key: cord-0008997-xpeiyi6v
authors: nan
title: Abstracts of Papers Presented at the Session of the Virology Section of the Deutsche Gesellschaft für Hygiene und Mikrobiologie, Freiburg, October 1–4, 1986
date: 2011-11-08
journal: Zentralbl Bakteriol Mikrobiol Hyg A
DOI: 10.1016/s0176-6724(87)80192-x
sha: 478532306e2fdcf86f4fc610d1fd14e5fa21e3de
doc_id: 8997
cord_uid: xpeiyi6v

nan

were stimulated in vitro with virus particles, 3 out of 49 IgG producing hybridomas produced antibodies which neutralized the homologous virus. The antibodies reacted only with type 1, Mahoney, and did not recognize the Lsc 2ab (Sabin) strain of type 1. Neutralization, binding of the virus and immunoprecipitation of VP1 are inhibited by preincubation of these antibodies with a synthetic peptide representing the amino acid sequence 93-104 of VP1 of poliovirus type 1, Mahoney. These highly specific antibodies therefore recognize neutralization epitopes located between amino acid 93-104 of VP1.

Neutralizing Antibodies Against Poliovirus Type 1, Mahoney in Rabbits and Mice Using a Synthetic Peptide of Sequence 93-104 of VP 1 WETZ, K. and ULRIKE GRAVENHORST-MUNTER Heinrich-Pette- Inst. f. Exp. Virologie u. Immunologie, Univ., D-2000 Hamburg 20 A peptide corresponding to sequence 93-104, immunodominant in poliovirus type 1 (Mahoney) has been synthesized with two additional cysteines at both ends of the peptide. A rigid conformation of the peptide by formation of a closed circle should induce more homogeneous antibodies. The peptide was linked to carrier proteins keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) by glutardialdehyde and such emulsified in: I Freund's adjuvans, II freshly precipitated aluminiumhydroxide (Aloxide) and III monophosphoryl lipid-A containing trehalose dimicholat (RAS). Mice and rabbits were injected five times subcutaneously every ten days. All animals produced antibodies, binding to peptide in an enzyme-linked immunosorbent assay (ELISA). Most of them also bound to virus with moderate titers compared to antiserum to virus. Three out of five rabbit antisera neutralized virus to serum dilutions of up to 1 to 42 and two mice sera up to 1 to 16 with respect to 100 TCID so · Induction of Coxsackie B -Specific Human T4+ Lymphocyte Lines is a Useful Tool in Diagnosing to whcih Virus Type the Individual is Reactive SKLENAR, I., K. BIENZ, P. ERB, and W. BERGER' Inst f. Mikrobiologie u. Hygiene, Depart. f. Innere Medizin', Univ., CH-4000 Basel Coxsackie B (CB) viruses are gaining increasing attention as multipotent pathogens causing e.g. Bornholm disease, aseptic meningitis, myocarditis and possibly 30-60% of the type 1 diabetes cases. Virus-specific IgM-ELISA-techniques are being often used for the diagnosis. However, frequently cross-reactive antibodies to two or more virus types are found. In this work a comparison between cellular immune responses and the humoral immunity to gradient purified CB-viruses was performed. In three individuals cross-reactive CB 3 and CB 4 IgG antibodies were found using ELISA. However, their lymphocytes proliferated predominantly with only one virus type. In addition, study of T-cell lines (OKT 4+) in one individual revealed that only stimulation with one virus type (CB3) led to induction of Tcells which were specific to the same virus type. Using the CB 4 virus it was impossible to induce type-specific T-cell lines. -These data suggest that T-cell memory tests in vitro may be more specific than tests of antibodies by ELISA. HERRLER, G. and H.-D. KLENK Inst. f. Virologie, Justus-Liebig-Univ., D-6300 GielSen, Inst. f. Virologie, Philipps-Univ., D-3550 Marburg 9-0-Acetyl-N-acetylneuraminic acid (Neu5, 9Ac2) has been shown to be a high-affinity receptor determinant for attachment of influenzaa C virus to erythrocytes. We present evidence that Neu5, 9Ac2is the primary receptor determinant required for influenza C virus in order to attach to tissue culture cells and to initiate an infection. Bovine brain gangliosides which contain this type of sialic acid were found to be potential receptors for influenza C virus. Several cell lines, which are resistant to infection by this virus, become susceptible after incubation with bovine brain gangliosides prior to infection. This result indicates that lack of appropriate receptors on the cell surface is a major reason for the restricted cell tropism of influenza C virus.

DURKOP, I., H.-D. KLENK, and G. HERRLER Inst. f. Virologie, Inst. f. Virologie, Philipps-Univ., D-3550 Marburg

We have isolated fifteen hybridomas secreting monoclonal antibodies directed against the surface glycoprotein gp of influenza C virus (Johannesburg/l/66). The antibodies were analyzed for their ability to inhibit the hemagglutination; the acetylesterase and the hemolytic activity of influenza C virus. The results obtained indicate that the surface glycoprotein of influenza C virus in addition to fusion activity also has hemagglutination and acetylesterase activity.

KURODA l , K., C. HAUSER 2, R. GRONER 3 , R. ROTT!, W. DOERFLER 1, Univ. GieBen\ 0-6300 GieBen, Univ. Koln", Hoechst AG\ Univ. Marburg" A cDNA sequence of the FVP hemagglutinin gene has been inserted into the BamHI site of the pAc373 polyhedrin vector. Recombinant virus was obtained after cotransfection of this construct, pAc-HA651 and authentic AcNPV DNA. The hemagglutinin gene is located in the polyhedrin gene of the recombinant virus genome. -Immunofluorescent labelling, immunoprecipitation and immunoblot analyses revealed that cultures of Spodoptera frugiperda (SF) cells produced immune reactive hemagglutinin after infection with the re-combinant virus. -Digestion with endoglycosidases Hand F showed that the hemagglutinin is glycosylated and that the oligosaccharides are processed. The hemagglutinin is expressed at the cell surface and has hemolytic capacity that is activated by post-translational proteolytic cleavage. When chickens were immunized with SF-cells expressing hemagglutinin, they developed hemagglutination inhibiting and neutralizing antibodies and were protected from infection with FPV. The hemagglutinin gene has also been expressed in larvae of tobacco budworm, and the gene product was found to be biologically active. -These observations demonstrate that the hemagglutinin is synthesized and processed in cultured insect cells or in whole animals in a similar fashion as in FPV-infected vertebrate cells and that it has full biological activity.

KISTNER, O. and C. SCHOL TISSEK Inst. f. Virologie, Justus-Liebig-Univ., D-6300 GielSen

The nucleoprotein of influenza A viruses is a phosphoprotein, the in vivo phosphorylation of which is influenced by the phorbol ester TPA. Phosphopeptide patterns, obtained by tryptic mapping, show specific differences in the presence and absence of TPA. -TPA has been shown to stimulate the proteinkinase C and the enzyme also phosphorylates isolated nucleoprotein in vitro. -These results, combined with the calcium influx and an accumulation of diacylglycerides 3 hours after infection, suggest that that proteinkinase C plays a role in the replication cycle of influenza A viruses. ROTT, R. and M. TASHIRO Inst. f. Virologie, justus-Liebig-Univ., D-6300 Giefsen Several strains of Staphylococcus aureus (Staph.) have been found to secrete serine proteases that activate some influenza A viruses (IV) by proteolytic cleavage of the hemagglutinin. When mice were co-infected intranasally with an appropriate strain of IV and Staph. the animals came down with a fatal pneumonia, while infection with IV or Staph. alone did not induce relevant pathological changes. Only low titers of virus could be found in the lungs of mice infected with the virus alone. However, addition of trypsin to lung homogenates increased infectivity of such tissue samples. Co-infection with Staph. produced high virus titers in the lung that could not be increased by in vitro treatment with trypsin. Thus, co-infecting bacteia can play an essential role in the development of influenza pneumonia by providing a protease suitable for cleavage activation of the hemagglutinin.

Serological Influenza-Diagnosis: Comparison Between CFT and Subtype and Immunoglobulin Class Specific 1FT DOLLER, G., P. C. DOLLER, and H.-J. GERTH Hygiene-lnst., Abt, f. Med. Virologie u. Epidemiologie, D-7400 Tiibingen Low titers (20-40) obtained by CFT, the usual method for influenza serodiagnosis do not allow to differentiate between acute and remote infection. As a rule this differentiation is possible with subtype and immunoglobulin class specific 1FT. We show this by results from 127 patients with confirmed influenza-infection. The individual immune response after influenza-infection varied considerably: 19.7% of the patients had produced IgG-, IgA-, and IgM-antibodies, 62.2% IgG-and IgA-antibodies and 7.9% only IgG-antibodies. lFT-positive/CFT-negative reacted 3.1 %, CFT-positive/IFT-negative reacted 2.3%. In 4.7% of the patients, neither with 1FT nor with CFT antibodies were detected despite of virus isolation. Totally in 27.5% of the patients it was not possible to detect an acute influenza-infection by CFT, in Under these conditions no viral surface antigen was secreted. In vitro the HeLa cell produced and secreted all three surface proteins as filamentous particles. The data suggest i) that secretion of large protein requires an excess of small protein, ii) that a high proportion of large protein favours formation of filaments in addition to 20 nm particles, and iii) that expression of large protein is regulated in a cell-type specific way. To study immunity to hepatitis B surface antigen (HBsAg) at the cellular level, peripheral blood lymphocytes were isolated 2-20 days after the 1st, 2nd and 3rd vaccination with hepatitis B vaccine (H-B-Vax(®), MSD). The lymphocytes were cultured for 7 days in the absence and the presence of pokeweed mitogen(PWM). Culture supernatants were analysed for specific antibodies against HBsAg (anti-HBs), total IgG and total IgM by enzyme immunoassays. After the 1st vaccination, no anti-HBs was detectable, whereas anti-HBs could be measured 6 days after the 2nd and, in a higher extent, 6-15 days after the 3rd vaccination. PWM inhibited the synthesis of anti-HBs. The influence of PWM upon the synthesis of total IgG and total IgM varied considerably. Anti-HBs concentrations were followed in 1070 persons vaccinated against hepatitis B between 1980 and 1982. In 27% of vaccinees initially seroconverted antibody levels declined to or below 10 lUll within 4 years. As shown earlier in a smaller group the persistence of anti-HBs was dependent of the maximal antibody response measured 4 weeks after the third immunisation. The rate of decrease of anti-HBs seems to be similar in all persons, without being influenced by age, sex or dose of vaccine. Revaccination was done in 70 persons with anti-HBs levels below 10 lUll. Within 4-6 days after the booster dose anti-HBs increased rapidly leading in 85% of vaccinees to anti-HBs concentrations considerably higher than after the first booster.

Fibroblasts CHASTONA Y, J. DE and G. SIEGL lnst. for Hygiene and Med. Microbiology, CH-3010 Bern Under one step growth conditions, first signs of viral metabolic activity are detected 2-4 days after the onset of infection. Therefore, early events, from adsorption to the time viral synthesis is initiated, together or in part, are slow processes. Furthermore, HAV replication is highly protracted, with viral metabolic activity basically occurring during the first two weeks of infection. Thereafter, the cells remain persistently infected. -The synthesis of total viral RNA (i.e. negative strand template RNA, genomic RNA, and viral RNA), as well as synthesis and encapsidation of genomic RNA occur in parallel to the appearance and accumulation of progeny infectious virus. -The appearance of a subgenomic vRNA about 2000 nucleotides long in close association with ribosomes is yet another feature that distinguishes HAV from typical Picornaviruses. KRECH, S. and G. SIEGL lnst. for Hygiene and Med. Microbiology, CH-3010 Bern Hepatitis A Virus (HAV) shares many characteristics with the picornaviruses. Its genome consists of a linear, single-stranded RNA of messenger-sense polarity. However, unlike other picornaviruses, HAV fails to shut off host cell metabolism and, hence, analysis of viral protein synthesis meets with difficulties. To circumvent this situation, HAV~enomic RNA was translated in a rabbit reticulocyte system, optimized in respect to K+, Mg ", tRNA, and viral RNA concentrations. The kinetics of translation of HAV RNA and poliovirus RNA then proved to be similar over a period of 60 min. Translation products of HAV RNA consisted in at least 12 polypeptides with mwts between 15 and 200 kd. Seven of them with mwts between 15 and 46 kd became evident within the first 10 min of translation. The five larger ones accumulated after 30 to 60 min only. -Processing of the polypeptides larger than 46 kd has so far not been observed.

Role of "I-Interferon in Pathogenesis of Hepatitis A Virus Infection MAIER, K., A. VALLBRACHT, P. GABRIEL, and B. FLEHMIG Hygiene-lnst., Dept. Med. Virology, D-7400 Tiibingen Peripheral blood lymphocytes (PBL) were collected from patients, who had suffered acute Hepatitis A Virus (HAV) infection at different times after onset of icterus. This cell population expressed both cytotoxic activity against autologous HAV-infected skin fibroblasts and the capacity to release y-lFN upon stimulation with HAV-infected fibroblasts. PBL, incubated with uninfected fibroblasts and PBL of anti-HAY negative persons, incubated with autologous HAV-infected fibroblasts produced no y-lFN and specific lysis also could not be detected. Although the highest activity of HAV-specific lysis, demonstrable 2-3 weeks after the onset of icterus, is not strongly correlated with the height of lFN-production, the coincidence of both parameters indicates, that cytotoxic T-cells and its product y-lFN might attribute to the elimination of the HAV-infection in man. The hepatotropic DNA viruses HBV, DHBV, GSHV and WHY are characterized by a small circular DNA genome of about 3 kbp length and a mechanism of replication, the central feature of which is the reverse transcription of an RNA intermediate into minus strand DNA. For the specific detection of replicative intermediates and of viral transcripts, strand-specific DHBV probes were prepared. -DHBV F 1-6 DNA of 3.0 kbp length was ligated with pSP 65 DNA in the Eco Rl position. The recombinant DNA molecule was cloned after transformation of E. coli HB 101. From DHBV DNA positive transformants two were selected (21-2 and 24-4) which contain the DHBV DNA insert in different orientation, as determined by restriction enzyme analysis. RNA probes synthesized from these two clones were specific for the detection of viral mRNA (24-4) and for viral minus strand replication DNA species (21-2), respectively. The probes are useful for the study of viral replication and transcription of viral genes and should add to our understanding of the biology of DHBV in hepatocytes as well as in nonhepatocytes.

