Comparison of Direct and Indirect Solid-Phase Microradioimmunoassays for the Detection of Viral Antigens and Antiviral Antibody

Viral antigens were fixed to the surface of microtiter wells, and serial dilutions of antiviral antibody were added. The amount of antiviral antibody bound to viral antigens was determined by measuring the extent to which the antiviral antibody either inhibited the specific binding of (125)I-labeled antiviral immunoglobulin G (IgG) (direct technique) or enhanced the specific binding of (125)I-labeled anti-IgG (indirect technique). Immune complexes composed of viral antigens and antiviral antibody (human) could be detected by the binding of (125)I-labeled rheumatoid factor. Specific binding was influenced by the concentration of protein in the diluents used during the different steps of the procedure. A high concentration of protein in the diluent used with the viral antigens decreased specific binding, whereas a high concentration of protein in the diluent used with (125)I-labeled anti-IgG increased specific binding by decreasing nonspecific attachment of the labeled anti-IgG. Under the conditions employed, the titer of a given antiviral serum was several hundredfold greater by the indirect than by the direct technique.     

The role of interferon-beta 1 and the 26-kDa protein (interferon-beta 2) as mediators of the antiviral effect of interleukin 1 and tumor necrosis factor

This study confirms our earlier finding that human interleukin (IL)-1 beta exerts an antiviral effect on diploid fibroblasts and on MG-63 osteosarcoma cells. It also extends the observation in that a similar effect was noted on aged but not freshly trypsinized HEp-2 cells, and that not only IL-1 beta but also IL-1 alpha and tumor necrosis factor (TNF)-alpha exerted similar antiviral effects on cells. The antiviral effects of these cytokines were neutralized by addition to the assay system of an antibody that was specific for interferon (IFN)-beta 1, indicating that IFN-beta 1 or a structurally or functionally related substance is involved in the antiviral activity observed. Both IL-1 and TNF were able to induce production of the 26-kDa protein, also known as IFN-beta 2, hybridoma/plasmacytoma growth factor (HPGF) or B-cell stimulatory factor-2 (BSF-2) and previously proposed as an alternative to IFN-beta 1 for mediating the antiviral effect of TNF. However, no good correlation was found between the antiviral effects of TNF and its potential to induce production of the 26-kDa protein. Furthermore, the anti-IFN-beta 1 serum which neutralized the antiviral activity of IL-1 and TNF did not cross-react with the 26-kDa protein. Conversely, the antiviral effect of IL-1 and TNF was only weakly neutralized by an antibody that did react with the 26-kDa protein and showed low cross-reactivity with IFN-beta 1. These observations, together with the low specific activity of the 26-kDa protein as an antiviral agent (less than 10(5) U/mg protein) provide strong arguments against this protein and in favor of IFN-beta 1 (or still another IFN-beta 1-related molecule) as the ultimate mediator of the antiviral effect of IL-1 and TNF.     

SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 μM) and La Crosse virus (EC(50)=0.27 μM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).     

Analysis of antigenic domains on natural and recombinant human IFN-beta by the inhibition of biologic activities with monoclonal antibodies

Human IFN-beta (HuIFN-beta) is a biologically potent protein with both antiviral and antiproliferative activities. To understand better its mode of action, a number of murine mAb were developed against a recombinant (serine-17) HuIFN-beta (rHuIFN-beta ser) and screened by ELISA and neutralization of antiviral activity. The panel of antibodies, composed of both IgA and IgG immunoglobulins, were specific for HuIFN-beta and did not crossreact with HuIFN-alpha or gamma. Furthermore, three functionally distinct epitopes (designated as sites I, II, and III) were identified based on mAb neutralization of antiviral and antiproliferative activities of recombinant and natural HuIFN-beta. Antibodies directed to sites I and II neutralized the antiviral and antiproliferative activities of rHuIFN-beta ser, though the antiviral neutralization potency of the mAb to site II was approximately 10-fold less than mAb to site I. Antibodies directed to site I neutralized both recombinant and natural HuIFN-beta, although the antiviral neutralization potency was approximately 10-fold higher against rHuIFN-beta ser than the native protein. The mAb directed to site II did not demonstrate any significant neutralization of the antiviral or antiproliferative activity of natural HuIFN-beta but neutralized a recombinant HuIFN-beta containing the native sequence. Antibodies recognizing site III did not neutralize the biologic activity of either recombinant or natural HuIFN-beta. Thus, three epitopes on HuIFN-beta have been identified, two of which are associated with both antiviral and antiproliferative activities.     

