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Antiviral Research (v.66, #1)
The efficacy of cidofovir treatment of mice infected with ectromelia (mousepox) virus encoding interleukin-4 by Samantha J. Robbins; Ronald J. Jackson; Frank Fenner; Sandra Beaton; Jill Medveczky; Ian A. Ramshaw; Alistair J. Ramsay (pp. 1-7).
Improved vaccines and therapies for virulent poxvirus infection are required, particularly in the light of recent threats of bioterrorism. Cidofovir (HPMPC) is an acyclic nucleoside analog with proven efficacy against poxviruses. Here, we evaluated HPMPC in mice given a recombinant ectromelia virus (ECTV) encoding interleukin-4 (ECTV-IL-4) that is highly immune suppressive. Mousepox-sensitive BALB/c mice given HPMPC for five consecutive days after infection were protected against the lethal effects of a control ECTV recombinant, although they suffered a chronic form of mousepox disease. High doses of the drug resulted in a milder localized disease. In contrast, HPMPC failed to protect mousepox-resistant C57BL/6 mice against ECTV-IL-4, although its lethal effects were delayed by five daily doses of 20mg/kg or a single dose of 100mg/kg. Higher daily doses further delayed mortality, although the majority of animals eventually succumbed to infection. It appears that HPMPC inhibited ECTV-IL-4 replication without clearance, with the virus having a lethal effect when the drug was removed. Resistance of ECTV-IL-4 to HPMPC treatment may relate to the virus's ability to inhibit antiviral cell-mediated immunity. Interestingly, ECTV-IL-4-mediated immune suppression was not accompanied by a reduction in systemic IFN-γ expression, suggestive of an alternative or highly localized suppressive mechanism.
Keywords: Cidofovir; Mousepox infection; Interleukin-4; Immune suppression
Cranberry juice constituents affect influenza virus adhesion and infectivity by E.I. Weiss; Y. Houri-Haddad; E. Greenbaum; N. Hochman; I. Ofek; Z. Zakay-Rones (pp. 9-12).
Cranberry juice contains high molecular weight materials (NDM) that inhibit bacterial adhesion to host cells as well as the co-aggregation of many oral bacteria. Because of its broad-spectrum activity, we investigated NDM's potential for inhibiting influenza virus adhesion to cells, and subsequent infectivity. Hemagglutination (HA) of red blood cells (RBC) caused by representatives of both influenza virus A subtypes (H1N1 and H3N2) and the B type was inhibited by NDM at concentrations of 125μg/ml or lower, which is at least 20-fold lower than that usually found in cranberry juice. A dose–response effect of NDM on HA was demonstrated. The infectivity of the A and B types was significantly reduced by preincubation with NDM (250μg/ml), as reflected by the lack of cytopathic effect on Madine-Darby canine kidney (MDCK) cells and the lack of HA activity in the media of infected cells. The effect of NDM was also tested after A or B type viruses were allowed to adsorb to and penetrate the cells. Various levels of reduction in virus tissue culture infective dose TCID50 were observed. The effect was most pronounced when NDM was added several times to the infected MDCK cells. Our cumulative findings indicate that the inhibitory effect of NDM on influenza virus adhesion and infectivity may have a therapeutic potential.
Keywords: Influenza; Cranberry; NDM; Antiviral effect
Anti-gene peptide nucleic acid targeted to proviral HIV-1 DNA inhibits in vitro HIV-1 replication by Caterina D. Pesce; Francesca Bolacchi; Barbara Bongiovanni; Federica Cisotta; Marcella Capozzi; Silvia Diviacco; Franco Quadrifoglio; Ruggiero Mango; Giuseppe Novelli; Giuseppe Mossa; Claudio Esposito; Domenico Ombres; Giovanni Rocchi; Alberto Bergamini (pp. 13-22).
