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Antiviral Research (v.77, #2)
Inhibition of human parainfluenza virus type 3 infection by novel small molecules by Hongxia Mao; Chandar S. Thakur; Santanu Chattopadhyay; Robert H. Silverman; Andrei Gudkov; Amiya K. Banerjee (pp. 83-94).
Human parainfluenza virus type 3 (HPIV3) is an important respiratory tract pathogen of infants and children. There are no vaccines or antivirals currently approved for prevention or treatment of HPIV3 infection. Towards developing an antiviral therapy to combat HPIV3 infection, we have established a green fluorescent protein (GFP)-tagged HPIV3 infected-cell assay and used it for screening of a small molecule library obtained from ChemBridge Diver. Two novel small molecules (C5 and C7) which shared structural similarities were identified and their inhibitory effects on HPIV3 were confirmed in CV-1 and human lung epithelium A549 cells by plaque assay, Western blot and Northern blot analyses. C5 and C7 effectively prevented the cytopathic effect in cells infected with HPIV3, achieving IC50 values of 2.36μM and 0.08μM, respectively, for infectious virus production. The inhibition appears to be at the primary transcriptional level of HPIV3 life cycle based on sequential time course test, binding and internalization assays, and finally by a minigenome transcription assay in cells as well as measuring viral transcripts in cells in the presence of anisomycin. Interestingly, vesicular stomatitis virus (VSV), another member of mononegavirales order, was also inhibited by these compounds, whereas poliovirus–a picornavirus was not. Use of these inhibitors has a strong potential to develop novel antiviral agents against this important human pathogen.
Keywords: HPIV3; Small molecules; Primary transcription; Mononegavirales
Peptide-conjugated morpholino oligomers inhibit porcine reproductive and respiratory syndrome virus replication by Deendayal Patel; Tanja Opriessnig; David A. Stein; Patrick G. Halbur; Xiang-Jin Meng; Patrick L. Iversen; Yan-Jin Zhang (pp. 95-107).
Porcine reproductive and respiratory syndrome (PRRS) has been devastating the global swine industry for more than a decade, and current strategies to control PRRS are inadequate. In this study we characterized the inhibition of PRRS virus (PRRSV) replication by antisense phosphorodiamidate morpholino oligomers (PMO). Of 12 peptide-conjugated PMO (PPMO), four were found to be highly effective at inhibiting PRRSV replication in cell culture in a dose-dependant and sequence-specific manner. PPMO 5UP2 and 5HP are complementary to sequence in the 5′ end of the PRRSV genome, and 6P1 and 7P1 to sequence in the translation initiation regions of ORF6 and ORF7, respectively. Treatment of cells with 5UP2 or 5HP caused a 4.5log10 reduction in PRRSV yield, compared to a control PPMO. Combination of 6P1 and 7P1 led to higher level reduction than 6P1 or 7P1 alone. 5UP2, 5HP, and a combination of 6P1 and 7P1 inhibited PRRSV replication in porcine alveolar macrophages and protected the cells from PRRSV-induced cytopathic effect. Northern blot and real-time RT-PCR results demonstrated that the effective PPMO led to a reduction of PRRSV RNA level. 5UP2 and 5HP inhibited virus replication of 10 other strains of PRRSV. Results from this study suggest potential applications of PPMO for PRRS control.
Keywords: Porcine reproductive and respiratory syndrome virus; PRRSV; Morpholino; Antisense; Antiviral; PRRSV RNA
In vitro efficacy of ribavirin against canine distemper virus by Gabriella Elia; Chiara Belloli; Francesco Cirone; Maria Stella Lucente; Marta Caruso; Vito Martella; Nicola Decaro; Canio Buonavoglia; Paolo Ormas (pp. 108-113).
Despite vaccination, canine distemper virus (CDV) remains one of the important pathogen of dogs with worldwide distribution. Ribavirin (RIB) inhibits replication of measles virus (MV), a morbillivirus closely related to CDV, both in vitro and in vivo. In this report the antiviral activity of RIB against CDV in cell cultures was assessed. Quantitative real-time RT-PCR was used to measure viral RNA in VERO cells infected by CDV and to evaluate the inhibitory effects of RIB. RIB caused a dose- and time-dependent decrease in accumulation of CDV RNA when added after virus adsorption. RIB was highly effective in preventing CDV replication at low concentrations with 50% virus-inhibitory concentrations ranging from 0.02 to 0.05mM. Such low values were comparable to values displayed by highly susceptible strains of MV. In addition, CDV was passaged sequentially in VERO cell monolayers in the presence of RIB to trigger viral extinction. The virus was no longer detected after three passages, suggesting that error catastrophe is one of the modes of action of RIB against CDV. These findings suggest RIB as a promising tool for the therapy of CD in dogs.
