European Virology 2000 — oral abstracts

O-01

 

 Interaction between lyssavirus glycoprotein and the rabies neuronal receptor P75NTR 

EMMANUEL DESMEZIERES¹, J. Bénéjean², A. Flamand², N. Tordo¹, P. Perrin¹ and C. Tuffereau^{2 

 1}Institut Pasteur, Laboratoire des Lyssavirus, 75724 Paris, France

 ²CNRS, Laboratoire de Génétique des Virus, 91198 Gif sur Yvette, France

 

Rabies and rabies-related viruses which make up the Lyssavirus genus induce a fatal encephalomyelitis. They propagate mainly into neurons but can multiply on cells of non-neuronal origin after adaptation. They are currently divided into seven genotypes (GT) and the most common members are: rabies virus (RV: GT1), Lagos Bat virus (LB: GT2), Mokola virus (Mok: GT3), Duvenhage virus (Duv: GT4), European Bat viruses 1 and 2 (EBL1: GT5 and EBL2: GT6) and Australian Bat lyssavirus (ABL: GT7). Geographic distribution, vectors, as well as their pathogenicity by peripheral route and the crossreactivity of virus neutralizing antibody varies among Lyssaviruses.

The low-affinity nerve-growth factor receptor (P75NTR) was recently characterized as a ligand for a soluble form of the rabies glycoprotein (GTI) and a receptor for rabies virus. P75NTR is a type I glycoprotein and belongs to the superfamily of cystein rich domain proteins which includes the TNF receptor.

We investigate the ability of P75NTR to interact with different lyssavirus glycoproteins expressed after infection of fibroblastic cells (BSR) or transient transfection of neuroblastoma cells (Neuro-2a). For this purpose, we develop a specific binding assay between infected or transfected cells and Spodoptera frugiperda cells expressing P75NTR at their surface after infection by a recombinant baculovirus. Under these conditions, cells expressing P75NTR interact with RV (GT1: CVS, PV and street rabies) or to the closely related EBL2 (GT6) glycoproteins. On the contrary, no interaction was evidenced with glycoproteins from lyssaviruses belonging to other GT (2, 3, 4 and 5).

 

 

O-02

 

 Expression of the herpesvirus receptor gene encoding HVEC in nervous and ocular tissue from adult mouse 

LARS HAARR^1,2, Deepak Shukla¹, Eyvind Rødahl³ and Patricia G. Spear^{1 

 1}Dept. of Microbiology–Immunology, Northwestern University, Chicago, USA

 ²Centre for Research in Virology, Dept. Microbiol. & Immunol., University of Bergen, Norway

 ³Dept. of Ophthalmology, Haukeland University Hospital, Bergen, Norway

 

Several receptors for herpes simplex virus (HSV) 1 and 2 and other alphaherpes-viruses have been detected recently. HveA is a member of the TNF/NGF receptor superfamily, while HveB and HveC belong to the immunoglobulin superfamily. Both human and mouse forms of these proteins serve as entry receptors for one or more of the alphaherpesviruses. Most sudies of these receptors have been performed in cell cultures. The purpose of the present work was to focus on HveC and investigate its expression in various mouse tissues by in situ hybridization. Northern blot hybridization using a specific probe had indicated expression in murine epidermal and nervous tissue.

The murine and human HveC genes have previously been shown to have a high degree of homology. In the present work, sense and antisense probes were digoxigenin (DIG)-labelled by in vitro transcription of a 291bp fragment from the middle region of the mouse HveC gene cloned into an expression vector. Hybridized probe was detected with an anti-DIG antibody conjugated to alkaline phosphatase. Specific signals were detected in the cytoplasm of neurons from spinal ganglia, superior cervical ganglia, trigeminal ganglia, pterygopalatine ganglia, brain stem, cerebellum, brain cortex and hippocampus. Furthermore, strong signals were observed in cornea and retina. No hybridization was detected in 16 days old mouse embryos.

The experiments demonstrate that the HveC gene is widely expressed in nervous tissue of adult mouse, and also in corneal epithelial cells and in retinal cells.

 

 

O-03

 

 8.5 A structure of HSV-1 capsid 

WAH CHIU², Hong Zhou¹, Matthew Dougherty², Joanita Jakana², Jing He² and Frazer Rixon^{3 

 1}University of Texas, Houston Medical School, Houston, Texas, USA

 ²Baylor College of Medicine, Houston, Texas, USA

 ³MRC Virology Unit, University of Glasgow, Scotland, UK

 

Human herpes simplex viruses, which cause a variety of important diseases, are among the largest and structurally most complex viruses. The capsid shell of HSV-1, with a total molecular mass of 0.2 billion Daltons, is formed by 4 sequence unrelated proteins: VP5 (149 kDa), VP26 (12 kDa), VP23 (34 kDa) and VP19C (50 kDa). An asymmetric unit within the icosahedral capsid shell has a molecular mass of ∼3.2 MDa and consists of 1 penton subunit, 15 hexon subunits and 51/3 triplexes. We have imaged ice-embedded HSV-1 capsids in a JEOL 4000 electron cryomicroscope with a LaB₆ gun operated at 400 kV. Despite the low contrast of the images, they contain data extending beyond 7 Å. By merging 5860 particle images from 130 micrographs, we have determined the molecular structure to 8.5 Å resolution. Using computational graphics tools, more than 30 putative α helices were identified in the four shell proteins. Some of these helices in VP5 are located at domains that are known to undergo conformational changes during capsid assembly and DNA packaging. Triplexes are characteristic features of HSV-1 capsids that link adjacent capsomeres. Each triplex was believed to be a heterotrimer of one VP19C and two VP23 molecules. Our structure shows the unique spatial arrangement of the heterotrimer at the local 3-fold positions. Their structural organization accounts for the asymmetric interactions with adjacent capsid components and the unusual co-dependant folding of its subunits.

 

 

O-04

 

 Probing virus-host interaction, essence of supermolecular symmetry 

HOLLAND CHENG

Dept. Biosciences Novum, Karolinska Institute, Huddinge 14157, Sweden

 

Viral macromolecules arise as orderly structures, which are often composed of subunits arranged in a symmetrical manner, thereby providing the tool for enhancement of single-molecule signalling and networking into larger supermolecules. The fundamental concept of crystalline or symmetry arrays is the applicability of strict geometry—the subunits, the asymmetric units, can be superimposed on itself by operations in taking such geometry into account. This requires not only the specific interactions among protein motifs, but also a build-in flexibility to accommodate conformational changes. For instance, the capsid proteins of phytoreovirus are found not only to assemble into a T=13 quasi-equivalent lattice in the virion, but preferably to utilize similar hexagonal array to form an elongated tube configuration without the presence of inner cores (e.g., Wu et al., 2000). In addition, the same capsid proteins can be extracted to conform a monolayer crystal in vitro, but to reconstitute a native-like double-shell particle in the presence of the inner core particles. Thus, here, the viral proteins provide the capacity to function both in assembly and in disassembly by a built-in structural flexibility to be triggered by conformational changes with particular external or internal cellular event (Hammar et al., 1999). Models of human pathogens in vitro systems have been established, for instance, virus-like particles of human hepatitis E virus expressed in insect cells (Xing et al., 1999). Critical macromolecular interactions have been analyzed in their ‘native’ forms in order to understand the interplay among viral components with cryo-electron microscopy (Cheng, et al., 1992, 1994, 1995). Fine details of protein folding can be revealed in a virus particle as a whole, without the need of heavy-metal derivatives or crystal arrayed packing. Images of virus particles are collected with Helium cooled, field emission gun, electron microscopy at low-dose condition of electron beams in vitreous ice. Various assemblies of such large-latticed particles are studied with this rapid-frozen and low-dose imaging procedure (Cheng, 2000). We are also able to localise the function-specific domains based on the mutagenesis and the difference imaging analysis (e.g., Xing et al., 2000). With symmetry built in the intermediates, the image process takes a great advantage in building up 3D reconstruction with Fourier averaging (Baker and Cheng, 1996, Cheng, 2000). Functional relevance will be examplified by ligands expected to interfere with the infection cycle designated from sequence information. Fundamentally, the metastability principles learned from our study should provide not only the biotechnological base for viral defense strategies, but also insights into an array of biological functions.

 

Baker TS and Cheng RH (1996) J Struct Biol 116, 120–30.

Cheng RH et al (1992) Virology 186, 655–68.

Cheng RH et al (1994) Structure 2, 271–82.

Cheng RH et al (1995) Cell 80, 621–30.

Cheng RH (2000) In ‘Simulation and Visualization on the Grid’, in press, Springer-Verlag New York.

Hammar, L et al (1998) Bia J 5, 22–23.

Wu, B et al (2000) Virology, 271, 18–25.

Xing L et al (2000) EMBO J. 19, 1207–1216.

Xing L et al (1999) Virology 265, 35–45.

The study is sponsored by Natural Science Research Council (NFR-11691), Medical Research Council (MFR-12175), and Structural Biology Network in Sweden. http://www.csb.ki.se/kisv/ 

 

 

O-05

 

 Icosahedral organization and functions of the flavivirus envelope glycoproteins 

 

FRANZ X. HEINZ, Steven L. Allison and Karin Stiasny

Institute of Virology, University of Vienna, Kinderspitalgasse 15, Vienna A-1095, Austria

 

X-ray crystallographical (F.A. Rey et al., Nature 375, 1995) and cryo-EM studies (Fuller et al., EMBL Heidelberg, in preparation) of the flavivirus tick-borne encephalitis virus have revealed that flaviviruses are distinct from the ‘spiky’ type of enveloped viruses. The basic subunit of the envelope glycoprotein E is a head to tail dimer that is oriented parallel to the virion membrane. These dimers interact in a specific way to form an icosahedral network that undergoes dramatic structural and oligomeric rearrangements at the acidic pH required for fusion. This includes the dissociation of the E-protein dimer, the exposure of an internal fusion peptide, and the irreversible formation of trimers. The flavivirus fusion machinery is the fastest and most efficient one known so far while being structurally completely different from that of many other enveloped viruses including influenza virus and HIV. In these cases fusion is mediated by trimeric spikes with alpha-helical coiled coils, whereas in flaviviruses the same function is carried out by an icosahedral network of flat, head-to-tail dimers of mostly beta-sheet proteins. Despite these structural differences there are a number of similarities between flaviviruses and other enveloped viruses. The use of acidic pH in the endosome to trigger fusion, the conversion of the fusion protein to a kinetically trapped metastable state by a proteolytic cleavage event, and the use of protein oligomerization as a mechanism for controlling these steps are all principles used by other viruses as well, although many of the details are different.