Highly active antiretroviral therapy (HAART) is unlikely to affect reservoirs of HIV in latently infected cells. Anti-gene compounds, such as peptide nucleic acids (PNAs), which block transcriptional activity via sequence-specific invasion of double-stranded DNA may be an effective strategy to target cells harbouring proviral HIV DNA. Here we show that a PNA oligomer (PNAHIV), 15 bases in length, linked to a nuclear localization signal (NLS), substantially suppressed HIV-1 replication in chronically infected lymphocytes and macrophages and efficiently prevented mitogen-induced HIV-1 reactivation in lymphocytes, as determined by HIV-p24 antigen production in supernatants and FACS analysis for intracellular HIV accumulation. In contrast, a mismatched PNA did not show any effect on HIV expression. Semi-quantitative RT-PCR and quantitative real-time RT-PCR demonstrated a decrease of HIV RNA expression in infected cells treated by PNAHIV indicating that inhibition of HIV-1 replication occurred at the transcription step. In conclusion, the use of anti-gene PNA to target the HIV-1 proviral DNA in the quest for new antiretroviral agents appears quite promising.
Keywords: Peptide nucleic acid (PNA); Proviral DNA; Latent infection; HIV
Effects of IL-12 and IL-18 on HBcAg-specific cytokine production by CD4 T lymphocytes of children with chronic hepatitis B infection by Andrzej Szkaradkiewicz; Aleksandra Jopek; Jacek Wysocki (pp. 23-27).
The influence of IL-12 and IL-18 was evaluated on hepatitis B core antigen (HBcAg)-specific cytokine production (IFN-γ, IL-4, IL-5 and IL-10) by CD4 T lymphocytes isolated from peripheral blood of children with chronic hepatitis B. CD4 T cells were isolated from peripheral blood of 20 children with chronic active hepatitis B, cultured for 48h in presence of rHBcAg and of co-stimulators, IL-12 or IL-18 or IL-12+IL-18 or in their absence (control). Production of studied cytokines was examined using the ELISPOT assay. Co-stimulation with IL-12 or IL-18 was found to significantly augment the HBcAg-specific secretion of IFN-γ. However, the most pronounced stimulatory effect was observed in the presence of IL-12+IL-18 and resulted in peak levels of IFN-γ production. The obtained results allowed concluding that the anti-HBV activity of Th1 lymphocytes is strongly induced by IL-12+IL-18 and may contribute to viral clearance in children with chronic hepatitis B infection.
Keywords: Hepatitis B virus; Th lymphocytes; Interferon-γ; Interleukin-12; Interleukin-18
Ethanol extract of Polygonum cuspidatum inhibits hepatitis B virus in a stable HBV-producing cell line by Jung-San Chang; Hong-Wen Liu; Kuo-Chih Wang; Mei-Chun Chen; Lien-Chai Chiang; Yi-Cheng Hua; Chun-Ching Lin (pp. 29-34).
Chronic hepatitis B virus (HBV) infection is endemic in Asia and its consequences are among the major public health problems in the world. Unfortunately, the therapeutic efficacies of present strategies are still unsatisfactory with a major concern about viral mutation. In search of effective antiviral agent, we examined the efficacy of extracts of Polygonum cuspidatum Sieb. et Zucc. ( P. cuspidatum) against HBV in HepG2 2.2.15 cells by quantitative real time polymerase chain reaction. The expressions of viral antigens, HBeAg and HBsAg, were also determined by enzyme linked immunosorbent assay. The ethanol extract of P. cuspidatum could inhibit dose-dependently the production of HBV ( p<0.0001) with an effective minimal dosage of 10μg/ml. The water extract of P. cuspidatum might also inhibit the production of HBV at a higher dosage. The expression of HBsAg was significantly increased by both ethanol extract and water extract of P. cuspidatum dose-dependently ( p<0.0001) and time-dependently ( p<0.0001). Higher dose of water extract of P. cuspidatum (30μg/ml) could inhibit the expression of HBeAg ( p<0.05). The extract of P. cuspidatum might contain compounds that would contribute to the control of HBV infection in the future. However, its promoting effect on the expression of HBsAg and its cytotoxicity should be monitored. Further purification of the active compounds, identification and modification of their structures to improve the efficacy and decrease the cytotoxicity are required.
