Attachment of coxsackievirus B3 variants to various cell lines: mapping of phenotypic differences to capsid protein VP1.

The coxsackievirus B3 (CVB3) strain Nancy P establishes a persistent carrier-state infection without visible cytopathic effect in primary human fibroblasts (HuFi H), whereas the derivative variant PD induces a complete lysis of the cell monolayer. To define the molecular basis of this exceptional growth property, the complete genomes of both viruses were sequenced and compared to all published sequences of CVB3. As a result, six unique amino acid substitutions in the VP1 capsid protein were observed. Via hybrid virus construction, the lytic phenotype was transferred to a nonlytic cDNA-generated CVB3. Mapping experiments indicate that the presence of amino acid residues K78, A80, A91, and I92 in VP1 is sufficient to induce "lytic" infections in HuFi H cells. Binding assays demonstrate that CVB3 Nancy P preferentially binds to the human coxsackievirus-adenovirus receptor (CAR), while PD exhibits a very weak interaction with CAR but strong binding to the decay accelerating factor (DAF). These results suggest that the mutated amino acid residues in VP1 are involved in receptor recognition/binding. Moreover, the lytic replication of CVB3 PD and the hybrid virus in various nonpermissive rodent cell lines indicates that cell surface molecules other than CAR and DAF may be involved in attachment of this variant to cell surfaces.

Coxsackievirus B3-induced chronic myocarditis in outbred NMRI mice.

OBJECTIVES: The pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis was investigated in adult Han:NMRI mice. The outbred model, in comparison with inbred models, represents better the natural variable susceptibility of the human population. STUDY DESIGN/METHODS: We analyzed the replicating virus titer, the antibody response in the acute and chronic phase of disease, the histology of myocardial injury, and the persistence of viral RNA. RESULTS: NMRI mice infected with 5000 plaque-forming units (PFU) of the CVB3 variant "P"D, a lytic variant to human fibroblast lines, showed a peak of virus replication at day 14 and developed a severe acute myocarditis. The chronic myocarditis was characterized by progressive fibrosis, small foci of infiltrates, persistent viral RNA in the heart, and detectable anti-CVB3 IgG production and neutralizing antibody response up to day 98 postinfection. CONCLUSIONS: CVB3"P"D is able to induce chronic myocarditis in NMRI mice. This model provides a method for examining and proving the mechanisms of myocardial pathogenesis and of developing therapeutic strategies.

Permissiveness of human embryonal fibroblasts for coxsackieviruses B3. Investigations on virus genetic markers in vitro and localization of virus receptor distribution by immunogold replica technique.

In our previous paper (29) we could show, that the replication of four Coxsackie B3 virus strains in human fibroblast lines from different origin was dependent on both the virus strain and the cell line used. Although generally no cytopathic effect could be observed out of one virus strain more pathogenic virus variants could be selected able to destroy virus-sensitive as well as insensitive fibroblasts. The present study was designed to characterize these virus strains by using several in vitro genetic markers. Differences were found concerning plaque size and the temperature marker, whereas the other markers (d, DD, DEAE-D) remained constant. Furthermore, these models of persistent as well as lytic CVB3 infection were analysed by immunoelectron microscopy to study the interaction of viral ligands with cellular receptors. The qualitative and quantitative differences in adsorption of the CVB3 strains to two human fibroblast lines as well as to HeLa cells corresponded well with the virological results. They underline that even in vitro in human cell lines of different origin changes in distribution, quantity and quality of receptors were demonstrable forming the base for the various virus sensitivity.

Replication and persistence of coxsackieviruses B3 in human fibroblasts.

Although in some cases a carrier state of Coxsackieviruses in human fibroblasts was described, the persistence mechanism has remained unknown. Our results demonstrate a replication of Coxsackievirus B3 (CVB3) in diploid human fibroblasts that is dependent on the virus strain as well as on the cell line involved. Two CVB3 Nancy strains could be multiplied over more than 10 passages in cell line H, whereas CVB3 strains SH"C" and SH"W" did not longer form infectious virus following several passages in these fibroblasts. None of the CVB3 strains replicated in cell lines J and K after a few passages. These results were not influenced by variations of culture conditions such as duration of incubation (3 or 7 days), temperature (33 or 37 degrees C) or application of trypsin. In line H, only 3-5 per cent of human fibroblasts were virus-infected. This was demonstrated by 1. antigen evidence by immunofluorescence, 2. determination of infectious centres, and 3. virus reproduction in dependence on MOI of the virus. We observed a carrier state of CVB3 Nancy strains over 16 cell passages in line H fibroblasts infected once. As shown in virus passage experiments, only few cells were productively infected. An addition of specific anti-CVB3 antiserum terminated this persistent infection. Generally, no cytopathic effect was observed. However, in one case a cell destruction by CVB3 Nancy "P" at the end of the life-time of the carrier cells could be found. This virus caused a complete cytopathic effect during more than 10 passages in line H and could still be neutralized by CVB3-specific antiserum.