BOSCH, VALERIE, G. RADZIWILL, and H. SCHALLER 2MBH, D-6900 Heidelberg

The P-frame of hepatitis B virus presumably codes for the proteins involved in viral replication. However, neither the nature of these putative products nor their mode of biosynthesis have been elucidated. To approach this problem, 7 segments, in sum 80%, of the duck hepatitis virus (DHBV) P-frame have been expressed as fusion proteins in E. coli and antisera prepared against them and against 4 peptides. These anti-fusion protein sera contain a significant titre towards the respective P-segment and could also imrnunoprecipitate Pvspecific polypeptides synthesized in vitro (SP-6). Intact viral cores containing DHBV-DNA and endogenous polymerase activity were immunoprecipitated from infected duck liver using anti-capsid (C) serum. Neither Western blotting of large amounts (20-40 IlgC)of this material, nor immunoprecipitation after radioiodination have revealed in vivo synthesized P-products. The reason for this negative finding is still unclear. The Pekin duck model of hepatitis B is a convenient system for the study of viral replication and antiviral strategies. For the detailed characterization of viral nucleic acid species, the DHBV genome was cloned from serum. DHBV was isolated by ultracentrifugation of serum; the viral genome was extracted, linearized with Eco RI and ligated with pBR 322 DNA. E. coli HB 101 was transformed with the recombinant DNA. From DHBV DNApositive colonies plasmid-DHBV DNA was prepared. The 3.0 kbp DHBV DNA insert was purified; its physical structure was established by restriction enzyme analyses. The restriction pattern of DHBV DNA F 1-6 is identical to DHBV DNA 16-t (Mandart et aI., 1984) but differs significantly from DHBV DNA 3 (Sprengel et aI., 1985) . Using cloned viral DNA as a probe, viral DNA species from infected serum and liver were analyzed by molecular hybridization techniques. Congenitally infected ducks have high levels of viral DNA in serum with a gradual decline over months. The mechanism of this reduction of viral particles in serum and its correlation with viral replication in the liver are presently being investigated. FUCHS, Transcription of Woodchuck Hepatitis virus (WHV), a member of the Hepadna-virus family, was studied in liver carcinomas of chronically infected woodchucks (Marmota monax). Replication of WHV-DNA was studied in tumors and non-tumor livers by Southern blots. In ten tumors with and without viral replication the transcription of WHY was studied by Northern blots. RNA of these tumors was hybridised with subgenomic fragments of the WHV-DNA cloned in the gemini 2 vector. These clones contain a single reading frame or part of it. In seven tumors the two major RNA's were transcribed (3.7 and 2.1 kb), as it has been shown in chronically infected livers by Moray et al., 1985 . Sometimes larger transcripts were observed. No transcription despite of presence of WHV-DNA could be detected in one tumor. In two tumors with integrated WHV-DNA only one major transcript (3.5 ego 2.5kb) could be seen. Hybridisation with the subgenomic clones revealed that in both tumor transcripts in contrast to chronically infected liver the C-gene and a part of the preSI-gene are deleted. Hepatitis delta virus (HDV) proteins were identified by immunoblot using HD patients sera as source of antibody. Two proteins, P27 and P29, copurified with HDV particles from the serum of an acutely infected chimpanzee. The same proteins were also found in sera from viremic HD patients and from HD infected woodchucks. Detection of HD proteins by immunoblot as marker of HD viremia is superior to detection of HD antigen by ELISA because it is possible even in the presence of excessive HD antibody. P29 was also found in HD infected liver but P27 was missing. However, variable patterns of other HD reactive proteins (P26, P22, P16, P15, P13) were observed. These proteins were predominantly localized in the nucleus. P22 from a woodchuck liver was found to be bound to RNA. RASSHOFER, R., C. PAHLKE, M. BUIl, and M. ROGGENDORF Max V. Pettenkofer Inst., Univ., Ciutat Sanitaria, Vall d'Hebron, Barcelona, Spain The Hepatitis Delta Virus (HDV) is a defective virus which needs HBV as a helper. HDV is composed of a circular RNA of 1.75 kb and the Delta Antigen. The coat of the HDV is the HB,Ag. -HDV is a powerful pathogen. In both simultaneous infection with HBV and superinfection of chronic HBsAgcarriers it can cause serious liver-damage. Superinfection of chronic HBsAg carriers with HDV often results in chronic active hepatitis and cirrhosis. -HDV infection is diagnosed by detection of HD Ag, Anti-HD-IgM and IgG and, since recently, by nucleic acid hybridisation of its RNA with specific probes. -In this study, hybridisation-conditions for HDV-RNA were optimized. Strand-specific probes like M13 primer-extension-probe or SP6-Riboprobe showed a much higher sensitivity than nicktranslated probes. However, when riboprobe is used, the problem of background hybridisation arises. Characterization of the HDV-RNA by Northern-blotting showed the typical band at 1.75 kb, which contained both genomic and antigenomic RNA, and some minor species of RNA, the predominant one of these banding at 0.8 kb. This species only contained genomic RNA.-HDV-RNA was found in the serum of 68% of patients with chronic active hepatitis or cirrhosis and Anti-HD-IgM. All of these patients had HD Ag in the liver. 50% of the HDV-RNA-negative patients had also HD-Ag in the liver and 67% of these Anti-HD-IgM in the serum. Only 5 of 11 HDV RNA-positive patients had HBV-DNA in the serum.

Characterization of Proteins Associated with the Hepatitis Delta Virus (HDV) ROGGENDORF, M., B. BOHM, R. RASSHOFER, and C. PAHLKE Max v. Pettenkofer Inst., Univ., D-8000 Munchen

The number and size of proteins associated with HDV from serum and liver of a chimpanzee at the acute stage of HDV infection were analyzed by immunoblotting. HDV positive serum was sedimented through a sucrose gradient (10-30%) and HDV-associated antigens (HDAg) and HBsAg determined in each fraction by ELISA or RIA. Peak fraction of HDAg were pooled and subjected to SDS PAGE and subsequently blotted on nitrocellulose. HDV-associated proteins were detected by incubation with human anti-HD positive serum and 125 1 Protein A. Two proteins of 30 and 28 kd were visualized by immunoblotting. Immunoblots of HDAg extracts from liver tissue (chimpanzee) with 7 M guanidinium HCI resulted in two proteins of 28 and 15 kd. Extracts of a human liver from a patient with chronic HDV infections were 13 and 12 kd in size. However, direct homogenation of chimpanzee and human liver in boiling mix subjection to PAGE and subsequent Western blotting resulted in two major protein bands of 30 and 28 kd. -These data indicate that HDV proteins are unstable and rapidly degrade to smaller fragments.

Structural Analysis of p19 and p24 Core Polypeptides of Primate Lymphotropic Retroviruses (PLRV) JURKIEWICZ l , E., H. NAKAMURA 2 , J. SCHNEIDER l , M. HAYAMI 2 , and C. HUNSMAN l Deutsches Primatenzentrum, Kellnerweg 4, D-3400 G6ttingen Inst. of Med. Science, Univ., Tokyo, Japan Several monkey species have antibodies crossreacting with human T-cell leukemia virus type I (HTLV-I). The main core polypeptides of these primate Iymphotropic retroviruses (PLRV) have very similar molecular weights. To discriminate individual PLRVs we have compared two dimensional tryptic peptide maps of 125I-Iabelled core polypeptides p19 and p24 of ten isolates originating from humans and six simian species. Peptide maps showed homologies between all HTL V-I related isolates. According to the relationship of their peptide maps we have classified the PLRVs into three groups, the human and chimpanzee isolates, macaque viruses, and green monkey virus. In contrast p24 and p18 of HIV (Human immunodeficiency virus) and related simian isolates (SIV) were completely different to those of HTLV-I related isolates, therefore they represent a separate group of PLRV. With respect to p24 and p18, the isolate of an African green monkey (SIVagm) is more closely related to HIV than to the viruses of sooty mangabey (SIVsm) and rhesus monkey (SIVmac). However, those of SIVsm and SIVmac are more closely related to each other than to SIVagm and HIV. CASSER-BETTE, M., V. ERFLE, and] . SCHMIDT Abt, f. Molekulare Zellpathologie, Gesellsch. f. Strahlen-u. Umweltforschung, D-8042

The target cell for OA MuLV and the effects of virus infection on bone cells were studied using primary skeletoblastic cell cultures of neonatally OA MuLV infected NMRI mice, in vitro infected primary bone cell cultures, and a permanent osteoblastic cell line (MC 3T3-El). -Seven days after infection of NMRI mice cells were obtained by fractionated dissociation of calvaria. Cultures from early fractions containing lesser differentiated osteoblastic cells showed a higher rate of infection than those from late fractions (differentiated osteoblastic cells). The osteoblastic differentiation followed by alkaline phosphatase activity (ALP) was higher in cultures from infected mice than in those from controls. In vitro infection of primary cultures from periosteum resulted in a decrease of cell growth associated with an increase of osteoblastic differentiation. The differentiation of calfaria cells was not influenced by virus infection. Infection of MC 3T3-El cells was followed by reduction of ALP activity associated with an increased cell growth. However, the cells were still responsive to chemical induction of differentiation. -These data indicate that in bone tissue, undifferentiated progenitor cells are the target for OA MuLV and virus infection increases osteoblastic activity. This fact possibly explains the critical role of OA MuL V in bone tumorigenesis. In contrast, infection of the permanent MC 3T3-El osteoblastic cells was followed by a reversible block of differentiation.

SCHMIDT, j., A. LUZ", E. LIVNE" "", M. SILBERMANN"", and V. ERFLE Abt. f. Molekulare Zellpathologie; " Inst. £. Pathologie, Gesellsch. £. : ." Israel Inst. of Technologie, Technion, Haifa, Israel Mandibular condyles of neonatal mice represent a so-called secondary type of cartilage, containing well-defined cell layers of different states of differentiation. In vitro, bipotential precursor cells, present in such tissues shift their differentiation program. They express alkaline phosphatase, collagen type I, bone sialoprotein and osteonectin, and finally become bone cells. -Twenty-four hours after in vitro infection of mandibular condyles with FBR MSV only cells of the progenitor cell layer were found to be infected. Further cultivation resulted in increasing loss of cellular organization, polymorphism, mineralization of remnant cartilage matrix and invasive growth of fibroblastic perichondreal cells into the condylar tissue and underlaying collagen sponge. Seven days after infection the tissue acquired the appearance of an osteosarcomatous lesion. After implantation of infected mandibular condyles into syngeneic mice, a transplantable fibro-chondrosarcoma tumor line was established. Tumor induction could not be observed after FBR MSV infection of similar tissue such as Meckels cartilage, or Xiphoid. -The data suggest that the presence of chondroprogenitor cells in condylar tissue, inhabiting the capacity of bidirectional differentiation, is a prerequisite for FBR MSV-induced osteosarcomagenesis in vitro. This system faciliates a detailed morphological and molecular analysis of early steps in viral osteosarcomagenesis.

Inst. of Immunobiology, Univ., D-7800 Freiburg Friend murine leukemia virus (F-MuLV) causes erythroleukemia in mice. In order to study the biology of this virus and the functions of its genes we have sequenced the genome of an highly infectious F-MuLV. The polymerase gene of this virus codes for a protease, the reverse transcriptase and an endonuclease. The sequence obtained showed a high degree of homology to Moloney murine leukemia virus and to AKV in the sequences coding for the protease and the reverse transcriptase. In contrast, the sequence coding for the carboxyterminus of the endonuclease was much less conserved. This might represent the specificity of the viral integration in the host genome. The knowledge of the sequence now allows to study the functions of defined regions of the genome in leukemogenesis.

SCHULZ, A., N. HESS 1, and R. FRIEDRICH Inst. of Immunobiology, Univ., D-7800 Freiburg 1 Heinrich Pette Inst., Univ., Hamburg Induction of disease by the murine leukemia viruses is assumed to involve insertional activation of cellular genes. Localization of newly integrated virus is hampered by the presence of the large number of endogenous viruses in the cellular genome. As a tool to study the localization of possible common integration sites of Friend murine leukemia virus and to determine a possible activation of cellular genes we have constructed a virus containing a selectable marker. A bacterial supressor tRNA gene (sup-F) was inserted into the LTR without destroying its biological function. Rat-1 cells were transfected with this DNA. We obtained several cell clones producing infectious virus. The virus collected from these cells has been injected into susceptible mice which, after tumor development, will be used for the preparation of genomic libraries. These will then be used for screening of sup-F harbouring proviruses which should facilitate determination of possible common integration sites.

WERNER, A., M. BAIER, J. LOWER, and R. KURTH Paul-Ehrlich-Inst., D-6000 Frankfurt 70

During the course of our studies of antibodies, in human sera, against HIV, we occasionally observed a reactivity against the viral p24 in persons without any risk of having contracted a corresponding virus infection. Reactivity with viral p24 was only detectable in sensitive Western Blots and usually not in ELISAs. -In extension of these initial observations, HTLV I and retroviruses of other mammalians (Baboon endogenous virus, BEV; Friend murine leukemia virus, FLV) and chicken (Rous sarcoma virus, RSV) were employed as antigens. Both sucrose density-gradient banded viruses as well as purified major gagantigens(p27, p30) were used. -It could easily be demonstrated that the gag protein of mammalian retroviruses is recognized by antibodies not only from people suffering from HIV infection, but also from patients with multiple sclerosis and other neurological diseases or with teratocarcinomas and even from healthy blood donors. In contrast, p27 of RSV was not recognized. -We are presently determining what proportion of the human sera react with animal retrovirus gag-antigens and whether a disease-specific patterns can be observed. The origin of these gag-reactive antibodies remains obscure at present. We either observe a biologically trivial true cross-reaction, which may, however, playa practical role in sensitive serodiagnostic procedures, or the reactivity of "interspecies-specific antibodies" formed in response to a yet unknown endogenous or exogenous human retrovirus strain. We have investigated the expression of the proto-oncogene c-src, the cellular homology of the Rous sarcoma virus transforming gene v-src. Our previous results on c-src expression during ontogenesis of vertebrates suggest that the physiological function of c-src appears to be more closely related to differentiation processes than to proliferation processes. This statement is supported by data obtained from experiments with the pro myelocytic human cell line HL-60. Induction of monocytic and granulocytic differentiation of HL-60 cells by 12-0-tetradecanoyl-phorbol-13-acetate (TPA), retinoic acid (RA) and dimethyl-sulfoxide (DMSO) is associated with an activation of the pp60 c , src kinase, but not with increased c-src gene expression. Control experiments exclude an interaction of TPA and DMSO themselves with the tyrosine-specific pp60 c , src kinase. Using embryonal carcinoma cell lines, which also can be induced to differentiate in vitro, we are currently analyzing whether the differentiation-dependent expression of the c-src gene product is restricted to monomyelocytic cells or can be generally observed during cellular differentiation processes. To study the expression of c-src and of c-src related genes, a chicken cDNA library was established and screened with the 0.8 kb PvuII fragment of v-src, 10 positive clones were obtained. Restriction analysis and hybridization experiments revealed that all clones are derived from the same genetical locus, which is however different from the locus of the c-src gene. The largest src-related cDNA clone (3,4 kb) was sequenced and shows up to 72% homology to v-src. Further comparison to published DNA sequences revealed homology to all known tyrosine kinases, especially to Isk, a tyrosine kinase isolated from the LSTRA cell line. -In a Northern Blot study the 3.4 kb clone hybridized to a 3.8 kb mRNA from chicken embryo fibroblasts. A signal of the same size ws obtained by Bishop with a Pvull fragment of v-src and assumed to be the c-src mRNA. -On the basis of the results presented here, the 3.4 kb cDNA clone represents a novel tyrosine kinase gene. Furthermore the expression uf (src requires further investigation.

Attenuation in the Control of c-myc Gene Expression? EICK D. and G. W. BORNKAMM Inst. f. Virologie, Zentrum f. Hygiene, Univ., D-7800 Freiburg DMSO (dimethylsulfoxide), a potent inducer of differentiation in HL60 cells, causes a rapid decrease of cytoplasmic steady state c-myc RNA. This decrease is regulated mainly at the level of transcript elongation. Elongation is blocked within the untranslated c-myc leader. We propose that the c-rnycleader is able to undergo two alternative conformations, an one-stem-and-Ioop structure allowing readthrough of the RNA polymerase into the coding part of the c-rnyc gene, and a two-stern-and-loop structure causing transcription arrest. We compare the attenuation model for the VP1 gene of the simian virus 40 (SV40) Restriction endonuclease and electron microscopic heteroduplex analyses of cloned DNA sequences of the ]ijoye and M-ABA strains of Epstein-Barr virus (EBV) had revealed several regions of partial or non-homology in the EBNA-2 open reading frame located in U2 (1), a feature which allowed the differentiation of 2 types of EBV (2) . This report describes an additional region of divergence caused by a 105 base pair (bp) insertion, 100 bp downstream of the ]ijoye EBNA-2 exon when compared with the B95-8 EBV DNA sequence. The insertion is flanked by 19 bp direct repeats and constitutes a truncated duplication (18% mismatch) of an adjacent sequence on the 3' side. The structural characteristics, both primary and secondary, of the insert and the surrounding genomic region indicate that it is derived from the Alu-family of human interspersed repeated sequences.

HARTL, P. and M. LIPP Inst. f. Biochemie, Ludwig-Maximilians-Univ., 0-8000 Miinchen 2

We have anaylzed the Burkitt lymphoma line BL64 in which a reciprocal translocation joins the immunoglobulin kappa light chain locus on chromsome 2 to the c-myc gene on chromosome 8. The breakpoints on the two marker chromosomes Sq" and 2p-occurred 5' of the ]5-segment within the conserved nonamer and heptamer recombination sequences. Both signals were detected directly adjacent to the breakpoints in sequences of chromosome 8 suggesting that the translocation in BL64 was catalyzed by enzymes normally involved in V-] recombinant. The distance between the c-myc gene and the breakpoint in Y amounts to at least 70 kb on the DNA level. In one allele of the c-myc gene somatic mutations were found in the promoter-leader region. This allele is trancribed and supposed to be involved in the translocation. Using transient expression assays, no functional differences of the c-myc promoters isolated from both alleles could be detected in various cell types. These results suggest that in Burkitt lymphoma the translocation occurs during an early stage of B-cell differentiation and that c-myc is activated by its location in the Ig chromatin structure. Epstein-Barr-Virus (EBV) infected cells can be induced by the treatment with TPA or other chemical or biological inducers to express early antigens (EA) and to produce virus. The induction of Raji cells, a nonproducer cell line derived from an African Burkitt's lymphoma leads only to the expression of EA. The activation of a strong induced region, which is in part duplicated in the EBV genome "DR", was studied in detail. It could be shown by 'nuclear run-on' assay, that the treatment of Raji cells with TPA and IUdR activates transcription of the DR-Region. The 2.8 kb RNA encoded by this region is detectable about 5 hours after induction as shown by Northern blot analysis. This induction is protein synthesis dependent indicating, that other gene functions (either EBV or cellular) are required for activation. -DR-Promoter-CAT-Constructs were transiently transfected into EBV positive and negative lymphoid cells and tested for CAT activity after treatment with various EBV inducing agents. The construct could only be activated in EBV positive cells. This indicates, that at least part of the functions required for the activation of the OR-Region are encoded by EBV. In many cases of Burkitt's lymph oma with t (8; 14) translocat ion and most murine plasmacytom as the c-rnyc gene is truncated suggesting that sepa ration of the bod y of the gene from its physiological prom oters and upstream regulatory sequences is one of the impo rtant mechan isms lead ing to c-myc deregulation. The fact that c-rnyc mRNA disappears earl y after induction of differentiation in various cell systems, suggests a regulatory ro le of the c-myc gene product for the option of the cell either to prol iferate or to undergo differentiation. Burkitt's lymphoma cells show morphological and phen otypical changes cha racteristic for B-cell differenti ation in response to sodium butyrate, a potent inducer of differentiation in various cell systems. We report here, that the treatment of a variety of different Burkitt's lymphoma cell lines with sodium butyrate, irrespective of the type of translocation or of the association with Epstein-Barr Virus, leads to a rapid decrease of cmyc steady state mRNA. This is due to a reduced trans cripti on rate of the c-rnyc gene, as shown by "nuclear run-on" ana lysis. The fact that sodium but yrate is capab le of downregulating an intact as well as a trun cated c-myc gene indicates that an important target site of tra nscriptio nal regulat ion is located downstream of the dual prom oters and the first exon . The Jijoye Epstein -Barr virus (EBV) strain is characterized by a substitution of 1.8 kb in the C-termina l part of EBN A 2 gene compared to B95-8 or M-ABA virus. Protei n immunoblot analysis using human EBNA 2 positive sera indicated that an immunological var iant to the EBNA 2 of B95-8 (type A) is encoded by the Jijoye virus (type B). In order to generate a specific EBNA 2B antiserum the NaeIlNsiI DNA fragment of the ]ijoye virus containing 237 bp of the C-terminus from th e EBNA 2B gene was cloned in an E. coli expression vector (pM E3). The resulting fusion prote in contained 79 C-term inal amino acids of the viral prot ein and a 37000 Dalton part of the bacterial anthranilate synt hase. Rabbit antisera generated against this fusion protein reacted specifically with two prot eins of 73000 and 77 000 Dalton from Jijoye cells and three other cell lines carr ying type B viru s while no prot eins could be identified in the type B cell line BL 29. In addition, using the se sera directed against the pME 3 fusion prote in no reacti on could be observed with the EBNA 2A prot ein from the B95-8 and several ot her cell lines containing type A virus . Therefore, these rabbit sera seem to be a useful and specific too l for fur ther investigations.