Characterization of translational inhibitors from Phytolacca americana. Amino-terminal sequence determination and antibody-inhibitor conjugates

Two translational inhibitors (pokeweed antiviral protein and pokeweed antiviral protein II) isolated from the leaves of the pokeweed plant, Phytolacca americana, were characterized as to their behavior during reverse-phase HPLC and their amino-terminal sequences. Alignment of the sequences demonstrated that a substantial degree of homology was present (10 of 29 identical residues). Pokeweed antiviral protein was shown by reverse-phase chromatography to be composed of at least two components, pokeweed antiviral proteina and pokeweed antiviral proteinb, which comigrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, shared identical N-terminal amino-acid sequences through residue 31, and had similar specific activities in a cell-free translation inhibition assay. Pokeweed antiviral protein II was covalently coupled to a monoclonal antibody that recognizes the transferrin receptor (anti-transferrin receptor). The disulfide-linked conjugate inhibited protein synthesis in the human breast tumor cell line MCF-7, whereas anti-transferrin receptor, pokeweed antiviral protein II, or an immunotoxin composed of an irrelevant antiserum and pokeweed antiviral protein II, were nontoxic. The inhibitory dose 50% of anti-transferrin receptor-pokeweed antiviral protein II for MCF-7 cells was 0.7 nM, whereas the corresponding ricin A chain conjugate (anti-transferrin receptor-ricin A chain) was more potent with a inhibitory dose 50% of 0.1 nM. Pokeweed antiviral protein II can be added to the growing list of translation inhibitors that are effective as components of immunotoxins in vitro. Additional studies will be needed to determine whether pokeweed antiviral protein II immunotoxins provide advantageous properties for in vivo applications.     

Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.     

Studies on antiviral glycosides. Synthesis and biological evaluation of various phenyl glycosides.

A variety of analogues and derivatives of phenyl glycosides were synthesized for examination of their biological activities and of the relationship between structure and antiviral activity. For antiviral activity, a 6-deoxy-6-halogeno-D-glucose residue was most suitable for the carbohydrate moiety and p-alkylphenyl groups for the aglycone moiety. Based on these results, p-(sec-butyl)phenyl 6-chloro-6-deoxy-beta-D-glucopyranoside and p-(sec-butyl)phenyl 6-deoxy-6-iodo-beta-D-glucopyranoside were prepared, and the former compound was found to be the most potent antiviral substance, in this series, against influenza and Herpes simplex virus. The anomeric configuration of phenyl glycosides did not contribute to the antiviral activity.     

Correlation between the lipopolysaccharide response of mice and the capacity of mouse peritoneal cells to transfer an antiviral state. Role of endogenous interferon

Freshly harvested mouse peritoneal cells, from normal lipopolysaccharide (LPS)-responsive (Lpsn) mice, were capable of transferring an antiviral state (to vesicular stomatitis virus) to "in vitro aged" mouse macrophages permissive for viral replication. The transfer of the antiviral state was completely abrogated by addition of antibody to interferon (IFN)-alpha/beta in the co-culture medium. In contrast, even large numbers of donor peritoneal cells from LPS-hyporesponsive (Lpsd) C3H/HeJ and C57BL/10ScCR mice did not transfer an antiviral state to target cells. Although peritoneal macrophages from Lpsd mice did not transfer an antiviral state to target cells, they were nevertheless found to be in an antiviral state when first placed in culture. Injection of mice with antibody to mouse IFN-alpha/beta rendered peritoneal macrophages from both Lpsn and Lpsd mice permissive for vesicular stomatitis virus. The decay of this initial antiviral state in peritoneal macrophages during in vitro culture was far more rapid for Lpsd mice than for normal mice. Addition of antibody to mouse IFN-alpha/beta markedly enhanced the in vitro decay of the antiviral state of peritoneal macrophages. Treatment of total peritoneal cells from Lpsn mice with LPS resulted in IFN production, whereas IFN was not detected in the cellfree medium of LPS-treated peritoneal cells from Lpsd C3H/HeJ and C57BL/10ScCR mice. Genetic studies with F1 hybrids between Lpsn and Lpsd mice and with Lpsn and Lpsd recombinant inbred strains revealed a striking correlation between the capacity of peritoneal cells to transfer an antiviral state and their capacity to produce IFN after stimulation with LPS, suggesting that closely linked, if not identical, genes are in some way involved in the transfer of antiviral state as well as in the LPS response by peritoneal cells of normal mice.     