On the antiviral activity of diffusomycin (oxazolomycin).

The effect of the beta-lactone antibiotic diffusomycin (oxazolomycin) was investigated against vaccinia (Lister), herpes simplex type 1 (Kupka), influenza A (WSN; H1N1), and Coxsackie A9 viruses. Diffusomycin reduced significantly the plaque formation of enveloped DNA and RNA viruses by more than 90% in the range of the maximally tolerated dose. As could be shown with vaccinia virus, the antiviral action was not caused by virucidal effect on virions or by interaction with virus adsorption and penetration. In one-step growth cycle assays diffusomycin prevented the replication of herpes simplex type 1, vaccinia and influenza A viruses in a dose-dependent manner. The replication of influenza A viruses was blocked immediately after addition of the compound during zero to six hr p.i. Partial reversibility of the antiviral action was established by washing off the antibiotic from chicken embryo cells (CEC) infected with influenza A virus. Finally, replication of Coxsackie A9 virus was not inhibited by diffusomycin. Electron-optical studies revealed a reduced synthesis of HSV-1 nucleocapsids in dependence on the concentration of the compound.

[Protein adsorption at the tips from synthetic polymers as the cause of incorrect titer analysis of interferons in antiviral bioassays]. / Proteinadsorption an Tips aus synthetischen Polymeren als Ursache für fehlerhafte Titerbestimmungen von Interferonen im antiviralen Bioassay.

The determination of the biological activity of interferons are carried out on the basis of the inhibition of the cytopathic effect of vesicular stomatitis virus on MDBK cells. Using microtiter-plates and tips made of synthetic polymers incorrect data may be observed due to the adsorption of the protein interferon to the tips and, thus, carryover of activity. The following recommendations may help to avoid this kind of mistakes: 1. Generally changing of the tips, at least for the highest concentrations. 2. Reduction of high initial activities by an appropriate pre-dilution. 3. Addition of the detergent Triton X 100 in concentrations between 50 to 100 mg/l to the dilution media for interferon.

[SEM studies on IFN-receptor dynamics of cultured cells from mammals]. / REM-Untersuchungen zur IFN-Rezeptordynamik von Säugerkulturzellen.

Visualization of rIFN-alpha 1 and -alpha 2 receptors on MDBK-cells was done by means of a two step method with gold-labelled monoclonal anti IFN-antibodies in the SEM. This binding showed a marked dependency from the temperature, concentration of the rHu-IFN, prefixation of the MDBK-cells, and time of incubation. This results are in agreement with findings of classical virological methods. By means of SEM was shown that the distribution and amount of the IFN-receptors on MDBK-cells depends strongly on the cell cycle and that not each was labelled. The results were discussed in connection with structural-functional alterations of the cytoskeleton.

[Sensitivity of different cell lines to interferons: the relative antiviral activity as a function of the interferon subtype]. / Interferonsensitivität verschiedener Zellkulturen: Untersuchungen zur Abhängigkeit der relativen antiviralen Aktivität vom Interferon-Subtyp.

The phenomenon that rHuIFN-alpha1(D) displays an apparently higher antiviral activity when assayed on bovine cells as compared to human cell lines was applied to the elucidation of the nature of recombinant HuIFN prepared in our institute. These investigations were carried out by using a microtitre test, which defines biological activity as the IFN concentration leading to 50% inhibition of the cytopathic effect of vesicular stomatitis virus (VSV). In addition, the ability of IFN to diminish the reproduction of infectious viruses was monitored. The two methods yielded similar results. With bovine cells, antiviral activities of the same order of magnitude were observed, regardless of the interferon types applied, i.e. rHuIFN-alpha 1, rHuIFN-alpha 2 and human leukocyte interferon. On human fibroblasts, however, rHuIFN-alpha 1 had an apparently 45 to 165 times lower activity than the other two interferons. On human WISH cells, the differences in apparent activity between the respective IFNs were even greater, with factors of up to 212 fold being observed. Still more distinctive were the effects on murine L 929 cells where an antiviral effect could be confirmed only for rHuIFN-alpha 1 whereas the other two interferons proved completely inactive.