Keywords: Antivirals; Ribavirin; Canine distemper virus; Real-time RT-PCR; VERO cells
Imidazo[4,5-c]pyridines inhibit the in vitro replication of the classical swine fever virus and target the viral polymerase by R. Vrancken; J. Paeshuyse; A. Haegeman; G. Puerstinger; M. Froeyen; P. Herdewijn; P. Kerkhofs; J. Neyts; F. Koenen (pp. 114-119).
Selective inhibitors of the replication of the classical swine fever virus (CSFV) may have the potential to control the spread of the infection in an epidemic situation. We here report that 5-[(4-bromophenyl)methyl]-2-phenyl-5 H-imidazo[4,5-c]pyridine (BPIP) is a highly potent inhibitor of the in vitro replication of CSFV. The compound resulted in a dose-dependent antiviral effect in PK15 cells with a 50% effective concentration (EC50) for the inhibition of CSFV Alfort187 (subgroup 1.1) of 1.6±0.4μM and for CSFV Wingene (subgroup 2.3) 0.8±0.2μM. Drug-resistant virus was selected by serial passage of the virus in increasing drug-concentration. The BPIP-resistant virus (EC50: 24±4.0μM) proved cross-resistant with VP32947 [3-[((2-dipropylamino)ethyl)thio]-5 H-1,2,4-triazino[5,6-b]indole], an unrelated earlier reported selective inhibitor of pestivirus replication. BPIP-resistant CSFV carried a T259S mutation in NS5B, encoding the RNA-dependent RNA-polymerase (RdRp). This mutation is located near F224, a residue known to play a crucial role in the antiviral activity of BPIP against bovine viral diarrhoea virus (BVDV). The T259S mutation was introduced in a computational model of the BVDV RdRp. Molecular docking of BPIP in the BVDV polymerase suggests that T259S may have a negative impact on the stacking interaction between the imidazo[4,5-c]pyridine ring system of BPIP and F224.
Keywords: Antiviral agent; Flaviviridae; Pestivirus; Inhibition of classical swine fever virus; RNA-polymerase; Imidazopyridine
Inhibition of murine AIDS by pro-glutathione (GSH) molecules by A. Fraternale; M.F. Paoletti; A. Casabianca; C. Orlandi; G.F. Schiavano; L. Chiarantini; P. Clayette; J. Oiry; J.-U. Vogel; J. Cinatl Jr.; M. Magnani (pp. 120-127).
Antioxidant molecules can be used both to replenish the depletion of reduced glutathione (GSH) occurring during HIV infection, and to inhibit HIV replication. The purpose of this work was to assess the efficacy of two pro-GSH molecules able to cross the cell membrane more easily than GSH. We used an experimental animal model consisting of C57BL/6 mice infected with the LP-BM5 viral complex; the treatments were based on the intramuscular administration of I-152, a pro-drug of N-acetylcysteine and S-acetyl-β-mercaptoethylamine, and S-acetylglutathione, an acetylated GSH derivative. The results show that I-152, at a concentration of 10.7 times lower than GSH, caused a reduction in lymph node and spleen weights of about 55% when compared to infected animals and an inhibition of about 66% in spleen and lymph node virus content. S-acetylglutathione, at half the concentration of GSH, caused a reduction in lymph node weight of about 17% and in spleen and lymph node virus content of about 70% and 30%, respectively. These results show that the administration of pro-GSH molecules may favorably substitute for the use of GSH as such.
Keywords: MAIDS; GSH; Pro-GSH molecules; Antiretroviral
Altered sensitivity of an R5X4 HIV-1 strain 89.6 to coreceptor inhibitors by a single amino acid substitution in the V3 region of gp120 by Yosuke Maeda; Keisuke Yusa; Shinji Harada (pp. 128-135).
The replication of several R5X4 strains is blocked by single CXCR4 inhibitors such as AMD3100 or T140 although the target cells express both CXCR4 and CCR5 in vitro. To identify which region(s) of the Env are involved in the increased sensitivity to CXCR4 inhibitors, we isolated a T140-escape mutant using R5X4 HIV-1 strain 89.6. An isolated mutant harbored a single amino acid substitution in the V3 region of the Env (arginine 308 to serine R308S). Luciferase-reporter HIV-1 pseudotyped with the mutant Env showed that the substitution conferred total resistance to CXCR4 antagonists but increased sensitivity to a CCR5 antagonist TAK-779 in the infection of the cells expressing both CCR5 and CXCR4. Analyses using the cells expressing a single coreceptor showed that the mutant Env predominantly and efficiently utilized CCR5 rather than CXCR4 while retaining R5X4 phenotype. These results indicated that the sensitivities of the R5X4 strain to coreceptor inhibitors were altered by a single amino acid substitution in the V3 region of gp120.
