Cytoplasmic interactions between decay-accelerating factor and intercellular adhesion molecule-1 are not required for coxsackievirus A21 cell infection.

Coxsackievirus A21 (CAV-21) employs a cell receptor complex of decay-accelerating factor (DAF) and intercellular adhesion molecule-1 (ICAM-1) for cell infectivity. In this study, the nature of potential extra- and/or intracellular interactions between DAF and ICAM-1 involved in picornaviral cell entry was investigated. Firstly, it was shown that intracellular interplay between DAF and ICAM-1 is not required for CAV-21 infection, as CAV-21 lytic infection mediated via the DAF/ICAM-1 receptor complex is not inhibited by replacement of the transmembrane and cytoplasmic domains of ICAM-1 with those from an unrelated cell surface molecule, CD36. By immunoprecipitation, chemical cross-linking and picornaviral binding assays, the existence of a close spatial association between DAF and ICAM-1 on the surface of ICAM-1-transfected RD cells was confirmed. Furthermore, it was shown that potential extracellular DAF/ICAM-1 interactions are likely to occur in an area on or proximal to DAF SCR3 and may influence the route of CAV-21 cell entry.

Antibody binding to individual short consensus repeats of decay-accelerating factor enhances enterovirus cell attachment and infectivity.

Decay-accelerating factor (DAF), a widely expressed membrane complement-regulatory protein, is utilized as a cellular receptor by many human enteric pathogens. We show here that the binding of two enteroviruses to individual short consensus repeats (SCR) of DAF on the cell surface is greatly augmented by mAb binding to an alternate SCR: Coxsackievirus A21 binding to the SCR1 of DAF is increased by Ab binding to SCR3 and, conversely, Echovirus 7 binding to SCR3 is enhanced severalfold by Ab binding to SCR1. These Ab-induced increases in viral binding also resulted in increased viral infectivity. Using purified soluble DAF in a solid phase assay it was found that Ab binding to SCR1 is increased greatly in the presence of an Ab against SCR3 and, reciprocally, Ab against SCR1 greatly increases Ab binding to SCR3. In contrast to the results obtained with the larger viral particles, however, this reciprocal Ab-induced enhancement of binding is not seen when measuring Ab binding to membrane-bound DAF SCR on the cell surface. These findings provide a possible explanation for functional differences between membrane-bound and soluble DAF with implications for a potential role for DAF-binding molecules in regulating DAF function. This is the first demonstration of enhancement of viral infectivity mediated by Ab against the viral receptor.

A decay-accelerating factor-binding strain of coxsackievirus B3 requires the coxsackievirus-adenovirus receptor protein to mediate lytic infection of rhabdomyosarcoma cells.

The composition of the cellular receptor complex for coxsackievirus B3 (CVB3) has been an area of much contention for the last 30 years. Recently, two individual components of a putative CVB3 cellular receptor complex have been identified as (i) decay-accelerating factor (DAF) and (ii) the coxsackievirus-adenovirus receptor protein (CAR). The present study elucidates the individual roles of DAF and CAR in cell entry of CVB3 Nancy. First, we confirm that the DAF-binding phenotype of CVB3 correlates to the presence of key amino acids located in the viral capsid protein, VP2. Second, using antibody blockade, we show that complete protection of permissive cells from infection by high input multiplicities of CVB3 requires a combination of both anti-DAF and anti-CAR antibodies. Finally, it is shown that expression of the CAR protein on the surface of nonpermissive DAF-expressing RD cells renders them highly susceptible to CVB3-mediated lytic infection. Therefore, although the majority of CVB3 Nancy attaches to the cell via DAF, only virus directly interacting with the CAR protein mediates lytic infection. The role of DAF in CVB3 cell infection may be analogous to that recently described for coxsackievirus A21 (D. R. Shafren, D. J. Dorahy, R. A. Ingham, G. F. Burns, and R. D. Barry, J. Virol. 71:4736-4743, 1997), in that DAF may act as a CVB3 sequestration site, enhancing viral presentation to the functional CAR protein.

Coxsackievirus A21 binds to decay-accelerating factor but requires intercellular adhesion molecule 1 for cell entry.