Revelation of Antiviral Activities by Artificial Sulfation of a Glycosaminoglycan from a Marine Pseudomonas

Sulfated derivatives of a glycosaminoglycan containing l-glutamic acid produced by a marine Pseudomonas species, No. 42 strain, were prepared by the method of dicyclohexyl-carbodiimide-mediated reaction. Both low and high degrees of sulfation of the polysaccharides (products A1 and A2, respectively) were investigated for their antiviral activities against influenza virus type A (FluV-A) and B (FluV-B) in MDCK cells. Both preparations showed antiviral activity against FluV-A at the 50% antiviral effective concentration of 17.3 and 5.2 μg/ml, respectively, whereas they had no antiviral activity against FluV-B. No cytotoxicity of either product was noted against MDCK cells at the 50% cytotoxic concentration of 100 μg/ml. Received April 4, 1998; accepted July 24, 1998.     

Antiviral Potential of Lactic Acid Bacteria and Their Bacteriocins

Emerging resistance to antiviral agents is a growing public health concern worldwide as it was reported for respiratory, sexually transmitted and enteric viruses. Therefore, there is a growing demand for new, unconventional antiviral agents which may serve as an alternative to the currently used drugs. Meanwhile, published literature continues shedding the light on the potency of lactic acid bacteria (LAB) and their bacteriocins as antiviral agents. Health-promoting LAB probiotics may exert their antiviral activity by (1) direct probiotic–virus interaction; (2) production of antiviral inhibitory metabolites; and/or (3) via stimulation of the immune system. The aim of this review was to highlight the antiviral activity of LAB and substances they produce with antiviral activity.     

含硒抗病毒多肽的半胱氨酸缺陷型表达及鉴定

目的：在大肠杆菌中进行含硒抗病毒多肽的可溶性半胱氨酸缺陷型融合蛋白的表达,并对其纯化蛋白进行生物学活性的初步鉴定。方法：将含硒抗病毒多肽Se-GBVA10基因克隆到GST融合表达载体pGEX-2T中,转化至半胱氨酸缺陷型大肠杆菌BL21 cysE51 后进行半胱氨酸缺陷型表达,通过谷胱甘肽Sepharose 4B 亲和柱纯化目的蛋白质,并对其含硒量、抗病毒活力等性质进行初步鉴定。结果：利用半胱氨酸缺陷型表达法成功地可溶性表达了sjGST-Se-GBVA10和sjGST-GBVA10融合蛋白,并经凝血酶切后获得了纯化的Se-GBVA10和GBVA10抗病毒多肽。测定Se-GBVA10的含硒量为0.974 mol/mol peptide,GPX活力为47.52 U/μmol,其EC₅₀为21.73μmol/L,CC₅₀为849.41μmol/L。结论：Se-GBVA10具有与GBVA10相同的抗病毒活性,并具有一定的抗氧化活力。     

Antiviral substance from silkworm faeces: characterization of its antiviral activity

The antiviral activity of a substance (L4-1) purified from silkworm faeces was examined in an HVJ (Sendai virus)-LLC-MK2 cell system. Its antiviral effect depended on the period of light irradiation and was inhibited by sodium sulfite and anaerobic conditions. These results indicate that the antiviral activity of L4-1 is associated with active oxygen species produced from the substance. SDS-polyacrylamide gel electrophoretic analysis showed that viral proteins were damaged by this substance under light irradiation. The results suggest that the antiviral activity is due to damage to viral protein(s) caused by active oxygen species produced from L4-1.     