[Griseochelin methyl ester, a new polyether derivative with antiviral activity]. / Griseochelinmethylester, ein neues Polyether-Derivat mit antiviraler Wirksamkeit.

The methylester of griseochelin (1) is a new chemically-made antiviral derivate of the antibiotic griseochelin isolated from fermentations of Streptomyces griseus. It belongs to the polyether group and possesses antiviral activity against enveloped RNA and DNA viruses cultivated in chicken embryo cells (CEC), namely influenzavirus A/WSN, vesicularstomatitis virus (Indiana), vaccinia virus (Lister) and herpes simplex hominis virus type 1 (Kupka). The methylester of griseochelin failed to show virucidal effects on extracellular influenza vacciniavirus particles or to influence virus adsorption and penetration processes. The antibiotic in concentrations of 125-15 micrograms/ml inhibited the virus-induced cytopathic effect of the above mentioned viruses and caused over 90 per cent plaque reduction. Addition of 1 during a one-step growth cycle of influenzavirus A at 4 and 6 h p.i. resulted in complete suppression of virus multiplication at the control niveau of the virus yield accumulated to the same time point. A partial reversibility of the antiviral action against influenzavirus A could be achieved. Coxsackie A9 virus growth in human fibroblast cells was not affected by the inhibitor. Electron-optical observations showed a failure of the formation of the viral capside proteins of HSV type 1 at the second halftime of the replication cycle in CEC-infected and 1-treated cultures.

Expression and secretion of interferon-alpha 1 by Streptomyces lividans: use of staphylokinase signals and amplification of a neo gene.

A gene coding for mature human interferon, IFN-alpha 1, fused to the expression and secretion signals of a staphylokinase gene (sak) derived from Staphylococcus aureus phage 42D, was inserted into the Streptomyces promoter probe vector pIJ487. Streptomyces lividans transformed with the recombinant plasmid (pMG341) secreted biologically active IFN-alpha 1 into the culture medium. Expression of the IFN-alpha 1 gene was at least on the translational level directed by the sak signals since numerous upstream stop codons would have prevented the formation of a fusion protein. Long-term continuous chemostat cultivation under various limitation conditions was used to select clones with an IFN-alpha 1 yield increased about 60-100-fold (1-2 x 10(5) IU/ml). The increase in IFN-alpha 1 formation was accompanied by spontaneous amplification of the adjacent neo gene, but not of the remaining plasmid DNA. Examination of the DNA sequence around the endpoints of the amplified region revealed almost identical stem-loop structures followed by an octanucleotide direct repeat.

Expression of the human interferon-alpha 1 gene under transcriptional and translational control of the speA gene.

The human gene for mature interferon-alpha 1 (IFN-alpha 1) was inserted in a new transcription-translation fusion vector system based on the expression and secretion signals of the gene for type A streptococcal pyrogenic exotoxin, speA. As deduced from the known nucleotide sequences of the component elements, the encoded IFN-alpha 1 was a fusion protein carrying an N-terminal extension of 17 amino acids. When inserted in appropriate vectors capable of replication in Escherichia coli, Bacillus subtilis and Streptococcus sanguis, this expression configuration directed the synthesis of antiviral activity in all 3 organisms, as judged by the cythopathic effect inhibition assay of Vesicular Stomatitis Virus. In E. coli JM101, IFN activity was found mainly in the cytoplasmic protein fraction whereas in the gram-positive hosts, it was completely secreted into the culture medium.

[Biological effects of coordinated compounds of transitional metals. 7. Antiviral activity of 4-methyl-2-aminopyridine palladium chloride against herpes simplex virus type 1 and type 2 in vitro and in vivo]. / Uber biologische Wirkungen von Koordinationsverbindungen der Ubergangsmetalle. 7. Zur antiviralen Wirksamkeit von 4-Methyl-2-amino-pyridinpalladiumchlorid gegenüber Herpes simplex-Virus Typ 1 und Typ 2 in vitro und in vivo.

4-Methyl-2-amino-pyridine-palladium chloride (MAP) showed an antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in a serum-free medium under in vitro conditions. The replication of these viruses on primary rabbit testes cells was completely suppressed by 10(-5) M/l MAP. In animal tests using ABD2-mice the course of HSV-1 and HSV-2 encephalitis was not influenced by MAP indicated by mean survival time and lethality.