Keywords: Abbreviations; α-SMA; α-smooth muscle actin; CC; 50; cytotoxic concentration of 50%; DMSO; dimethyl sulfoxide; ELISA; enzyme linked immunosorbent assay; HBV; hepatitis B virus; HBeAg; e antigen of HBV; HBsAg; surface antigen of HBV; HCC; hepatocellular carcinoma; IC; 50; concentration of 50% inhibition of viral replication; IFN; interferon; NF-κB; nuclear factor-κB; OD; optical density; PCE; the ethanol extract of; P. cuspidatum; P. cuspidatum; Polygonum cuspidatum; Sieb. et Zucc; PCW; the water extract of; P. cuspidatum; SI; selectivity index; Th; helper T cell; TNF; tumor necrosis factorAntiviral assay; Chronic B hepatitis; HepG2; Polygonum cuspidatum; Polymerase chain reaction; TaqMan
Antiviral activity of serum from the American alligator ( Alligator mississippiensis) by Mark E. Merchant; Melanie Pallansch; Robin L. Paulman; Jay B. Wells; Aysegul Nalca; Roger Ptak (pp. 35-38).
Serum from wild alligators was collected and tested for antibiotic activity against three enveloped viruses using cell-based assays. Alligator serum demonstrated antiviral activities against human immunodeficiency virus type 1 (HIV-1; IC50=0.9%), West Nile virus (WNV; IC50=4.3%), and Herpes simplex virus type 1 (HSV-1; IC50=3.4%). The inhibitory concentration (IC50) is defined as the concentration of serum that inhibits 50% of viral activity. The antiviral effects of the alligator serum were difficult to evaluate at high concentrations due to the inherent toxicity to the mammalian cells used to assay viral activities. The TC50 (serum concentration that reduces cell viability to 50%) values for the serum in the HIV-1, WNV, and HSV-1 assays were 32.8, 36.3 and 39.1%, respectively. Heat-treated serum (56°C, 30min) displayed IC50 values of >50, 9.8 and 14.9% for HIV-1, WNV and HSV-1 viruses, respectively. In addition, the TC50 values using heat-treated serum were substantially elevated for all three assays, relative to untreated serum (47.3 to >50%). Alligator serum complement activity has been shown to be heat labile under these conditions. HIV-1 antiviral action was heat-sensitive, and thus possibly due to the action of serum complement, while the anti-WNV and anti-HSV-1 activities were not heat labile and thus probably not complement mediated.
Keywords: Alligator; Antiviral; Complement; Crocodilian; Serum
Differential profile of genes expressed in hemocytes of White Spot Syndrome Virus-resistant shrimp ( Penaeus japonicus) by combining suppression subtractive hybridization and differential hybridization by Nanhai He; Qiwei Qin; Xun Xu (pp. 39-45).
White Spot Syndrome Virus (WSSV) is the major viral pathogen of culture shrimp. Although remarkable progress has been made in characterizing the WSSV genome, information concerning the antiviral process of host is still limited. To identify the genes differentially expressed along with their expression profile in the hemocytes of the virus-resistant shrimp, suppression subtractive hybridization (SSH) and differential hybridization (DH) were employed. Relying on the sequences identified in the subtractive cDNA library, 30 genes were characterized to be involved in the antiviral process as defense-relevant, among them, 22 are found for the first time in penaeid shrimp. The most interesting finding is that the interferon-like protein (IntlP) and (2′–5′) oligo(A) synthetase-like protein (data not shown) known as the antiviral factors showed increased expression in virus-resistant shrimp and the non-specific antiviral activity of IntlP protein was verified by cytotoxicity experiment. A number of proteins with certain similarities to the components of the complement and cytokines system in vertebrates were also found in the subtracted library. The high expression of redox-related factors (NADH dehydrogenase, glutathione peroxidase and transcription factor AP-1 precursor), plasma defensive protein (C-type lectin and laminin-like protein) and translationally controlled tumor protein (TCTP) in the virus-resistant shrimp suggested that they are essential components participating in the antiviral process. Our work provides a wide array of genes differentially expressed in the virus-resistant shrimp, and a framework for further studies aimed at antiviral mechanism in shrimp.
Keywords: Antiviral factor; Virus resistance; Differential profile; Penaeus japonicus
Involvement of HIV-1 protease in virus-induced cell killing by Iván Ventoso; Joaquín Navarro; Mª A. Muñoz; Luis Carrasco (pp. 47-55).