BOOS, H., R. BERGER, C. KUKUK-ROOS, andN. MUELLER-LANTZSCH Inst. f. Virologie, Zentrum f. Hygiene, D-7800 Freiburg

The BNLFlc reading frame of the EBV genome is known to code for a part of a membrane protein of 60000 Daltons expressed in latently infected cell lines (LMP) and an unidentified protein of presumably 28000 Daltons. Antisera against both proteins were generated in rabbits. These sera reacted with a protein varying in size between 60000 and 65000 Daltons found in all EBV harbouring cell lines examined so far. In addition, a second protein of 49000 Daltons on SDS-PAGE in B95-8, P3HR-l, and M-ABA cells was identified. Furthermore, these sera exhibited a positive cytoplasmic immunofluorescence in 1 to 10% of the cells depending on the cell line examined. Analysing the non-producer cell line Raji, the number of immunofluorescence positive cells and the amount of the 60000 Dalton protein was rapidly increased by addition of fresh medium with 10% fetal calf serum, by the tumour promoter TPA or to an even higher extent by n-butyrate. No synergistic effect of TPA and butyrate could be observed. The kinetics of induction reached a maximum at 24 h after addition of medium with 10% fresh serum. TPA orland n-butyrate and decreased after 24 to 48 h. Because of lack of the synergism mentioned above and because of a difference in expression kinetics of LMP and EA-komplex following induction of EBV lytic cycle LMP seems not to be a member of the EA-complex despite of its inducibility. Using a computer program that predicts the secondary structure of proteins and superimposes values for hydrophilicity, surface probability and flexibility, we identified potential membrane proteins derived from open reading frames encoded in the EB viral genome. Antigenic sites from four of those membrane proteins encoded by the open reading frames BLLFl, BILFl, BILF2 and BALF4 were selected and oligopeptides were synthesized by solid phase peptide synthesis. The pep tides were used as antigens in ELISA assays to screen sera from NPC and infectious mononucleosis patients and from healthy individuals. Sera from NPC and infectious mononucleosis patients showed elevated reactions to all peptides; the highest reactions were found to BALF4-derived peptide in NPC patients and to the BLLFladerived peptide in fresh infections. Only very low antibody titers could be identified against the peptide from reading frame BILFl. For immunization of rabbits, the Nl-lj-terminal amino acids of the peptides were covalently linked with palmitic acid and high-titered antisera against the individual oligopeptides could be obtained. Those rabbit sera were used for the identification of the corresponding EB viral proteins on Western blots, by imrnunoprecipitation and immunofluorescence. -Supported by SFB 217 TP B3. For diagnosis of EBV-correlated diseases we chose five relevant antigens of EBV: p150 VCA BcLF1, pB8 EA BALF2, p54 EA BMRFI, gp250/350 MA BLLF1, EBNA-1 BKRFl. The coding sequences of these antigens, analysed by computer programs to predict antigenic sites, were cloned in E. coli or -as in the case of gp250/350 -in CHO (Chinese hamster ovary) cells. The products were tested for their antigenicity and stability by immunoblotting. The identified recombinant antigens were purified by gel filtration and ion exchange chromatography (HPLC). The purified EBV proteins were evaluated in Western blots following antibody class specific staining and used in ELISAtests in order to screen sera from EBV-negative individuals, patients with acute EBV infection, reconvalescents and from patients with nasopharyngeal carcinoma. From the results combined with diagnostic significance suggestions of antigen/antibody class specific test combinations were derived for the various conditions. -Supported in part by BMFT 01ZR 85069 and 01ZR 102/5. , 1985) . The open reading frame BVRF2 spans the BamHI fragments, V, d and I in rightward orientation. A late active promoter was mapped in the BamHI d fragment (Baer et al., 1984) . Single-stranded DNA of a MB clone containing several hundred base pairs of BVRF2 selected the mRNA for the 49kD protein proving the transcription of the p49 mRNA in rightward orientation. By Northern blotting we further analyzed the transcription of this gene. To prove that p49 is encoded by BVRF2 and to further charaterize the protein we expressed the 3' end of BVRF2 as a fusion protein with -galactosidase in E. coli. The latter was partially purified and used to immunize a rabbit.

The fusion protein was recognized by a pool of sera from patients with nasopharyngeal carcinoma in Western blots. Using the rabbit serum we were able to characterize the 49kD protein as late protein in Western blots and immunoprecipitations. -Supported by DFG W0227/4. The two major envelope proteins of EBV (gp250/350) are encoded by the same reading frame (BLLF 1). The smaller variant gp250 is generated by a partial splice event which other tumors and in normal individuals no IgG/MA antibodies were detectable. Our data removes an internal part of the transcript encoding gp350 to yield the mRNA encoding gp250. These two proteins are candidates for a EBV-subunit vaccine. -The yield of gp250/ 350 expression in recombinant CHO cells is not very high and amplification of the MA BaLLFl encoding sequences in these cells is not possible, perhaps due to toxic effects. We now present a eukaryotic expression plasmid construct where the sequences encoding the transmembrane and anchor region are removed. Following transfection of CHO cells, this plasmid results in secretion of the EBV membrane proteins into culture medium. Furthermore, amplification by methotrexate selection was successful, yielding high levels of gp250/ 350 expression.

Max v. Pettenkofer-Inst., D-8000 Munchen

The protein encoded by the open reading frame BNLF1 (BNLF1-MA) has been connected with the target for cytotoxic T-cell reaction directed against EBV-infected lymphocytes; due to Northern and dot blot experiments, this gene is transcribed in latently infected cells in addition to the nuclear antigens (EBNAs) and is the only one of these polypeptides with characteristics of membrane proteins. Using sera against synthetic oligopeptides derived from the amino acid sequence of BNLF1-MA we could show that this protein is synthesized in Burkitt lymphoma cell lines in a truncated form lacking 138 amino acids at the amino terminal end (Modrow and Wolf, PNAS 83, 1986) . Using those antipeptide sera in immunofluorescence tests on latently infected cell lines, a positive reaction in about 20-30% of the cells was obtained. A similar amount of BNLF1-MA producing cells could be identified by in situ hybridization using 3H-cytidine labeled DNA probes. Since latent viral products (EBNA 1-3) should be detectable in more than 90% of the cells, we suggest that BNLF1-MA may be not a typical product of the latent EBV infection but a protein expressed in 20-30% of the cells respectively. In vivo those cells are eliminated by cytotoxic T-cells; in rare cases and in combination with additional factors only the truncated form of BNLF1-MA may be produced and those cells may develop to lymphomas. The presence of IgA antibody to membrane antigen (MA) of Epstein Barr-virus was tested in sera from nasopharyngeal carcinoma patients, patients from other tumors than NPC and normal individuals. Only 54% of the sera from NPC-patients showed a positive reaction, sera from control groups were negative. After adsorption of the sera with Staphylococcus aureus Protein A, 100% of the NPC-patients had IgA/MA antibody titers, in patients with indicate, that preadsorption of sera with Staph. aureus Protein A renders the diagnostic test significantly more sensitive for the detection of NPC and can be used for trials on the prognosis of patients. Furthermore our data show the importance of antibody tests to EBviral membrane antigens, especially when those are available in larger amounts by recom- In pevious experiments we correlated the proteins encoded in the BamHI region to single open reading frames. By Northern blotting we further substantiated the model of the transcription of this DNA area derived from hybrid-selected translations, sequence data (Baer et al., 1984) and mapped active promoters. When the protein synthesis was blocked simultaneously with the superinfection, the BamHI M fragment was still transcribed in different cell lines. A 2.6 kb mRNA transcribed from the left part of the fragment was detectable. From this mRNA, the BMRF1 reading frame is translated into a 47 kD protein which is posttranslationally modified into a 54 kD phosphoprotein. A second 1.9 kb mRNAs is transcribed from the right part of the BamHI M fragment where the BMLF1 reading frame is located. To identify the protein, we expressed large parts of the BMLF1 reading frame in E.

coli as a fusion protein with~-galactosidase. The fusion protein was recognized by a pool of sera from NPC patients on Western blots and used to immunize rabbits. The sera can be used to characterize the BMLF1 protein. -Supported by DFG Wo227/4. The 2.0 kb HindlII K fragment, located in the B95-8 deletion, but not the neighbouring fragments hybridized in Northern blots with a 1.8 kb mRNA, isolated from induced P3HRl and Raji cells and from Raji and X50-7 cells superinfected with P3HRl virus. When the protein synthesis is blocked simultaneously with the superinfection by cycloheximid or anisomycin, only a few mRNAs characteristic for the switch from latency to the lytic cycle can be expressed. Under these conditions the 1.8 kb mRNA is transcribed. The Hind III K fragment consists mainly of the single open reading frame BJ'LF4. Therefore BJ'LF4 seems to encode a protein which is expressed immediately after the superinfection of latently infected cells. We expressed the 3' half of the reading frame as fusion protein with~ galactosidase in E. coli. The protein was partly purified and used to immunize rabbits. Using the rabbit serum we were able to immunoprecipitate a 68 kD protein from Raji and X50-7 cells superinfected with P3HRl virus early after infection. Several groups have already mapped the coding region of EBNA 1 in the Bam HI Kfragment of the EBV-DNA. In order to characterize some natural epitopes of the EBNA 1 protein the Hind III-Il-fragment of the EBV strain M-ABA was cut into fragments of about 200 bp by using DNase. These fragments were then cloned into the E. coli expression vector according to all three reading frames in both directions at the 3' end of the lac Z protein.

After transformation the bacterial colonies expressing a specific epitope were identified. The method used was colony immunoblotting. -The results were verified by the protein immunoblotting method. Two different epitopes of the EBNA 1 protein could be detected. The sequence analysis localized the epitopes to different regions of repetitive sequences in the region coding for the EBNA 1 protein. After immunization of rabbits with the EBNA 1 epitopes an antiserum was obtained which reacts specifically with the EBNA 1 protein of different EBV strains in the immunoblots. Cell surface density and molecular structure of HLA class I and II antigens of Burkitt's lymphoma (BL) cells and Epstein Barr Virus (EBV) transformed lymphoblastoid cell lines (LCL) established from the same patient were examined. Using a radioimmuno assay with a monoclonal antibody against a common determinant of class I molecules we found a 1,2-25 fold higher density of HLA class I molecules on LCLs than on BLs in all cell pairs studied. In 6 of 8 cell pairs, class II antigens were also expressed in higher amounts on LCLs. In addition, immunoprecipitation of radiolabeled components and subsequent analysis on SDS-polyacrylamide gel electrophoresis revealed qualitative differences between the class I molecules of BL and LCL cells in all 6 cell pairs studied. The observed changes in HLA class I expression may well playa role for the immunological growth control of EBVtransformed cells, allowing BL cells to escape from immune surveillance mechanisms. Monensin, at concentrations which dependet on the multiplicity of infection, was found to prevent DNA replication of human cytomegalovirus (HCMV) as well as production of viral progeny. The effect of the drug on HCMV DNA-synthesis was fully reversible. Delayed addition was effective only until 24 h postinfection. Induction of viral DNA polymerase was not impeded by the inhibitor. Monensin did not affect, on the other hand, DNA replication of herpes simplex virus. Analysis of protein-and glycoprotein synthesis revealed that monensin interfered with the production of a number of HCMV-specific polypeptides. By the use of a monospecific antiserum, evidence was obtained that the drug interfered with the processing of a 135 kd glycopolypeptide. Herpesviruses differ in the number of their ie gene products, but at least one IE protein has trans activating properties. -The mode of IE protein-DNA interaction, resulting in promoter activation, is unclear. The following possibilities can be envisaged. 1. IE proteins bind DNA. 2. IE protein complexes bind DNA. 3. IE proteins bind DNA after interaction with cellular facors. -The iel gene products of MCMV have been demonstrated to activate promotors in trans. We examined DNA-binding properties of the major IE protein (pp89) and of its processing product (pp76) using CT-DNA-cellulose-chromatographie. pp89 revealed a high affinity for DNA, whereas pp76 showed very little DNA-binding properties. DNA-binding of pp89 however required the presence of a cellular DNA-binding factor(s). Thus, if direct or indirect DNA-binding is a prerequisite for regulation, only pp89 but not pp76 can activate promotors. Regulative activity is either direct after pp89-factor interaction or indirect after factor modification by pp 89. To examine how the iel gene product, pp89, is involved in the induction of early gene expression, early genes must be identified and isolated. For this purpose monoclonal antibodies (mAb) against early proteins of MCMV were prepared. -One mAb stained antigen located in the cytoplasm and immunoprecipitated a 52 K and 67K early protein. Staining with a second mAb resulted in faint homogenous nuclear fluorescence during the early phase, while at late times the antigen accumulated in large aggregates in the nucleus. This mAb precipitated a 40K and a 47K early protein. The third mAb produced speckled nuclear staining in the early phase, whereas at late times the distribution of antigen resembled that of the second antigen. The immunoblot with the third mAb showed a 36K, 37K, and 39K early protein. Immunoprecipitation with the mAb after in vitro translation of early RNA revealed the 36K and 39K proteins. Gene expression of MCMV is subdivided into three coordinately regulated phases: immediate-early (IE), early and late. The ie1 proteins transactivate promoters, but it is not clear whether this function is associated with the 89K gene product (pp89) or the processed 76K protein (pp76). Because in regulatory viral proteins complex formation and function is often associated, the sedimentation patterns of pp89 and pp76 were investigated. When early gene expression was allowed for less than three hours, both proteins sedimented as monomers. In the "delayed" early (more than 6 h p.i.) or late phase, only pp76 was detectable as complex. Cellular proteins appeared not to be involved in complex formation, a possible participation of early proteins is presently under study. Events of the "delayed" early phase are responsible for complex formation of pp76. A role in transactivation is unlikely, since only pp89 was found to bind to DNA. The potential recognition of pp76 by cytolytic T lymphocytes requires further analysis.