 

 

O-06

 

 The membrane protein of the transmissible gastroenteritis coronavirus binds to the internal core through the carboxy-terminus and stabilizes core structure 

JAVIER ORTEGO, D. Escors, J. Ortego, and L. Enjuanes

Centro Nacional de Biotecnologla, CSIC, Deparment of Molecular and Cell Biology. Campus Universidad Autonoma, Madrid, Spain

 

The Transmissible Gastroenteritis Coronavirus (TGEV) is an enveloped virus with an internal core made of RNA, N and M proteins. The presence of the M protein in purified cores was unexpected since this is an integral membrane protein.

In this work it has been demonstrated that a significant proportion of the M protein integrated within the virus membrane interacts with the core through ionic interactions. Purified cores were treated with a collection of caiotropic agents, and the removal of the M protein was correlated with the destabilization of the structure of the core. The structure of the core was stabilized by the presence of divalent cations and high pH, and was partially resistant to non-ionic detergents and reducing agents. It was demonstrated that the interaction between the M protein and the cores was specific and mediated by the carboxy-terminus, based on three types of evidence. (i) The interaction domain was mapped in 16 residues located at the carboxy-terminus by an in vitro binding assay of M protein mutants with purified nucleocapsids; (ii) the presence of the interaction domain in the carboxyterminus was confirmed by inhibiting the binding using synthetic peptides mimicking the binding domain; (iii) the binding of the M protein to nucleocapsids was inhibited with M-specific mAbs directed to the C-terminus of the M protein. These data consolidates the structure and composition of TGEV internal core.

 

 

O-07

 

 HLA-class I alleles and heterozygosity influence the outcome of HTLV-I infection 

Asna A Siddiqui¹, Mike Bunce², Alun L. Lloyd³, Alison M. Vine¹, Aviva D. Witkover¹, Shuji Izumo⁴, Koichiro Usuku⁵, Kenneth I Welsh², Mitsuhiro Osamell⁶, Charles RM Bangham¹ and KATIE JEFFREY^{6 

 1}Department of Immunology, Imperial College School of Medicine, St. Mary's, Norfolk Place, London, UK

 ²Oxford Transplant Centre, Nuffield Department of Surgery, Churchill Hospital, Oxford, UK

 ³Institute for Advanced Study, Einstein Drive, Princeton, USA

 ⁴Division of Molecular Pathology, Centre for Chronic Viral Diseases, Department of Medical Informatics, and the

 ⁵Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Japan

 ⁶Microbiology Department, John Radcliffe Hospital, Headley Way, Headington, Oxford OX3 9DU, UK

 

The inflammatory disease human T-cell leukaemia virus type-l (HTLV-1) associated myelopathy (HAM/TSP) occurs in only 1-2% of HTLV-l infected individuals, and is associated with a high HTLV-I provirus load. We hypothesise that the risk of developing HAM/TSP depends upon the efficiency of the immune response to the virus, which may differ between individuals because of polymorphisms in genes influencing this response. In a case-control association study, we have previously demonstrated that possession of HLA-A*02 was associated with a lower risk of HAM/TSP, and with a lower provirus load in healthy carriers (HCs) of HTLV-l (1). However, HLA-A*02 did not account for all the observed difference in the risk of HAM/TSP. Additional HLA-typing shows that HLA-Cw*08 is also associated with disease protection (P=0.002), and with a lower proviral load in HCs. In contrast, HLA-B*5401 is associated with a higher susceptibility to HAM/TSP (P=0.0003), and with a higher provirus load in HAM/TSP patients. We suggest that the association of HLA-B*5401 with susceptibility to HAM/TSP is most likely to be due to a pro-inflammatory gene in linkage disequilibrium with B*5401. In addition, individuals who are heterozygous at all three HLA Class-1 loci have a lower HTLV-I provirus load than those homozygous at one or more loci. These results are consistent with the proposal that a strong Class-I-restricted cytotoxic T cell (CTL) response to HTLV-1 reduces the proviral load and hence the risk of disease.

 

K.J. Jeffery et al., Proc Natl Acad Sci USA 96, 3848–53 (1999).

 

 

O-08

 

 Hepatitis C virus-specific cytotoxic T cells in acute and chronic infection quantified with MHC tetramers: relationship with viral clearance and escape mutations 

X.-S. He¹, Y. Lu¹, J. Boisvert¹, R. Cheung¹, M.M. Davis¹, H.B. Greenberg¹, M. Berenguer², T. Wright², F.X. LÓPEZ-LABRADOR³, X.-S. He³, M. Berenguer⁵, Y. Lu³, J. Boisvert³, R. Cheung³, B. Rehermann⁴, M.M. Davis³, T. Wright⁴ and H.B. Greenberg^{3 

 1}VAMC Palo Alto/Stanford University, Stanford, CA, USA

 ²VAMC/University of California, San Francisco, USA

 ³VAMC Palo Alto/Stanford University School of Medicine, Pl. Ballester-Fandos 2, E-D 5-10, Valencia 46013, Spain

 ⁴NIDDK, NIH, Bethesda, MD, USA

 ⁵VAMC/Uni of California, San Francisco, CA, USA

 

 Background: Hepatitis C virus (HCV)-specific CD8+ cytotoxic T lymphocytes (CTL) are thought to be involved in viral pathogenesis.

We designed MHC tetramers based on epitopes of the NS3 protein (NS3-1, aa1073-1081: CVNGVCWTV and NS3-2, aa1406-1415: KLVALGINAV) to directly quantify HCV-specific CTLs (PNAS 1999;59:5692-7).

 Aims/methods: We analysed the effect of HCV-specific CTLs, on the on the disease course of a patient with acute infection, and on the aminoacid sequence of the viral epitope in 48 HLA-A2+ chronically infected patients.

 Results: In the patient with acute infection, the NS3-specific CTL response was detected at a much higher level than that in chronic infection, and it was associated with clearance of virus and resolution of disease. Twenty-two (46%) of the chronically-infected patients had detectable HCV-specific CTLs with the tetramer NS3-2.

Aminoacid variations were detected in the viral coding sequences for the NS3-2 epitope. The majority of these variations were genotype-related differences. In few patients, potential viral CTIL escape mutants were identified.

The HLA-A2 anchor residues of the epitope in the infecting virus were conserved.

 Conclusions: These findings suggest that HCV-specific CD8+ T cells are one of the factors playing a role in restraining and eliminating HCV. Although CTL escape may occur during HCV infection, it does not seem a common event.

 

 

O-09

 

 Temporary inactivation of follicular dendritic cells delays neuroinvasion of scrapie 

Fabienne Mackay Garvan¹, Fiona Minns², Moira Bruce² and NEIL MABBOTT^{2 

 1}Insitute of Medical Research, Darlinghurst NSW, Australia

 ²Institute for Animal Health, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK

 

Creutzfeldt–Jakob disease (CJD) of humans, sheep scrapie and bovine spongiform encephalopathy are members of a group of neurological diseases known as transmissible spongiform encephalopathies (TSEs) or ‘prion diseases’. Natural TSE infections are usually acquired by peripheral exposure, for example by ingestion or transplantation of contaminated tissues. After experimental peripheral exposure, TSE agents usually accumulate in lymphoid tissues long before infection is detected in the central nervous system. Studies of a mouse scrapie model have shown that mature follicular dendritic cells (FDCs), expressing the I ‘prion protein’ (PrP), are critical for replication in lymphoid tissues and their absence neuroinvasion is impaired. As FDCs require lymphotoxin alpha/beta signals from B-cells to maintain their differentiated state, blocking this signalling with a lymphotoxin beta-receptor/immunoglobulin fusion protein (LTBR-1g) temporarily inactivates FDCs within 72 hours, for approximately 28 days. These observations lead to the prediction that treatments that interfere with the integrity or function of FDCs will also interfere with TSE pathogenesis. Here we demonstrate that temporary inactivation of FDCs by treatment with LTBR-Ig, blocks scrapie replication in lymphoid tissues and delays neuroinvasion. These results suggest a new potential approach to prophylaxis in the TSEs.

 

 

O-010

 

 Impairment of antigen presentation to hiv-specific CD8+ T lymphocytes through down regulation of MHC Class I on human dendritic cells 

MURIEL ANDRIEU², Dorine Chassin¹, Jean-François Desoutter¹, Isabelle Bouchaert², Marjorie Baillet¹, Daniel Hanau³, Jean-Gérard Guillet¹ and Anne Hosmalin^{1 

 1}Laboratoire d'Immunologie des Pathologies Infectieuses et Tumorales, Paris, France

 ²Service commun de cytométrie, U445 ICGM, 27, rue du Fauborg St. Jacques, Paris 75014, France

 ³INSERM EP 99-08, Biologie des Cellules Dendritiques Humaines, Etablissement Français du Sang-Alsace, Strasbourg, France

 

The HIV-1 early regulatory Nef protein is involved in the impairment of the immune response by downregulating surface expression of Major Histocompatibility Class I molecules on different immortalized cell lines and on T lymphocytes, thus decreasing lysis by cytotoxic cells. Induction of primary immune responses requires dendritic cells, and DC expressing CD4 and CCR5 or DC-SIGN are major candidates as the first cells that can internalize the virus and present it to T cells in mucosal contamination. To test whether Nef impaired MHC-I restricted antigen presentation by dendritic cells, we used recombinant vaccinia viruses to induce the expression of Nef and control proteins in human monocyte-derived dendritic cells. Flow cytometric analysis of double labeling for a vaccinia protein and Major Histocompatibility Class I showed that HIV-1 Lai Nef indeed down-regulated MHC class I surface expression on dendritic cells. Major Histocompatibility Class I-restricted presentation to a Nef-specific CD8+ cell clone from an infected patient was decreased in an Interferon-g ELISpot assay. Presentation of a reverse transcriptase epitopic peptide on sorted Nef-infected cells was decreased in a peptideconcentration dependent way, confirming the role of MHC I downregulation in the impairment of the CD8+ specific response. Therefore, Nef downregulates Major Histocompatibility Class I expression on dendritic cells, impairing presentation to HIV-specific CD8 cells. This action of Nef, which probably induces a deleterious delay in the early CD8+ responses during the first days of infection and at the onset of new viral mutants, can be triggered by several therapeutic options.

