detected in the same HCMV construct, virus-drug resistance might be enhanced in comparison to that of the single mutants studied separately. To our knowledge for the first time, seven of 12 amino acid changes (F102L, D118V, M330T, T400A, R507P and C511R and I533V) in the UL97 gene of an isolate are herein reported.
Keywords: MMT isolate; Human cytomegalovirus; GCV resistance; Polymorphism
Single and multiple mutations in the human cytomegalovirus UL97 gene and their relationship to the enzymatic activity of UL97 kinase for ganciclovir phosphorylation by Silvia I. Sanchez Puch; Verónica L. Mathet; Margarita Porta; María L. Cuestas; José R. Oubiña; Cristina M. Videla; Horacio E. Salomón (pp. 194-198).
In this study we determined that the double mutant M460V/D605E in the UL97 gene of an HCMV isolate from an immunocompromised patient (MMT isolate) is related to resistance to ganciclovir (GCV) therapy. Our results suggest that the aspartic acid-to-glutamic acid substitution at codon 605 may be associated with a natural polymorphism of the UL97 gene, and not with positive selection pressure exerted by the antiviral drug. We also determined that GCV resistance due to the M460V mutation in the HCMV UL97 gene is not offset by a second mutation (D605E) at codon 605. Furthermore, we showed that when the two mutations related to GCV resistance were simultaneously detected in the same HCMV construct, virus-drug resistance might be enhanced in comparison to that of the single mutants studied separately. To our knowledge for the first time, seven of 12 amino acid changes (F102L, D118V, M330T, T400A, R507P and C511R and I533V) in the UL97 gene of an isolate are herein reported.
Keywords: MMT isolate; Human cytomegalovirus; GCV resistance; Polymorphism
Amantadine-resistant influenza A viruses isolated in South Korea from 2003 to 2009 by Woo-Young Choi; SuJin Kim; NamJoo Lee; Meehwa Kwon; InSeok Yang; Min-Ji Kim; Seul-Gi Cheong; Donghyok Kwon; Joo-Yeon Lee; Hee-Bok Oh; Chun Kang (pp. 199-202).
To investigate the frequency of amantadine resistance among influenza A viruses isolated in Korea during the 2003–2009 seasons, 369 (16.8%) 2199 A/H1N1 viruses and 780 (14.8%) of 5263 A/H3N2 viruses were randomly selected. The M2 and HA1 genes of each isolate were amplified by reverse transcription-polymerase chain reaction and followed by nucleotide sequencing. The results showed that the resistance rate to amantadine among A/H1N1 viruses increased significantly from 2004–2005 (33.3%) to 2007–2008 (97.8%) and then decreased dramatically in 2008–2009 (1.9%). The A/H1N1 isolates recently detected in 2008–2009 turned amantadine-sensitive containing two new substitutions at specific sites (S141N, G185A) in HA1. Compared with A/H1N1 viruses, the amantadine resistance among the A/H3N2 viruses increased from 2003–2004 (9.7%) to 2005–2006 (96.7%) and decreased in 2006–2007 (57.4%). During 2006–2007, both of amantadine-resistant and -sensitive A/H3N2 viruses co-circulated but clustered in different branches phylogenetically. All of A/H3N2 isolates tested during 2007–2009 appeared to cluster in the same group being resistant to amantadine.
Keywords: Amantadine resistance; Influenza A virus; Phylogenetic analysis; South Korea
Amantadine-resistant influenza A viruses isolated in South Korea from 2003 to 2009 by Woo-Young Choi; SuJin Kim; NamJoo Lee; Meehwa Kwon; InSeok Yang; Min-Ji Kim; Seul-Gi Cheong; Donghyok Kwon; Joo-Yeon Lee; Hee-Bok Oh; Chun Kang (pp. 199-202).
To investigate the frequency of amantadine resistance among influenza A viruses isolated in Korea during the 2003–2009 seasons, 369 (16.8%) 2199 A/H1N1 viruses and 780 (14.8%) of 5263 A/H3N2 viruses were randomly selected. The M2 and HA1 genes of each isolate were amplified by reverse transcription-polymerase chain reaction and followed by nucleotide sequencing. The results showed that the resistance rate to amantadine among A/H1N1 viruses increased significantly from 2004–2005 (33.3%) to 2007–2008 (97.8%) and then decreased dramatically in 2008–2009 (1.9%). The A/H1N1 isolates recently detected in 2008–2009 turned amantadine-sensitive containing two new substitutions at specific sites (S141N, G185A) in HA1. Compared with A/H1N1 viruses, the amantadine resistance among the A/H3N2 viruses increased from 2003–2004 (9.7%) to 2005–2006 (96.7%) and decreased in 2006–2007 (57.4%). During 2006–2007, both of amantadine-resistant and -sensitive A/H3N2 viruses co-circulated but clustered in different branches phylogenetically. All of A/H3N2 isolates tested during 2007–2009 appeared to cluster in the same group being resistant to amantadine.
Keywords: Amantadine resistance; Influenza A virus; Phylogenetic analysis; South Korea
Corrigendum to “The transport of anti-HIV drugs across blood–CNS interfaces: Summary of current knowledge and recommendations for further research” [Antiviral Res. 82 (2) (2009) A99–A109]
by Lavanya Varatharajan; Sarah A. Thomas (pp. 203-203).
Corrigendum to “The transport of anti-HIV drugs across blood–CNS interfaces: Summary of current knowledge and recommendations for further research” [Antiviral Res. 82 (2) (2009) A99–A109]
by Lavanya Varatharajan; Sarah A. Thomas (pp. 203-203).