SCRIBA, M., L. OSTBERG, and E. PURSCH

Human monoclonal antibodies were made by fusion of the Spaz 4 cell (a mouse x human myeloma) with either peripheral blood lymphocytes from patients with an acute CMV infection or spleen cells stimulated in vitro with CMV antigen prior to fusion. -With both methods we could obtain several hybridomas secreting anti-CMV antibodies. Four of these hybridomas were established as stable producers, secreting between 20-60 !-tg/ml of antibodies since more than 2 years now. Three of these antibodies are neutralizing antibodies of the IgGl subtype; the 4 th antibody is an IgG3, recognizing an internal structural protein of the virus. -Clinical trials for prophylaxis of CMV infection in bone marrow transplantation patients have been initiated recently with 2 of the neutralizing antibodies. The number of immediate-early (IE) genes differs among different herpesviruses. At least one IE gene product is necessary to initiate the early phase of the viral gene expression. The product of the IE gene 1 (ie1) of the murine cytomegalovirus (MCMV) activates promoters in trans. The transcription of ie1 is controlled by a strong enhancer which also influences the expression of another IE gene, ie2. The ie2 codes for a 43K protein which is not immunoprecipitated by murine antisera against MCMV IE proteins. It could only be detected after in vitro translation of hybrid-selected RNA. The function of the ie2 gene product is unknown. Therefore, as a first step, the ie2 gene was analyzed in more detail. -The 43K protein is translated from a 1.75 kb mRNA, a spliced molecule which contains 3 exons of 75 n, 109 n, and 1277 n. The 1277 n exon at the 3' end of the 1.75 kb mRNA has an open reading frame of 1173 n, which could encode a polypeptide of 391 amino acids with a calculated molecular weight of 43.8K. Human cytomegalovirus (HCMV) particles contain a phospho-protein of about 150 K (pp150) in their matrix; the protein appears particularly reactive in Western blot analyses with human antisera. The gene for pp150 was mapped by screening a bacteriophage Lambda gt11 eDNA expression library with mono specific rabbit antisera. Subsequent hybridization of eDNA with cosmid and plasmid clones containing the entire HCMV strain AD169 genome mapped the gene to HindIIIfragments J and N. The genomic segment is transcribed into an abundant late 6.2 kb RNA. The nucleotide sequence of this region was determined, and transcription initiation and polyadenylation sites of the transcript were located by primer extension and nuclease protection experiments. Polypeptide secondary structure analysis revealed multiple~-pleated sheets in hydrophilic clusters, providing a possible explanation for the immunogenic properties of the polypeptide. ,. Ernst-Rodenwaldt-Inst., D-5400 Koblenz An RNA-class of about 5kb is encoded within the immediate-early (IE) region of human cytomegalovirus strain Ad169. This transcript is distinct from all other RNAs of HCMV thus far described. Extensive Northern-blot analyses demonstrated that this RNA is present in RNA preps from all phases of the infectious cycle. Through Sj-analyses, RNase-protec-tion and primer extension using synthetic oligonucleotides, 3'-end and 5'-end were determined. In addition the 3'-coding sequence was established thus providing the whole nucleotide sequence of the coding region. A typical polyadenylation signal was found at the 3'-end. Upstream to the putative initiation site, however, no typical TAT A-box was detected. Since no longer open reading frame was found in the sequence, this RNA might have some regulatory rather than a protein-coding function.

Antibody to Envelope-Proteins of Human Cytomegalovirus (HCMV) SCHMITZ, H. and A. BRUEHMANN Bernhardt-Nocht-Inst., D-2000 Hamburg 4 We have compared the immune response to HCMV using both an ELISA and a neutralization test (NT). When antibody titers in human sera and in hyperimmune-gamma globulins were tested in parallel a relatively low correlation coefficient (r s=052) was found. -In addition, we analyzed the immune response to HCMV glycoproteins in human sera using monoclonal antibodies which had been characterized by immunofluorescence, by immunoblot and by NT. These monoclonals were used to obtain enzyme-labelled antigens (ELAs). With these glycoprotein ELAs we again looked for specific antibody activity in human sera. This time a close correlation with the NT-titers was found. Therefore, an enzyme immunoassay can now be used for the selection of blood donations for HCMV hyperimmune-gamma globulins. A NIH 3T3 subclone, clone 1, was found to be deficient with respect to the antiproliferative response as well as to the antiviral effect against lytic RNA-viruses, e.g. encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV). Induction of 2-5 A synthetase and dsRNA dependent protein kinase by IFN as well as inhibition of retrovirus production e.g. murine leukemia virus (MMuLV), indicate a defect other than the lack of a functional IFN receptor. A very low level of 2-5A dependent RNase was discussed to be the reason for the defects described. Development of a selection system, based on IFN treatment and subsequent virus challenge by EMCV allowed the isolation of protected colonies derived from the unprotected cell population. Characterisation of these subclones led to a dose response comparable to the IFN sensitive L 929 cells with respect to EMCV. In contrast, VSV protection as well as inhibition of cell growth was much less extensive resembling the original NIH 3T3 line. This implicates at least partial independent mechanisms leading to each of the effects. No difference in the level of 2-5A dependent nuclease could be demonstrated, implying another defect besides a nonfunctional 2-5A system, responsible for the partial IFN resistance of the original NIH 3T3 line. Infection of cultured mouse bone-marrow macrophages with Newcastle Disease Virus (60 HAU/l0 6 cells) resulted in high titers of IFN -1,5 to 3 X 10 41U/mlthat reached peak levels 8-10 h following induction. This multiplicity resulted in infection of 100% of the cells as determined by immunofluorescence. Analysis by in situ hybridization revealed the presence of IFN-a and p mRNA 2 h after infection in 50-60% or 25-30% of the cells, respectively. 6 h after infection specific hybridization signals were seen in 60-80% of the cells and a strong increase in the number of grains per cell was observed. A great heterogeneity in grain densities per cell was observed following hybridization with both IFN cDNA probes; however the cells were labeled homofeneously, when hybridization was done with a probe coding for the MHC antigen H-2K . In addition, specific hybridization signals were found in 5-15% of non-induced control cells in the absence of detectable antiviral activity in the corresponding culture supernatants, suggesting that very low amounts of IFN a and~are synthesized constitutively by cultured mouse bone-marrow macrophages.

DOMKE-OPITZ, I., P. STRAUB, P. HAGENDORN, and H. KIRCHNER Inst. of Virus Research, German Cancer Research Center, Splenic macrophage cultures from HSV resistant C57BL/6 mice survived HSV infection in vitro. In contrast, macrophages from HSV susceptible DBA/2 mice were completely lysed by the virus. Resistance was mediated by the production of interferon early after infection. During prolonged culturing macrophages from C57BL/6 mice continued to produce infectious virions, indicating establishment of a persistent infection. At this time, interferon was undetectable. However, as shown by the addition of an anti-interferon serum, interferon was involved in the maintenance of the persistent infection. During the acute phase of virus infection, induction of viral proteins and DNA replication were identical in macrophages from resistant or susceptible mice. Later on, viral DNA content and the number of cells expressing HSV antigens decreased in macro phages from C57BL/6 mice. However, single cells remained to express viral proteins and to produce infectious particles. The results show that macrophages can be persistently infected with HSV due to their genetically controlled properties. Fourty-five patients with virologically confirmed dendritic keratitis were treated in a randomized, double-blind controlled study with a basic therapy of trifluorothymidine (TFT) eye drops. In addition they received different human recombinant interferon (rHu IFN) eye drops. The following results were obtained for average healing times: TFT plus one drop daily of rHu IFN-alpha 2 arg (30 million iu/ml): 3.3 days, TFT plus rHu IFN-gamma (30 million iu/rnl): 3.9 days, TFT plus a mixture of alpha plus gamma (0.3 million iu/ml each): 6.1 days. TFT plus a mixture of alpha plus gamma (1.5 million iu/ml each): 3.3 days. Hightiter gamma interferon did not significantly differ from high-titer alpha interferon in the combination therapy of dendritic keratitis. A mixture of alpha plus gamma at a moderate titer (1.5 million iu/ml each) was as effective as a high-titer monopreparation. Adding a lowtiter interferon mixture gave no better therapeutic results than antiviral monotherapy. Thus it seems possible to save about 90% of interferon commonly used in the combination therapy of dendritic keratitis by applying a mixture of different suitable interferons instead of interferon mono species.

GR U N , J. , BARBARA ZOLLER , ER NA KROON, and C. JUNGWIRTH

The molecular mechani sm of interferon action on pox virus specific immediate earl y prot ein synthesis was studied in interferon treated chick cells. Under conditions of over 90% inhibition of pro xvirus specific thymidine kinase induction RNA for , this earl y enzyme is synthesized but does not accumulate. Northern blot analysis reveals strong degradation of residual thymidine kinase RNA . Blot hybrid ization analysis using tot al vaccinia DNA and restr iction fragment N as pro bes demonstrate a generally reduced steady state amount of vaccinia specific early RNA's in int erferon treated chick embr yo fibroblasts. Expression of the chloramphenicol acetyltransferase gene inserted into an infectiou s vaccinia recombinant is inhibited in the interferon tr eated cells.

BR ANDNER ! , G. , B. ALTINKILl C I , and M. LlPp 2 lnst. f. Virologie, Univ., D-7800 Freiburg Biochemisches lnst. , Univ., D-8000 Miin chen HSV replication is sensitive to IFN in cell culture and in vivo. We have claimed (1972 -1985) We observed antiviral activitiy of hu rTNF on certain cell lines (HEL, WI-38, HEp-2) in a typical in vitro antiviral assay. Pretreatment with TNF led to protection from infection with VSV, EMCV or HSV. We observed inhibition of the cytopathic effect, virus-yield reduction and suppression of formation of viral proteins. (2'-5') (A)n-synthetase, an enzyme induced by IFN, was also induced by TNF in confluent HEp-2-cells. No IFN-mRNA could be detected in Northern blot analysis of RNA from TNF-treated cells. Anti-IFN~-antibodies, however, present during pretreatment with TNF led to a partial reduction of the antiviral TNF-effect, while anti-IFNa or anti-IFNy did not. The amount of this induced IfNp-like activity was not sufficient to account for the observed virus-yield reductions suggesting an antiviral activity of TNF itself. Mouse macrophages grown from spleen cells of mice were found to be very sensitive to interferon a/~[IFN] activity against Herpes simplex virus type 1 . Therefore, we have used these cells to investigate the level at which IFN blocks the replication of HSV-1. -HSV a,~and y protein syntheses was strongly reduced in IFN a/~acts at a very early step of the viral replication cycle. Isolation of infected cell nuclei prior to the begin of viral replication showed equal levels of HSV-DNA in pretreated and control cells. These results rule out IFN action on virus uptake. Run-on transcription experiments revealed a delay of transcription from the ICP4, ICPO and the thymidinkinase-gene, In contrast, we found inhibition of total ICP4-RNA steady state levels throughout the replication cycle. These results suggest effects of IFN a/~on both transcription and stability of viral RNA. In chronically HSV type 1 infected BL cells (B]AB, Raji) viral proteins are synthesized continuously in variable amounts during the growth cycle. Reisolated viruses of these chron-ically infected cell lines show a changed glycoprotein pattern and syncytia formation in Vero cells after several months of persistence. -After addition of human anti HSY sera to the culture media, the infection becomes latent. During establishing latency viral protein synthesis stops rapidly; the contents of viral DNA decrease in 14 days to less than one copy per cell. After removal of the antibodies the cells show again the characteristics of a chronic infection. Five exoglycosidases and three endoglycosidases were used to determine the glycosylation pattern and its relevance for the infectivity of HSY-l. Digestion with exoglycosidases yielded a reduction of infectivity up to 4.5 log units. By control experiments it could be shown that the proteolytic activity detected in some of the enzyme preparations had no effect on fully glycosylated virions. SDS·PAGE analysis yielded decreases of the rel, mol. wts. of glycoproteins to be expected from glycosidase-treatment. Digestion with endo H did not result in a decrease of infectivity but a decrease of the rel., mol. wt. of gB. This suggests that i) gC and gD are "complex-type"-glycosylated, ii) gB is to 30% glycosylated in a "highmannose-type" -manner and iii) the removal of these carbohydrates does not decrease the infectivity of HSY-1. Digestion with endo F decreased the infectivity by 90% and resulted in a decrease of the rel. mol. wts. of the viral glycoproteins. Experiments with PEG could not restore the ceased infectivity suggesting that endo F caused a block of infectivity at the level of adsorption. The data presented provide evidence that O-linked carbohydrates might play a role in HSV-1 infectivity. In the present study the protein specificity of antibodies (abs) against HSV-1 structural and nonstructural proteins has been analyzed by WBA and RIPA-PAGE. Sera containing anti-HSY IgM were tested class-specifically in WBA and RIPA-PAGE for the reactivity of IgG and IgM abs. It could be demonstrated that IgG and IgM abs are directed against viral immediate-early (IE), early (E) and late (L) proteins. -Following acute primary HSY-1 infection the earliest IgM ab response was found to be directed against viral glycoproteins, subsequently viral nonglycosylated structural and nonstructural proteins were recognized by IgM abs. Already early in infection IgG abs against viral glycoproteins and other viral structural proteins with an apparent mw of 56,45 and 39 kD could be detected. Viral IE and E proteins were poorly recognized by IgG abs in acute primary HSY-1 infections. In acute recurrent HSY-1 infections IgM abs exhibited a less complex pattern of reactivity with viral proteins, predominantly viral nonglycosylated structural, proteins were recognized by IgM abs.lgG abs from patients with acute recurrent HSV-l infections reacted strongly with a variety of viral structural and nonstructural proteins. In latently infected, a long lasting and prominent ab response against gB and gD could be shown, whereas abs against other viral structural and nonstructural proteins seemed to be produced temporarily. From seven adults and two neonates with mortal diseases unrelated to HSV, DNA samples of brain tissue (1 g) were separated in viral (high density) and cellular (low density) fractions by two cycles of CsCI density gradients. Bam HI restrictograms were hybridized with HSV type 1 fragments: U L (Bam HI-C) and tk gene (-Q), junction (-K), origin of Us (-N) and ICP4 gene (-Y). -Neonatal brains were free of HSV DNA. All seven temporal grey and white matter of adult brains and only two brainstems contained HSV DNA sequences, found in both (viral and cellular) fractions. Only one adult brain sample is consistent with a linear organization of its HSV genome; in six adult brains HSV DNA exists in an incomplete form.

Novel Small RNAs in HSV Infected Cells In eucaryotic cells, small nuclear-and small cytoplasmic RNAs (sn-or scRNAs) are associated with distinct proteins, forming SNRNPS or SNCRNPS. In the present study we analyzed the protein composition as well as the small RNA pattern in noninfected and Herpes simplex virus type 1 (HSV-1) infected vero cells. -We found that concomitantly with the shut off of host cell messenger RNA synthesis, synthesis of U-SNRNAS is stopped. Due to their stability, however, U-SNRNAs are still present in HSV infected cells 36 h p.i. Besides these RNAs two novel small RNAs were detected in infected cells, which we termed HVRI and HVR2. Based on the relative mobilities in urea gels, the apparent chain length of these newly synthesized RNAs were determined to be 255 and 154 nucleotides, respectively. Libraries of small fragments of BHV-2 and HSV-l DNAs were estabished with the expression vector lambda gtll and screened with monoclonal antibodies directed against cross-reacting epitopes present on a 130K glycoprotein of BHV-2 (gB-BHV-2) and glycoprotein gB of HSV-l. Using one of the lambda gtll-gB-BHV-2 clones as a probe it could be shown that the gB-BHV-2 gene maps colinearly to the gB gene of HSV-1 in the UL segment of the genome. It could be demonstrated that one group of cross-reacting epitopes is clus-tered in a region of approximately 100 amino acids. Nuceotide sequencing revealed a highly conserved region in the gB genes of BHV-2 and HSV-l. The determination of the nucleotide sequences coding for the common epitopes is in progress. The attenuated live vaccine strain Rac-H was developed from a fetal isolate by continuous propagation in porcine embryonic kidney cells (256 passages). Strain Rac-H (256 th p.) DNA proved to have a restriction enzyme pattern different from other EHV-l isolates and strains. For comparison of DNA preparations from high and low passaged Rac-H strain we used digestion with the restriction enzyme Bam HI. At least up to the 185 th passage DNA patterns showed to be similar to those obtained from field isolate DNA. We assume, that mutations resulting in the altered fragment pattern of the vaccine strain do not correspond to the loss of virulence, which was observed during propagation before the 185 th passage. 

