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Antiviral Research (v.73, #3)

Editorial Board (pp. ii).

Notes to Authors (pp. co3).

Bovine lactoferrin inhibits echovirus endocytic pathway by interacting with viral structural polypeptides by Maria Grazia Ammendolia; Agostina Pietrantoni; Antonella Tinari; Piera Valenti; Fabiana Superti (pp. 151-160).

Lactoferrin, an 80kDa bi-globular iron-binding glycoprotein belonging to the transferrin family, is a pleiotropic factor with potent antimicrobial and immunomodulatory activities, present in breast milk, in mucosal secretions, and in the secondary granules of neutrophils. Recently, we have shown that bovine lactoferrin prevents the early phases of echovirus infection and also acts as a survival factor inhibiting viral-induced apoptosis. In the present research we investigated the mechanism of bovine lactoferrin anti-echoviral effect demonstrating that echovirus enters susceptible cells by an endocytic pathway and that lactoferrin treatment is able to prevent viral genome delivery into the cytoplasm. It is likely that lactoferrin interaction with echovirus capsid proteins induces alterations that stabilize the conformation of the virion making it resistant to uncoating.Taken together, the results of our study show that the inhibition of echovirus 6 infectivity by lactoferrin is dependent on its interaction not only with cell surface glycosaminoglycan chains but also with viral structural proteins demonstrating that this glycoprotein targets the virus entry process.

Keywords: Echovirus 6; Lactoferrin; Endocytosis

2′- C-Methylcytidine as a potent and selective inhibitor of the replication of foot-and-mouth disease virus by Goris Nesya; De Palma Armando; Toussaint Jean-François; Musch Ina; Neyts Johan; De Clercq Kris (pp. 161-168).

We report on the potent and selective in vitro antiviral activity of 2′- C-methylcytidine (2′- C-MetCyt) against foot-and-mouth disease virus (FMDV). FMDV belongs to the Picornaviridae and has the potential to cause devastating epidemics in livestock. The 50% and 90% effective concentrations (EC₅₀ and EC₉₀) for inhibition of the FMDV-induced cytopathic effect (CPE) formation were 6.4±3.8 and 10.8±5.4μM. Comparable EC₅₀ values for inhibition of viral RNA synthesis were observed. Treatment of FMDV-infected BHK-21 cells with 77μM 2′- C-MetCyt resulted in a (1.6–3.2)×10³-fold reduction of infectious virus yield. Time-of-drug addition experiments suggest that 2′- C-MetCyt interacts with viral replication at a time point that coincides with the onset of intracellular viral RNA synthesis. In contrast to emergency vaccination, a potent and selective antiviral agent may provide almost immediate (prophylactic/therapeutic) protection against infection and thus constitute an important alternative/supplementary option to contain outbreaks such as those caused by FMDV.

Keywords: Foot-and-mouth disease; Swine vesicular disease; 2′-; C; -Methylcytidine; Ribavirin; Disease control

Antiviral activity of HPMPC (cidofovir) against orf virus infected lambs by A. Scagliarini; C.J. McInnes; L. Gallina; F. Dal Pozzo; L. Scagliarini; R. Snoeck; S. Prosperi; J. Sales; J.A. Gilray; P.F. Nettleton (pp. 169-174).

( S)-9-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPC, cidofovir, CDV, Vistide^®) is an acyclic nucleoside analogue with a potent and selective activity against a broad spectrum of DNA viruses including the poxviruses. In this study we present the results of different treatment regimens in lambs experimentally infected with orf virus with different cidofovir formulations prepared in Beeler basis and Unguentum M. Our results show that choice of excipient, concentration of cidofovir and treatment regimen were all important to the clinical outcome of the therapy. Whilst one particular regimen appeared to exacerbate the lesion, treatment with 1% (w/v) cidofovir cream, prepared in Beeler basis, for 4 consecutive days did result in milder lesions that resolved more quickly than untreated lesions. Furthermore the scabs of the treated animals contained significantly lower amounts of viable virus meaning there should be less contamination of the environment with virus than would normally occur.

Keywords: Orf virus; Contagious ecthyma; Cidofovir; HPMPC; Topical treatment; Formulation

Targeting the NF-κB pathway through pharmacological inhibition of IKK2 prevents human cytomegalovirus replication and virus-induced inflammatory response in infected endothelial cells by Patrizia Caposio; Tiziana Musso; Anna Luganini; Hiroyasu Inoue; Marisa Gariglio; Santo Landolfo; Giorgio Gribaudo (pp. 175-184).