 

 

O-011

 

 Strong impairment of the stimulatory function of monocyte-derived dendritic cells found in chronic carriers of hepatitis C virus but not in long term responders to therapy 

CHRISTINE BAIN¹, A. Fatmi¹, Z. Zoulim¹, C. Trepo¹, G. Inchauspe¹ and J.P. Zarski^{2 

 1}INSERM, INSERM U 271, 151 Cours Albert Thomas, Lyon 69003, France

Chu Grenoble, Grenoble, France

 

The mechanisms underlying the progression to a chronic carrier state following HCV infection are not well understood. Dendritic cells (DC) are professional antigen presenting cells required for the initiation of primary immune responses but have also been involved, in many viral infections, in the establishment of viral persistence. Here, we report that in vitro monocyte-derived DCs from 40% of chronic carriers can harbor HCV genomic sequences at different stages of maturation of the DCs. During DC cultures, sequences of a unique nature, distinct from those identified in the same patient's serum, liver biopsy and peripheral blood mononuclear cells, were selected, not only within the hypervariable region 1 (HVR1), but also within regulatory domains important for the expression of viral proteins. At the immature and mature stages, these DC display a normal morphology and phenotype, expressing molecules important for antigen presentation (i.e. MHC antigens and costimulatory molecules). The capacity of immature DC to capture antigen via the mannose receptor was comparable to that of DC derived from naive individuals. However, their ability, at the mature stage, to stimulate the proliferation of allogeneic T cells was dramatically impaired in comparison with DC from nafve controls (p=0.0013). An active immunosuppressive mechanism was excluded since DC derived from chronic patients did not exert an active inhibitory effect on allogeneic MLR induced by DC from seronegative individuals. We could neither show a dysregulation of the production of interleukin-12 (IL-12) or IL-10 after stimulation of DC derived from chronic patients with lipopolysaccharide when compared with normal individuals. However, the allostimulatory function of DC derived from HCV patients was clearly correlated with the disease state since this function was restored in long term responders to antiviral therapy (p<0.0001). Overall, these results suggest that, independent of the viral load and infecting genotype, during natural infection with HCV, monocyte-derived DC appear to constitute an extrahepatic reservoir for specific HCV variants. Impairment of their antigen presenting cell (APC) function could play a critical role in the development of viral persistence in vivo.

 

 

O-012

 

 Functional mucosal immune response to HIV-1 mediated by IGA 

Pia Skott^1,2, Mariethe Ehnlund^1,2, Erik Lucht³, Joakim Dillner¹ and EWA BJÖRLING^{1,2 

 1}Microbiology and Tumorbiology Center, Karolinska Institute, Theorells vag 3, Stockholm S-171 77, Sweden

 ²Research Center, The South hospital, Karolinska Institute, Stockholm, Sweden

 ³Department of Infectious Diseases, Huddinge University Hospital, Sweden

 

Human immunodeficiency virus (HIV) infection is transmitted predominantly through sexual intercourse and transmission requires the dissemination of virus from sites of infection at mucosal surfaces to T cell zones in secondary lymphoid organs. In mucosal transmission there are a number of barriers that need to be passed before the infection is disseminated by blood. The first barrier is the epithelia surface where viral adhesion occurs may be prevented by sIgA and IgG antibodies to the HIV envelope glycoprotein gp 120.

In this study we have investigated the in vitro function and capacity of HIV-1 specific immune responses in saliva in comparison to the immune responses in serum during natural infection. By affinity chromatography we have purified antibodies of both IgG and IgA isotypes from pairwise sequential saliva and serum samples. The variation between different HIV-1 positive individuals are considerably high, suggesting that some patients have a broad spectra of sIgA specificities, while others do not express any HIV-1 specific sIgA mediated response.

Purified IgA antibodies were then used in in vitro neutralization assays of HIV-1 infection of both T-cell adapted strains (TCLA) and primary isolates of TCLA of syncytial inducing (SI) and non-syncytial inducing (NSI) phenotypes.

IgA-mediated neutralization was recorded in this study against both TCLA strains and the tested primary isolates. A great interindividual variance in neutralizing capacity was observed, indicating that the presence of potent anti-HIV-1 IgA antibodies is not a general phenomenon but can be observed on an individual basis, even in late stages of disease.

 

 

O-013

 

 Surrogate animal models for HCV 

HELEN BRIGHT, Debbie Mesoghoti, Mathew Cleveland, Nigel Parry and Tony Caroll

Department of Immunology and Virology, GlaxoWellcome Research, Gunnells Wood Road, Stevenage SG1 2NY, UK

 

The lack of an in vitro replication system and a small animal model has hindered the discovery of novel therapies for HCV. However, the recent molecular characterisation of GBV-B, a closely related flavivirus isolated from infected tamarins (Sanguinus sp), has opened up new opportunities to study HCV disease using GBV-B as a surrogate virus. GBV-B is classified in the same virus family as HCV. This study describes the infection of four red-bellied tamarins (Sanguinus labiatus) with GBV-B infectious sera and the characterisation of the disease by monitoring viral and liver enzyme levels in the serum. One animal was culled 32 days post infection and its sera used as an infectious stock (titre 1.0 E+10 genome equivalents per ml (ge/ml)). The remaining 3 animals were monitored for 100 days p.i. These animals showed viral loads of 1.0 E+08 to 1.0 E+10 ge/ml by 10 days p.i. and these levels were maintained for 60–70 days. Two of the three infected tamarins showed raised levels of ALT and GLDH between 30 and 90 days pi. Both these animals cleared virus by 100 days pi. and had anti-GBV-B antibody. The remaining animal maintained normal liver enzyme levels throughout the infection and had high levels of viral RNA in the serum at 100 days pi. No anti-GBV-B antibodies were detected in the serum of this animal. The livers of all the infected tamarins showed evidence of hepatitis with periportal lymphocytic infiltration and a qualitative increase in collagen deposition around portal tracts typical of early stage fibrosis. Further work is ongoing to validate this model.

 

 

O-014

 

 Engineering coronavirus RNA genome as an infectious bacterial artificial chromosome 

LUIS ENJUANES¹, Fernando Almazan¹, Jose M Gonzalez¹, Zoltan Penzes¹, Ander Izeta¹, Enrique Calvo¹, Juan Plana-Duran^{2 

 1}Centro Nacional de Biotecnologia, CSIC Department of Molecular and Cell Biology, Campus Universidad Autonoma, Madrid 28049, Spain

 ²Fort Dodge Veterinaria, Olot, Spain

 

The construction of cDNA clones encoding large size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. We have shown that the application of two strategies: cloning of the cDNAs into a bacterial artificial chromosome (BAC) and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a BAC. The rescued coronavirus conserved all the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one and no splicing was observed. The cDNA was derived from an attenuated isolate that exclusively replicates in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike (S) gene of the virus was replaced by the S gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus Gan be modified. This opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine and feline species susceptible to group 1 coronaviruses. In fact, TGEV cDNA has been successfully used to express large amounts (>40 mg/10⁶ cells) of green fluorescent protein.

 

 

O-015

 

 Recognition of RNA encapsidation signal by the yeast L-A double-stranded RNA virus 

TSUTOMU FUJIMURA and Rosa Esteban

Inst. Microbiología Bioquímica CSIC/Univ. Salamanca, Edificio Deparamental. Avda. del Campo Charro s/n, Salamanca 37007, Spain

 

The yeast L-A virus has a linear dsRNA genome with two open reading frames. The 5′ORF is Gag and encodes the major coat protein. The 3′ORF is Pol and encodes RNA-dependent RNA polymerase which is expressed only as Gag-Pol fusion protein by a −1 ribosomal frameshifting mechanism. The Pol region of the fusion protein has single-stranded binding activity and is responsible for encapsidation of the viral RNA. The encapsidation signal of L-A contains a 24 nucleotide stem-loop with an A bulged at the 5′ side of the stem. Opened empty particles specifically bind to this encapsidation signal in vitro. We found that binding to empty particles protected the bulged A and the surrounding two nucleotides from cleavage by Fe(II)-EDTA-generated hydroxyl radicals. Interestingly the loop sequence (4190-GAUCC-4194) was not protected. However, RNase-protection and in vitro mutagenesis experiments indicated that G-4190 of the loop is essential for binding. Although the sequence of the other four nucleotides of the loop is not essential, data from RNase-protection and chemical modification experiments suggested that base C at 4194 was also directly involved in binding to empty particles, rather than indirectly through its potential base-pairing with G at 4190. These results, therefore, suggest that the Pol domain of the fusion protein contacts the encapsidation signal at two sites: one, the bulged A and the other, G and C bases at the opening of the loop. These two sites are conserved in the encapsidation signal of M1, a satellite RNA of the L-A virus.

 

 

O-016

 

 RNA binding properties of Bunyamwera virus nucleocapsid protein 

JANE OSBORNE and Richard M. Elliott

Institute of Virology, University of Glasgow, Church Street, Glascow G11 5JR, UK

 

The genome of Bunyamwera (BUN) virus (family Bunyaviridae, genus Bunyavirus) comprises three negative-sense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5′ terminus of each segment. The BUN N protein was expressed as a 6-histidine tagged fusion protein in E. coli and purified by metal chelate chromatography. An RNA probe containing the 5′ terminal 32 and 3′ terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter-binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 μg/ml RNase A. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5′ terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.

 

 

O-017

 

 Intra-cellular retrotransposition of an exogenous primate retrovirus 

AXEL RETHWILM¹ and Martin Heinkelein^{2 

 1}Technische Universitaet Dresde, Institut fuer Virologie, Gerichtsstr. 5, Dresden 01069, Germany

 ²Institut fuer Virologie und Immunbiologie, Versbacher Str.7, 97078 Wuerzburg, Germany

 

The foamy virus (FV) subgroup of Retroviridae reverse transcribe their RNA (pre-)genome late in the replication cycle before leaving an infected cell. We studied whether a marker gene-transducing FV vector is able to reshuttle to the nucleus and integrate into host cell genomic DNA. While a potential intra-cellular retrotransposition of vectors derived from other retroviruses was below the detection limit of our assay, we found that up to 5% of cells transfected with the FV vector were stably transduced and harboured up to 10 vector integrates. The generation of the integrates depended on the expression of functional capsid, reverse transcriptase, and integrase proteins and did not involve an extra-cellular step. PCR analysis of the U3 region of the 5′ long terminal repeat and determination of proviral integration sites showed that a reverse transcription step had taken place to generate the integrates. Coexpression of a mutated envelope allowing particle egress and avoiding extra-cellular infection resulted in a significantly increased rescue of cells harbouring integrates suggesting that accumulation of proviruses via intra-cellular retrotransposition is an integral strategy of FV replication.

 

 

O-018

 

 Response to foot-and-mouth disease virus to increased mutagenesis. Influence of viral load and fitness in loss of infectivity 

SALETA SIERRA ARAGÓN¹, Mercedes Dávila¹, Pedro Lowenstein² and Esteban Domingo^{1 

 1}Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Campus de Cantoblanco, Facultad de Ciencias, E-28049 Madrid, Spain

 ²University of Manchester, Department of Medicine, Molecular Medicine Unit, Stopford Building, Oxford Road, Manchester, M13 9PT, UK

 

Passage of foot-and-mouth disease virus (FMDV) in cell culture in the presence of the mutagenic base analogs 5-fluorouracil or 5-azacytidine resulted in decreases of infectivity and occasional extinction of the virus. These mutagenic treatments led to 2-to-6-fold increases in mutation frequency. The highest increase observed corresponded to the 3D (polymerase)-coding region, which remained very conserved in nonmutagenized FMDV populations. The mutant spectrum heterogeneity for the 3D-coding region became very similar to that of the variable VP1-coding-region for mutagenized populations, but not for populations passaged in parallel in the absence of mutagen.