Acute infection of human CD4+ cells with cytopathic strains of HIV-1 causes rapid cell death. The role played by HIV-1 protease (PR) in virus-induced cell killing was investigated by subjecting C8166 cells to a single round of infection. The presence of HIV-1 PR inhibitor saquinavir from 24h post-infection prevented virus-induced cell lysis. This inhibitor caused only a small reduction in the number of infected cells and in the expression of HIV-1-specific proteins. Moreover, treatments that block HIV-1 reinfection, such as AZT or the anti-CD4 antibody leu3.a, exerted little effect on virus-induced cell death. Thus, the specific inhibition of HIV-1 protease reduced the extent of both necrosis and apoptosis in C8166 cells such that most cells survived HIV-1 infection. Continued treatment of the infected cells with saquinavir led to the progressive suppression of HIV-1 expression; no viral proteins being detected 10 days after primary infection. Notably, reactivation of HIV-1 protease in these cells by removing the saquinavir triggered virus replication and cell lysis. These findings may contribute towards a better understanding of HIV-1 pathogenesis, and emphasise the potential of the virus protease as a key therapeutic target in AIDS treatment.
Keywords: HIV-1 protease; Saquinavir; Cell lysis; Apoptosis; Cytopathogenicity
Development of a cotton rat–human metapneumovirus (hMPV) model for identifying and evaluating potential hMPV antivirals and vaccines by Philip R. Wyde; Srikrishna N. Chetty; Alan M. Jewell; Shauna L. Schoonover; Pedro A. Piedra (pp. 57-66).
Hispid cotton rats were inoculated with two different human metapneumovirus (hMPV) subtype A strains and one subtype B hMPV. Although no overt disease was seen in any virus-inoculated animal, following an eclipse phase, significant pulmonary virus titers were observed in every hMPV-inoculated animal through day 7 post virus inoculation (p.i.) and in most through day 10. Peak virus titers occurred four days p.i., while virus-induced histopathology was most evident in lung sections obtained from animals 7 to 10 days p.i. The latter consisted primarily of desquamating and hypertrophic columnar epithelial cells lining the bronchi and bronchioles and the presence of large numbers of leukocytes in and around the bronchi and bronchioles. In fluorescent antibody studies, virus antigen-specific fluorescence was most evident in the desquamating tall columnar epithelial cells lining bronchi and bronchioles, in pneumocytes lining alveoli and in single or small groups of free cells, most probably leukocytes, present in the lumen of alveoli, bronchi and bronchioles. Virus was generally not detected in inoculated animals >10 days p.i. Although the pattern of virus replication in cotton rats was similar for all the three virus stains, the B subtype consistently grew to lower levels than the two A strains. Regardless, these findings indicate that hMPV replicates in cotton rats and that these animals may be used as a small animal model of hMPV infection and to facilitate the identification and development of vaccines and antivirals for preventing and/or ameliorating infections caused by this virus.
Keywords: hMPV; Human metapneumovirus; Cotton rats; Animal model; Antiviral testing; Vaccine evaluation
The fd phage and a peptide derived from its p8 coat protein interact with the HIV-1 Tat-NLS and inhibit its biological functions by A. Krichevsky; M. Rusnati; A. Bugatti; E. Waigmann; S. Shohat; A. Loyter (pp. 67-78).
Filamentous fd bacteriophages are used to construct phage-display peptide libraries, which have been instrumental in selecting peptides that interact with specific domains within target molecules. Here we demonstrate that the fd bacteriophage itself, as well as NTP8 – a synthetic peptide derived from it and bearing amino acids 1–20 of the phage p8 protein – interact with the nuclear localization signal (NLS) of the HIV-1 Tat protein. Accordingly, fd bacteriophage and the NTP8 peptide inhibit binding mediated by the Tat-NLS to the nuclear-import receptor importin β and Tat-NLS-mediated translocation into cell nuclei. The NTP8 peptide, at 100μM concentration, also caused about 50% inhibition of HIV-1 propagation in cultured cells. The fd bacteriophage prevents heparan sulfate proteoglycans-mediated uptake of extracellular Tat by target cells and consequently transactivation of a chloramphenicol acetyltransferase (CAT) reporter gene. A BSA-NTP8 conjugate inhibits Tat-NLS-mediated binding to heparin immobilized on a BIAcore surface. BLAST analysis of the NTP8 amino-acid sequence revealed similarity to sequences in several human proteins, including ADA2 and CD53.
Keywords: HIV-1; Tat; Fd bacteriophage; Inhibitory peptide; Nuclear-import