Endothelial cells are important reservoirs for human cytomegalovirus (HCMV) replication, dissemination and persistence. HCMV infection of endothelial cells has been associated with a proinflammatory response characterized by an increased expression of chemokines and adhesion molecules and modulation of angiogenesis. Many of the host proinflammatory genes augmented in HCMV-infected endothelial cells are regulated, at least in part, by the NF-κB pathway. HCMV is a potent activator of NF-κB through the IKK-IκB signaling axis. To explore whether inhibition of HCMV-induced NF-κB activation may interfere with the onset of virus-associated inflammatory response, we measured the effects of the specific IKK2 inhibitor AS602868 on the expression of a panel of proinflammatory genes in HUVEC cells infected with a clinical isolate. Treatment of infected HUVEC with AS602868 was shown to impair HCMV-induced NF-κB activity, IE gene expression, viral replication and to prevent HCMV-induced upregulation of ICAM-1, IL-8, RANTES, IP-10, I-TAC and COX-2 gene expression. Consistent with these results, HCMV-mediated upregulation of another NF-κB-dependent gene, the plasminogen inhibitor type-1, a regulatory factor of endothelial proliferation and angiogenesis, was abrogated by AS602868. These results suggest that inhibition of HCMV-induced IKK-NF-κB activation may be of interest to limit the virus-induced inflammatory response of infected endothelial cells.

Keywords: HCMV; Endothelial cells; Inflammatory response; Gene expression; NF-κB; IKK2

Highly selective action of triphosphate metabolite of 4′-ethynyl D4T: A novel anti-HIV compound against HIV-1 RT by Guangwei Yang; Ginger E. Dutschman; Chuan-Jen Wang; Hiromichi Tanaka; Masanori Baba; Karen S. Anderson; Yung-Chi Cheng (pp. 185-191).

2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T), is a recently discovered nucleoside reverse transcriptase inhibitor (NRTI) showing a 5- to 10-fold greater anti-human immunodeficiency virus type 1 (HIV-1) activity and less cellular and mitochondrial toxicity than its parental compound, stavudine (D4T). It is also active against a variety of NRTI-resistant HIV-1 mutants under non-cytotoxic concentrations. In this study, the effects of 4′-Ed4TTP, which is the triphosphate metabolite of 4′-Ed4T, on HIV-1 reverse transcriptase (RT) activity were investigated. We found that 4′-Ed4TTP was a substrate of HIV-1 RT serving as a DNA chain terminator, and it inhibited the DNA polymerase activity of RT more efficiently than D4TTP. The value of K_{i(4′-Ed4TTP)}/ K_m(dTTP) is 0.15 for DNA/RNA primer/template duplex (P/T), but 0.7 for DNA/DNA P/T, suggesting 4′-Ed4TTP inhibits RT more efficiently during RNA-dependent DNA synthesis than DNA-dependent DNA synthesis. 4′-Ed4TTP was also found to inhibit the 3TC (Lamivudine)-resistant RT mutant, M184V, with 3-fold less efficiency than the wild type (wt) RT. 4′-Ed4TTP showed much less inhibitory effects toward major host DNA polymerases. Overall, our results suggest that 4′-Ed4TTP is the active form for anti-HIV-1 activity via its inhibitory effect against RT.

Keywords: Abbreviations; NRTI; nucleoside reverse transcriptase inhibitor; NNRTI; non-nucleoside reverse transcriptase inhibitor; D4T; 2′,3′-didehydro-3′-deoxythymidine (also known as stavudine); 4′-EdT; 4′-ethynyl D4T; AZT; 3′-azido-3′-deoxythymidine (also known as zidovudine); 3TC; β-; l; -2′,3′-dideoxy-3′-thiacytidine (also known as lamivudine); P/T; primer/template duplex2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T); HIV-1; RT; Inhibition; NRTI

Ribavirin and mycophenolic acid markedly potentiate the anti-hepatitis B virus activity of entecavir by Chunxiao Ying; Richard Colonno; Erik De Clercq; Johan Neyts (pp. 192-196).