Keywords: HIV; Coreceptor antagonist; CXCR4 usage; Envelope; V3 region
Comparison of the efficacy of thymosin alpha-1 and interferon alpha in the treatment of chronic hepatitis B: A meta-analysis by Yong-Feng Yang; Wei Zhao; Yan-Dan Zhong; Yi-Jun Yang; Ling Shen; Ning Zhang; Ping Huang (pp. 136-141).
Chronic hepatitis B virus (HBV) infection is a serious problem because of its worldwide distribution and possible adverse sequelae, such as cirrhosis and hepatocellular carcinoma. Thymosin alpha-1 (Tα1) is an immune modifier that has been shown to be effective for chronic hepatitis B (CHB) in some trials. But the trials comparing Tα1 vs. interferon alpha (IFNα) treatment in CHB have been small and the results have been inconsistent. So we conducted a meta-analysis to compare the efficacy of Tα1 and IFNα in the treatment of CHB. Generally, four randomized controlled trials including 199 CHB patients who received Tα1 or IFNα treatment were identified through MEDLINE and EMBASE online search. Virological (for hepatitis B e antigen (HBeAg) positive patients, loss of HBV DNA and HBeAg; for HBeAg negative patients, loss of HBV DNA), biochemical (normalization of transaminases) and complete responses (fulfill criteria of biochemical and virological response simultaneously) were analyzed using the intention-to-treat method. The odds ratio (OR) was used to measure the magnitude of the efficacy. The ORs (95% confidence interval) of the virological response, biochemical response and complete response of Tα1 over IFNα at the end of 6 months treatment were 0.62 (0.35, 1.10), 0.60 (0.34, 1.05) and 0.54 (0.30, 0.97), respectively. The ORs (95% confidence interval) of the virological response, biochemical response and complete response of Tα1 over IFNα at the end of follow-up (6 months post-treatment) were 3.71 (2.05, 6.71), 3.12 (1.74, 5.62) and 2.69 (1.47, 4.91), respectively. These data showed that compared with IFNα, the benefit of Tα1 was not immediately significant at the end of therapy, but virological, biochemical and complete response had a tendency to increase or accumulate gradually after the therapy. For three of the four trials that studied HBeAg-negative patients, the results are mostly applicable to HBeAg-negative CHB.
Keywords: Thymosin; Interferon; Hepatitis B; Meta-analysis
Inhibition of red seabream iridovirus (RSIV) replication by small interfering RNA (siRNA) in a cell culture system by Lua T. Dang; Hidehiro Kondo; Ikuo Hirono; Takashi Aoki (pp. 142-149).
Small interfering RNAs (siRNAs), mediators of a process of sequence-specific gene silencing called RNA interference, have been shown to have activity against a wide range of viruses and are considered to be potential antiviral tools. Here, we describe an antiviral activity of a siRNA that targets the major capsid protein (MCP) gene of red seabream iridovirus (RSIV), a marine fish-pathogenic virus, in a cell culture system. Inhibition of RSIV replication was demonstrated by reduced MCP expression level and reduced RSIV titer. MCP-targeted siRNA (siR-MCP) dose-dependently inhibited the expression of MCP gene in cells that either transiently expressed or stably expressed the MCP gene. At 84 and 96h after viral infection, siR-MCP reduced the expression of MCP gene by 55.2% and 97.1%, respectively. Transfection with siR-MCP reduced the production of RSIV particles in supernatants of samples infected with RSIV, while the corresponding mismatched siR-MCP (MsiR-MCP) and nsRNA controls did not exhibit this effect. These results show that MCP-targeted siRNA can effectively and specifically inhibit the expression of the target gene and hinder RSIV replication during an in vitro infection, providing a potential approach for the control of viral diseases in aquaculture.
Keywords: RNA interference (RNAi); Small interfering RNA (siRNA); Major capsid protein; MCP; Red seabream iridovirus; RSIV
Inhibition of human coronavirus 229E infection in human epithelial lung cells (L132) by chloroquine: Involvement of p38 MAPK and ERK by Masakazu Kono; Koichiro Tatsumi; Alberto M. Imai; Kengo Saito; Takayuki Kuriyama; Hiroshi Shirasawa (pp. 150-152).
The antiviral effects of chloroquine (CQ) on human coronavirus 229E (HCoV-229E) infection of human fetal lung cell line, L132 are reported. CQ significantly decreased the viral replication at concentrations lower than in clinical usage. We demonstrated that CQ affects the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK). Furthermore, p38 MAPK inhibitor, SB203580, inhibits CPE induced by HCoV-229E infection and viral replication. Our findings suggest that CQ affects the activation of MAPKs, involved in the replication of HCoV-229E.