GERRITZEN, A. and K. E. SCHNEWEIS Inst. of Med. Microbiology and Immunology, Univ., 0-5300 Bonn 1

In mice genitally infected with herpes simplex virus (HSV) no infectious virus can be isolated from the inflamed draining lymph nodes, contrary to the positive results in the lumbosacral ganglia. Attempts to reactivate an abortive infection eventually established in lymph node or spleen cells by stimulation with phythaemagglutinin or lipopolysaccharide rendered no positive results, not even when adult, but immunodeficient mice were used as test animals. Isolation of infectious virus from lymph node and spleen cells was successful in immature 4 to 6-week old mice, particularly when these had undergone pretreatment with cyclophosphamide, silica, antimacrophage serum and/or cortison; 5 days p.i. being the date of optimum virus yield. HSV-1 infected mice were more frequently positive than those with HSV-2, and genetically sensitive animals more so than resistant mice. The data indicate that the lymphohaematogenous spread of the virus is inhibited in favour of the neural dissemination by means of an active defence mechanism, probably by macrophages and/or NK-cells. Clin. Microbiol. 5 (1977) 250) in which SV40 has integrated once in the genome as a partial tandem is specially suited for further detailed studies on this amplification process. Elona is not permissive for SV40, but T-antigen and the origin of replication are functional. Monoclonal antibodies (mABs) directed against a 130K glycoprotein of the bovine herpesvirus type 2 (BHY-2) cross-react in several serological tests with the human herpes simplex virus types 1 and -2 (HSY-1, -2). The antigen of HSY involved in the cross-reaction was identified as the glycoprotein gB. Indirect immunofluorescence using 0.5 urn cryosections of HSY-1 infected cells and the mABs revealed either a patchy intranuclear or a cytoplasmic membrane labelling. lmmuno-gold lEM of ultra thin cryosections of infected cells showed that two antibodies detect epitopes on the capsids and in distinct areas in the nuclei, whereas other stain envelopes of viruses in the cytoplasm and after egress. Western blot analysis of capsid preparations isolated from different compartments of infected cells are in agreement with the hypothesis that gB is transported into the nucleus were it attaches to the capsids. Further investigations will show whether biochemical differences are detectable between gB molecules localized in different cellular compartments. Patients after organ transplantation have a higher risk to get infected by e.g. cytomegalovirus, herpes simplex virus, varicella zoster. -We studied the course of 30 kidney transplanted patients during the first 3 months after transplantation; immunosuppressive therapy with ciclosporin. Additionally to the virus-specific titers (ELISA) of the IgG and IgM class against CMV, HSV, VZV the ratio of T 4/T8 lymphocytes in the peripheral blood was estimated. -So far this T 418 ratio shifted to lower values -in some cases below 0.5 -if infectious diseases caused by e.g. CMV, HSV occurred days to weeks later. This would mean that the T 418 ratio earlier indicates to an outcoming infectious disease apparent or inapparent in clinical course than it could be seen by e.g. seroconversion of virus-specific IgM and IgG. The T 418 ratio doesn't seem to be influenced by ciclosporin medication. Liebig Univ., D-6300 Gielsen Sera collected 13 years ago from 348 residents of the Republic of Liberia were tested for antibody to Marburg-and Ebola-virus using Elisa and immunoblot techniques. Antibody to Marburg virus (MV) was found in 18.1 % and to Ebola virus (EV) in 10.6%. Distribution of seropositives was completely independent of sex and of tribal affiliation. The incidence of antibody was only 4.1 % in people who had lived in the Savannah region for more than ten years and amounted to 23.2% in the rest of the population. In certain divisions of a rubber plantation as many as 52% of people were seropositive for either virus. The highest prevalence of infection was seen in the age groups between 20 and 39 years. There was no indication of a professional risk. Antibody against both viruses was found in 7.4% of sera. EV antibody occurred five times as often in MV positive individuals than in the whole population and in EV positive sera antibody to MV was present 10 times as often as in the whole population. These positive correlations indicate that there must exist a common epidemiological risk factor for both infections. C129-mice were intracerebrally infected with Sendai-virus (D52). 85 days after infection the brain cells were cocultivated with uninfected mice brain cells. From this persistently infected cells infectious and temperature sensitive (ts) mutants were isolated (MG-Ia). The protein pattern of the purified MG-Ia particles differs from the wildtyp pattern. The hemagglutination (HN) protein band was more prominent and of lower molecular weight. In contrast to the other viral proteins the HN-band was not recognised by an anti-Sendai serum, but by an anti-HN-monoclonal antibody. That the differences are due to genomic mutations may be concluded from the weak hybridisation signal with HN-mRNA-specific cDNA probes, which doesn't correlate with the HN-protein abundance: -To study therole of HN in viral persistence we isolated ts-mutants of Sendai wildtyp virus after mutagenesis with 5' -Fluoruracil. The HN-protein of three ts-mutants show the same immunogenic properties as MG-Ia in immuno-blot. We'll use these mutants to study the course of infection in mice and different cell culture systems. Only one showed an additional reaction with the NE-Hallnas strain. Also 287 sera from laboratory rats collected during the last ten years in Europe from different breeding colonies were tested against 4 Hantavirus strains. 22 sera (7.7%) were positive, titers ranging between 1 : 10 and 1 : 1280. Further work is necessary to locate the natural foci of Hantavirus in Clethrionomys glareolus in Western Germany. Different cell lines persistently infected with Sendai virus were investigated for viral infectivity: at the beginning of persistency an increase of infectivity was detected, which could be explained by adaptation of the virus to the host cells. However, although compared to an acute infection the infectivity is about four magnitudes lower, the virus particles are capable of multiple-cycle replication in non-permissive cells. In a later stage of persistency, a lack of viral infectivity was observed. In contrast to acute Sendai virus infection, no activation was obtained by trypsin treatment. On the other hand a small number of these virus particles are activated by elastase and thermolysin. From these firidings it appears that the virus population is heterogeneous and consists of F protein mutants. It could be concluded that infectivity is lost during Sendai virus persistency due to lack of selection leading to a heterogeneous virus population. We have shown recently that the budding of measles virus is connected with a polar growth of actin filaments. Calcium ions are known to playa key role in the regulation of assembly and disassembly of actin filaments as well as in the regulation of contractility. Therefore calcium ions may also influence the budding process of measles virus. We modulated the calcium concentration in measles virus infected cells by use of the calcium ionophore A23187. In the presence of calcium, but not of magnesium or barium the ionophore induced (l) a disappearance of virus structures and microvilli from the cell surface, (2) a random redistribution of virus hemagglutinin at the cell surface, (3) a dissociation of nucleocapsids from the protoplasmic membrane face, and (4) a significant reduction of the cell bound infectivity. The data indicate, that calcium ions are able to influence the morphology of budding virus particles at the plasma membrane and may playa role in virus morphogenesis. -Supported in part by Gemeinniitzige Hertie-Stiftung, Frankfurt/M. inserted into the ER membrane and set free from the precursor by cellular signalase. The remaining non-structural proteins are set free by a different protease which is possibly virus specific. -3. All proteins contain hydrophobic sequences which could function as transmembrane sequences. This finding is in accord with the well known fact that all Flavivirus proteins are membrane associated. The mechanism(s) mediating positive and negative regulation of transcription by Adenovirus early region lA proteins is at present unknown. Whether ElA gene products interact with RNA-Polymerase II, specific DNA nucleotide sequences (enhancer elements) or counteract a cellular repressor bound to the viral or cellular genome, all remains formal possibilities. -In order to verify one of the possible interactions of the E1A proteins mentioned above, we have looked for a direct or indirect binding capability of Ad12 E1A proteins to double-stranded (ds) DNA. Nuclear protein extracts, prepared from infected KB cells, have been passed through columns of viral and cellular ds DNA-cellulose. After elution with salt gradients, each fraction was screened for E1A polypeptides by imrnunoprecipitation using E1A-specific antipeptide antibodies. We have detected an E1A protein with a MW between 38K and 45K, which elutes from the column at 0.2 to 0.3 M KCI. A second column run of the fractions eluted at 0.2-0.3M salt showed no or very little binding of the E1A polypeptide to ds DNA. However, incubation of the E1A protein containing fractions (eluted from the first column) with nuclear extracts of uninfected cells, restores the capability of this E1A polypeptide to interact with viral or cellular DNA, suggesting that the binding of the viral protein is possibly mediated by (a) cellular protein(s). We are currently characterizing the binding conditions, a possible sequence specificity and the cellular component(s) of the DNA-bound viral/cellular protein complex. -(Supported by the Deutsche Forschungsgemeinschaft through SFB 102-All). The E1B 58K protein of adenovirsus (Ad) is involved in oncogenic transformation of cells and exhibits essential viral functions during productive infection e.g. DNA replication, transport of viral mRNA, shut off of host cell protein synthesis. Since only limited amounts of E1B proteins are synthesized in infected cells, we decided to express the E1B 58K gene in E. coli. Such an expression system also offer the possibility to synthesize parts and to raise antibodies against various domains of the proteins and to investigate possible functions in more detail. -The expression vector which has been used (piWiT15) directs transcription from a synthetic promoter controlled by the Lac operator. The clone pB58N-gal codes for the N-terminal first 200 amino acids of the Ad12 E1B 58K protein fused to B-galactosidase. Total protein extracts from pB58N-gal transformed bacteria contain a fusion protein of expected MW (150kD) and in addition fusion proteins of 140kD and 120kD. Expression clone pB58, a derivative of pB58KN-gal, codes for the whole 58K protein. As the described proteins are specifically degraded in E. coli, we are currently testing the T4 pin gene function in respect to stabilization of these proteins. Results about the expression of the corresponding adenoviral proteins in E. coli and the specificity of antibodies, directed against these proteins will be dicussed. -(Supported by the Deutsche Forschungsgemeinschaft through SFB 102-All). We have established human cell lines, which express the E3/19K protein of adenovirus 2 by using a transfection system. We found that the E3/19K protein binds to human histocompatibility antigens (HLA) thereby blocking their terminal glycosylation and transport to the cell surface (1) . The reduced level of class I antigens on the cell surface impairs T-cell recognition of E3/19K+ -cells in vitro (2) . The same effects are found in normal human cells infected with adenovirus 2. The observed mechanism might be important for the establishment of persistent infections in vivo. -The construction and expression of hybrid proteins allowed us now to identify the domains of class I antigens necessary for E3/19K association. E3/19K binds to the domains which are crucial for T-cell recognition. In order to identify target sequences within the Ad12 Ela promoter which are required for efficient transcription, the activities of different promoter fragments were compared in a transient expression assay. So far two important positions have been detected: 1. Deletion of the leftmost 152 bp of the AdI2 DNA reduces El a promoter activity 5 to 10fold, deletion of the leftmost 170 bp at least 10fold. The deleted DNA includes the Inverted Terminal Repetition (ITR, -164 bp) and is functionally important for transcription from both TATA boxes as well as from the first one alone. The presence of early Ad12 gene products does not overcome this reduction. ITR fragments at the 3' end of the CAT gene in either orientation restrict the control element to DNA sequences between positions 144 and 170 and demonstrate that they serve as a transcriptional enhancer. -2. Transfection of increasing amounts of DNA of the CAT constructs containing a promoter fragment from position 0-525 results in maximaL CAT activity at 15 rtg of transfected DNA followed by a rapid promoter inactivation. Smaller promoter fragments yield increasing CAT activity accoding to increasing amounts of transfected DNA. Northern analysis suggests that this control mechanism occurs at the level transcription initiation. Two in limited amounts occurring nuclear proteins of BHK cells binding between positions 400 and 494 can be identified by "Gel Shift" tests. These factors are stimulated in an Ad12 transformed hamster cell line. The target for these factors could be mapped to the El a CAP site.

Reconstituted SV40 Promoter GARBRECHT, SABINE and INGE KRUCZEK Inst. f. Biochemie, In order to isolate cellular transcriptional control sequences, we transfected BHK cells with selectable plasmids containing the neo gene under the control of a defective SV40 promoter (p892 neo) or without any eukaryotic promoter sequence (pSVO neo). G 148 resistant cell clones were isolated and genomic analysis showed multiple insertions of the neo gene into the BHK genome. In order to obtain single copy integration, BHK cells were transfected with DNA fragments of resistant cell clones. Northern analysis of the new clones showed that comparable amounts of neospecific RNA according to wt RNA were synthesized. Therefore we conclude, that cellular sequences have functionally completed the defective promoter of p892 neo. To facilitate the cloning of the single copy insertion sites, we tried an amplification by fusion with COS I cells (SV40 transformed monkey cells containing the permissive factor and T-Antigen which are necessary for SV40 replication). In most cases an amplification up to 1000 fold was achieved. The amplified DNA fragments were cloned into pUC 12 and are under investigation.

Inst. for Biochemistry, D-800 Miinchen 2

Simian virus 40 (SV40) large T antigen exists in multiple molecular forms, some of which are separable by zone velocity sedimentation of soluble extracts of infected monkey cells. Three subclasses from infected cells have been separated and characterized: 5S, 7S and 14S. Newly synthesized T antigen occurs in the 5S form, a monomer. The 14S form represents a tetra mer and the 7S form, a dimer. The rate of oligomerization of newly synthesized 5S T antigen into tetramers in vivo was greater at early times after infection than at late times. -The DNA binding properties of each subclass were investigated after immunopurification. The affinities of the 3 forms for SV40 DNA and for a synthetic 19 bp sequence from binding site I are very similar (K D 0.4 nM). The specific activity of DNA binding was greatest for the 5S and 7S subclasses and least for the 14S subclass. Moreover, the specific activity of the 5S and 7S forms increased sharply at about 40 h after infection, whereas that of the 14S form remained at a constant low level throughout infection. The binding stoichiometry of T antigen was consistent with the idea that stable tetramers do not bind to viral DNA at all, and that the observed low binding activity derives from contamination with dimers. A model relating oligomerization and DNA binding of T antigen in infected cells will be presented. During lytic infection SV40 T antigen binds specifically to three different regions of the SV40 DNA to initiate DNA replication and to regulate early and late transcription. We constructed plasmids containing either 22 basepair synthetic oligonucleotides representing site I or II or combinations of binding site II and III with or without the SV40 specific flanking regions. T antigen bound to site I with very high affinity, whereas isolated site II was not bound specifically. Measurable specific binding could be restored to some extent by combining site II with the 3' SV40 flanking region. Binding to site III remained weak with or without the 3' and 5' flanking sequences. Binding to DNA fragments containing both sites II and III was higher than binding to fragments with either II or III added together. Thus flanking regions not bound directly by T antigen could influence binding affinity probably by changing the DNA structure of the nearby binding site.

MONTENARH 1 , M., C. VESC0 2 , GUDRUN KEMMERLING!, DOROTHEE MULLER!, and R. HENNING l 1 Dept. of Biochemistry, Univ., D-7900 Ulm; 2 Inst. di BioI. Cellulare del CNR, 00196 Roma, Italy

The SV40 large T antigen is composed of 708 amino acids. It occurs in monomers and various oligomers as well as in complexes with the cellular oncoprotein p53. To detect distinct regions on the polypeptide chain of large T antigen which are essential for the formation of these homo-and heterologous forms we have analyzed T antigen from various SV40 deletion mutants. We found that an area between amino acids 110 and 152 and additionally a C-terminal region between amino acids 591 and 634 are essential for oligomerization. Only this C-terminal region between amino acid 591 and 634 but not the N-terminus up to amino acid 152 seems also to be critical for T-p53 complex formation. Analyzing the potential influence of the phosphorylation of T antigen we found that only phosphorylation of amino acid 124 seems to be important for oligomerization but not forT-p53 complex formation. Adenovirus type 12 transcriptional complexes were isolated from the infected cells during early phase. Sedimentation analysis identified a fast sedimenting complex type I and a slow sedimenting complex type II. Both complexes made viral specific RNA and contained viral DNA, which in type II but not in type I had nucleosomal configuration. Analysis of the proteins, of the complexes with antiserum against Ad12 EIA-~-galactosidase fusion protein expressed in E. coli (12-1A-FP, demonstrated the following; a) type I complex contained EIA 45k protein which coprecipitated with cellular proteins of mol. wt. 42, 58 and 60k. (b) type II complex contained EIA 48k protein, which coprecipitated with major cellular proteins of 35, 42-43k and minor proteins of 58,60,68,86 and 120-150k. The association of EIA specific and cellular proteins to transcriptional complexes, was sensitive to both IM-NaCI and DNAse I establishing the deoxyribonucleoproteins nature of the complexes. Treatment of transcriptional templates with IMNaCI or DNAse I released E1A proteins which still remained strongly bound to a conglomerate of cellular proteins. These findings indicate that E1A specific antigens do bind to viral DNA but this binding is indirect and mediated by some of the cellular proteins described above. The molecular mechanism of persistence of adenoviruses (Ad) in the organism is still unknown. Therefore, we have tested human tonsillar tissue for infectious virus and for viral DNA-sequences by DNA-DNA hybridization with genomic Ad 2 DNA and cloned Hind III fragments. We hereby observed cases in which infectious virus could neither be isolated directly from ground tissue probes nor in cell cultures established therefrom and propagated for weeks or months. Yet, after application of the "In situ hybridization" we were able to demonstrate adenoviral sequences in cells located in the periphery of cryostat sections of such tonsillar tissue. Only part of the cells harbored viral sequences. Cell cultures established from such tonsillar material exhibited positive hybridization signals, too, but again in only a portion of the cells. -In Southern-Blot hybridizations "off-size" bands were detectable besides bands comigrating with adenoviral marker fragments. In some cases viral DNA fragments were missing. Additionally, different intensities of various bands suggest that parts of the viral genome are overrepresented.