The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase

It is not yet clear to what extent depletion of intracellular GTP pools contributes to the antiviral activity of ribavirin. Therefore, the antiviral activities of (i) ribavirin, (ii) its 5-ethynyl analogue, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), and (iii) mycophenolic acid (MPA) (a compound that inhibits only cellular IMP dehydrogenase activity) were studied on the replication of flaviviruses and paramyxoviruses. In addition, the effects of these three compounds on intracellular GTP pools were assessed. A linear correlation was observed over a broad concentration range between the antiviral activities of ribavirin, EICAR, and MPA and the effects of these compounds on GTP pool depletion. When the 50% effective concentrations (EC50s) for the antiviral activities of ribavirin, EICAR, and MPA were plotted against the respective EC50 values for GTP pool depletion, a linear correlation was calculated. These data provide compelling evidence that the predominant mechanism of action of ribavirin in vitro against flavi- and paramyxoviruses is based on inhibition of cellular IMP dehydrogenase activity.     

Biological characterisation of a recombinant Atlantic salmon type I interferon synthesized in Escherichia coli

Type I (alpha/beta) interferons (IFNs) are a family of cytokines that stimulate the expression of numerous proteins that mediate an antiviral state in uninfected cells. Two Atlantic salmon (Salmo salar) IFN-alpha (SasaIFN-alpha1 & 2) genes have previously been cloned and both were found to contain a putative N-linked glycosylation site. Recombinant SasaIFN-alpha1 (rSasaIFN-alpha1) produced in eukaryotic systems has repeatedly been shown to confer antiviral properties. However, different IFN-alpha subtypes may exhibit differential antiviral activities and be subject to glycosylation. To evaluate the potential therapeutic impact of a rSasaIFN-alpha, the mature form of the SasaIFN-alpha2 protein was produced in a high-level Escherichia coli expression system. Expression of the rSasaIFN-alpha2 was detected by SDS-PAGE and Western blot, and its identity was confirmed by mass spectrometry. In the homologous Chinook salmon embryonic (CHSE-214) cell line, the rSasaIFN-alpha2 incited early expression of the IFN-induced Mx protein and exhibited high antiviral activity of about 2.8 x 10(6) U mg(-1) against infectious pancreatic necrosis virus (IPNV). Conversely, antiviral protection by rSasaIFN-alpha2 was not observed in a heterologous Japanese flounder embryo (HINAE) cell line. Hence, a biologically active form of rSasaIFN-alpha2 was successfully produced using a glycosylation-deficient prokaryotic system and purified to homogeneity, suggesting that glycosylation is not required for its antiviral activity.     

Sendai Virus C Proteins Counteract the Interferon-Mediated Induction of an Antiviral State

We have studied the relationship between the Sendai virus (SeV) C proteins (a nested set of four proteins initiated at different start codons) and the interferon (IFN)-mediated antiviral response in IFN-competent cells in culture. SeV strains containing wild-type or various mutant C proteins were examined for their ability (i) to induce an antiviral state (i.e., to prevent the growth of vesicular stomatitis virus [VSV] following a period of SeV infection), (ii) to induce the elevation of Stat1 protein levels, and (iii) to prevent IFN added concomitant with the SeV infection from inducing an antiviral state. We find that expression of the wild-type C gene and, specifically, the AUG114-initiated C protein prevents the establishment of an antiviral state: i.e., cells infected with wild-type SeV exhibited little or no increase in Stat1 levels and were permissive for VSV replication, even in the presence of exogenous IFN. In contrast, in cells infected with SeV lacking the AUG114-initiated C protein or containing a single amino acid substitution in the C protein, the level of Stat1 increased and VSV replication was inhibited. The prevention of the cellular IFN-mediated antiviral response appears to be a key determinant of SeV pathogenicity.     