It is becoming increasingly apparent that many viruses employ multiple receptor molecules in their cell entry mechanisms. The human enterovirus coxsackievirus A21 (CAV21) has been reported to bind to the N-terminal domain of intercellular adhesion molecule 1 (ICAM-1) and undergo limited replication in ICAM-1-expressing murine L cells. In this study, we show that in addition to binding to ICAM-1, CAV21 binds to the first short consensus repeat (SCR) of decay-accelerating factor (DAF). Dual antibody blockade using both anti-ICAM-1 (domain 1) and anti-DAF (SCR1) monoclonal antibodies (MAbs) is required to completely abolish binding and replication of high-titered CAV21. However, the binding of CAV21 to DAF, unlike that to ICAM-1, does not initiate a productive cell infection. The capacity of an anti-DAF (SCR3) MAb to block CAV21 infection but not binding, coupled with immunoprecipitation data from chemical cross-linking studies, indicates that DAF and ICAM-1 are closely associated on the cell surface. It is therefore suggested that DAF may function as a low-affinity attachment receptor either enhancing viral presentation or providing a viral sequestration site for subsequent high-affinity binding to ICAM-1.

Mouse cells expressing human intercellular adhesion molecule-1 are susceptible to infection by coxsackievirus A21.

Competitive viral binding assays have revealed previously that coxsackievirus A21 (CAV21) and human rhinovirus 14 (HRV14) share a common cell surface receptor. More recently, intercellular adhesion molecule-1 (ICAM-1) has been identified as the cellular receptor for HRV-14. Also, anti-ICAM-1 monoclonal antibodies (MAbs) blocked infection by HRV14, CAV13, CAV18, and CAV21, suggesting that these viruses share this receptor; however, this has never been established by more direct methods. In this study we show conclusively that CAV21 binds to ICAM-1 and that MAbs directed against the N-terminal domain of the molecule inhibit this attachment. Furthermore, we show that the specific interaction between ICAM-1 and 160S CAV21 virions induces formation of 135S A particles. Finally, we show transfection of normally nonsusceptible mouse L cells with human ICAM-1 cDNA renders them susceptible to infection by CAV21.

Integrin alpha v beta 6 enhances coxsackievirus B1 lytic infection of human colon cancer cells.

Viral entry into host cells depends upon specific interactions between virus attachment proteins and cell surface receptors that enable virus binding and internalization of virus and/or the virus-receptor complex. We have recently reported that the ubiquitous cell surface molecule, decay-accelerating factor (DAF), is a major cell attachment receptor for Coxsackieviruses B1, B3, and B5. However, DAF permits only virus binding and not virus internalization, invoking the presence of secondary or accessory receptors. Among the known receptors for enteroviruses are members of the cell adhesion molecule family known as integrins. In the present study, we found that expression of the epithelial-restricted integrin, alpha v beta 6, on colonic epithelial cells significantly enhanced Coxsackievirus B1-mediated cell lysis. Importantly, the viral-mediated cell killing required the presence of the 11-amino-acid C-terminal cytoplasmic extension unique to the beta 6 subunit, providing the first evidence of regulation of viral infectivity by integrin cytoplasmic domains. These results indicate that alpha v beta 6 expression on intestinal epithelial cells critically affects Coxsackievirus B1 infectivity. This may be essential in the conversion of asymptomatic enterovirus infection into clinically apparent disease.

Characterization of recombinant Eschericha coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase: analysis of enzymatic activity and substrate specificity.

Recombinant E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (EC 3.2.2.9) was used to study the potential for this enzyme to serve as a target for chemotherapeutic intervention. An examination of the parameters required for enzymatic activity indicate that the nucleosidase functions over a broad range of pH and temperature, with acidic conditions and temperatures of 37-45 degrees C being optimal. Analogs of 5'-methylthioadenosine and adenosine were assessed as potential enzyme inhibitors and to provide details regarding substrate specificity and reaction mechanism. The 5'-arylthio analog, 5'-(p-nitrophenyl)thioadenosine, was the most potent enzyme inhibitor studied, with a Ki of 20nM. A mutant of the nucleosidase lacking the first 8 amino acids was engineered to determine the contribution of these conserved residues toward enzyme specificity. The truncated enzyme exhibited a K(m)[MTA] of 1.43 microM, approximately 3 fold higher than the K(m) reported for the full-length nucleosidase.