Low viral fitness enhanced the frequency of extinctions in the presence of mutagens, as shown with closely related FMDV clones differing up to 10⁵-fold in relative fitness. Passages at low multiplicity of infection increased the frequency of extinctions, while large viral loads allowed extinction mutagenesis of low fitness viruses only. These results suggest that strategies combining reductions of viral load and viral fitness with increased mutagenesis could be effective as a potential antiviral strategy.

 

 

O-019

 

 Follicular dendritic cells support replication of the ME7 strain of scrapie 

KAREN BROWN, Diane L. Ritchie and Moira E. Bruce

Institute for Animal Health, Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK

 

The immune system is central to the pathogenesis of scrapie and other transmissible spongiform encephalopathies. After infecting by peripheral routes most TSE agents replicate in spleen and lymph nodes before neuroinvasion. Characterisation of the cells supporting replication in these tissues is important for understanding early pathogenesis and may indicate potential targets for therapy. The host protein PrP is required for TSE agent replication and accumulates in modified forms in infected tissues. Abnormal PrP is detected on follicular dendritic cells in lymphoid tissues of mice experimentally infected with scrapie (1), in sheep with natural scrapie and in humans with ‘new-variant’ Creutzfeldt–Jakob disease (CJD).

Previous studies using ionising radiation (2) and severe combined immunodeficient mice (SCID) (3) produced strong evidence for an involvement of FDCs in scrapie replication. To clarify the role of FDCs we produced chimaeric mice with a mismatch in PrP status between FDCs and other cells of the immune system by grafting PrP+/+ or PrP-/- bone marrow into PrP+/+/PrP-/- or SCID PrP+/+/PrP-/- mice. Using these models we obtained strong evidence that replication of the ME7 strain of scrapie depends on PrP-expressing FDCs (4). Confocal analysis of spleen from SCID-chimaeric mice demonstrated co-localisation of PrP and FDCs only when the recipient of the bone marrow graft carried a functional PrP gene suggesting that FDCs themselves, which are not bone-marrow derived, produce PrP protein.

 

(1) McBride, P.A. et al. PrP protein is associated with follicular dendritic cells of spleens and lymph nodes in uninfected and scrapie infected mice. Journal of Pathology 168, 413–418 (1992).

(2) Fraser, H. & Farquhar, C.F. Ionising radiation has no influence on scrapie incubation period in mice. Veterinary Microbiology 13, 211–223 (1987).

(3) Fraser, H. et al. Replication Of Scrapie In Spleens Of Scid Mice Follows Reconstitution With Wild-TypeMouse Bone-Marrow. Journal Of General Virology 77, 1935–1940 (1996).

(4) Brown, K.L. et al. Scrapie replication in lymphoid tissues depends on prion protein expressing follicular dendritic cells. Nature Medicine 5, 1308–1312 (1999).

 

 

O-020

 

 Human retrovirus-5: a novel retrovirus in inflammatory disease 

DAVID GRIFFITHS, Alex Brand^1,2, Cécile Voisset¹, Chris Hervé², Patrick J.W. Venables² and Robin A. Weiss^{1 

 1}Wohl Virion Centre, Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1P 6DB, UK

 ²Kennedy Institute of Rheumatology, 1 Aspenlea Road, London W6 8LH, UK

 

We have previously reported the discovery of a novel retroviral sequence in human tissues. This sequence is not endogenous in the human genome and so appears to represent a novel infectious human retrovirus, provisionally designated human retrovirus-5 (HRV-5). Preliminary studies suggested that HRV-5 might be a common infection in humans but with an extremely low viral load (Griffiths et al, (1997) J Virol, 71:2866–2872). More recently, we have shown that the HRV-5 load is increased in patients with arthritis and systemic lupus erythematosus. Using a nested PCR assay, HRV-5 DNA was detected in 50% of synovial membrane biopsies from patients with arthritis and 12–16% of peripheral blood samples from patients with arthritis or lupus were also positive for the virus. In contrast, HRV-5 was detected only very rarely in normal tissues or in other inflammatory conditions (Griffiths et al (1999) Arthritis Rheum, 42:448–454). Our current work is focusing on the development of a virus culture system and on the development of serological assays for HRV-5 infection. Recent data will be presented.

 

 

O-021

 

 Infection of monocyte-derived dendritic cells with human cytomegalovirus leads to suppressed allostimulation and downregulation of MHC class I and CD80 molecules 

FRANK T. HUFERT, Ursula Meyer-König, Kerstin Beck, Christian Nern

Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Hermann-Herder-Str. 11, 79104 Freiburg, Germany

 

Herpesviruses, such as the human cytomegalovirus (HCMV) establish a persistent infection within the host and have developed different mechanisms to escape the host immune defence. Here, we analyzed whether antigen presentation mediated by dendritic cells (DC) is disrupted by HCMV infection. First, we analyzed whether DC are permissive for the HCMV. DC were generated from monocytes in the presence of IL-4 and GM-CSF and infected with endothelial cell-(ECV) or human foreskin fibroblastadapted (FFV) clinical isolates. An increase in viral DNA was measured by quantitative real time TaqMan PCR in ECV-infected DC, only. Immediate early (IE) protein was found in FFV-infected DC by double immunofluorescence, whereas IE as well as the matrix proteins (pp65, pp150) were detected in ECV-infected DC. These data show that DC are fully permissive for EC-adapted HCMV strains, only, whereas HCMV infection with FFV abrogates at the level of IE gene expression. Furthermore, we could show that HCMV infected DC downregulate MHC class I molecules as well as the cosignal CD80, thus leading to an impaired T cell response during antigen presentation. This was documented by a suppressed allogeneic mixed lymphocyte reaction (MLR). In conclusion, we could show that HCMV has developed a mechanism to impair the initiation of the immune response.

 

 

O-022

 

 Antigenic drift in the influenza a virus (H3N2) nucleoprotein and escape from recognition by cytotoxic T lymphocytes 

ALBERT OSTERHAUS, J.T.M. Voeten, T.M. Bestebroer, N.J. Nieuwkoop, R.A.M. Fouchier and G.F. Rimmelzwaan

Dept. of Virology, Erasmus University Rotterdam, Dr. Molewaterplein 50, 300 DR Rotterdam, The Netherlands

 

Viruses exploit different strategies to escape immune surveillance, including the introduction of mutations in cytotoxic T lymphocyte (CTL) epitopes. The sequence of these epitopes is critical for their binding to MHC class-I molecules and recognition by specific CTI-s, both of which interactions may be lost by mutation. Sequence analysis of the nucleoprotein gene of influenza A viruses (H3N2) isolated in The Netherlands from 1989 to 1999 revealed two independent amino acid mutations at the anchor residue of the HLA-1327 specific CTL epitope SRYWAIRTR (383–391). A R384K mutation was found in influenza A viruses isolated during the influenza season 1989-1990 but not in subsequent seasons. In the influenza season 1993–1994, a novel mutation in the same CTL epitope at the same position was introduced. This R384G mutation proved to be conserved in all influenza A viruses isolated from 1993 onwards. Both mutations R384K and R384G abrogated MHC class-I presentation and allowed escape from recognition by specific CTI-s.

 

 

O-023

 

 Multimeric humanised anti-varicella zoster virus (VZV) antibody fragments neutralise VZV 

ANDY PORTER¹, P.D. Drew¹, M. Moss¹, C. Grose², K. Duus², C. Hatfield² and W.J. Harris^{1 

 1}University of Aberdeen, Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK

 ²University of Iowa, Department of Microbiology, Iowa City, IA 52242, USA

 

Viral coat glycoproteins are the sites of attachment and fusion of varicella-zoster virus (VZV) to the host cell and are important for cell to cell spread. Murine monoclonal antibody 206 (mAb mu206) binds to glycoprotein H, neutralising VZV in vitro in the absence of complement and inhibiting cell to cell spread of VZV in cultured cells. We have humanised this antibody to generate mAb hu206 by complementarity determining region grafting. MAb hu206 retains binding and in vitro neutralising activity, and cross-reactivity with ten clinical isolates. The biological activity of the humanised antibody is around 2400 times that of standard varicella-zoster immunoglobulin (VZIG) preparation on a mg for mg basis. For shingles treatment, topical delivery of this antibody may be preferable for penetration to neural tissues, and to this end singlechain antibody fragments (scAb hu206) were produced in Escherichia coli. These antibody fragments retain the binding properties of the whole antibody, however, monomeric scAb exhibited significantly reduced neutralising activity compared to the bivalent mAb hu206. The neutralising activity of scAb hu206 could be enhanced by the inclusion of a secondary antibody specific for the human constant kappa chain of the scAb. Shortening the peptide linker joining the VH to the VK domain from 14 to 5 or even 0 residues encourages multimerisation. These diabodies had neutralising efficacy closer to that of the parental monoclonal. These results may be explained by the need for viral receptor cross-linking for neutralisation, or that the antibody must have sufficient size for steric inhibition of the fusion process.

 

 

O-024

 

 A spliced C-type lectin-like gene is encoded by rat cytomegalovirus 

SEBASTIAN VOIGT, Gordon R. Sandford and William H. Burns

Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA

 

We have found the first C-type lectin-like homologue in herpesviruses. Interestingly, this gene in the English strain of rat cytomegalovirus (RCMV) is spliced into five exons and shows a splicing pattern with exon boundaries similar to that of the human differentiation antigen CD69. Genomic sequence analysis of the clone KPN R of RCMV with the BLAST programme revealed significant homology to several other C-type lectin-containing molecules implicated in natural killer (NK) and T cell interactions, as well as genes from four poxviruses and the unclassified African swine fever virus. The cap site of the gene was mapped by 5′-RACE and primer extension experiments and splicing patterns confirmed. The core promotor of the RCMV lectin-like gene contains a GATA rather than a TATA box. Also unusual, translation of the protein does not seem to be initiated by a methionine since there is none in the open reading frame. The gene is expressed in the delayed early and late phases of the viral replication cycle. 293T cells transiently transfected with a KPN R-FLAG fusion plasmid stained positive upon indirect detection with FLAG and FITC-conjugated antibodies whereas control-transfected cells did not, indicating the expression of a protein. Western blot analysis showed a protein of 36 kDa that diminished to 22 kDa when glycosylation was inhibited. To study its role in virus replication and pathogenesis, a recombinant virus was constructed in which the gene is interrupted, and in vivo experiments are under way.