Direct evidence for the quantum theory of the cell transformation by use of the nucleoproteide equivalents' staining.

The separation of the nucleoproteide equivalents of human Fogh and Lund [FL] cells by means of sucrose gradient centrifugation devoted 5 separate distinguished fractions. A treatment of the cells by pancreatic RNase and/or extraction of RNA by hypertonic 0.9% sodium chloride solution eliminated the last 3 fractions. An increase of the acidification of the intercalating thiazine dyes by acetic acid at 2.9 less than or equal to pH less than or equal to 5.5 accumulated the complete diffuse dispersed nucleoproteide pool in the endoplasmic reticulum and nucleus gradually over formation of chromosomal aberrations and cluster into a heap of nucleoproteide crystals with empty cytoplasmic zones.

[The biological effects of coordination compounds of transitional metals. 6. Effect of 4-methyl-2-aminopyridine-palladium chloride and cis-dichlorodiammine-platinum(II) on retroviruses and the virus-associated RNA polymerase of the influenza virus]. / Uber biologische Wirkungen von Koordinationsverbindungen der Ubergangsmetalle. 6. Wirkung von 4-Methyl-2-aminopyridin-palladiumchlorid und cis-dichloro-diammin-Platin(II) auf Retroviren und die virus-assoziierte RNS-Polymerase von Influenzavirus.

The effect on retroviruses of two transition metal complexes of known antiviral activity, 4-methyl-2-amino-pyridine-palladium-chloride (MAP) and cis-dichloro-diammine-platinum(II) (cis-DDP) has been investigated. The experiments included the evaluation of the action of compounds on virus particle-associated reverse transcriptase in exogenous assays, on virus propagation in persistently infected cell cultures and on virus infectivity in mice. In disrupted viruses and in the absence of excess protein, the reverse transcriptase was inhibited by MAP but not by cis-DDP. The same results were obtained when examining the activity of the virus-associated RNA polymerase of influenza virus A/WSN. Both compounds did not inhibit the replication of retroviruses in cell cultures, except at high dose levels which exerted toxic action on both cells and virus formation. The leukemogenicity of Rauscher murine leukemia virus was strongly inhibited when the virus had been incubated with MAP before inoculation. A similar treatment with cis-DDP did not influence viral leukemogenicity. Despite somewhat different results with both compounds tested, we conclude from the present results that the above mentioned compounds cannot be considered as antiretroviral drugs.

[Interferon sensitivity of various cell lines]. / Interferonsensitivität verschiedener Zellkulturen.

An economic assay to determine the yield of interferon is necessary for the induction, production and purification of interferon. Using the inhibition of cytopathic effect of vesicular stomatitis virus in a microtitre system, we compared the susceptibility of different cell lines against an internal IFN alpha standard obtained from Sendai virus-induced human leukocytes. Human fibroblasts carrying trisomy G 21 and bovine MDBK cells showed the highest sensitivity followed by normal human fibroblasts. Human RH kidney cells exhibited a susceptibility similar to that of human WISH amnion cells frequently used by others. The human amnion cell line FL and monkey Vero kidney cells, as described here, were unsuitable for the determination of IFN yields.

[In vitro effectiveness of new antiviral substances. 1. Results of screening studies against selected picornaviruses]. / Uber die In-vitro-Wirksamkeit neuer antiviraler Substanzen. 1. Mitteilung. Ergebnisse von Screeninguntersuchungen gegenüber ausgewählten Picornaviren.

A number of 3108 new synthetic compounds were subjected to a screening for antiviral activity. The screening resulted in the selection of numerous drugs active against mengovirus in vitro. Some drugs were also active against Coxsackie viruses A9 and B1; most of the drugs efficient against Coxsackie virus A9 also showed activity against mengovirus. Quantitative investigations--such as the determination of a therapeutic index or of the inhibition of infectious virus yields in one-step growth cycle experiments--allowed the selection of the compounds with the highest in vitro efficacy.

[In vitro effectiveness of new antiviral substances. 2. studies with enteroviruses and rhinovirus lB]. / Uber die In-vitro-Wirksamkeit neuer antiviraler Substanzen. 2. Mitteilung. Untersuchungen mit Enteroviren und Rhinovirus 1B.

The antiviral efficacy of several compounds was demonstrated against Coxsackie viruses type A9, B1, B3, B4 and B5, echoviruses type 6, 11, 30, and 33, attenuated polioviruses and rhinovirus 1B. In one-step growth cycle experiments virus multiplication was clearly inhibited by application of the compounds immediately after virus adsorption.