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Antiviral Research (v.69, #2)

Prelim II: Editorial Board (pp. ii).

Prelim V: Guide for Authors (pp. v-xi).

Inside Back Cover: Notes to Authors (pp. co3).

Ribavirin and cysteinyl leukotriene-1 receptor blockade as treatment for severe bronchiolitis by Cynthia A. Bonville; Helene F. Rosenberg; Joseph B. Domachowske (pp. 53-59).

In this work we have evaluated the clinical responses of pneumovirus-infected mice to combination therapy with the antiviral agent, ribavirin, and the CysLT1 cysteinyl leukotriene receptor antagonist, montelukast. We observed substantial virus replication in our mouse model of pneumovirus infection and significant accumulation of cysteinyl leukotrienes in lung tissue, the latter detected at levels that correlate directly with granulocyte recruitment to the airways. While administration of the nucleoside analog, ribavirin, reduced virus replication ∼2000-fold, the clinical outcomes as measured by morbidity and mortality, in response to ribavirin monotherapy were indistinguishable from those of the no-treatment controls. Similarly, montelukast therapy alone did not reduce granulocyte recruitment nor did it improve the clinical outcome. However, combined therapy with ribavirin and montelukast resulted in a significant reduction in morbidity and a substantial reduction in mortality (50% survival at t=14 days and onward, compared to 10–20% survival in response to montelukast alone or to ribavirin alone, respectively, p<0.01). These findings define further the independent contributions made by virus replication and by the ensuing inflammatory response to the detrimental sequelae of pneumovirus infection in vivo.

Keywords: Pneumovirus; Chemokine; Montelukast; Ribavirin; Mice

The anti-malaria drug artesunate inhibits replication of cytomegalovirus in vitro and in vivo by Suzanne J.F. Kaptein; Thomas Efferth; Martina Leis; Sabine Rechter; Sabrina Auerochs; Martina Kalmer; Cathrien A. Bruggeman; Cornelis Vink; Thomas Stamminger; Manfred Marschall (pp. 60-69).

Treatment of human cytomegalovirus (HCMV) infections with any of the currently available antiviral agents is frequently associated with the occurrence of severe complications, seriously threatening the successful outcome of treatment. Therefore, the development of novel antiviral strategies is a challenging goal of current investigations. Previously, we reported that artesunate (ART) is an effective, non-cytotoxic inhibitor of HCMV in vitro. Here, we demonstrate that the efficacy of the antiviral effect of ART is augmented by co-treatment of HCMV-infected fibroblasts with ferrous iron, i.e. Ferrosanol™, and/or the iron transfer-mediating molecule holo-transferrin. This could alleviate the HCMV-induced modulation of cell surface expression of adhesion molecule Thy-1, suggesting that ART might be able to prevent pro-inflammatory effects of infection. The iron-enhanced, antiviral effect of ART could also be demonstrated in cultured cells infected with rat cytomegalovirus. Experiments using the RCMV/rat model showed that both the viral DNA load and virus titers in the salivary glands from infected rats were significantly reduced upon treatment with ART. Furthermore, an additive antiviral effect for ART together with each one of conventional anti-HCMV drugs, i.e. ganciclovir, cidofovir or foscarnet, was detected in HCMV-infected fibroblasts. These findings might open new perspectives regarding the use of ART in clinical trials.

Keywords: Cytomegalovirus; Drug development; Artesunate; Ferrous iron; Combined antiviral therapy

Design and biological activities of novel inhibitory peptides for SARS-CoV spike protein and angiotensin-converting enzyme 2 interaction by Tin-Yun Ho; Shih-Lu Wu; Jaw-Chyun Chen; Yen-Chiao Wei; Shin-Ei Cheng; Yung-Hsien Chang; Hsu-Jan Liu; Chien-Yun Hsiang (pp. 70-76).

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus (SARS-CoV). The binding of SARS-CoV spike (S) protein to cellular angiotensin-converting enzyme 2 (ACE2) is the first step in SARS-CoV infection. Therefore, we assayed the inhibitory effects of small peptides derived from S protein on the binding of S protein to ACE2 and on the S-protein-pseudotyped retrovirus infectivity. SP-4 (residues 192–203), SP-8 (residues 483–494), and SP-10 (residues 668–679) significantly blocked the interaction between S protein and ACE2 by biotinylated enzyme-linked immunosorbent assay, with IC₅₀ values of 4.30±2.18, 6.99±0.71, and 1.88±0.52nmol, respectively. Peptide scanning suggested the region spanning residues 660–683 might act as a receptor-binding domain. SP-10 blocked both binding of the S protein and infectivity of S protein-pseudotyped retrovirus to Vero E6 cells. In conclusion, this is the first report of small peptides designed to disrupt the binding of SARS-CoV S protein to ACE2. Our findings suggest that SP-10 may be developed as an anti-SARS-CoV agent for the treatment of SARS-CoV infection.