Coxsackieviruses B1, B3, and B5 use decay accelerating factor as a receptor for cell attachment.

Receptor binding and subsequent cell-mediated internalization or disassembly are the initial steps in virus replication. Cell surface molecules that participate in this process are the primary determinants of virus tissue tropism. Monoclonal antibody blockade, immunoprecipitation, and DNA transfection were used to identify decay accelerating factor as a major cell attachment receptor for coxsackieviruses B1, B3, and B5. However, expression of human decay acceleration factor on the surface of nonpermissive murine fibroblasts led only to virus attachment without subsequent replication, and it was concluded that an additional cellular cofactor(s) is required to facilitate cell entry and subsequent replication.

A study of respiratory infections in a healthy adult population during the 1987 Australian winter.

During the 1987 Australian winter, respiratory illness patterns were studied in a population of 454 healthy adults, aged 18-59, over a period of 45 days. These patterns were matched with data obtained from laboratory diagnoses for respiratory viruses, Mycoplasma pneumoniae and bacteria. Influenza B/1/86 was by far the most prevalent pathogen but other viruses including influenza A, paramyxoviruses, respiratory syncytial virus and coronavirus OC-43 were also present, either alone or in combination during the sampling period. Overall, 92 males and 101 females experienced one episode, 12 males and 22 females experienced two episodes and four females experienced three episodes. However, there were only 52 instances of viral or M. pneumoniae infections, of which 37 had a defined aetiology, while the remainder were clinically silent. No bacterial pathogens could be detected from throat swabs taken from 15 of 37 volunteers in whom a viral infection was detected, or from 43 of 70 volunteers who did not experience such infections. The study indicates that major deficiencies in our understanding of the aetiology of respiratory viral illness are probably due to methodological problems in obtaining laboratory diagnoses for many respiratory viruses, and that great difficulties exist in establishing an aetiology for respiratory infections based upon clinical symptoms alone.

Western blot analysis of antibody responses to influenza virion proteins.

An immunoblotting procedure was developed to detect antibody responses in mice and humans to influenza virion proteins. The technique was capable of detecting 1.5 micrograms of haemagglutinin (HA) on nitrocellulose strips at a 1:5000 dilution of a mouse serum with an initial haemagglutination inhibition titre of 20. The effects of the use of the blocking agent Tween-20 on virion proteins were also studied. The commonly used concentration of 0.05% (v/v) Tween-20, when included in blocking and incubation buffers, greatly reduced the amount of detectable matrix protein but caused no detectable loss of HA and neuraminidase/nucleoprotein proteins. If virion proteins were separated by polyacrylamide gel electrophoresis under reducing conditions, antibody bound to HA2 more strongly than to HA1. Under non-reducing conditions, more antibody bound to the uncleaved HA protein than to other proteins. IgG1 and IgG2a antibody responses in mice to each protein were stronger than IgG2b and IgG3 responses.

Improved colorimetric assay for detecting influenza B virus neutralizing antibody responses to vaccination and infection.

An automated neutralization test for influenza B virus is described in which antibody titers are determined according to the release of neutral red from infected or uninfected cells of the Madin-Darby canine kidney line. Endpoints are determined in a standard enzyme-linked immunosorbent assay reader. The test requires no expensive immunologic reagents and was used to evaluate responses to both vaccination and natural infection against influenza B virus. Overall responses to vaccination were comparable with those obtained by hemagglutination inhibition, using Tween-ether-split influenza B/Ann Arbor/1/86 virus as the antigen (the HI-TE test). The sensitivities of neutralization responses compared with those obtained by the HI-TE test for two vaccines were 88 and 89%; the specificities were lower at 61 and 60%, respectively. Responses to vaccination, measured by hemagglutination inhibition, were significantly higher with split virus compared with whole virus. However, seroconversion by both the HI-TE and neutralization tests was observed in 5 of 10 individuals from whom virus was detected by either culture of nasal or throat washings or the presence of antigen from immunofluorescence in cells from nasal washings.