MPA [the active metabolite of the immuno-suppressive agent CellCept] and ribavirin markedly potentiate the anti-HBV activity of the guanine-based nucleoside analogue entecavir (ETV) against both wild-type HBV and a lamivudine-resistant variant. Ribavirin (in its 5′-monophosphate form) and MPA are inhibitors of IMP-dehydrogenase and cause depletion of intracellular dGTP pools. The active triphosphorylated form of ETV may inhibit more efficiently the priming reaction, reverse transcription and DNA-dependent DNA polymerase activity of the HBV polymerase in the presence of reduced levels of dGTP. The potential for enhanced ETV activity is supported by the observation that exogenously added deoxyguanosine reversed the potentiating effect of ribavirin and MPA. Our observations may have important implications for those (liver) transplant recipients that receive MMF as part of their immunosuppressive regimen and who, because of a de novo or a persistent infection with HBV need antiviral therapy such as ETV. Further studies will need to be conducted to determine if combining ribavirin (a compound used for the treatment of HCV infections) with ETV could have an advantage for the treatment of HBV infections, in particular in patients co-infected with HCV.

Keywords: HBV; Entecavir; CellCept; Ribavirin

Strand-specific silencing of a picornavirus by RNA interference: Evidence for the superiority of plus-strand specific siRNAs by Steffen Schubert; Diana Rothe; Denise Werk; Hans-Peter Grunert; Heinz Zeichhardt; Volker A. Erdmann; Jens Kurreck (pp. 197-205).

RNA interference triggered by small interfering RNAs (siRNAs) can be used to effectively contain viral spread. Here, we report on the mechanism of action of siRNAs targeting the medically important coxsackievirus B3 (CVB-3) as a typical representative of viruses with a non-segmented RNA genome in positive-strand orientation. Antiviral siRNAs can be designed to target the genomic (+)-strand, the (−)-strand that occurs as a replication intermediate, or both. In the present study, two complementary and systematic approaches are presented providing direct evidence that silencing of the viral (+)-strand is the key to inhibit CVB-3: first, we used rational siRNA design to direct silencing activity specifically against either of the two viral strands. As a second approach, we employed siRNA containing modified nucleotides to render them specific for one of the virus RNAs. Experiments with infectious coxsackievirus revealed that the inhibitory efficiency correlates exclusively with the activity of the siRNAs directed against the viral (+)-strand. Our finding that only (+)-strand specific siRNAs exert significant antiviral potency may hold true for other RNA viruses with (+)-stranded genomes as well and may therefore be helpful in the development of efficient strategies to inhibit virus propagation.

Keywords: Coxsackievirus; Enterovirus; (+)-RNA strand specificity; Locked nucleic acids

Antiviral activity of tiazofurin and mycophenolic acid against Grapevine Leafroll-associated Virus 3 in Vitis vinifera explants by A. Panattoni; F. D’Anna; E. Triolo (pp. 206-211).

The ability to control plant viral diseases with chemicals has great potential value for agriculture, but few chemicals are available to date due to the difficulty in obtaining effective drugs. IMP dehydrogenase is an enzyme which catalyzes the conversion of inosine 5′-monophosphate to xanthosine 5′-monophosphate in the de novo purine nucleotide synthetic pathway, and is considered a sensitive target for antiviral drugs. Two IMPDH inhibitors, tiazofurin (TR) and mycophenolic acid (MPA), were tested for their inhibitory effect on Grapevine leafroll-associated virus 3 (GLRaV-3) in in vitro grapevine explants. TR administration produced plantlets characterized by negative ELISA readings. No PCR products were obtained from these samples. This was confirmed by the absence of viral particles. MPA was essentially ineffective against GLRaV-3 replication in Sangiovese explants. This is the first report of GLRaV-3 eradication in grapevine explants following TR administration.

Keywords: Vitis vinifera; explant; GLRaV-3; Tiazofurin; Mycophenolic acid

Oral 1- O-octadecyl-2- O-benzyl- sn-glycero-3-cidofovir targets the lung and is effective against a lethal respiratory challenge with ectromelia virus in mice by Karl Y. Hostetler; James R. Beadle; Julissa Trahan; Kathy A. Aldern; Gelita Owens; Jill Schriewer; Lora Melman; R. Mark Buller (pp. 212-218).

