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Antiviral Research (v.91, #1)
Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication by Dae-Gyun Ahn; Wooseong Lee; Jin-Kyu Choi; Seong-Jun Kim; Ewan P. Plant; Fernando Almazán; Deborah R. Taylor; Luis Enjuanes; Jong-Won Oh (pp. 1-10).
The programmed −1 ribosomal frameshifting (−1 PRF) utilized by eukaryotic RNA viruses plays a crucial role for the controlled, limited synthesis of viral RNA replicase polyproteins required for genome replication. The viral RNA replicase polyproteins of severe acute respiratory syndrome coronavirus (SARS-CoV) are encoded by the two overlapping open reading frames 1a and 1b, which are connected by a −1 PRF signal. We evaluated the antiviral effects of antisense peptide nucleic acids (PNAs) targeting a highly conserved RNA sequence on the – PRF signal. The ribosomal frameshifting was inhibited by the PNA, which bound sequence-specifically a pseudoknot structure in the −1 PRF signal, in cell lines as assessed using a dual luciferase-based reporter plasmid containing the −1 PRF signal. Treatment of cells, which were transfected with a SARS-CoV-replicon expressing firefly luciferase, with the PNA fused to a cell-penetrating peptide (CPP) resulted in suppression of the replication of the SARS-CoV replicon, with a 50% inhibitory concentration of 4.4μM. There was no induction of type I interferon responses by PNA treatment, suggesting that the effect of PNA is not due to innate immune responses. Our results demonstrate that −1 PRF, critical for SARS-CoV viral replication, can be inhibited by CPP-PNA, providing an effective antisense strategy for blocking −1 PRF signals.
Keywords: SARS-CoV; Frameshifting; Peptide nucleic acids; RNA replication
Development and characterization of a stable luciferase dengue virus for high-throughput screening by Gang Zou; Hao Ying Xu; Min Qing; Qing-Yin Wang; Pei-Yong Shi (pp. 11-19).
To facilitate dengue virus (DENV) drug discovery, we developed a stable luciferase reporter DENV-2. A renilla luciferase gene was engineered into the capsid-coding region of an infectious cDNA clone of DENV-2. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated high titers (>106PFU/ml) of luciferase reporter DENV-2. The reporter virus was infectious to a variety of cells, producing robust luciferase signals. Compared with wild-type virus, the reporter virus replicated slower in both mammalian Vero and mosquito C6/36 cells. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. All passaged viruses stably maintained the luciferase gene, demonstrating the stability of the reporter virus. Furthermore, we found that the passaged virus accumulated a mutation (T108M) in viral NS4B gene that could enhance viral RNA replication in a cell-type specific manner. Using the reporter virus, we developed a HTS assay in a 384-well format. The HTS assay was validated with known DENV inhibitors and showed a robust Z′ factor of 0.79. The Luc-DENV-2 HTS assay allows screening for inhibitors of all steps of the viral life cycle. The reporter virus will also be a useful tool for studying DENV replication and pathogenesis.
Keywords: Abbreviations; DENV; dengue virus; HTS; high-throughput screen; C; capsid protein; prM; premembrane; E; envelope; NS; non-structural protein; VLP; virus-like particle; Rluc; Renilla; luciferase; FMDV2A; foot-and-mouth disease virus 2A autocleavage siteDengue virus; Luciferase reporter; High-throughput screening; Antiviral drug discovery
Mixed influenza A and B infections complicate the detection of influenza viruses with altered sensitivities to neuraminidase inhibitors by Peter G. Mohr; Henriette Geyer; Jennifer L. McKimm-Breschkin (pp. 20-22).
Previously, three influenza A(H3N2) isolates with a reduced susceptibility to the neuraminidase inhibitors (NAIs) zanamivir and oseltamivir were identified during screening by the Neuraminidase Inhibitor Susceptibility Network (NISN). The isolates were from untreated patients from the first three years post licensure of the NAIs. We plaque-purified progeny from each of these isolates and determined the NAI sensitivity of each plaqued population. Sequencing and serology for each population revealed that the isolates contained a mix of wild type influenza A(H3N2) and influenza B. The NAI susceptibility reductions that had originally been reported were a consequence of influenza B neuraminidases that have lower relative NAI sensitivities, rather than being due to resistant influenza A(H3N2) viruses. Our study highlights the need to check for mixed influenza infections when isolates with potentially lower sensitivities to NAIs are identified.