Enzymatically active APOBEC3G is required for efficient inhibition of human immunodeficiency virus type 1

APOBEC3G (APO3G) is a cellular cytidine deaminase with potent antiviral activity. Initial studies of the function of APO3G demonstrated extensive mutation of the viral genome, suggesting a model in which APO3G's antiviral activity is due to hypermutation of the viral genome. Recent studies, however, found that deaminase-defective APO3G mutants transiently expressed in virus-producing cells exhibited significant antiviral activity, suggesting that the antiviral activity of APO3G could be dissociated from its deaminase activity. To directly compare the antiviral activities of wild-type (wt) and deaminase-defective APO3G, we used two approaches: (i) we titrated wt and deaminase-defective APO3G in transient-transfection studies to achieve similar levels of virus-associated APO3G and (ii) we constructed stable cell lines and selected clones expressing comparable amounts of wt and deaminase-defective APO3G. Viruses produced under these conditions were tested for viral infectivity. The results from the two approaches were consistent and suggested that the antiviral activity of deaminase-defective APO3G was significantly lower than that of wt APO3G. We conclude that efficient inhibition of vif-defective human immunodeficiency virus type 1 requires catalytically active APO3G.     

Enhancement of the antiviral and interferon-inducing activities of poly r(A-U) by carminic acid

Experiments have been designed to systematically examine the effects of carminic acid (CAR) on the antiviral/interferon-inducing activity of poly r(A-U), using the human foreskin fibroblast-vesicular stomatitis virus bioassay system. Modulation of the antiviral/interferon-inducing activity of poly r(A-U) by carminic acid was examined at fixed poly r(A-U) concentrations of 0.05 mM or 0.2 mM while varying the carminic acid concentrations to produce variable CAR/ribonucleotide ratios ranging from 1:16 to 2:1. Carminic acid and poly r(A-U) were tested individually at the concentrations employed in the CAR/poly r(A-U) combinations. Neither the carminic acid alone nor poly r(A-U) alone were effective antiviral agents/interferon inducers. The antiviral/interferon-inducing activity of poly r(A-U) was potentiated twelve-fold at CAR/ribonucleotide ratios in the region of 1/6 to 1/4. These results suggest a synergism between the poly r(A-U) and the carminic acid at the concentrations employed in this study.     

Anti-influenza virus activities of 2-alkoxyimino-n-(2-isoxazolin-3-ylmethyl)acetamides.

A series of 2-alkoxyimino-N-(2-isoxazolin-3-ylmethyl)acetamides and related compounds were synthesized and their antiviral activities against human influenza A virus were assessed. Studies of the structure-activity relationships revealed the strongest antiviral activity when position-5 of the isoxazoline ring was substituted with a tert-butyl group. When the alkoxyimino moiety was substituted with a methyl, ethyl, isopropyl or allyl group, good antiviral activity was obtained. Among the geometrical isomers at the oxime moiety, the E-isomers were more active than the Z-isomers. Among the compounds examined, (E)-2-allyloxyimino-2-cyano-N-(5-tert-butyl-2-isoxazolin-3-ylmethyl)acetamide (1j) was the most active inhibitor with an EC(50) of 3 microg/mL in vitro.     

Inhibition of enterovirus 71 (EV-71) infections by a novel antiviral peptide derived from EV-71 capsid protein VP1

Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50) values ranging from 6-9.3 μM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.     

Antiviral Activity of Antisense Oligonucleotides Against Various Targets of Herpes Simplex Virus 1 (Hsv1) and Coxsackievirus B3 (Cvb3) Genome

Abstract

The influence of chemical modification on the antiviral activity of oligonucleotides was studied on Green monkey kidney cells (GMK) using a known antisense oligodeoxynucleotide (AS-ODN) directed against the IE-110 gene of HSVl. The highest antiviral activity was observed with ODNs carrying exclusively phosphothioate internucleotide linkages. CVB3-specific ODNs of this type were synthesized and successfully tested for antiviral activity on HeLa cells.