 

 

O-025

 

 The natural course of varicella zoster virus (VZV) DNA load in blood during chickenpox 

MENNO D. DE JONG, T. Schuurman, W.R. Boom, P.M.E. Wertheim-van Dillen and J.F.L. Weel

 ¹Dept. of Medical Microbiology, section Clinical Virology, Academic Medical Centre, University of Amsterdam, AMC L1-104, Postbox 22660, Amsterdam 1100 DD, The Netherlands

 

 Background and methods: Although it is assumed that viremia during chickenpox peaks around the onset of the skin rash, this has never been fully documented due to lack of quantitative assays. Using an internally controlled quantitative PCR assay targeted at gene 29 (J. Clin. Microbiol., in press), we measured VZV DNA levels in single plasma, serum or whole blood specimens, obtained within 7 days prior to 10 days after the onset of the rash, from 26 untreated immunocompetent chickenpox-patients. The course of viremia was estimated by analyzing VZV DNA levels in relation to the number of days since onset of the rash using linear regression.

 Results: VZV DNA was detected in 14/16 plasma or serum specimens, and 9/10 whole blood samples, at levels ranging from 60 to 100 000 copies/ml. Negative samples were only obtained beyond 8 days after rash onset. Highest VZV DNA levels were observed around the onset of the rash. Linear regression showed a significant inverse relation between VZV DNA levels in plasma or serum and the number of days after rash onset (slope: −0.27; r: −0.68; p=0.005). Highly similar findings were observed in whole blood.

 Conclusions: Despite the assumed cell-associated nature of VZV viremia, high levels of VZV DNA are detectable in plasma and serum during chickenpox. While the origin of VZV DNA in these compartments requires further study, they provide convenient matrices for PCR diagnosis. Our results lend support to the assumed course of viremia during chickenpox. Quantitative analyses during treatment of VZV infections are ongoing.

 

 

O-026

 

 Comparative analysis of human papillomavirus infections in cervical scrapes and biopsy specimens by general SPF10 PCR and LiPA genotyping 

BERNHARD KLETER¹, L.J. van Doorn¹, G. Scholte², P.H.M. Smits³, J Lindeman² and W.G.V. Quint^{1 

 1}Delft Diagnostic Laboratory, R. de Graafweg 7, Delft 2625 AD, The Netherlands

 ²Department of Pathology, Slotervaart Hospital, Amsterdam, The Netherlands

 ³Department of Molecular Biology, Slotervaart Hospital, Amsterdam, The Netherlands

 

The broad-spectrum SPF10 PCR primers were used to analyze the HPV-status in 174 women with a history of either two consecutive cervical smears, classified as ASCUS or mild dysplasia, or with one cervical smear showing moderate dysplasia. Cervical scrapes and biopsies were obtained on the same day (group 1), or with an interval of up to more than 2 years (group 11, mean 97 days). HPV-positive cases were genotyped by reverse hybridization of the same amplimer on an INNO-LiPA HPV prototype research assay, detecting 25 different genotypes.

In group 1, HPV-DNA was detected in both the cervical smear and the biopsy specimen (n=100), 64 (64%) yielded concordant genotyping results, whereas genotypes in 4 (4%) were discordant. Among the 32 (32%) cases with non-identical but compatible genotypes (at least one common genotype), 24 (24%) contained additional genotypes in the cervical scrape, as compared to the corresponding biopsy specimen. Conversely, in 6 (6%) cases, additional genotypes were found in the biopsy specimen, and in 2 (2%) of the patients, both the scrape and the biopsy specimen contained additional genotypes. Results in group II were highly similar. In both groups, multiple HPV genotypes were more frequently detected in cervical scrapes (35–43%) as compared to the corresponding biopsy specimens (17–24%).

In conclusion, analysis of cervical scrapes accurately reflects the spectrum of HPV genotypes in the patient's cervical region. SPF10 PCR-LiPA offers a valuable tool for clinical and epidemiological studies and the development of type-specific vaccines.

 

 

O-027

 

 A novel predictor for response to therapy of Epstein–Barr virus (EBV) associated lymphoproliferative disease after allogeneic stem cell transplantation (allo-SCT) 

HUBERT G.M. NIESTERS¹, J.W.J. van Esser², S.F.T. Thijsen³, E. Meijer⁴, A.M. van Loon³, J.W. Gratama⁵, L. Budel⁶, R. van der Holt⁷, B. Löwenberg², L.F. Verdonck⁴, A.D.M.E. Osterhaus¹ and J.J. Comelissen^{2 

 1}Department of Virology, University Hospital Rotterdam, Dr Molewaterplein 40, Rotterdam 3015 GD, The Netherlands

 ²Department of Hematology, University Hospital Rotterdam, Rotterdam, The Netherlands

 ³Department of Virology, University Hospital Utrecht, Utrecht, The Netherlands

 ⁴Department of Hematology, University Hospital Utrecht, Utrecht, The Netherlands

 ⁵Department of Medical and Tumorimmunology, University Hospital Rotterdam, Rotterdam, The Netherlands

 ⁶Department of Pathology, University Hospital Rotterdam, Rotterdam, The Netherlands

 ⁷Department of Statistics, University Hospital Rotterdam, Rotterdam, The Netherlands

 

Epstein–Barr virus lymphoproliferative disease (EBV-LPD) is a very serious complication of allo-SCT with an extremely poor prognosis. Recently we developed a sensitive real-time Taqman assay for quantitative detection of EBV-DNA in plasma. Using that assay we set out to assess the value of closely following EBVDNA plasma levels for monitoring and predicting response to treatment and subsequent survival. Fourteen patients from two centers, who received a sibling (n=4) or matched unrelated donor (MUD, n=10) graft were monitored frequently at the time of EBV-LPD diagnosis, during therapy until assessment of clinical response. Therapy modalities included: no therapy (n=l) interruption of immunosuppression (SI, n=7), acyclovir (ACV, n=7), chemotherapy (CTX, n=2), anti-CD 20 monoclonal antibody (MA, n=6) and donor lymphocyte infusion (DLI, n=8). Seven patients had a complete response to therapy. Survival at six months from EBV-LPD diagnosis was 3/14 (21%). Causes of death included progressive EBV-LPD (n=7), graft versus host disease (GVHD, n=3) and relapsed AML (n=l). Absolute levels of EBV-DNA at the time of EBV-LPD diagnosis did not predict for response or survival. In contrast, the pattern of EBV-DNA level (50% decrease versus increase) within 72 hours from the start of therapy significantly predicted for clinical response to therapy. No association was observed between the patterns of LDH-levels and clinical response. Lymphopenia was associated with poor outcome (p=0.001). We conclude that quantitative monitoring of EBV-DNA levels from the start of and during therapy for EBV-LPD predicts for response to therapy and allows for individual tailoring of different treatment modalities.

 

 

O-028

 

 Worldwide evaluation of HIV-1 drug resistance genotyping quality using the ENVA-2 panel 

ROB SCHUURMAN¹, Don Brambilla³, Tom de Groot², Ireen Benders² and Charles Boucher^{2 

 1}Dept. of Virology, University Medical Center, Heidelberglaan 100, Utrecht 3584 CX, The Netherlands

 ²ENVA, University Medical Center, Utrecht, The Netherlands

 ³NERI, Boston, USA

 

Genotypic HIV-1 drug resistance determinations currently applied in the absence of any inter-laboratory standardization. A study was performed to evaluate the accuracy of sequencing protocols in detecting mutations and sequence heterogeneities in the HIV-1 RT and protease genes.

A coded panel of five plasma samples (ENVA-2 panel), infected with different proportions (0–100%) of two HIV-1 strains, was sent to 60 experienced genotyping laboratories worldwide and analysed by nucleotide sequencing. A total of 38 datasets were received from 35 laboratories. Samples that were 100% wild type over five resistance-associated sites in each gene were correctly identified in most laboratories (RT: 100%; protease: 94%). Fewer completely correct sequences were obtained when samples were 100% mutant at these sites (RT: 66%; protease: 71%). All five mutations were detected in mixtures of 50% wild type and 50% mutant in less than half of the laboratories for each of the genes, independent of the sequencing technology used.

In samples harboring an equal mixture of wildtype and mutant, as well as in completely mutant samples, the presence of a mutant genotype was underestimated. This may have implications for clinical application of genotyping results. Ongoing quality assurance monitoring is warranted.

 

 

O-029

 

 Kaposi's sarcoma associated herpesvirus gene expression during latnecy and lytic replication as revealed by DNA arrays 

PAUL KELLAM², Chris Boshoff^1,2 and Richard Jenner^{2 

 1}Cancer Research Campaign Viral Oncology, Department of Oncology, University College London, London, UK

 ²Wohl Virion Centre, Dept. of Molecular Patholgy, Windeyer Inst. University College London, 46 Cleveland Street, London W1P 6DB, UK

 

Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) is associated with three human tumors, Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV encodes a number of homologs of cellular proteins involved in the cell cycle, signal transduction and modulation of the host immune response. Of the virus complement of over 85 open reading frames (ORFs), the expression of only a minority has been characterised individually. We have constructed a nylon membrane based DNA array which allows the expression of every known ORIF of KSHV to be measured simultaneously. A PEL-derived cell line, BC-3, was used to study the expression of KSHV during latency and after the induction of lytic replication with Tetradecanoylphorbol Acetate (TPA). Multiple samples were taken over 72 hours of lytic replication and the expression of all HHV8 genes was determined. Cluster analysis, a bioinformatics; tool that arranges genes according to their expression profile, revealed a correlation between expression and assigned gene function that is consistent with the known stages of the virus life cycle. In addition, because of the global nature of such analysis much information is gained into the functions of viral genes.

 

 

O-030

 

 Kaposi's sarcoma associated herpesvirus (KSHV) vflip protein is expressed from a bicistronic message via an internal ribosome entry site (IRES) 

SIMON TALBOT

University of Edinburgh Laboratory of Clinical & Molecular Virology Royal (Dick) Veterinary School, Summerhall, Edinburgh EH9 1QH, UK

 

We have previously examined the transcription and splicing of open reading frames (orfs) 71 (K13), 72 and 73 of Kaposi's sarcoma associated herpesvirus (KSHV) in the primary effusion lymphoma cell line BCP-1 (latently infected with KSHV) (Virology 257, 84–94 [1999]). The three genes encoded by these orfs (v-FLIP, v-Cyclin, LNA) are transcribed from a common transcription start site in BCP-1 cells during both latency and lytic cycles. The resulting transcript is spliced to yield a 5.32 kb message encoding LNA, v-Cyclin and vFLIP, and a 1.7 kb bicistronic message encoding v-Cyclin and v-FLIP. To investigate whether the v-FLIP protein could be expressed from this v-Cyclin/v-FLIP message we utilised a bicistronic luciferase reporter system. The genes for Renilla and Firefly luciferase (which utilise different substrates) were cloned in tandem downstream from a T7 RNA polymerase promoter. Fragments of DNA immediately upstream from the initiating codon of v-FLIP were cloned between the two luciferase genes. The relative expression of the two luciferases, one directed by the putative IRES sequences and the other by cap-dependent ribosome scanning were used to compare the activities of the different DNA fragments. A minimum fragment, 495bp immediately upstream from the v-FLIP start codon was found to direct efficient expression of the downstream (Firefly) luciferase. The activity of this IRES was orientation dependent and enhanced by methods used to inhibit cap-dependent translation. This is the first demonstration of an IRES element encoded by a DNA virus and may represent a novel mechanism whereby KSHV controls protein expression.