Keywords: Influenza A; Influenza B; Oseltamivir; Zanamivir; Mixed infections
Effect of telbivudine therapy on the cellular immune response in chronic hepatitis B by Yu Chen; Xuefen Li; Bo Ye; Xianzhi Yang; Wei Wu; Baode Chen; Xiaoping Pan; Hongcui Cao; Lanjuan Li (pp. 23-31).
Weak T-cell reactivity to the hepatitis B virus (HBV) is believed to be the dominant cause of chronic HBV infection. Several lines of experimental evidence suggest that treatment with telbivudine increases the rate of HBV e antigen (HBeAg) loss, undetectable HBV DNA, and normalization of serum alanine aminotransferase (ALT) in chronic hepatitis B patients (CHB). However, it is still unclear how early antiviral therapy affects cellular immune responses during sustained telbivudine treatment. In order to investigate this issue, we measured detailed prospective clinical, virological, and biochemical parameters, and we examined the frequency of T cell subgroups as well as the ability of peripheral blood mononuclear cells (PBMC) to respond to stimuli at five protocol time points for 51 CHB patients who received telbivudine therapy for one year. The preliminary data from this study revealed that effective-treated patients showed an increased frequency of peripheral blood CD4+T lymphocytes, an augmented proliferative response of HBV-specific T-cells to the hepatitis B core antigen (HBcAg), and the induction of cytokines, such as interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α) release at the site of infection compared to non-responsive patients. Enhanced HBV-specific T-cell reactivity to telbivudine therapy, which peaked at treatment week 12, was confined to a subgroup of effective-treated patients who achieved greater viral suppression.
Keywords: Chronic hepatitis B; Lymphocyte subgroups; Telbivudine; Antiviral therapy
Genotypic characterization of human cytomegalovirus UL97 phosphotransferase natural polymorphism in the era of ganciclovir and maribavir by David Boutolleau; Sonia Burrel; Henri Agut (pp. 32-35).
The molecular mechanisms of human cytomegalovirus (CMV) resistance to both ganciclovir and maribavir reported so far rely predominantly on the presence of mutations within UL97 phosphotransferase. The accurate interpretation of genotypic antiviral resistance assay results requires the clear distinction between resistance mutations and natural interstrain sequence variations. The objective of this work was to extend the catalog of CMV UL97 phosphotransferase natural polymorphisms. The full-length UL97 gene sequence analysis from 4 laboratory strains and 35 clinical samples from patients who had not received any previous anti-CMV treatment was performed. At the nucleotide level, the interstrain identity was >98%. At the amino acid level, ten natural polymorphisms never previously described were identified. Together with all previous results reported in the literature, a new map of UL97 phosphotransferase natural polymorphism could be settled in the era of ganciclovir and maribavir.
Keywords: Cytomegalovirus; UL97 phosphotransferase; Natural polymorphism; Genotypic antiviral resistance testing; Ganciclovir; Maribavir
Inhibition of porcine reproductive and respiratory syndrome virus infection in piglets by a peptide-conjugated morpholino oligomer by Tanja Opriessnig; Deendayal Patel; Rong Wang; Patrick G. Halbur; Xiang-Jin Meng; David A. Stein; Yan-Jin Zhang (pp. 36-42).
Porcine reproductive and respiratory syndrome (PRRS) causes substantial economic losses to the swine industry in many countries, and current control strategies are inadequate. Previously, we explored the strategy of using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to inhibit PRRS virus (PRRSV) replication. PPMOs are nuclease-resistant and single-stranded DNA analogs containing a modified backbone conjugated to a cell-penetrating peptide and can act as antisense agents through steric blockade of complementary messenger RNA. A PPMO (designated 5UP2) targeting highly conserved sequence in the 5′-terminal region of the PRRSV genome was found to produce multi-log10 inhibition of PRRSV replication in cultured cells. To evaluate 5UP2 in vivo, we here administrated the PPMO to 3-week-old piglets via intranasal instillation at 24h before, and 2 and 24h after infection with PRRSV (strain VR2385). Blood samples were collected at 6, 10 and 14days post-infection (dpi) for detection of PRRSV RNA and antibodies. Necropsy was performed at 14dpi. Monitoring weight gain in all piglet groups throughout the experiment indicated that PPMO was well tolerated at the doses used. PPMO 5UP2 treatment significantly reduced PRRSV viremia at 6dpi. On day 14, piglets receiving 5UP2 had significantly less interstitial pneumonia and lower level of anti-PRRSV antibodies than untreated piglets. In alveolar macrophages isolated at the time of necropsy, the expression of antiviral genes in PPMO-treated piglets was elevated in comparison with untreated. This study provides further data indicating that the 5UP2 PPMO can be considered a candidate component for a novel PRRS control strategy.