Keywords: SARS-CoV; Spike protein; Angiotensin-converting enzyme 2; Peptide; Vero E6 cells; Pseudovirus

Treatment of intravaginal HSV-2 infection in mice: A comparison of CpG oligodeoxynucleotides and resiquimod (R-848) by Michael J. McCluskie; Janna L.M. Cartier; Amy J. Patrick; Dusan Sajic; Risini D. Weeratna; Kenneth L. Rosenthal; Heather L. Davis (pp. 77-85).

The mammalian innate immune system recognizes pathogens via a series of pattern-recognition receptors such as the toll-like receptors (TLR) that interact with pathogen-associated molecular patterns (PAMPs) and lead to the rapid activation of innate immune cells. In this study, we compared the efficacy of CpG ODN (a TLR9 agonist) and resiquimod (R-848; a TLR7/8 agonist) for topical immunoprophylaxis or immunotherapy of vaginal herpes simplex virus type 2 (HSV-2) infection in mice. Efficacy against HSV infection was observed with CpG ODN but less so with R-848, even after repeated administrations. Intravaginal (IVAG) administration of CpG ODN resulted in strong local but relatively weak systemic immune activation, as determined by levels of the chemokines IP-10, MIG and I-TAC in vaginal tissue and plasma, respectively. In contrast, IVAG administration of R-848 resulted in high levels of plasma IP-10, similar to those seen after parenteral administration, but overall, weaker or shorter-lived local immune responses than obtained with CpG ODN. These findings suggest that differences in biodistribution and sites of immune activation between CpG ODN and R-848 after IVAG delivery account for differences in efficacy, and demonstrate the need for local mucosal innate activation for protection against HSV-2.

Keywords: CpG ODN; R-848; Innate; TLR; HSV-2; Vaginal

Identification and characterization of potent small molecule inhibitor of hemorrhagic fever New World arenaviruses by Tove′ C. Bolken; Sylvie Laquerre; Yuanming Zhang; Thomas R. Bailey; Daniel C. Pevear; Shirley S. Kickner; Lindsey E. Sperzel; Kevin F. Jones; Travis K. Warren; S. Amanda Lund; Dana L. Kirkwood-Watts; David S. King; Amy C. Shurtleff; Mary C. Guttieri; Yijun Deng; Maureen Bleam; Dennis E. Hruby (pp. 86-97).

Category A arenaviruses as defined by the National Institute of Allergy and Infectious Diseases (NIAID) are human pathogens that could be weaponized by bioterrorists. Many of these deadly viruses require biosafety level-4 (BSL-4) containment for all laboratory work, which limits traditional laboratory high-throughput screening (HTS) for identification of small molecule inhibitors. For those reasons, a related BSL-2 New World arenavirus, Tacaribe virus, 67–78% identical to Junín virus at the amino acid level, was used in a HTS campaign where approximately 400,000 small molecule compounds were screened in a Tacaribe virus-induced cytopathic effect (CPE) assay. Compounds identified in this screen showed antiviral activity and specificity against not only Tacaribe virus, but also the Category A New World arenaviruses (Junín, Machupo, and Guanarito). Drug resistant variants were isolated, suggesting that these compounds act through inhibition of a viral protein, the viral glycoprotein (GP2), and not through cellular toxicity mechanisms. A lead compound, ST-294, has been chosen for drug development. This potent and selective compound, with good bioavailability, demonstrated protective anti-viral efficacy in a Tacaribe mouse challenge model. This series of compounds represent a new class of inhibitors that may warrant further development for potential inclusion in a strategic stockpile.

Keywords: Arenavirus; Antiviral; Tacaribe; Jun�n; Hemorrhagic fever

Bovine lactoferrin peptidic fragments involved in inhibition of Echovirus 6 in vitro infection by Agostina Pietrantoni; Maria Grazia Ammendolia; Antonella Tinari; Rosa Siciliano; Piera Valenti; Fabiana Superti (pp. 98-106).

Bovine lactoferrin is a multifunctional glycoprotein folded in two symmetric globular lobes (N- and C-lobes), each being able to bind one ferric ion. We have previously demonstrated that this protein is able to prevent echovirus-induced apoptosis. In the present study, we have investigated both the role of tryptic fragments of bovine lactoferrin and the mechanism of lactoferrin effect on echovirus infection. Results obtained showed that bovine lactoferrin inhibits echovirus-induced cytopathic effect and antigen synthesis in a dose-dependent manner and that this protein is able to prevent viral replication when added not only during the entire cycle of infection but also before, during or after the viral adsorption step. The N-terminal cationic peptide was sufficient to prevent viral binding. Our data suggest that lactoferrin inhibition of echovirus attachment to cell receptors could be mediated by the cluster of positive charges at its N-terminus (lactoferricin).