A study of intranasally administered interferon A (rIFN-alpha 2A) for the seasonal prophylaxis of natural viral infections of the upper respiratory tract in healthy volunteers.

The efficacy of interferon A (rIFN-alpha 2A), an Escherichia coli-derived interferon, in the prophylaxis of acute upper respiratory tract infection, was evaluated in a community-based double-blind placebo-controlled study in the Australian winter of 1985. The trial population of 412 healthy volunteers (190 males and 222 females, aged 18-65 years) self-administered 1.5, 3.0 and 6.0 megaunits (MU) of interferon A per day or a placebo, intranasally for 28 days. The period of study coincided with an outbreak of H3N2 influenza A (detected in 35 of the 107 acute specimens) as well as substantial numbers of respiratory syncytial virus and adenovirus infections. Rhinoviruses were isolated from only three specimens. In many cases, subjects had laboratory and clinical evidence of having had more than one respiratory tract infection during the period of the study. Viruses were detected in 54 or 107 acute specimens (49%). No statistically significant differences were noted between the various treatment groups in the incidence of laboratory-proven viral infection (virus isolation and/or antibody response). Analysis of reported symptoms indicated that blood-tinged mucus and nasal stuffiness occurred more frequently with higher doses of interferon. There appeared to be no clinical benefit from the use of interferon A in the amelioration of symptoms.

The development of Akabane virus-induced congenital abnormalities in cattle.

A prospective study of the incidence and severity of congenital deformities of calves, attributable to maternal infection by Akabane virus, was carried out on a population of 174 susceptible animals that were between one and nine months pregnant at the time of infection. The study was carried out in the Hunter Valley of New South Wales during 1983, after an epidemic of Akabane virus infection in late February to early March 1983. The incidence of virus-induced abnormalities in calves and fetuses was 17.8 per cent (31/174). The highest incidence of abnormalities occurred during the third and sixth months of gestation (27 to 29 per cent). The earliest abnormality was observed after infection at 76 days of gestation, and the last after infection at 249 days. The development of the pathological entities of hydranencephaly/porencephaly and arthrogryposis were found to be quite distinct. Cases of hydranencephaly and porencephaly developed after infection between 76 and 104 days of gestation whereas arthrogryposis developed after infection between 103 and 174 days of infection. It was concluded that the type of congenital deformity produced by maternal infection with Akabane virus was dependent on the stage of fetal development at the time of infection. The data suggest that the infection was transplacental and that fetuses of less than two months of age were protected from infection.

Immunization against influenza by the ocular route.

The immunogenicity of influenza A strain A/Northern Territory/60/68 for CSL mice when delivered by the ocular, nasal and subcutaneous routes was determined according to the median protective dose, PD50, i.e. the dose of infectious virus required to induce inhibition of multiplication of a standard intranasal challenge dose of 10(4.5) median tissue-culture-infectious doses (TCID50) of homologous virus three weeks after vaccination (PD50). For mice inoculated by the ocular route, an immunizing dose of 10(2.89) TCID50 per animal was required. For anaesthetized mice vaccinated intranasally and unanaesthetized mice vaccinated subcutaneously these figures are less than 10(2.00) and greater than 10(6.00) TCID50 per animal, respectively. The lower immunogenicity of virus delivered by the ocular route compared with the intranasal route can be correlated with a lowered capacity of ocularly administered virus to replicate in the murine respiratory tract. The immunogenicity of A/Ann Arbor/6/60-ca administered in two identical doses, was also determined for (a) the intraocular route, (b) the intranasal route with anaesthetized animals and (c) the intranasal route with unanaesthetized animals, using the parental A/Ann Arbor/6/60 as the challenge virus. Two doses were required because ca viruses have been shown to be poor immunogens in the same animal model. The PD50 for the ocular route was 10(2.83) TCID50 per animal compared with 10(2.71) for the intranasal route using unanaesthetized animals and 10(1.36) for the intranasal route using anaesthetized animals. Administration of living attenuated vaccine viruses by the ocular route is thus an effective means of inducing immunity to influenza viruses in the respiratory tract of mice.