Keywords: Porcine reproductive and respiratory syndrome virus; PRRSV; Morpholino; Antisense; Antiviral; PPMO
Treatment of spatial memory impairment in hamsters infected with West Nile virus using a humanized monoclonal antibody MGAWN1 by Cynthia A. Smeraski; Venkatraman Siddharthan; John D. Morrey (pp. 43-49).
In addition to functional disorders of paresis, paralysis, and cardiopulmonary complications, subsets of West Nile virus (WNV) patients may also experience neurocognitive deficits and memory disturbances. A previous hamster study has also demonstrated spatial memory impairment using the Morris water maze (MWM) paradigm. The discovery of an efficacious therapeutic antibody MGAWN1 from pre-clinical rodent studies raises the possibility of preventing or treating WNV-induced memory deficits. In the current study, hamsters were treated intraperitoneally (i.p.) with 32mg/kg of MGAWN1 at 4.5days after subcutaneously (s.c.) challenging with WNV. As expected, MGAWN1 prevented mortality, weight loss, and improved food consumption of WNV-infected hamsters. The criteria for entry of surviving hamsters into the study were that they needed to have normal motor function (forelimb grip strength, beam walking) and normal spatial reference memory in the MWM probe task. Twenty-eight days after the acute phase of the disease had passed, MGAWN1- and saline-treated infected hamsters were again trained in the MWM. Spatial memory was evaluated 48h after this training in which the hamsters searched for the location where a submerged escape platform had been positioned. Only 56% of infected hamsters treated with saline spent more time in the correct quadrant than the other three quadrants, as compared to 92% of MGAWN1-treated hamsters ( P⩽0.05). Overall these studies support the possibility that WNV can cause spatial memory impairment and that therapeutic intervention may be considered.
Keywords: West Nile virus; MGAWN1; Hamsters; Spatial memory; Monoclonal antibody
Immune responses of chickens inoculated with a recombinant fowlpox vaccine coexpressing HA of H9N2 avain influenza virus and chicken IL-18 by Hong-Ying Chen; Yan-Hong Shang; Hui-Xia Yao; Bao-An Cui; Hong-Ying Zhang; Zi-Xin Wang; Ya-Dan Wang; An-Jun Chao; Ting-Yun Duan (pp. 50-56).
Control of the circulation of H9N2 avian influenza virus (AIV) is a major concern for both animal and public health, and H9N2 AIV poses a major threat to the chicken industry worldwide. Here, we developed a recombinant fowlpox virus (rFPV-HA) expressing the haemagglutinin (HA) gene of the A/CH/JY/1/05 (H9N2) influenza virus and a recombinant fowlpox virus (rFPV-HA/IL18) expressing the HA gene and chicken interleukin-18 (IL-18) gene. Recombinant plasmid pSY-HA/IL18 was constructed by cloning chicken IL-18 expression cassette into recombinant plasmid pSY-HA containing the HA gene. Two rFPVs were generated by transfecting two recombinant plasmids into the chicken embryo fibroblast cells pre-infected with S-FPV-017, and assessed for their immunological efficacy on one-day-old White Leghorn specific-pathogen-free chickens challenged with the A/CH/JY/1/05 (H9N2) strain. There was a significant difference in HI antibody levels ( P<0.05) elicited by either rFPV-HA or rFPV-HA/IL18. The level of splenocyte proliferation response in the rFPV-HA/IL18-vaccinated group was significantly higher ( P<0.05) than that in the rFPV-HA group. After challenge with 106.5ELD50 H9N2 AIV 43days after immunization, rFPVs vaccinated groups could prevent virus shedding and replication in multiple organs in response to H9N2 AIV infection, and rFPV-HA/IL18 vaccinated group had better inhibition of viruses than rFPV-HA vaccinated group. Our results show that the protective efficacy of the rFPV-HA vaccine could be enhanced significantly by simultaneous expression of IL-18.