HERLT 1 , M., E. GAUL', R. NEUMANN 2 , and H. J. EGGERS 2 I lnst. f. Genetik, Univ., D-5300 Bonn 1 2 lnst. f. Virologie, Univ., D-5000 Koln 41

A modern approach to study DNA-binding in a mixture of proteins consists of fractionation of proteins by gel electrophoresis and transfer to nitrocellulose filters by electro-or diffusion-blotting. We describe the use of nick translated biotin-labeled DNA-probes in DNA-binding studies. -Phosphorus-or biotin-labeled DNA-probes were applied to the filter by a specially designed vacuum-filtration apparatus. It hereby became possible to detect DNA-binding activity by even 10 ng of calf thymus core histones dotted on nitrocellulose filters. Furthermore, chromosomal proteins with a lower affinity to DNA (histone HI and HMG-nonhistone proteins) are detectable among other DNA-nonbinding proteins. -In summary our results demonstrate that the DNA-binding capacity of proteins transferred to nitrocellulose filters can be tested with phosphorus-as well as with biotin-labeled DNAprobes. The sensitivity of both labelling systems appears equal. During the last years, large amounts of DNA and protein sequences became available due to the rapid progress in sequencing techniques; three-dimensional structures, however, will be available for few proteins. For most purposes it is sufficient to obtain data on the secondary structure, hydrophilicity, flexibility, surface probability and modification of the amino acid sequence. Those predictions are mainly important for immunological questions. Consequent application however, may result in valuable information for three-dimensional arrangements of proteins. In this paper we present a computer program with access to sequence libraries which calculates secondary structures of proteins, superimposes those predictions with additional values and creates a two-dimensional graph. A further algorithm was developed and included which combines structural and other parameters in order to recognize antigenic sites. This algorithm was used to predict epitopes on proteins which had been characterized by X-ray cristallography (lysozyme, myoglobin, poliovirus VPl) and on experimentally well characterized viral polypeptides (EBV, HlV, FMDV); a good coincidence in antigenicity profiles could be obtained. This program can be directly combined with the UWGCG program collection. The immune response of Pseudorabies virus (PRV; herpes suid 1) -infected swine was found to be directed against the major glycoproteins gI, gIl and gIll. Monoclonal antibodies against gIl neutralized PRY in vitro in the presence and absence of complement. Individual MCA were also able to protect mice against lethal challenge with PRY. Thus, the gllcomplex is important in virus neutralization in vitro and in vivo. Viral mutants (MAR) resistent to neutralization with individual anti-gIl MCA were selected. One mutant was only resistent to neutralization with that MCA used for selection, which also did not recognize the expressed gIl. Other MAR mutants were neither neutralized by the MCA used for selection nor by different other MCA against gIl, although gIl was expressed in all these MAR mutants. The activity of the genome of Pseudorabies virus (PRV) was investigated in two latently infected pigs (60 weeks p.i.). PRY-specific antigen(s) could be detected in the brain by immunohistochemical staining of thin sections. For this purpose a polyclonal goat antiserum was used, which recognizes immediate early (IE) protein of PRY. Monoclonal antibodies specific for the major glycoproteins (gI, gIl, gIll) and for the major capsid protein of PRY did not react. In situ cytohybridization with strand-specific probes (cRNA synthesized with SP6-or T7-Polymerase) revealed the presence of PRY IE-specific RNA in the brain. In some other organ tissues of both animals, PRY-specific DNA sequences could be also detected by Southern blot hybridization, which, however, did not represent the complete viral genome. Transfection of permissive cells with DNA extracted from these organs lead to the production of PRY. The structural gene of the glycoprotein complex gIl of Pseudorabies virus (PRV; Herpes suid 1) was recently mapped in the unique long part of the genome on BamHI/SalI-fragments lA and IG (Mettenleiter et al., Virology 152 (1986) 66-75). The 3'-end of the gll-mRNA is located in fragment IG. Southern blot analysis of DNA derived from different PRY strains revealed a heterogeneity in size of fragment IG ranging between 0.6 to 1.2 kbp. After subcloning in M13 the DNA sequence of different IG fragments of 3 PRV strains (Phylaxia, Ka, Dessau) was compared. All the fragments analysed exhibited nearly identical DNA sequence, except of single point mutations and a duplication of 6 nucleotides unique for strain Ka. The fragments differed in the presence of variable copy numbers (3 to 50) of a direct repeat sequence of 15 nucleotides. This variability was observed both between the different virus strains and between different IG fragments from a given virus population. We conclude that the size heterogeneity of fragment IG depends on the present copy number of the repeat unit. Finally this repeat unit seems not to be located in the coding region of the gIl gene. Glycoprotein complexes were identified in pseudorabies virus. The formation of the gIlcomplex will be shown. One precursor protein (pgIl, 1l0K) of the three disulfide-linked glycoproteins of the complex (gIla, gllb, gIlc) could be immunoprecipitated as a monomere under non-reducing and reducing conditions; e.g. no disulfide bridge-linked complex of the precursor could be identified. After processing of pgIl to the mature forms glla, gllb and gIlc these proteins were identified as monomers in infected cells. The gIl-complex was immunoprecipitated not before 6 h p.i. Electronmicroscopical studies of petri dishes of the same experiment show the first envelope particles in the cytoplasm of the infected cells at the same time (6 h p.i.) as the formation of the glycoprotein complex. This indicates that the nuclear membrane fraction plays an important role in the formation of gIl-complex of the virus. The infectibility of porcine bone marrow cells (BM) was studied after in vitro-and in vivo-infection with Pseudorabiesvirus (PRV). PRY infection was shown by viral multiplication, immunofluorescence (IF), electronmicroscopic examinations, Western blotting and radioimmuno-precipitation of PRY-protein as well as by PRY-specific hybridization of viral DNA and RNA. Age-dependent decreasing frequencies of PRY replicating BM between 1 in 3-35 (a) and 1 in 3S-101 (b) were shown by Infectious Center Assays with PRY-infected cultured BM of pigs less than 12 weeks (a) and more than 5 months (b) of age. IF-studies detected a nearly constant ratio of 65-70% BM positive for immediate early, early and late viral proteins and 30% PRY-major capsid protein-positive BM. PRY was isolated from BM of experimentally infected pigs between dpi. 1 and 3 (max. 1/13 000 PRY replicating BM) and viral RNA was detected between dpi. 1 and 6. Thus infection of BM with PRY was shwon to be established before infection of CNS. PRY-infection of BM was corroborated by studies on naturally infected pigs. The human polyomavirus j C persists in systemic organs without cell damage and causes in rare cases progressive multifocalleucoencephalopathy (PML) a disease associated with a lytic lev infection of the CNS. Since the highly variable lytic leV genomes are known to contain a celltype specific enhancer/promoter element we asked whether lCV genomes cloned from the kidney and the brain of one PML case are virus isolates identical in sequence and structure. Protein coding sequences of both genomes were identical as shown by sequence analysis and the control region revealed corresponding base sequences and structural elements in the "core origin of replication". The putative enhancer/promotor element, however, showed a complex pattern of insertions and structural changes suggesting a celltype specific rearrangement of transcriptional control elements. The 3.5 KB BamHI to HindIII genome fragment of the low virulent ADV Pullman and the high virulent Utah strains, the cell culture-adapted lymphotropic ADV-SL3 isolate and the apathogenic ADV-G variant were cloned in pUC18 and pUC19. The ADV Pullman and Utah DNA was derived from virions, isolated from infected mink, whereas the ADV RF DNA from the latter strains originated from cell culture. After induction of lac operondependent protein expression, capsid-specific antigens were only detected in recombinants of pUC19. The expressed proteins of ADV Utah and Pullman had a MW of 57 and 34 KD, the corresponding proteins of ADV-G were each 2 KD smaller and those of ADV-SL3 showed an additional loss of 2 KD. -These results are discussed with emphasis on the pathogenicity and persistence of ADV. The growth of ADV in feline kidney cells is characterized by a reduced production of infectious virus at 37°C. After 3 consecutive passages of ADV at 3rC, the virus titer drops below the detection limit of the fluorescence focus assay (10 4 FFU/ml). The production of viral antigen and viral RNA as well as the replication of viral replicative form DNA was found to be not defective at 37°C. However, the synthesis of viral progeny DNA was diminished. By 50 h p.i., about threefold less progeny DNA was synthesized at 37°C compared to 32°C. The reduction of the synthesis of new viral DNA strands may account for the reduced production of infectious virus. In order to clarify the prognostic significance of the demonstration of HPV sequences in cells taken from cervical swabs for the ultimate development of cervical cancer routinely taken swabs were screened for HPV nuclei acid sequences using phosphorus -as well as biotinlabeled HPV 16/18 DNA-probes. So far, cells from 516 patients have been tested. Out of this group 18.9% (98/516) exhibited positive hybridization results and 67.6% (349/516) were negative. Results from 69 patients (13.5%) were equivocal. Positive hybridization signals were registered in all groups of the Papanicolaou rating system. In the group with non pathological smears 17.6% (48/273) exhibited positive hybridization results. When taking follow-up swabs we confirmed the first hybridization result in 10 out of 17 cases. Correlating histological and hybridization data we detected positive signals in 2 of 4 squamous cell carcinomas and in 1/2 adenocarcinomas. In cases rated carcinoma in situ 9 out of the 15 specimens analyzed (59.9%) and one out of five dysplasias were also positive. We detected viral sequences in 42.8% (317) specimens without any histological indication of malignancy.

FUCHSl, P. G., F. GIRARDI 2 , A. ENGELHARDT', and H. PFISTER 1 Inst. f. Klin. Virologie, Univ., D-8520 Erlangen, and Ceburtshilflich-gynakologische Univ.-Klinik, A-8036 Graz Colposcopically directed cervical punch biopsies from 233 Austrian patients were screened by Southern blot hybridization for the presence of human papillomavirus (HPV) DNA. The biopsies represented different stages of cervical intraepithelial neoplasia (CIN), invasive carcinomas, as well as samples from histologically normal cervical epithelium. HPV DNA was detected in 53.4% of the tumors and in 37.5% of the biopsies from healthy epithelium, respectively. The most frequent HPV type in tested biopsies was HPV 16 (34.3%), followed by HPV 6 andlor 11 (15.9%) and HPV 18 (6.9%). HPV 10-related sequences have been identified in 3 invasive carcinomas. HPV 16 prevailed especially in biopsies of invasive carcinomas (48.4%), CIN II and CIN III (39%). Interestingly, HPV 16 DNA was also detected in 22.5 % of the histologically normal epithelia. HPV 6 andlor 11 dominated only in samples from mild dysplasias (26.3%). In about 14% of HPV-positive biopsies mixed infections have been detected. The incidence of HPV 6 andlor 11 in CIN I and HPV 18 in cervical cancers as compared with data from other continents indicates the possibility of considerable geographical fluctuations.

SCHOLZ, J., H. REISNER, J. BDGERT, and G. DARAI Inst. f. Med. Virologie, Univ., Virions of the molluscum contagiosum virus (MCV), a member of the poxviridae, were isolated directly from lesions of individual patients of two countries (Germany and Hongkong) and characterized by restriction enzyme analysis The comparative analysis of the DNA cleavage patterns of 40 independently isolated virus samples revealed that MCV isolates can be classified into two different types. The majority of MCV isolated from clinically typical skin lesions showed similar DNA cleavage patterns and were termed MCV type 1, whereas one isolate derived from a vaginal lesion showed a completely different DNA cleavage pattern and therefore was termed MCV type 2. To confirm these results DNAs of MCV type 1 and 2 were separately labelled with 32p and were cross-hybridized by the Southern blot hybridization method to nonradioactive DNAs of MCV type 1 and 2. This study revealed that both viral DNAs hybridized to each other, indicating their close relationship. For further investigation a defined gene library of MCV DNA sequences was established. The BamHI DNA fragments of the viral genome of MCV type 1 prototype isolate 1/ 80 and MCV type 2 were inserted into the bacterial plasmid vector pAT153. All cloned BamHI DNA fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNAs to viral DNA. Using this defined gene library the relatedness of the DNA sequences of MCV genome to the genome of other members of the poxvirus family can now be investigted in more detail. This study is in progress. -This study was supported by a grant from the Forderverein zur Bekampfung der Viruskrankheiten e.V. SAGNER, G., G. STEGER, and H. PFISTER Inst. f. klin. Virologie, Human Papillomaviruses (HPV) induce benign skin tumors in patients with Epidermodysplasia verruciformis (E.v.) . About 90% of the E. v. associated skin carcinomas contain HPV5 or HPV8 DNA. Although the viral DNA was shown to persist extrachromosomally in high copy number, no viral proteins could be identified. -To raise antisera against proteins from HPV8, we cloned DNA-fragments from open reading frames (ORF) E1, E2, E4, E6, E7 and Ll in procaryotic expression vectors. With the resulting HPV8/B-gal fusion proteins we immunized rabbits and guinea pigs. The sera were used to test protein extracts from an HPV8-induced E.v.-wart and from a plantar wart in immunoblot assays. The anti-Ll serum recognized an HPV8-specific protein in the E.v.-lesion. The molecular weight of 60kD correlates with the size of the expected Ll-product. No early proteins could be detected so far. Using the chloramphenicol acetyl transferase (CAT) system subgenomic fragments of HPV 8 DNA were tested for enhancer activity. Such activity could be demonstrated neither in mouse fibroblasts (C127 cells) or in human epithelial cells (HeLa). In BPV1 transformed C127 cells, however, the 1406 bp EcoRI-PvuII-fragment which covers the noncoding region led to a clear increase of CAT gene expression. The fragment was active independent of its orientation both upstream and downstream of the CAT gene. Cotransfection experiments using expression vectors for different BPV1 genes as well as transfection experiments of C127 cells transformed with BPV1 deletion mutants suggest that the E2 gene product of BPV1 has a' trans activating effect on the HPV 8 enhancer. Qualitative and quantitative differences were noted between HPV 8 specific transcription in C127 and BPV1 transformed C127 cells indicating that BPV1 influences authentic HPV 8 transcription.

KOHLER, E., J. KUHN, K. MUNK, and R. BRAUN Inst. f. Virusforschung, Deutsches Krebsforschungszentrum, Cross-hybridizing experiments revealed hybridization between the cloned Herpes simplex virus type 2 "joint" region and a 15 kb Hind III fragment of Human Embryonal Lung Cell (HEL-) DNA, persisting under stringent conditions. This cellular fragment was rescued out of a A-library, and was subcloned into pBR322. The region homologous to viral DNA was first localized on a 1920 bp Sst II subfragment which was subcloned into pN01523, then on a 230 bp subfragment between a Bgl I and Hpa II restriction site. This fragment, together with adjacent regions, was sequenced by the Maxam-Gilbert method. Sequence analysis revealed a 65% GC-content, an open reading frame of 210 bp, and four "stemloop"structures. A 120 bp region could be determined, representing a 70% homology to the HSV-2 L-S inversion region sequence. -The most intense cross-hybridization with the viral "joint" region was observed within the Bam HI-G fragment on a 520 bp Sst II subfragment. The nucleotide sequence of this fragment was analyzed. The GC-content totaled to 81%; futhermore 11 "stem-loop"-structures and a 16 X directly repeated sequence of 15 nucleotides was detected. A 3' 120 bp DNA-sequence, corresponding to the HSV-2 L-S inversion region, showed the highest homology with the cellular DNA.

A Mutation in Bovine Papillomavirus 1 (BPV-1) Open Reading Frame E4 Affects the Persistence of Viral DNA DIETRICH-GOTZ, W. and H. PFISTER Inst. f. klin. Virologie, Univ., We have analysed the functions of the overlapping open reading frames E2 and E4 of BPV-1 by mutagenesis with an XbaI linker carrying a TAG termination codon. Mutations in E2 (NcoI) or E2 and the 3' end of E4 (KpnI) have following features in a C127 cells transfection assay: reduced focus forming activity, integration of the viral DNA and stability of the transformed phenotype. Embryonic C57bl mouse fibroblasts immortalised by these mutants could be grown in soft agar. A mutation near the 5' end of E4 (TthlllI) had contrary effect: about 50% focus forming activity of wild type, extrachromosomal persistence of the mutant DNA in varying, partially very high copy number and instability of the transformed phenotype correlated with the loss of the viral DNA. No asynchrony of viral and cellular DNA replication could be observed. We assume that E4 plays a role in long term maintenance of extrachromosomally persisting viral DNA molecules.

Molecular Cloning and Characterization of a Novel Papillomavirus Type from a Patient with Hodgkin's Disease ELLINGER l , K., G. FUCHS l , G. GROSS 2 , and H. PFISTER l 1 Inst. f. Klin. Virologie, Univ., D-8520 Erlangen 2 Univ.-Hautklinik, D-7800 Freiburg A patient with Hodgkin's disease developed macular skin lesions 12 years after treatment with X-rays and chemotherapy. DNA was extracted from the lesions and a 7.9 kb papillomavirus genome was cloned into the vector pIC 20H after partial Hind III digest. As seen by Southern blot analysis this isolate showed homology with human papillomavirus (HPV) 14 and to lesser extent with several viruses, which occur in skin lesions of patients with epidermodysplasia verruciformis (EV). The pattern of DNA homology between the cloned HPV and other HPV types differed from that of HPV 14 and the extent of cross-hybridization with HPV 14 as measured by reassoziation in liquid phase at T m-20 was below 20%. Both viruses differed in their physical maps of restriction enzyme cleavage sites. This data infer, that the cloned DNA represents a new HPV type belonging to the group of EV-viruses.