 

 

O-031

 

 An AU-rich inhibitory sequence on the late human papillomavirus type 1 mRNAs 

STEFAN SCWARTZ, Marcus Sokolowski, Margaret Rush and Anette Carlsson

Department of Medical Biochemistry & Microbiology, BMC, Uppsala University, Husargatan 3, Box 582, Uppsala 751 23, Sweden

 

The human papillomavirus (HPV) life cycle is strictly linked to the epithelial differentiation program and production of virus particles is dependent on terminal cell differentiation. The virus structural proteins Ll and L2 are therefore detected only in the upper layers with differentiated cells in the infected epithelium. The abundance of virus particles detected in vivo may vary between the various HPV types. Our research group is interested in the regulation of expression of the HPV structural proteins Ll and L2. We found that the HPV-1 late mRNAs; contain an inhibitory sequence in the 3′ UTR. It was mapped to a 57 nucleotide, AU-rich sequence with two AUUUA- and three UUUUU-motifs as the functional elements. Replacing two Us with two Cs in each of the five elements inactivated the inhibitory sequence. The element acts via a bimodal mechanism and the presence of the inhibitory sequence on the mRNA results in rapid degradation and inhibition of translation. These two effects are studied separately. Our results demonstrate that the AU-rich element inhibits translation of capped and polyadenylated mRNAs, suggesting that factors interacting with the inhibitory sequence disturb the interaction between the polyA tail and the cap structure. A cytoplasmic 50 kDa protein that binds specifically to the AUUUA elements has been identified in addition to the nuclear HuR and hnRNP C proteins. The interplay between the cellular factors and the AU-rich inhibitory RNA element may regulate the Ll and L2 expression levels in a cell differentiation dependent manner.

 

 

O-032

 

 Expression of transcriptional units engineered with coronavirus derived minigenomes and full-length genomes 

S. Alonso, I. Sola, C.M. Sánchez, A. Izeta and LUIS ENJUANES

Centro Nacional de Biotecnología (CSIC) Department of Molecular and Cell Biology, Campus Universidad Autonoma, Madrid 28049, Spain

 

The expression of an engineered transcriptional unit has been studied using a helper dependent expression system based on transmissible gastroenteritis virus (TGEV)-derived minigenomes. RNA expression levels have been increased using a two-step amplification system based on CMV promoter that couples polll driven transcription in the nucleus to replication supported by helper virus polymerase in the cytoplasm. A collection of TGEV transcription regulatory sequences (TRSs) including a variable number of nucleotides flanking the core sequence CUAAAC, at both the 5′ and 3′ ends, was introduced in the minigenome. The expression of the engineered mRNA encoding the b-glucuronidase (GUS) was evaluated in vitro and in vivo. The expression of the GUS gene was stable for about 10 passages in tissue culture and expression levels of around 8 μg per 10⁶ cells of GUS protein were obtained under optimal transcription regulatory sequences.

To overcome the limitations of the helper-dependent system, a single-genome expression vector has being developed by promoting targeted RNA recombination between the helper virus and the minigenome carrying the foreign transcriptional unit. Also an infectious cDNA clone derived from TGEV has been engineered. Expression of high levels (>40 mg/10⁶ cells) of green fluorescent protein (GPT) has been achieved with this cDNA. The infectious cDNA is very promising as a vaccinial vector to induce secretory immunity and for gene therapy.

 

 

O-033

 

 Characterization of novel human i-mfa domain-containing protein (HIC) that regulates tax-mediated HTLV-1 promoter activation 

SABINE THÉBAULT, C. Devaux and J.-M. Mesnard

Laboratoire ‘Infectious Retrovirales et Signalisation Cellulaire’, Institut de Biologie, 4 Bd. Henri IV, 34060 Montpellier, France

 

Regulation of the viral genome expression is the result of complex cooperations between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the bHLH-repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40 respectively. We show that the HIC protein isoforms present difference subcellular localizations, p32 being mainly distributed throughout the cytoplaxm whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T cells the expression of a luciferase reporter gene driven by the HTLV-I long terminal repeat (LTR) in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Lastly, we also show that HIC is able to down-regulate the luciferase expression from the HIV-1 LTR induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of the new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain.

 

 

O-034

 

 The United Kingdom national external quality assessment scheme for microbiology HIV-1 RNA quantification proficiency scheme 

V.L.A. JAMES, K. Gerrard, J. Hawkins, K. O'Hara and J.V. Parry

Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK

 

The United Kingdom National External Quality Assessment Scheme for Microbiology has piloted four proficiency test distributions for HIV-1 RNA quantification. Each distribution consisted of a pair of specimens derived from a single HIV-1 RNA positive plasma diluted in normal human plasma. Specimens were freeze-dried and tested in six reference laboratories before distribution. In the clinical interpretation of RNA quantification assay results, a difference of more than 0.5 log₁₀ copies/ml between sequential specimens is considered significant. The scheme was therefore designed to focus on the difference in log₁₀ HIV RNA copies/ml between the paired samples rather than absolute values. This would also avoid bias due to observed differences in absolute values obtained with different manufacturers’ kits. The dilutions employed in the preparation of the specimens resulted in a difference between the two specimens of approximately one log₁₀ copy/ml. For the first distribution the specimens were designed to have in the order of 2 log₁₀ copies/ml and 3 log₁₀ copies/ml. For the other three distributions specimens were designed to have in the order of 3 log₁₀ copies/ml and 4 log₁₀ copies/ml. The percentages of laboratories reporting a difference in log₁₀ concentration of 1±0.5 were 77%; 95%; 95% and 96% respectively.

 

 

O-035

 

 Laboratory variation in HIV viral load testing; the independent run control program 

ROB SCHUURMAN¹ and Jolanda Tijnagel^{2 

 1}Dept. of Virology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands

 ²International Molecular Services, Utrecht, The Netherlands

 

HIV quantification assays are widely applied to determine and monitor viral load in patients on antiviral therapy. Independent monitoring of inter-laboratory and inter-kit lot performance characteristics may be important tool when performing multicenter research and clinical trials.

In an attempt to set up such a monitoring system, an Independent Run Control (IRC) containing 10 000 RNA copies/ml was developed using a EM quantified HIV-1 viral stock. The IRC was aliquoted in single use 250 μl volumes and distributed among 35 participating laboratories using the Amplicor Monitor assay. The IRC was run in parallel to the regular kit controls in every viral load run and results of the IRC, the kit controls, and additional information on the kit lot number, kit version, assay type (normal or ultrasensitive), technician name were reported in real time to the central database, analysed and reported.

After 24 months follow-up >3000 viral load runs were analysed. Inter-laboratory differences were observed in the IRC results, as indicated both by differences in the mean values as well as SDs. Inter-kit-lot comparisons demonstrated no statistically significant differences between lot numbers. The control could also be used to evaluate the performance of the Ultrasensitive version of the Monitor assay. Ultrasensitive assay results highly correlated with IRC results obtained with the regular versions. A similar high level of correlation was observed between results obtained with the manual (microwell plate) assay and results obtained with the Cobas Amplicor.

The IRC demonstrates to be an important tool for independent, long term monitoring of viral load assay performance on a large scale.

 

 

O-036

 

 Multicenter proficiency testing for molecular detection and quantitation of blood-borne viruses 

ANTON M. van LOON¹, Jurjen Schirm, Elizabeth Valentine-Thon, Jim Reid, Paul E. Klapper and Graham M. Cleator

Department of Virology, University Medical Center Utrecht, Heidelberglaan 100 (G04.614), Utrecht 3584 CX, The Netherlands

European Union Concerted Action for Quality Control (EU-QCCA) of Nucleic Acid Amplification in Diagnostic Virology

 

The aim of the EU-QCCA is to establish an external quality control programme for assessment and evaluation of existing and evolving nucleic acid amplification procedures in diagnostic virology. For this purpose proficiency panels have been prepared for molecular detection and quantitation of various viruses, including blood-borne viruses (BBV's).

In May 1999 proficiency panels for HIV-RNA, HCV-RNA and HBV-DNA each were distributed to laboratories in up to 20 countries; in February 2000 a second distribution took place. Panels consisted of 2 negative samples and 6 positive samples with various concentrations and genotypes of virus. The majority of labs used commercial assays for qualitative or quantitative examination of samples; Roche assays were used most frequently. The false-positive rate was well below 3% in all panels, excluding the first HBV-DNA panel (5.3%). False-negative rates were difficult to determine because of large variation in detection limits of various assays. However, when limited to clearly positive samples, the false-negative rates were very low (<2%).

Analysis of quantitative results showed considerable variation in viral loads due to variability within and between assays of various manufacturers. In general, standard deviations of mean viral load values obtained by bDNA assays were significantly lower than those obtained by other types of assays.

Agreement between results from assays of different manufacturers was generally good to acceptable with the most frequently circulating genotype (industrialized countries!), but was less good with less common genotypes. Our results indicate that considerable improvements in molecular detection and quantitation of BBV's have been achieved in recent years, particularly through the use of commercial assays. Further standardization, including less common genotypes, however, is still needed.

 

 

O-037

 

 A panel for quality control of nucleic acid amplification of Herpes Simplex Virus (HSV) 

L. SCHLOSS¹, A Linde¹, F. Lundqvist¹, K.I. Falk¹, P. Cinque², P. Klapper³, T. Popow-Kraupp⁴, J. Schirm⁶, B.F. Vestergaard⁵, B. Niesters⁷ and G. Cleator^{3 

 1}Swedish Institute for Infectious Disease Control, Stockholm, Sweden

 ²Centro Riserca E Ospedale San Raffaele, Milano, Italy

 ³Central Manchester Healthcare Trust, Manchester, UK

 ⁴University of Vienna, Austria

 ⁵Statens Serum Institute, Copenhagen S, Denmark

 ⁶Regional Public Health Laboratory, Groningen, The Netherlands

 ⁷University Hospital, Rotterdam, The Netherlands

 

 Aim: External quality control (EQC) of nucleic acid amplification (NAA) for HSV-1 and -2.