Keywords: Coronavirus; 229E; Chloroquine; p38; MAPK; ERK
Genistein treatment of cells inhibits arenavirus infection by Eric M. Vela; Gavin C. Bowick; Norbert K. Herzog; Judith F. Aronson (pp. 153-156).
Arenaviridae is a family of enveloped viruses some of which are capable of causing hemorrhagic fever syndromes in humans. In this report, we demonstrate that treatment of host cells with the tyrosine kinase inhibitor genistein inhibits infection of cells with the New World arenavirus Pichindé (PICV). The greatest degree of inhibition was observed in pre-treated target cells, but modest inhibition of infection was also seen when drug was added to cultures up to 48h after infection. We show that PICV-induced phosphorylation of the activating transcription factor-2 protein (ATF-2) and cyclic adenosine monophosphate response element binding protein (CREB) is inhibited following genistein treatment. Lastly, genistein treatment also inhibited transduction of cells with pseudotyped retrovirus particles expressing envelope proteins of the Old World arenavirus Lassa virus. These results demonstrate that kinase activity is required for arenavirus infection and that therapeutics designed to inhibit kinase activity should be explored.
Keywords: Arenavirus; Lassa virus; Pichindé virus; Genistein; CREB; ATF-2
Synthesis of new benzimidazole–coumarin conjugates as anti-hepatitis C virus agents by Jih Ru Hwu; Raghunath Singha; Shih Ching Hong; Yung Hsiung Chang; Asish R. Das; Inge Vliegen; Erik De Clercq; Johan Neyts (pp. 157-162).
Nineteen new conjugated compounds were successfully synthesized by a one-flask method from benzimidazole and coumarin derivatives. A methylenethio linker was used to connect these two kinds of derivatives. In addition, substituted benzimidazol-2-thiones were also coupled with β-d-glucose peracetate; the resultant glucosides were further converted to the corresponding 2-(methylthio)coumarin derivatives. Their activity against the hepatitis C virus was tested; two of the most potent compounds 2-[(6′-bromocoumarin-3′-yl)methylenethio]-5-fluorobenzimidazole (4i) and its derivative 1-[(2″,3″,4″,6″-tetra- O-acetyl)glucopyranos-1″-yl]-2-[(6′-bromocoumarin-3′-yl)methylenethio]benzimidazole (7c) showed EC50 values of 3.4μM and 4.1μM, respectively. At a concentration of 5.0μM, compound7c inhibited HCV RNA replication by 90% and had no effect on cell proliferation. Given these data, a structure–activity relationship was established.
Keywords: Benzimidazole; Coumarin; Conjugate; N; -Glucosides; Anti-HCV
Activity of the neuraminidase inhibitor A-315675 against oseltamivir-resistant influenza neuraminidases of N1 and N2 subtypes by Yacine Abed; Benjamin Nehmé; Mariana Baz; Guy Boivin (pp. 163-166).
Clinical use of the neuraminidase inhibitor (NAI) oseltamivir has been associated with the emergence of viral resistance resulting from subtype-specific neuraminidase (NA) mutations. In this study, we evaluated the impact of the most frequent oseltamivir-resistant NA mutations including E119V, H274Y, R292K and N294S on the susceptibility profile to a novel NAI (A-315675) using recombinant NA proteins of N1 and N2 subtypes and also selected oseltamivir-resistant influenza H1N1 and H3N2 viruses. In the N1 subtype, recombinant NA proteins containing mutations H274Y and N294S previously associated with resistance to oseltamivir (754- and 197-fold increases in IC50 values, respectively, compared to WT) remained susceptible to A-315675 (2.5- and 2-fold increases in IC50 values, respectively). In the N2 subtype, NA proteins harboring mutations E119V and R292K conferring high levels of resistance to oseltamivir (1016- and >10,000-fold increases in IC50 values, respectively) had IC50 values that increased by only 1.5- and 13-fold, respectively, against A-315675. Similar susceptibility patterns to A-315675 were obtained when testing recombinant H1N1 mutant viruses (H274Y and N294S) and clinical H3N2 mutants (E119V). The V116A and I117V mutations, previously associated with oseltamivir resistance in H5N1 viruses, were susceptible to oseltamivir when tested in the H1N1 background suggesting a strain-specific impact of these mutations. These results confirm the potent inhibitory effect of A-315675 against oseltamivir-resistant influenza viruses of the N1 and N2 subtypes and support the clinical development of its bioavailable prodrug A-322278.
Keywords: Influenza; Neuraminidase inhibitors; A-315675; Oseltamivir; Resistance