KRAUS, j., TH. IFTNER, and H. PFISTER Inst. f. Klin. Virologie, Univ., Human papillomaviruses (HPV) 19 and 25 belong to the group of epidermodysplasia verruciformis (EV) associated HPVs. In contrast to HPV 5 and 8, HPV 19 and 25 were not detected in skin carcinomas of these patients. We sequenced the RRs of HPV 19 and 25. The sequence analysis of HPV 5, 8, 19 and 25 revealed a strong continuous homology between HPV 19 and 25, whereas HPV 5 and 8 showed insertions and rearrangements. Six sequence motifs are shared by the 4 EV specific HPVs. The palindrome ACCGNNNNCGGT, which is found within the RR of all PVs, occurs 3 times within the EV specific RR. It plays a role as binding site for the E2 protein, that acts as a controlling element for transcription U. Schiller, pers. comm.) . Intermingled with each palindrome we found SV40 late promoterlike sequences. A unique feature of HPV 19 and 25 is a block of over 20 alternating AT residues at the 3' end of the RR. Foamy viruses (spumavirinaes subfamily of the retroviridae) cause persistent infections in various mammalian species, probably including man. SFV strain LK-3 was isolated from an African green monkey and reveales T-lymphotropic properties. In various infected cells proviral LK-3 DNA was detected by hybridization to a 32P-labelled viral cDNA probe prepared by "in vitro" reverse transcription (J. Gen. Virol. 67 (1986 ) 1993 -1999 . It was characterized as non integrated, linear, double-stranded DNA of about 14 kb, containing "gaps" sensitive to nuclease S1. Probably two whole genomes are linked by single-stranded DNA to form a tandem structure as described for the visna-lentivirus. Proviral DNA from infected MoltA cells was cleaved with XhoI and cloned in lambda L.47.1. Positive plaques were identified by hybridization to the 32P-labelled viral cDNA. An LK-3-specific 3.1 kb insert was isolated and subcloned in pUC 8. With viral insert DNA as a probe, strong hybridization occurred to DNA from cells infected with SFV-3, whereas other SFV prototypes and human syncytium forming virus showed weak hybridization signals under less stringent conditions. -Supported by the Deutsche Forschungsgemeinschaft (Ne 213/4-2). HTLV-lIIB, LAV-2 and SIV were studied by IEM, surface replica, and thin section EM. During morphogenesis virus core components are assembled concomitant with budding at the cell surface; the spherical RNP-core complex 18 nm in thickness is apposed to the virus envelope. During "maturation" this complex rearranges into a tubular core shell with p24 antigenicity and the RNP-nucleoid. At the inner side of the envelope the p17 membrane protein is located. "Immature" HIV are densely studded with probably 72 surface knobs 15 nm in diameter. On the viral envelope gp41 and gp120 determinants are revealed. Sera from HIV infected persons show a close qualitative correlation between labelling density of the envelope and neutralizing capacity. During virus "maturation" knobs are lost progressively. This shedding is discussed as a possible pathomechanism. Three peptides corresponding to residues 500-511, 579-589, and 611-620 on LAV/ HTLV-III envelope glycoprotein precursor gp160 were synthesized. Criteria for the selection of the peptides were conservation of amino acid residues and hydropathic character of the region. 107 sera from AIDS-patients, LAVlHTLV-III positive persons and normal donors were tested for the presence of antibodies recognizing the peptides.91 % of the positive sera show reactivity against peptide 500-511, 23% against peptide 579-589, and 16% against peptide 611-620. The peptides were not recognized by sera from the control group. The limits of detection for peptide recognition of positive sera were characteristically below a serum dilution of 1:100,000. There is as yet no real animal model for AIDS. During the course of our studies ofSTLV-I in various primate species it was observed that some of the animals, notably among the African Green Monkey and Baboons, also possessed antibodies that reacted with HIV in ELISAs and Western blots. -The African Green Monkeys (Cercopithecus aethiops) raised in a closed colony at the Paul Ehrlich-Institute are more than 50% seropositive for HIV, but obviously healthy. Viruses were isolated from these animals (STLV-III AGM ) and were shown to be cytopathogenic for cultured T-lymphocytes of human and macaque origin. Thus far however, the isolates replicate in low titers only in vitro. Efforts to characterize STLV-III AGM proteins and DNA and in vivo inoculation of STLV-III AGM Human immunodeficiency viruses (HIV) obtained from AIDS-or LAS patients showed differences in cytopathic effects(CPE) which they induced in fresh peripheral lymphocytes (1) . -Two of the cytopathic isolates from German patients were used in this study to compare the case of infection, virus-production and cytopathic effects for different lines of T-lymphocytes (Molt 4, CEM, HUT 78), for HL 60, for freshly prepared cord lymphocytes as well as for cord lymphocytes which were immortalized by STLV I. -Some of the uninfected .cell lines produced spontaneous syncytia. As a consequence, the production of the cytopathic effects of the viruses was difficult to judge based on the morphology of the cells alone. Cell-vitality, therefore, was determined using a fluorescence technique which allowed to readily distinguish between live and dead cells. -All of the established cell lines proved to be much more resistant to infection by both virus strains than freshly prepared cord lymphocytes, but even between cell lines of similar origin, significant differences in infectability as well as virus production were observed. These experiments demonstrate that the production of HIV is strongly dependent on cellular events. -

SCHNEIDER 1, J., 1. WENDLER l, M. HA YAMI 2 , R. DESROSIERS 3 , and G. HUNSMANN l 1 Deutsches Primatenzentrum, D-3400 G6ttingen, 2 The Institute of Med. Science, Tokyo, Japan, 3 New England Regional Primate Research Center, Southborough, Mass., U.S.A.

Simian immunodeficiency viruses (SIV) are relevant to understand the origin of the human immunodeficiency virus (HIV) and to establish animal models for human AIDS. We have examined 856 sera of 34 nonhuman primate species for antibodies to HIV and SIV by a combination of ELISA and radioimmunoprecipitation (RIP). Sera of green monkeys and three closely related species were frequently positive in both assays. SIV isolates from green monkeys, sooty mangabeys, and rhesus monkeys were classified and compared with HIV by crossreactions of respective antisera in the RIP assay. With respect to the peripheral envelope glycoprotein the simian viruses represent a subgroup different from HIV since the simian sera reacted with the homologous and heterologous glycoproteins of the SIV isolates but not with gp120 of HIV. The core polypeptides were bidirectionally crossreactive between each of the four viruses with the exception that sera of green monkeys lacked antibodies to any core polypeptides. However all isolates were distinquishable by tryptic peptide maps of the core polypeptides p18 and p24. (see abstract Jurkiewicz et al.). Human immunodeficiency viruses (HIV= LAV, HTLV III, ARV or AAV) were cultured from either peripheral blood lymphocytes (PBL), plasma, whole blood or cerebrospinal fluid (CSF) of AIDS-, ARC-and asymptomatic patients mainly from Frankfurt. The isolates were shown to be LAV/HTLV III-related by reverse transcriptase tests, crossreactivity in immunofluorescence assays, electron microscopy and Southern blot analysis, probed with the HTLV III-specific probe lambda BH10. When the HIV isolates were grown on fresh human PBL striking differences in the morphology of the cytopathic effect induced in the PBL were found among them, which were also reflected in the virus titres and the speed of growth. In immunofluorescence assays the HIV isolates showed different sensitivities for detecting virus-antibodies from patients sera. Restriction analysis of a molecularyly cloned isolate revealed differences to the published restriction patterns of cloned HIV, and multiple variants within a given patient. -The observed variations between isolates from a confined area argue for a high mutation rate of HIY. The question is raised whether the very different clinical courses of infection with HIV correlate with particular subtypes. Furthermore the development of effective vaccines against HIV will depend on availability and characterization of a wide spectrum of variants. Further studies will have to show which epitop of the individual mutants are important for neutralizing antibodies. Independent isolates of human immunodeficiency virus (HIV) exhibit a striking genomic diversity, most of which is located in the viral envelope gene. Since this property of the HIV group of viruses may play an important role in the pathobiology of HIV, we analyzed the amino acid sequences of the envelope proteins of eleven different viral strains, three of which represent sequential isolates from a single patient. Using a computer program that predicts the secondary protein structure and superimposes values for hydrophilicity, surface probability and flexibility, we identified several potential antigenic sites in the envelope proteins of HIV. Interestingly, the majority of these predicted epitopes in the exterior envelope protein (gp120) were found in regions of high sequence variability among the independent and also in the sequential viral isolates which are interspread with highly conserved regions. gp41, the membrane-associated polypeptide, contains no highly variable regions; about 80% of the amino acids were found to be conserved and only two antigenic sites could be identified. These findings give insight into the secondary and possible tertiary structure of variant HIV envelope proteins and should facilitate experimental approaches directed towards identifying and fine mapping of HIV envelope proteins. Reverse transcriptase (RNA dependent DNA polymerase, RDDP) was isolated from density gradient banded Human Immunodeficiency Virus (HIV) by four chromatographic steps. Enzymatic (template primer and ion requirements) and kinetic parameters (Michaelis constant, Hill coeficient for several substrates) were determined. -In order to find inhibitors which are specific for HIV RDDP the effect on HIV RDDP and on the partially purified cellular DNA polymerases of a large number of drugs was tested and compared by the inhibitory dosis (ID so ), the concentration of the drug at which the enzymatic activity is inhibited by 50%. Most known RDDP inhibitors are equally or even more active on cellular polymerases. In contrast, tetracyclines exhibit a relatively specific effect on HIV RDDP. However, for therapeutic applications a substance has to be found, which shows a very specific inhibitory effect on HIV RDDP and nearly no effect on the cellular polymerases. Under all substances tested, only phosphonoformic acid and its derivatives which are examined in collaboration with ASTRA (Sweden) fulfills this requirement. Borna Disease Virus (BDV) has been regarded until recently as a strictly neurotropic agent that propagates along neur al path ways. The development of mon oclonal antibodies against viral antigens and of highly sensitive immunohistochemical techniques allowed the assessment of the exact distribution of viral antigens in the neural and non-neural tissues. In the nervous system (NS) virus specific antigen could be demonstrated beside neurons in astrocytes, oligodendroglial cells, ependyma, choroid plexus epithelium and in the pigment epithelium of the eye. In the peripher al NS axons were invariably positive, but the Schwann cells proved to be consequently non -permissive to BDV. Among non-neural tissues, the lacrimal, sebaceous and salivary glands, smooth muscle cells, epidermis, pituitary and adrenal glands and the brown fat were permissive to BDV-infection, while the liver, kidneys, lungs and skeletal muscle were not. For the elective vulnerabil ity of these tissues and cell types the specific distribution of surface receptors may offer a plausible explanation.

DOERING-MANTEUFFEL1 ,l , S. , G. GOSZTONYI 1 , and H. LUDWIG 1 Inst, f. Neuropathologie und Inst, f. Virologie, Freie Univ., D·1 000 Berlin Borna Disease is a slow virus infection. The distribution of the virus is mostly restr icted to the nervous system (neurons, glial cells). -Following ocular inoculation of rat s with Borna Disease Virus brain infection ensues by a centripetal spread of the agent mainly along the optic nerve. Borna specific ant igen can be determined immunohistochemically first 26 days in the nuclei of neurons of the central nervous system. -Despite the unilateral inoculation, immunoreactive cells appe ar to be localized symmetricall y in the diencephalon and the brain stem; this phase is followed by the rapid spread of Borna Disease Virus over the whole brain. Beside axonal and transsynaptic mechanisms oligodendrocytes are probably also involved in the propagation of infection. Prote in deposits which are host encoded and have all the prop erties of amyloids are found in brain s of scrapie infected hamsters. These socalled Scrapie-associated-fibrils (SAF) are absent from scrapie-spleens, when comp ared with brain mat erials of similar scrapie titer sv --With a delay of approximately 20 days the formation of SAF-protein in intraperitoneal infected hamsters follows a dramatic increase of scrapie titer up to a plateau in brains. Clinical signs start only after a considerable amount of SAF has accumulated. According to these observations infection with scrapie leads to a lethal, organ specific amyloidosis. Borna Disease (BD) virus infections lead either to fatal encephalomyelitides (horse, rabbit, adult rat) or life-long persistencies without disease (I-day old mouse, rat, chicken). Independently from disease or tolerant infection all BD virus infected animals react with a strong humoral immune response. The first and most prominent answer is directed against a complex of virus-induced proteins, the s (soluble)-antigen (s-ag). S-ag enriched supernatants can be prepared from infected brains by sonication and ultracentrifugation at 100,000 x g.

Immunogenic s-ag components were characterized by Western-blot-analyses. We found (l) that this antigen complex consists of two major proteins with apparent molecular weights of 60 and 40 KD, (2) that these proteins are recognized by polyclonal serum-as well as oligoclonal CSF-antibodies from all tested species (rabbit, horse, mouse, rat, rhesus monkey), and (3) that monoclonal antibodies equally well react with epitopes on s-ag proteins. Two out of three monoclonal antibodies bind to the 60 KD protein. The reactivity pattern to s-ag components could not be correlated with the presence or absence of the neutralizing capacity of those antibodies. We have reported an immunological relationship between human PDFG and the SSV transformation-specific glycopeptide (SSV-TrSgp) (Virology 136 (1984) 414-424) . SSV-TrSgp represents a proteoglycan-like molecule which is released from SSV-transformed cells. With regard to the putative biological activity of this molecule we found that nonfractionated cells, serum and virus free tissue culture supernatant shows growth-stimulating activity with PDGF-like characteristics on NIH 313 cells. Furthermore, the same tissue culture supernatant induces anchorage-independent growth of NRK cells and causes dramatic morphological alterations of various cell lines and non-established fibroblasts. Evidence that the SSV-TrSgp is involved in these processes comes from two additional findings: 1) After fractionation of supernatant from SSV-NP cells by gel filtration a major growthstimulating and transforming activity is present in the void volume (> 150 kD); 2) Using the same supernatant for preparative SDS-PAGE a high-molecular-weight (> 200 kD) growthstimulating activity can be electroeluted.

(IBDV) SPIES, U., H. MULLER, and H. BECHT lnst. for Virology, Univ., D-6300 GieBen IBDV is a member of the family Birnaviridae. Naked icosahedral virus particles are single-shelled; they are composed of several polypeptides and contain two segments of double-stranded (ds) RNA with mol. wts. of 2.2 x 10 6d and 1.9 x 10 6d. -An RNA dependent RNA polymerase activity is associated with infectious IBDV particles. To induce enzyme activity it is essential to remove Ca H -ions; however, Mg H -ions have to be present which are superior to Mn H . Enzyme activity is enhanced by non-ionic detergent and pH 8.5. At 41 DC, the reaction is linear with time for at least one hour, then reaches a plateau 2 to 3 h later, and finally ceases after about 5 h. Reaction products are 14 S dsRNA as virion RNA and 24S single-stranded RNA hybridizing to both genome segments. In view of the nature of the products, IBDV polymerase acts as transcriptase as well as replicase.

MULLER, H. and R. NITSCHKE Inst, f. Virologie, Justus-Liebig-Univ., D-6300 GieBen, and Bundesamt f. Sera u. Impfstoffe, Paul Ehrlich-Inst., D-6000 Frankfurt a.M.