 Materials and methods: Under the auspices of the European Union Concerted Action for Quality Control of Nucleic Acid Amplification in Diagnostic Virology, two external quality control proficiency panels were produced. HSV type 1 strain Macintyre (HSV1) and HSV type 2 strain MS (HSV2) obtained from ATCC were cultivated in African green monkey kidney cells, heat inactivated (56°C) and freeze-dried. End-point PCR positive results were determined by testing in 5 reference laboratories, the amount of HSV DNA in this dilution was determined by quantitative PCR in 2 reference laboratories. Panels containing 12 samples (HSV positive, Varicella-zoster virus positive and negative samples) were distributed to 68 European laboratories in 1999 and 90 in 2000.

 Results: In 1999, 66 laboratories submitted 76 data sets. 70% of the data sets recorded ‘satisfactory’ or better results. Most laboratories used qualitative ‘in-house’ PCR methods. Single PCR with gel-detection had a lower sensitivity than single PCR with probe-detection or nested PCR. A small number of datasets (6/76; 8%) reported false positive results. The results from the second panel will be presented.

 Conclusion: The results indicate that while most laboratories have satisfactory HSV PCR performance, further improvement is required. EQC proficiency testing is a most important tool for monitoring of diagnostic test quality.

 

 

O-038

 

 Different patterns of restriction to B19 Parvovirus replication in human blast cell lines 

GIORGIO GALLINELLA, Elisabetta Manaresi, Elisa Zuffi, Simona Venturoli, Laura Bonsi, Gian Paolo Bagnara, Monica Musiani and Marialuisa Zerbini

Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Via Masserenti 9, Bologna I-40138, Italy

 

B19 parvovirus can replicate in erythroid progenitor cells from bone marrow and foetal liver, and in a small number of human cell lines. In order to better understand and analyse B19 replicative cycle, we performed and compared the infection of bone marrow cells and of different erythroblastoid and mega karyobl astoid cell lines (UT7, TF-11, M07, and B1647). Following in vitro infection, B19-specific nucleic acids were characterised with respect to the genome replicative intermediates, to the transcription patterns and to the production of capsid proteins. While all cell lines tested proved to be susceptible to B19 virus infection, two different patterns of restriction to replication were observed. In a first pattern, observed in UT7 cells, the viral single-stranded (ss) DNA was converted to double-stranded (ds) replicative intermediates identical to those found in bone marrow cells and a full pattern of viral transcripts was observed. However, replication and transcription was restricted to a small subset of cells, and production of capsid proteins was not detected. In a second pattern, observed in TF-11, M07 and B1647 cells, the viral ssDNA was not converted to dsDNA replicative intermediates, and consequently, no transcription of the viral genome occurred.

In conclusion, our study provided evidence for the presence of two different levels of restriction to B19 virus replication. A first level of restriction occurs related to the uncoating of the viral genome and/or conversion of the viral ssDNA into replicating dsDNA, while a second level of restriction occurs related to the production of viral capsid proteins.

 

 

O-039

 

 Propagation of Hepatitis B virus in FT09.1 cells 

EDWARD S. DORNAN¹, A. Lackenby¹, M. Jazayeri¹, E. Depla², I. Ottavaere², K deVreese² and W.F. Carman^{1 

 1}Division of Virology, Institute of Biological and Life Sciences, University of Glasgow, Church Street, Glascow G11 5JR, UK

 ²Innogenetics N.V, Ghent, Belgium

 

The availability of a continuous cell line susceptible to infection by HBV would be a significant advance in the study of this virus allowing study of the early events of viral replication and analyses of the functional characteristics of HBV variants. Recently evidence that the phospholipid binding protein annexin V is a receptor for HBV has been accumulating. Additionally, infection of a rat cell line, stably transfected with the human AV gene, termed the FTO-9.1 cell line has been infected with HBV. We present here further evidence of the role of annexinV in HBV infection by demonstrating that HBV can be propagated in this cell line. HBV from both human sera and from culture supernatants (s/n) from HepG2 2.15 cells were used to infect FT09.1 cells. 3×10⁵ cells were plated on coverslips in 24 well plates. Infection was carried out with HBeAg +ve serum containing ∼5×10⁶ genome equivalents/ml or from five day culture supernatants from HepG2 2.15 cells (∼1×10⁷ gen.eq./ml). After overnight incubation at 37°C, cells were either washed thoroughly (at least five 5 times) with PBS or subjected to mild trypsinisation in the presence of chicken serum to remove residual innoculum.

The culture s/n at this stage became PCR negative. Cells and s/n were assayed at various times post infection by IF microscopy, (HBcAg staining in nucleus) PCR of culture s/n, and by cccDNA (generated only upon viral entry of cells) specific PCR of harvested cells. At day5 s/n and cells proved positive for HBV in all assays. At this time ∼10% of the cells were infected as assessed by IF. The presence of DMSO and, interestingly, recombinant AV augment the infectivity of the cells. This later observation may be explained by the binding of AV to phophatidyl serine on the HBV envelope, concomitant binding to HBsAg and subsequent fusion to the hepatocyte membrane. S/n from these initially infected cells were then used to infect fresh FTO 9.1 cells with similar outcome to primary infection. The virus has been further propagated in this manner in at least two further passages and can be reliably reproduced thus indicating the value of this cell line for in vitro studies of HBV. For example, we are now in a position to assess the replication competence of variants, both natural and drug (eg lamivudine) selected.

 

 

O-040

 

 Induction of papillomas in a mouse model using recombinant Bovine Papillomavirus type 2 (BPV2) DNA 

ANDREA PAWELLEK¹, G. Hewlett², J. Kreuter³ and H. Robsamen-Waigmann^{2 

 1}Bayer AG, Business Group Pharma, Antiinfective Research, PHR-AI2 Virology, Aprather Weg 18a, Wuppertal D-42096, Germany

 ²PHR-A12 Virologie Bayer AG, Wuppertal, Germany

 ³Institut for Pharmazeutische Technologie, Universitat Frankfurt am Main, Germany

 

Due to the fact that papillomaviruses cannot replicate in a monolayer cell culture, the classical animal models, for example using bovine papillomavirus, as well as transplantation models, have been used to study features of the papillomavirus replication cycle. Most of these models rely on infection with virus particles and do not allow genetic studies to be carried out with ease. Gene function can be analysed by manipulating cloned DNA so we decided to investigate whether recombinant BPV2-DNA can also induce warts in our recently established mouse xenograft model of papillomavirus infection.

Viral DNA was isolated from a bovine wart by phenol/chloroform extraction and identified as BPV2-DNA. The extracted BPV2-DNA was cut at its unique Hind III restriction site before cloning it into the pBluescript II (SK+) vector. After amplification in E. coli the viral DNA was recovered from the vector and religated. Calf scrotal skin was injected with the religated viral DNA before grafting it to the dorsum of severe combined immunodeficient (SCID) mice.

Five months after transfection, tumours developed that showed histological features of a papillomavirus infection. Southern blot hybridisation using BPV2 probes demonstrated the presence of BPV2 DNA in the transfected transplant. RT-PCR revealed the expression of genes typical for fibropapillomas.

In this model recombinant BPV2 DNA was shown to be infectious and to induce warts. This model should be useful for mutation analysis and therefore for the better understanding of the regulation of the papillomavirus infection.

 

 

O-041

 

 Naturally-occurring Human Papillomavirus type 16 E5 variants up-regulate entry into S & M phase 

JON BIBLE, Jennifer M. Best, Christine A. Mant, Barbara Kell, Ruth Gilchrist and John Cason

The Richard Dimbleby Laboratory of Cancer Virology, Department of Infection (Virology Section), St. Thomas’ Hospital, Kings College London, Block 9 Medical School, Lambeth Place Road, London SE1 7EH, UK

 

Human papillomavirus type-16 (HPV-16) is now accepted as the major causative agent for cervical intraepithelial neoplasia (CIN) and invasive carcinoma, however only a small percentage of HPV-16 infections result in cancer. Our investigations have shown that particular HPV-16 E5 DNA sequence variants have higher transcriptional activity, increased usage of common mammalian codons and are associated with neoplasia (Bible et al 2000 JGV in press).

Five naturally occuring HPV-16 E5 variants have been cloned into a pcDNA 3.1-Myc expression vector driven by a CMV promoter. Equivalent variants with a stop codon inserted were included as internal controls. All constructs were transfected into NIH-3T3 cells and stable cell-lines established after G418 selection. Cell proliferation assays, foci-formation assays and cell-cycle analysis by FACscan were performed on each cell line. Cell-cycle profiles of constructs (analysed in the presence of epidermal growth factor) indicated that all controls had similar percentages of cells in G1, S & M phases. In contrast, HPV-1 6 E5 caused statistically significant decreases in G1, and increases in S & M phases. All E5 variants differed markedly from the reference isolate of HPV-16 E5. Alteration of cell cycle profiles was accompanied by a marked increase in cell-growth and overall appears to be due to variants which substitute amino acids which stabilise the first (144®L) or third a-helices (165®V) of the E5 protein. Timecourse experiments are currently being analysed to determine whether E5 variants differentially effect the cyclin dependant kinase inhibitors 21waf/cip1 and p27, and levels of cyclin A & B. These data indicate that E5 variants have different biological activities which may contribute to the enhanced pathogenicity of particular HPV-16 variants.

 

 

O-042

 

 Adenovirus DNA polymerase: domains and residues contributing to activity in viral replication 

HUANTING LIU, James H. Naismith and Ronald T. Hay

Centre for Biomolecular Science, BMS Building, University of St. Andrews, North Haugh, St. Andrews KY16 9ST, UK

 

Adenovirus DNA polymerase is a member of the DNA polymerase a family and uses a protein primer for initiation of DNA synthesis. It contains motifs characteristic of a proofreading 3′–5′-exonuclease domain located in the N-terminal region and several DNA polymerase motifs located in the C-terminal region. To determine the mechanism and the roles of the conserved residues of adenovirus DNA polymerase in DNA replication, twenty-two site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region and the mutants were expressed in insect cells using a baculovirus expression system. Each mutant was tested for DNA binding activity, ability to interact with pTP, DNA polymerase catalytic activity and ability to participate in the initiation of adenovirus DNA replication. The mutant phenotypes identified functional domains within the adenovirus DNA polymerase and allowed discrimination between the role of conserved residues in the various activities carried out by the protein. Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (Wang, et al., 1997. Cell. 89:1087–99), the domain organisation of adenovirus DNA polymerase is envisaged as similar to that of RB69 DNA polymerase with the catalytic site composed of the conserved motifs YGDTD in Pol 1, DCGMYA in Pol 11, and KLLSNALYG in Pol 111. Addition residues lying outside the conserved regions are also required for DNA polymerase activity.