Hexadecyloxypropyl-cidofovir (HDP-CDV) has been shown to be orally active against lethal infection with orthopoxviruses including, mousepox, cowpox, vaccinia and rabbitpox. The alkoxyalkyl group provides oral absorption and reduces greatly the amount of drug reaching the kidney, the site of CDV's dose limiting toxicity. However, the amount of HDP-CDV detected in lung, an important site of early poxvirus replication, is low and the reduction of viral titers in surviving animals is reduced moderately compared with the liver where poxvirus titers are virtually undetectable. We synthesized a novel glycerol ester of CDV, 1- O-octadecyl-2- O-benzyl- sn-glycero-3-CDV (ODBG-CDV), and compared its oral pharmacokinetics with that of HDP-CDV. Surprisingly, ODBG-CDV levels in lung are much higher and liver levels are reduced, suggesting that the compound is transported in small intestinal lymph instead the portal vein. ODBG-CDV has excellent in vitro activity in cells infected with ectromelia virus (ECTV). In mice infected with a lethal aerosol or intranasal challenge of ECTV, HDP-CDV and ODBG-CDV are equally effective in preventing death from disease. Other drugs esterified to 1- O-octadecyl-2- O-benzyl- sn-glycerol or 1- O-octadecyl-2- O-benzyl- sn-glycerol-3-phosphate may provide lung targeting for treatment of microbial or neoplastic diseases while reducing first pass removal by the liver during oral absorption.

Keywords: Ectromelia virus; Cidofovir; Prodrugs; Lung targeting

Inhibition of SARS-CoV replication cycle by small interference RNAs silencing specific SARS proteins, 7a/7b, 3a/3b and S by Sara Åkerström; Ali Mirazimi; Yee-Joo Tan (pp. 219-227).

The severe acute respiratory syndrome coronavirus (SARS CoV) genome has 14 potential open reading frames (ORFs). The first ORF is translated from the full-length genomic mRNA while the remaining ORFs are translated from eight subgeomic RNAs (sgRNAs). In this study, we designed small interference RNAs (siRNAs) targeting sgRNA 2, 3 and 7 and tested their efficiency and specificity in silencing the protein translated from the targeted sgRNA. Our results demonstrated that siRNA 7 could inhibit sgRNA 7, which showed 19/19 nucleotides (nt) matching, and sgRNA 8, which showed 18/19nt matching; but, it did not inhibit the full-length genomic mRNA which showed 17/19nt matching. Overall, each of the siRNAs can inhibit the targeted sgRNA without affecting the full-length genomic mRNA or the other sgRNAs that showed mismatch of two or more nt. Thus, siRNA could be designed so as to knockdown the expression of viral protein(s) from a targeted sgRNA during viral infection, thereby allowing the contribution of individual viral proteins to viral infection to be delineated. When Vero E6 cells expressing siRNA 2, 3 or 7 were infected with SARS-CoV, a significant reduction in the yield of progeny virus was observed. Indirect immunofluorescence assays showed that in the infected cells expressing each of the siRNAs, there was aspecific silencing of S, 3a and 7a, respectively, but the expression of nucleocapsid protein was not affected. Thus, our data suggests that the accessory proteins, i.e. 3a and 7a, could play an important role during the replication cycle of the SARS-CoV.

Keywords: siRNA; SARS corona virus; 3a; 7a

Susceptibility of highly pathogenic A(H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes by A.C. Hurt; P. Selleck; N. Komadina; R. Shaw; L. Brown; I.G. Barr (pp. 228-231).

Since 2003, highly pathogenic A(H5N1) influenza viruses have been the cause of large-scale death in poultry and the subsequent infection and death of over 140 humans. A group of 55 influenza A(H5N1) viruses isolated from various regions of South East Asia between 2004 and 2006 were tested for their susceptibility to the anti-influenza drugs the neuraminidase inhibitors and adamantanes. The majority of strains were found to be fully sensitive to the neuraminidase inhibitors oseltamivir carboxylate, zanamivir and peramivir; however two strains demonstrated increased IC₅₀ values. Sequence analysis of these strains revealed mutations in the normally highly conserved residues 116 and 117 of the N1 neuraminidase. Sequence analysis of the M2 gene showed that all of the A(H5N1) viruses from Vietnam, Malaysia and Cambodia contained mutations (L26I and S31N) associated with resistance to the adamantane drugs (rimantadine and amantadine), while strains from Indonesia were found to be a mix of both adamantane resistant (S31N) and sensitive viruses. None of the A(H5N1) viruses from Myanmar contained mutations known to confer adamantane resistance. These results support the use of neuraminidase inhibitors as the most appropriate class of antiviral drug to prevent or treat human A(H5N1) virus infections.

Keywords: Influenza; A(H5N1); NA inhibitors; Adamantanes; Drug resistance

Contents of Volume 73 (pp. 232-233).

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Antiviral Research (v.73, #3)