Keywords: H9N2 avian influenza virus; Hemagglutinin gene; Chicken interleukin-18 gene; Recombinant fowlpox virus
Comparative inhibitory activity of the stilbenes resveratrol and oxyresveratrol on African swine fever virus replication by I. Galindo; B. Hernáez; J. Berná; J. Fenoll; J.L. Cenis; J.M. Escribano; C. Alonso (pp. 57-63).
Stilbenols are polyphenolic phytoalexins produced by plants in response to biotic or abiotic stress. These compounds have received much attention because of their significant biological effects. One of these is their antiviral action, which has previously been documented for two members of this class, namely resveratrol and oxyresveratrol. Here we tested the antiviral effect of these two compounds on African swine fever virus, the only member of the newly created family Asfarviridae and a serious limitation to porcine production worldwide. Our results show a potent, dose-dependent antiviral effect of resveratrol and oxyresveratrol in vitro. Interestingly, this antiviral activity was found for these synthetic compounds and also for oxyresveratrol extracted from new natural sources (mulberry twigs). The antiviral effect of these two drugs was demonstrated at concentrations that do not induce cytotoxicity in cultured cells. Moreover, these antivirals achieved a 98–100% reduction in viral titers. Both compounds allowed early protein synthesis but inhibited viral DNA replication, late viral protein synthesis and viral factory formation.
Keywords: Antiviral; Resveratrol; Oxyresveratrol; African swine fever virus
Inhibition of rabies virus propagation in mouse neuroblastoma cells by an intrabody against the viral phosphoprotein by Yoshihiro Kaku; Akira Noguchi; Kozue Hotta; Akio Yamada; Satoshi Inoue (pp. 64-71).
Rabies virus (RABV) is highly neurotropic and causes acute infection of the central nervous system. Death can be averted by prompt post-exposure prophylaxis; however, after clinical symptoms appear, the mortality rate is almost 100% and no reliable treatment is available. In this study, we investigated whether intracellular immunization using single-chain variable fragments (scFvs) against RABV phosphoprotein (RABV-P) could inhibit RABV propagation in neuronal cells. Of four scFv clones derived from an scFv phage-displayed library, scFv-P19 showed extremely high transfection efficiency and stable expression in mouse neuroblastoma (MNA) cells. The intracellular affinity and inhibition of RABV propagation were investigated using RABV-infected MNA cells pretransfected with the scFv-P19 gene. The specific interaction between scFv and RABV-P was confirmed by an immunoprecipitation assay and an indirect immunofluorescence assay showing that these molecules colocalized in the cytoplasm. Measurements of the spread of RABV in a culture well and the virus titer in the supernatant showed that RABV inhibition peaked 3 days after infection, at 98.6% and 99.9% inhibition, respectively. Although the mechanism of RABV inhibition by scFv-P19 is not clear, this scFv-based intracellular immunization could be a candidate for future RABV therapeutic studies if combined with appropriate delivery and application systems.
Keywords: Rabies; Phosphoprotein; Intrabody; scFv; Intracellular immunization
Development of Vaccinia reporter viruses for rapid, high content analysis of viral function at all stages of gene expression by Ken Dower; Kathleen H. Rubins; Lisa E. Hensley; John H. Connor (pp. 72-80).
Vaccinia virus is the prototypical orthopoxvirus of Poxviridae, a family of viruses that includes the human pathogens Variola (smallpox) and Monkeypox. Core viral functions are conserved among orthopoxviruses, and consequently Vaccinia is routinely used to study poxvirus biology and screen for novel antiviral compounds. Here we describe the development of a series of fluorescent protein-based reporter Vaccinia viruses that provide unprecedented resolution for tracking viral function. The reporter viruses are divided into two sets: (1) single reporter viruses that utilize temporally regulated early, intermediate, or late viral promoters; and (2) multi-reporter viruses that utilize multiple temporally regulated promoters. Promoter and reporter combinations were chosen that yielded high signal-to-background for stage-specific viral outputs. We provide examples for how these viruses can be used in the rapid and accurate monitoring of Vaccinia function and drug action.
Keywords: Orthopoxvirus; Antiviral; Gene expression; High-throughput; Drug discovery