A simple procedure for the analysis of the structural proteins of influenza and parainfluenza viruses involving adsorption to erythrocytes.

A simple procedure for the analysis of the structural proteins of influenza and parainfluenza viruses utilizing adsorption to erythrocytes is described. The method involves virus growth in the presence of [35S]methionine, adsorption of clarified culture medium with a 0.5% suspension of either guinea-pig or chicken erythrocytes and analysis of the virus-erythrocyte aggregates by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). All of the structural proteins can be detected using this procedure, and the protein profiles of virus-adsorbed erythrocyte complexes compare extremely well with those of sucrose density gradient purified virus preparations.

Relative immunogenicity of the cold-adapted influenza virus A/Ann Arbor/6/60 (A/AA/6/60-ca), recombinants of A/AA/6/60-ca, and parental strains with similar surface antigens.

The immunogenicity of several cold-adapted (ca) viruses was compared in CSL mice with that of wild-type parental viruses with similar surface antigens, according to the vaccinating dose required to clear a challenge consisting of 10(4.5) 50% tissue culture infective doses of the wild-type virus. All ca viruses were less immunogenic than their wild-type parental strains by a factor of 10(1.3) to 10(3.4), probably due to the restricted capacity of ca viruses to replicate in the respiratory tracts of mice. However, their immunogenicity was considerably enhanced when two quite small doses were administered 3 weeks apart. The immunogenicity of ca viruses when administered in two doses and wild-type viruses when administered as a single dose varied according to their surface antigens. It was highest for viruses with the H2N2 A/Ann Arbor/6/60 and H3N2 A/Queensland/6/72 surface antigens and lowest for those with H1N1 A/HK/123/77 surface antigens. When two doses consisting of 10(5.0) 50% tissue culture infective doses of A/Ann Arbor/6/60-ca were administered at an interval of 3 weeks, solid immunity was induced against the wild-type A/Ann Arbor/6/60 parental virus, two heterologous H3N2 strains, and an H1N1 strain.

Characterization of human parainfluenza viruses. I. The structural proteins of parainfluenza virus 2 and their synthesis in infected cells.

Two strains of human parainfluenza virus 2 (HPV2), P2 1972/6 and P2 1980, grow to high titre in MEK3 cells, and their structural proteins and virus-induced protein synthesis have been characterized by gel electrophoresis and immunoprecipitation. Purified viruses contain seven polypeptides, including cellular actin: L (175K mol. wt.), HN (72K to 74K), NP (66K to 67K), F1 (52K to 58K), P (49K), A (44.5K) and M (39K). Virus-induced polypeptide synthesis was first detected at 8 h post-infection with the appearance of NP; other major structural proteins were detected from 10 to 12 h after infection and onwards. The synthesis of both the structural glycoproteins was demonstrated, although proteolytic processing could not be detected. Reproducible differences in the gel migration of the HN, F1 and NP polypeptides were found in whole virus, in infected cells and cells subjected to immunoprecipitation. These differences may reflect genetic diversity within HPV2 and provide a means of probing the molecular epidemiology of these viruses.

Immunization against influenza by the ocular route. Brief report.

The immunogenicity of influenza A strain A/Northern Territory/60/68 for mice when delivered by the ocular, nasal and sub-cutaneous routes was determined according to the dose of infectious virus required to induce inhibition of multiplication of a standard intransal challenge dose in 50 per cent of animals per group 3 weeks after vaccination. For mice inoculated by the ocular route, an immunizing dose of 10(2.89) TCID50 per animal was required. For anesthetised mice vaccinated intranasally and unanaesthetised mice vaccinated sub-cutaneously these figures are less than 10(2.0) and greater than 10(6.0) TCID50 per animal, respectively. The lower immunogenicity of virus delivered by the ocular route compared with the intranasal route can be correlated with a lowered capacity of ocularly administered virus to replicate in the murine respiratory tract.