Keywords: Lactoferrin; Echovirus; N-lobe; Lactoferricin; Antiviral activity

Citrate-mediated disaggregation of rotavirus particles in RotaTeq^� vaccine by Susan E. Peterson; Shiyi Wang; Todd Ranheim; Katey Einterz Owen (pp. 107-115).

For the routine manufacture of live virus vaccines, virus is diluted into a formulation buffer to stabilize it for long-term storage, and to facilitate vaccine administration. The characteristics of this buffer are dependent on the storage temperature of the vaccine, as well as the desired characteristics of the product. The formulation buffer for RotaTeq^®, Merck's live, pentavalent, oral rotavirus vaccine to prevent rotavirus gastroenteritis was developed as a fully liquid solution that requires no pre-feeding prior to administration, and is stable for 24 months at refrigerated temperatures. In studying the effects of the formulation buffer on the live virus contained within RotaTeq^®, we observed that the formulation buffer also directly impacts the state of rotavirus aggregation. This observation, termed “the matrix effect,�? was first noted as an ∼50% increase in measured in vitro infectivity, following dilution of the virus into the buffer. Subsequent experiments confirmed that citrate in the formulation buffer facilitates the disaggregation of viral particles, likely through a carboxylic-acid mediated interaction. For vaccine manufacture, bulk virus is titered and subsequently diluted to a target concentration for dosing. Aggregation of the virus and subsequent inaccurate measurement of the amount of virus contained in either the bulk sample or in the final dosing container could lead to an inability to accurately predict final vaccine concentrations. Thus, discerning the nature and extent of the matrix effect was key principally for providing an accurate prediction of final virus concentration upon dilution, to ensure a robust manufacturing process. In addition, understanding potential contributions of the formulation buffer to clinical efficacy of the vaccine was critical. Clinical data have confirmed that the citrate-mediated disaggregation had no measurable impact on vaccine safety, immunogenicity, or efficacy.

Keywords: Abbreviations; MOI; multiplicity of infection; M-QPA; multivalent quantitative PCR-based cell infectivity assay; TNC; Tris, sodium chloride, calcium buffer; PFU; plaque forming unit; IU; infectious unit; R.S.D.; relative standard deviation; EM; electron microscopy; DLS; dynamic light scattering; S.D.; standard deviation; TLP; triple-layered particle; DLP; double-layered particleRotavirus vaccine; RotaTeq; ^�; Formulation; Citrate; Disaggregation

Fenretinide inhibits HIV infection by promoting viral endocytosis by Catherine M. Finnegan; Robert Blumenthal (pp. 116-123).

HIV fusion is mediated by the sequential interaction of the viral envelope glycoprotein with cellular receptors at the plasma membrane. We have previously reported that the upregulation of cellular ceramide levels following fenretinide treatment inhibits HIV fusion. As ceramide facilitates the internalization of a variety of microbes, we hypothesized that it may also promote the engulfment of HIV virions. Hence, we analyzed the effect of fenretinide treatment on virus binding and uptake. We observed that virus binding is not altered by fenretinide treatment. The distribution of HIV receptors was also unchanged. In contrast, virus uptake showed a significant increase. We have determined that fenretinide treatment promotes the internalization of virions from the plasma membrane and the accumulation of virus in the endocytic fraction of HeLa cells. This effect of fenretinide appears to be specific for virus as the endosomal accumulation of gp120, transferrin and horse-radish peroxidase was not increased. Notably, fenretinide increased the infectivity of influenza virus, which fuses in the endosomal compartment upon low pH activation. Our data suggest that fenretinide treatment effectively inhibits HIV infection by re-directing the virus to the endocytic pathway.

Keywords: HIV; Ceramide; Fenretinide; Endocytosis

Chemokine scavenging by the human cytomegalovirus chemokine decoy receptor US28 does not inhibit monocyte adherence to activated endothelium by J.M. Boomker; E.K. de Jong; L.F.M.H. de Leij; M.C. Harmsen (pp. 124-127).

The human cytomegalovirus has found smart ways to exploit the chemokine network in order to subvert immune attack. Chemokines trigger the arrest and firm adhesion of inflammatory cells to the vascular wall. Scavenging of chemokines by viral decoy receptors, such as US28, might prevent arrest of leukocytes to the vascular wall and impair an antiviral immune response. We determined the effect of chemokine scavenging by endothelium-expressed signaling mute US28 (US28R129A) on static monocyte adhesion. Despite the chemokine scavenging capacity of US28R129A, expression of this construct by endothelial cells was insufficient to disrupt leukocyte adhesion to cytokine-activated monolayers. Our results suggest that the concentrations of chemokines that trigger firm leukocyte adhesion are too high to be efficiently scavenged by viral chemokine decoy receptors like US28. From the results of this experimental model a role for US28 in viral immune evasion by chemokine scavenging would appear therefore unlikely.

Keywords: Immune evasion; Human cytomegalovirus; Leukocyte adhesion; Chemokine receptor; Chemokine

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Antiviral Research (v.69, #2)