The two double-stranded (ds) RNA segments of IBDV were released from virus particles by treatment with proteinase K in the presence of SDS and were then investigated with the electron microscope. Linear dsRNA molecules were observed, and their lengths were com-pared with those of the completely sequenced reovirus type 3 segments M3 and S2. Mol. wts.of 2.2 x 10 6 and 1.9 x 10 6d were calculated. These results indicate that the genomes of the other members of the family Birnaviridae are also smaller than those reported. As seen under the electron microscope, heating of IBDV particles in the presence of SDS without proteinase K results in dsRNA molecules which are circularized by protein. Electrophoresis of radioactively labeled particles in different gel systems indicates that this protein is the 90kd structural protein of IBDV which is supposed to represent all or part of the RNA dependent RNA polymerase. Aedesalbopictucs cells infected with SFV can be induced to fuse from within at pH 6. We have applied hydrophilic reagents to covalently modify the proteins at the cell surface. Each reagent was applied at pH 7 to cultures before the pH was lowered and to cultures after the cells had been exposed to pH 6 at 4 "C. Several chemicals reacting with amino-, guanidino-, sulfide-and disulfide groups inhibited the fusion only when applied after low pH exposure. On the basis of these results, which implicate that low pH induces a conformational change of a protein, radioactive reagents were utilized and the SFV E1 envelope protein could be identified as the protein undergoing the conformational change. The genome of insect iridescent virus type 6 -Chilo iridescent virus -(CIV) was shown to be circularly permuted and terminally redundant. Upon denaturation and reannealing of native linear CIV DNA (238 kbp), duplex DNA circles of a smaller size (211 kbp) with protruding tails were formed. A defined and complete gene library of the whole CIV DNA sequences was established using the insertion of 31 EcoRI DNA fragment (A to e'l into the corresponding site of the plasmid vector pACYCI84. Furthermore the CIV DNA was cleaved with restriction endonucleases BamHI, Ncol, Sail, Sphl, and/or double digested with BamHI/Sall, and the resulting DNA fragments were inserted in the BamHI, BamHI/Sall, and Sphl sites of the bacterial plasmid vector pATIS3 and in NcoI site of the plasmid vector pKm2. All cloned fragments were individually identified by hybridization experiments. The physical map of the viral DNA was constructed for restriction endonucleases Apal,Smal, BamHI,Sall, and Ncol. Although the CIV genome is linear, due to circular permutation the restriction maps are ciruclar, The defined and complete gene library of ClV DNA will be useful for further structural and functional analyses of the genome of this interesting virus.-This study was supported by the Deutsche Forschungsgemeinschaft, Project Da 142/2-1. The Human Parvovirus B19 is the causative agent of erythema infectiosum, During the viraemia viral DNA can be detected by DNA hybridisation, A 700 kb DNA fragment of the parvovirus genom, cloned in M13 and pGem was used as radioactive probe. With this technique a parvovirus positive blood probe could be detected which serves as antigen source for the establishment of an IgM/IgG and antigen capture assay. A flatbottom mirotiter plate was coated with anti-v resp. anti-A, incubated with test sera, antigen and a monoclonal anti-Parvovirus B19 antibody. After a further incubation with a peroxidase-conjugated antimouse IgG orthophenylendiamin and perhydrol were added, the enzymatic reaction stopped with IN H 2S04 and the optical density measured. For the detection of viral antigen a strongly anti-parvo positive serum was coated, test serum added and the procedure performed as described above. Comparing the antigen ELISA to the DNA-hybridisation, the DNA-hybridisation has a 1000 fold higher sensitivity and 0.06 pg/ml DNA could be detected. The detection of specific IgM7IgG enables a serological diagnosis of an acute and past parvovirus infection. Blood donations can be screened with the antigen capture assay. Using FMDY strain OjK, neutralizing monoclonal antibodies (nmab) with four different specificities were isolated. The nmab were first characterized with regard to their neutralization pattern. Two nmab showed a quite narrow but different neutralization pattern. In contrast, the third nmab reacted with all°subtypes tested, but not with the serotypes A and C. Crossneutralization with certain A and C subtypes was observed with the fourth nmab. The latter two nmab reacted with YPI by Western blotting, and by ELISAwitha a synthetic peptide whose sequence (AA 139-160 of YPI from OjK) has been shown to be important for the induction of neutralizing antibodies. The respective experiments with the two narrow reacting nmab were negative. Thus, two nmab may recognize a continuous sequence in a defined region of VPl. This hypothesis can now be tested directly since neutralization resistant variants have been isolated. Preliminary respective nucleic acid sequencing data will be presented.

Analysis of the HSV-2 Induced Suppression of Humoral Antibody Formation by B-Cell Enumeration NICK, S., B. METZGER, S. MULLER, and D. FALKE Div. expo Virology, Insr. Med. Microbiology, johannes-Gutenberg-Univ., D-6500 Maim HSY-2 but not HSY-l induces suppression of humoral antibody formation against HSY-1 and 2 as well as SRBC and HBs-antigen. !.P.-injection of silica partially release this suppression; therefore M0 are the first target (J. gen. Virol. 67 (1986) 1015). In order to analyse more exactly the mechanism of this suppression, we determined the number of HSY-specific IgM-or IgG producing B-cells. Primary or homologous and heterologous secondary infections (high and low dosis) with HSY-l induce a phase of "nonreactivity" of the spleen at day 8 against a high dose: Secondary infections by HSY-l at day 8 are manifested by no antibody secreting B-cells in the spleen, humoral antibody response how-ever is regular. At day 21 a typical B-cell IgG-booster response is manifested in the spleen. The HSV-2 induced suppression wanes more than 50 days p.i. This indicates that antigen presentation and memory cells are stable, only the switch to IgM or IgG production seems to be damaged by HSY-2. This was confirmed by analysis of the HSY-specific very low B-cell response: Some specific B-cells appear very late if compared to HSY-l. Some suppressor Tcells also seem to be produed by HSY-2. Suppression was demonstrated up to 50 days after infection with HSV-2. Generally HSY-2 induces a low total spleen cell response if compared to HSY-l.

JUST, W., B. WALTER, and G. KLOTZ Dept. of Virology, Univ., D-7900 Ulm MVL3 is a polyhedral virus with a diameter of about 60 nm. The viral genome is a linear double-stranded DNA of 26.10 6 mol wt, The infection is non-lytic but cytocidal. Circular permutation and terminal redundancy are shown by three different methods. -(1) Restriction patterns of L3 DNA show a nearly constant decrease in mol wt of some fragments. The difference equals 8% of the total genome size, indicating the shift of the permutation. (2) The determination of Eco RI binding sites on L3 DNA by electron microscopy reveals three populations of molecules. The ends from one class to the other were shifted by 1 urn, i.e. 8% of the genome size. (3) Treatment with Exonuclease III, generating protruding single-stranded ends, followed by renaturation, showed homology in the terminal regions.

The homology extends to about 8% of the genome length.

MAuELER, W., F. RAULF, and M. SCHARTL Max-Planck-Inst. for Biochemistry, Gene Center, D-8033 Martinsried

The genome of Xiphophorus contains several protooncogenes which are homologous to the transforming genes of certain retroviruses. Specific crossing between different genotypes of Xiphophorus lead to spontaneous formation of benign and malignant melanoma in the offspring. In order to obtain a better understanding of the functions of protooncogenes and oncogenes in nontransformed and in neoplastically transformed cells respectively, we studied the expression of c-src, e-ras, c-sis, c-erbA, c-erbB and c-mycduring embryogenesis, in adult tissue and in melanoma of Xiphophorus. During embryogenesis c-src, e-ras and csis are differential expressed. In adults e-ras and c-mycare tissue-specifically expressed and show no overexpression in melanoma. c-src is also expressed with tissue-specificity, the highest level was found in melanoma. High levels of c-erbB and c-sis related mRNA's were detected only in melanoma cells. This data indicate a normal function of protooncogenes more in differentiation than in proliferation and coordinated deregulation of several oncogenes during melanoma formation. and November 1985. All 27 seals got infested. In thin sections of bioptic skin specimens poxvirus inclusion bodies of type B were found. By negative staining of triturated suspensions of these bioptic specimens typical parapoxvirus particles were detected. So far all attempts failed to propagate the virus in embryonic cells of sheep skin and kidney. Inquiries in three other rearing stations along the North Sea coast revealed that similar skin alterations had been seen in at least two places, namely at Norden (summer 1985) and at PieterburenlNetherlands.

Characterization of a Poxvirus Isolated from a Child ROSEN l , A., J. PILASKI 2 , and G. DARAI l Inst. f. Med. Virologie, Univ., 0-6900 Heidelberg Med. Institut f. Umwelthygiene, Univ., 0-4000 Dusseldorf A 6-year-old girl at LiineburgiLower Saxony, who had not been vaccinated against smallpox, developed a skin lesion with central necrosis at the left hand in April 1985. An orthopoxvirus strain was isolated by Nasemann et al. (1986) . Since this strain produced efflorescences of 1.5 to 2.0 mm in diameter with a haemorrhagic center on the chorioallantoic membrane and cytoplasmic inclusion bodies of type A V+, it was incorporated into the group of cowpoxlike viruses and termed H-CP-LSax. The DNA restriction patterns of this virus strain were compared with those of other orthopoxviruses using the endonucleases BamH I, Hind III, Sma I, Sac I, and Kpn I. The Sma I and Hind III fragmentation patterns of the isolate H-CP-LSax were nearly identical with those of the cowpoxlike virus strain EP 267 which had been isolated from an Asian Elephant (Elephas maximus) at the zoological garden at Frankfurt in November 1977. The girl had no contact with zoo animals. She lived in close contact with some pet animals like cats, rabbits, a dog, and a guinea pig.

Determination of Viral Glycoprotein Antigen with the ELISA as an in-vitro Assay for the Potency of Rabies Vaccines THRAENHART, O. and C. RAMAKRSIHNA WHO Centre f. Reference and Research on Neurol. Zoonoses, Inst. of Med. Virology and Immunology, Univ., 0-4300 Essen

The content of rabies virus glycoprotein antigen (glp-ag) is correlated with the immunogenicity of tissue culture vaccines against rabies. An ELISA based on the determination of the glp-ag with monospecific, polyclonal anti-glp-antibodies directed against the rabies virus strains ERA, Flury LEP and PV therefore may replace the invivo protection test in the mouse (NIH-test). -Such an ELISA was standardized, which is based on the fixation of different vaccine dilutions starting at 1:16 at the solid phase of microtitre plates, reaction with an excess of anti glp-antiserum for 1 h at +3rc, a following incubation with a peroxidase coupled anti-IgG for 1 hat +3rc and measuring the reaction at 490 nm 30 minutes after addition of the substrate. -Under these conditions we were able to determine the potency of tissue culture rabies vaccines of different virus strain characteristic and procedure of production, tissue culture supernatants, vaccine concentrates, vaccines after heat treatment and after adsorption to Al(OHh. The glp-ag of unknown samples was tested in parallel with a reference vaccine of known potency and quantitated with the parallel line bioassay according to Finney (in "Statistical Method in Biological Assay", 3.ed., Ch. Griffin and Comp., London and High Wycombe, 1978) . -ELISA results were correlated with the results obtained in the NIH-test in a range of 0.3 to 700 IV. Glp-ag was expressed in BHKcells at 100 h and increased to a maximum at 200 h after infection. -Thermostability of lyophilized vaccines was demonstrated for the PCEC-and Vero-vaccines at +37"C for 120 days and at +56°C for 24 h. However, if vaccines were stored in liquid form potency decreased rapidly in less than 4 h at + 56 "C. Stability of the glp-ag at + 37 DC seemed to be influenced by the stabilizer of the vaccine. First results showed a stable glp-antigenicity of the resuspended PCEC vaccine for 120 days at + 37"C, but decreasing glp-antigenicity for the resuspended Vero vaccine after 30 days storage at +37"C. In tropics therefore prefer lyophilized vaccines. Human monoclonal antibodies (humab) directed against viral antigens were developed by combining immortalization of human primary B lymphocytes by Epstein-Barr virus (EBV) infection with consecutive fusion of selected immortalized lymphoblasts with an established, human lymphoblastoid cell line. Among the antiviral humab producing hybridomas thus generated, were three which produced anti-rubella humab. Rubella virus consists of three structural proteins, the core protein C and the two envelope proteins El and E2. By the Western blot technique we were able to show that two humab reacted with the core protein and the third humab with the envelope protein El. From the hybridomas grown in stationary cultures, highly purified humab preparations were obtained by subjecting the concentrated culture supernatant to immuno-affinity chromatography.

WINTER, J., H. WEGE, R. WATANABE, and V. TER MEULEN Inst. f. Virologie u. Immunbiologie, Univ., D-8700 Wlirzburg Coronavirus JHM infection of rodents can lead to diseases of the central nervous system accompanied by demyelination. This model is important for studies of pathogenesis. The outcome of infection depends on properties of the virus. Virulent and avirulentJHM-viruses are differentiable by the peplomer protein E2. Functions of E2 are attachment, binding of neutralising antibodies and fusion. -To learn more on relations between structure and virulence, we used monoclonal antibodies (MAB's) to select JHM-variants changed in de-finded E2-epitopes. Infection of hybridoma lines, which produce neutralising MAB's, leads to selection of JHM-variants which escape neutralisation. No other antigenic or structural changes are detectable. Four different groups of JHM-variants were selected corresponding to epitopes associated with neutralisation and cell fusion. Mice infected with variants changed in epitope E2-Ba develop predominantly chronic disease. JHM-variants changed in one of the other major epitopes cause acute disease like JHM-wild type virus. These data indicate that virulence is associated with defined domains of the peplomer protein E2. The main replicase in higher eukaryotic cells DNA polymerase a, contains a tightly associated DNA primase. It was demonstrated that this enzyme is able to initiate DNA replication in-vitro on multiple sites of cloned single-stranded SV40 DNA or on singlestrandes parvoviral DNA. In contrast we have used the naturally occuring single-stranded ciruclar porcine circo-virus (PCV) DNA to investigate initiation of DNA replication. Purified DNA polymerase a-holoenzyme from calf thymus primes specifically at one preferred site on this eukaryotic, in cells naturally coccuring as single-stranded DNA template. The structural characteristics of budgerigar fledgling disease (BFDV) were analyzed by biochemical and biophysical methods. The results obtained justify classification of BFDV as a member of the polyomaviruses, a papovavirus subgroup. BFDV is related to all members of this subgroup, but is not identical with anyone of them; therefore, it is a new and the first nonmammalian polyomavirus. The virus was able to transform hamster embryo cells in vitro which is a typical feature of polyomaviruses. However, while most polyomaviruses known until now only cause persistent infections without any clinical symptoms in their natural hosts, BFDV is the etiological agent of an acute disease resulting in high death rates of affected birds.

Barnkamm: A putative transforming gene of ]ijoye virus differs from that of Epstein-Barr virus prototypes

Phage DNA Restriction Enzyme Analysis as a Tool for Epidemiological Typing. C. diphtheriae as an Example CHASTONAY

CH-3000 Bern" and Inst. for Med. Microbiology and Virology

Dusseldorf Epidemiological classification of pathogens allows to monitor person to person spread. An increasingly used method in this context consists in analysis of DNA restriction enzyme patterns. Since toxigenic C. diphtheriae are always lysogenized, we applied this method to analyse phage DNA. -In one approach, DNA of UV-induced and replicated phages was digested with restriction enzymes. The obtained fragments were submitted to agarose gel electrophoresis and visualized by ethidium bromide staining. Another method consisted in digestion of prophage DNA. The various fragments then obtained by agarose gel electrophoresis were transferred to nitrocellulose and hybridized with a 32P-Iabeled phage DNA. -Prophage DNA analysis even allows to typify strains that are phage resistent and non lysogenic and therefore cannot be investigated by phage typing. Further studies with isolates from epidemics

Autoimmunity After Viral Infection: Production of Monoclonal Antibodies Against Golgi-Antigen After Infection of Mice with the Anti-Golgi Inducing Agent

D-7400 Tiibingen Recently we described Goigi autoantibodies induced by a transmissible agent in the mouse system. Further investigation showed, that Golgi antibodies were present in all infected animals already at day 8 p.i. with a maximum between two and four weeks p.i. Sometimes autoantibodies against cytoskeletal elements and mitochondrial structures could also be detected. In order to characterize the autoantigens, monoclonal antibodies were established. The highest yield of hybridomas producing Golgi autoantibodies was obtained 9-14 days after infection. We also succeeded in producing monoclonal antibodies against other cellular antigens. Using poly-and monoclonal antibodies in immunoelectron microscopy we were able to decorate the Golgi apparatus

A Parapoxvirus Infection in Harbour Seals (Phoca vitulina) in a Rearing Station at the North Sea Coast in Germany PILASKI 1

Heidelberg In a rearing station for Harbour Seals (Phoca vitulina) at the German North Sea coast a pox disease with proliferative skin alterations was observed during the time between

RICHT, J. and L. STITZ Inst. f. Virologie, Justus-Liebig-Univ., D-6300 GieBenThe fact, that class II MHC-antigen (Ia) is expressed in the brain of Borna disease virus (BDV)-infected rats, stimulated in vitro investigations employing astrocytes. Astrocytes can be induced in vitro to express la-antigen by y-Interferon (IFN-y) or, as it has recently been reported, after Coronavirus-infection (1). Therefore, we studied the expression ofIa-antigen on the surface of persistently BDV-infected astrocytes in vitro. The cells were obtained from the brain of newborn Lewis rats and were cultured for three weeks before being infected with BDV. They were found to be positive for both glial fibrillary acidic protein (GFAP) and BDV-specific intracellular antigens, using immunofluorescence and peroxidase-antiperoxidase technique. Infected astrocytes did not express la-antigen on their surface, but could be induced to express la-antigen after treatment with recombinant IFN-y or Concanavalin-A supernatant. We therefore conclude that induction of la-antigen on astrocytes is not a general consequence of viral infections.

BOHN, W., G . R UT T ER , H . H OH ENBER G , and K. MANNWE I LE R Heinrich-Pette-Inst. f. expoVirologie U. Immunologie, Univ., D-2000 Hamburg 20 We have shown recently that the budding of measles virus is connected with a polar growth of actin filaments. Calcium ions are known to play a key role in the regulation of assembly and disassembly of actin filaments as well as in the regulation of contractility. Therefore calcium ions may also influence the budding process of measles virus. We modulated the calcium concentration in measles virus infected cells by use of the calcium ionophore A23187. In the presence of calcium, but not of magnesium or barium the ionophore induced (1) a disappearance of virus structures and microvilli from the cell surface, (2) a random redistribution of virus hemagglutinin at the cellsurface, (3) a dissociation of nucleocapsids from the protoplasmic membrane face, and (4) a significant reduction of the cell bound infectivity. The data indicate, that calcium ions are able to influence the morphology of budding virus particles at the plasma membrane and may playa role in virus morphogenesis. -Supported in part by Gemeinniitzige Hertie-Stiftung, Frankfurt/M.