 

 

O-043

 

 A Herpes Simplex Virus type 1 UL12 null mutant packages replicated amplicon DNA relatively more efficiently than viral DNA 

IAIN M. PORTER and Nigel D. Stow

MRC Virology Unit, Church Street, Glasgow G11 5JR, UK

 

The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline nuclease. Although not absolutely essential for replication, UL12 null mutants produce 100–1000 fold less viable virus than wt HSV-1. It has been suggested that the alkaline exonuclease functions to remove branched structures from high molecular weight concatemeric DNA prior to its cleavage and the packaging of monomeric genomes into viral capsids. Failure to process the DNA results in unstable packaging of DNA into capsids which fail to egress from the nucleus. We have investigated the role of UL12 in the replication and packaging of plasmids (amplicons) containing HSV-1 replication origin and DNA encapsidation signals. Cells were transfected with the amplicon and then superinfected with either wt HSV-1 or the ambUL12 null mutant (A.H. Patel et al, J. Gen. Virol. 77, 199–209, 1996). Replication of both amplicon and viral genomic DNA in ambUL12 infected cells were similarly reduced by approximately 4-fold compared to wt HSV-1. In contrast, cells infected with ambUL12 exhibited a 50-fold reduction in the packaging of genomic DNA compared to only an 8-fold reduction in amplicon DNA packaging. The effects on amplicon replication and packaging could be complemented by the co-transfection of a plasmid expressing UL12 but not the overlapping UL-12.5 gene. We also demonstrated that the amplicons generated high molecular weight concatemers which recombine at high frequency, analogous to replicating viral DNA. Preliminary data additionally indicate that in the absence of UL12 an increased proportion of DNA lacking a packaging signal may be encapsidated.

 

 

O-044

 

 Nested multiplex polymerase chain reaction for the diagnosis of cutaneous herpes simplex and herpes zoster infections and a comparison with electron microscopy 

PETER COYLE, S. Jain, D. Wyatt, H.J. O'Neill and C. McCaughey

Regional Virus Laboratory, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BN, Northen Ireland, UK

 

Herpes simplex virus (HSV) and varicella zoster virus (VZV) are common causes of cutaneous and mucocutaneous vesicular eruptions. Laboratory diagnostic techniques include Tzanck smears, electron microscopy (EM), antigen detection and viral culture. This paper describes a nested multiplex polymerase chain reaction (NMPCR) with respective sensitivities of 0.0001, 0.01 and 0.1 TCID50 for VZV, HSV-1 and HSV-2. The assay was used in (a) a salvage capacity for slides already processed for EM, and (b) as a front-line assay for prospectively processed specimens. Sixty-two glass slides with vesicle lymph/scrapings from 58 patients with suspected cutaneous herpetic lesions were examined. The clinical presentations were described as atypical/not specified (24), VZV (20) or HSV (18), and involved eruptions from diverse anatomical sites, including the genitalia. Of the 62 specimens, 6 and 38 were positive by EM and NMPCR respectively, giving a comparative sensitivity of 16% for EM. NMPCR identified 15 VZV and 20 HSV-1 infections. Where the clinical details indicated either HSV or VZV (38/62), NMPCR was statistically likely to be reactive (26/38 versus 9/24) (χ² P=0.000004) whereas EM was not (4/38 versus 2/24) (χ² P=0.77). Where the clinical details indicated VZV (20/62) or HSV (18/62), NMPCR was statistically more likely to confirm VZV (10/20 versus 5/42) (χ² P=0.001) or HSV (9/18 versus 11/44) (χ² P=0.05) respectively. Two suspected HSV and 6 suspected VZV infections were shown to be VZV and HSV respectively by NMPCR.

 

 

O-045

 

 Simultaneous detection and identification of Rubella, measles and Parvovirus B19 viruses by multiplex PCR 

JUAN E. ECHEVARRÍA, Fernando de Ory and Mar Mosquera

Centro Nacional de Microbiologfa, Instituto de Salud Carlos 111, Ctra. Majadahonda-Pozuelo s/n, Majadahonda, Madrid 28220, Spain

 

Parvovirus B19 (B19V), measles virus (MV) and rubella virus (RV) are important causes of exanthematic diseases. Moreover, they cause several important complications, as congenital rubella syndrome; postinfectious encephalopathy and subacute sclerosing panencephalitis produced by MV; or transient aplastic crisis, chronic anemia and hidrops fetalis by B19V. Clinical features of the exanthemas caused by these viruses are similar and differential diagnosis is often required. In this report we describe a diagnostic method able to detect simultaneously viral genomes from all three viruses by multiplex RTPCR. Two pairs of primers (first and nested reaction) were designed for each virus. The target genes were El glycoprotein in the case of RV, nucleoprotein for MV and nucleoGapsid protein for B19V. Sizes of amplification fragments obtained after nested amplification were different for each virus and easily differentiated by agarose gel electrophoresis. Sensitivity was estimated on I DNA copy/tube for B19V genome and 10 cDNA copies of both MV and RV genomes. This technique allowed us to detect MV in 5 pharyngeal exudates from a measles outbreak and two cerebrospinal fluid of patients with post-measles encephalitis; and RV in two pharyngeal exudates and one urine of a newborn with congenital rubella syndrome and three pharyngeal exudates from post-vaccined children. Also, we detected B19V in 16 serum specimens out of 21 from an erythema infectiosum outbreak.

We conclude our technique seems to be useful as complement of serology for the diagnosis of these diseases, allowing the recovery of genomic material to use in subsequent studies. This is specially convenient for the surveillance activities of the measles eradication program.

 

 

O-046

 

 Prospective evaluation of the nuclisens basic kit NASBA for laboratory diagnosis of enterovirus infections 

JULIE FOX, S. Han¹, S. Corden² and D. Westmoreland^{2 

 1}Department of Medical Microbiology, Uuniversity of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK

 ²Virology PHL, UHW, Cardiff, UK

 

 Introduction and aim: ‘In house’ RT-PCR procedures described for investigation of enterovirus meningitis tend to be laborious and difficult to control. This project aimed to evaluate a recentlydeveloped ‘pan-enterovirus’ NASBA for diagnostic investigation of possible enterovirus infections.

 Method: The assay utilises kit-based reagents for extraction, amplification and detection of amplified products (NucliSens Basic Kit, Organon Teknika Ltd) together with ‘in house’ designed primers and probes (based on the 5′non-coding region).

 Results and conclusion: The specificity of the NASBA assay was very good. The sensitivity of the assay for detection of a wide range of enteroviruses was approximately 1 TC1 D50 input. The assay also picked up additional virus types not readily propagated in vitro. The Basic Kit NASBA gave an equivalent sensitivity to the more laborious ‘in house’ RT-PCR and NASBA assays on control and proficiency panel material. To date, results on prospectively-analysed clinical samples (n=380) have been concordant with ‘in house’ RT-PCR. Basic Kit results were available within 1 working day compared with 2–5 days for RT-PCR and culture, respectively. The Basic Kit NASBA is a suitable diagnostic molecular amplification method for analysis of enterovirus sequences in CSF, stool and respiratory specimens. The use of kitbased reagents and fast turn-around of results compared with ‘in house’ RT-PCR will enhance the costeffectiveness and quality control of our molecular-based diagnostic procedures for investigation of possible enterovirus meningitis.

 

 

O-047

 

 Development of real-time RT-PCR to detect viral infections in semen of boars 

LIESBETH JACOBS, R.W. Hakze-van der Honing, J. Boonstra and R. Moormann

Institute for Animal Science and Health (ID Lelystad), PO Box 65, Lelystad 0200 AB, The Netherlands

 

Nowadays artificial insemination is the main route for insemination of sows, making that A.I. stations can play a pivotal role in transmission of viral infections. During the last outbreak of classical swine fever (CSF) in The Netherlands it was proven that the virus could also be transmitted through semen. Porcine reproductive and respiratory syndrome virus (PPRSV) is causing reproductive failure in sows and respiratory distress in piglets. It is a relatively new disease that firstly was described in the US in 1987, and emerged in pigs in Europe in 1990. Since then has spread rapidly, and contributes significantly to economic losses in the pig industry. It is know that transmission of PRRSV can occur through boar semen. Typically, boars do not demonstrate significant clinical signs after infection, and seroconversion or viremia may not correlate with shedding of the virus in the semen. In this report we describe the development of a real-time PCR using the Light Cycler (Roche) technology to detect PRRSV or CSFV in semen. The main advantages of this system are i) the speed of the PCR which makes a high throughput possible ii) the use of specific hybridization probes iii) the PCR and analysis are done in a closed system which decrease the risk for false positives by amplication contamination. The PCR was validated using semen samples from boars experimentally infected with PPRSV or with CSFV. Semen samples were tested in the virus isolation a bioassay and PCR. We concluded that the PCRs described here were easy to perform, fast, reproducible and more sensitive than virus isolation or the bioassay.

 

 

O-048

 

 Mechanisms of Coxsackievirus induced damage to human pancreatic beta cells 

M. Roivainen¹, S. Rasilainen², P. Ylipaasto¹, R. Nissinen¹, J. Ustinov², L. Bouwensu³, D.L. Eizirika³, TAPAN HOVI¹ and T. Otonkoski^{2 

 1}Enterovirus Laboratory, Department of Virology, National Public Health Institute (KTL), Mannerheimintie 166, Helsinki FIN-00300, Finland

 ²University of Helsinki, Helsinki, Finland

 ³Vrije Universiteit Brussel, Brussels, Belgium

 

Insulin dependent diabetes mellitus (IDDM) is a severe chronic disease in which insulin producing betacells in Langerhans islets of the pancreas are gradually destroyed. By using primary human adult beta cells we are trying to find out the mechanisms by which different enteroviruses may induce or accelerate the process eventually leading to IDDM. According to our results significant functional impairment was seen after coxsackie B virus infections. Only a minority of infected cells became apoptotic, as shown by nuclear morphology and increased in situ DNA end labelling, while most of the infected cells died by pyknosis characterized by highly distorted nuclei with condensed but intact chromatin. In contrast to the CBVs, CAV-9, which is genetically closely related to CBVs, did not cause any immediate adverse effects in human beta cells despite replicating there well. When diabetogenic properties of other enteroviruses were studied we found that they can be divided into two major groups. For example, several serotypes of echovirus were cytolytic like the CBVs but there were some CAV-9-like less-cytolytic serotypes as well.

 

 

O-049

 

 The differentiating epithelial cells of the bronchiolar epithelium eliminate virally infected cells from the mouse lung by hyperplasia and sloughing 

KEITH GOTTLEIB and Luis P. Villarreal

Department of Molecular Biology and Biochemistry, University of California, Irvine, Biological Sciences II, Box 86, Irvine, CA 92697, USA

 

The lung is a resilient organ that must avoid overt tissue damage from virus infections and their induced immune responses to prevent loss of function of this vital component of the respiratory system. Our lab investigates the acute stage of infection in the mouse lung of viruses indigenous to wild mouse populations following the natural route of infection. Using polyomavirus (Py) as a model, we demonstrated an innate, non-immune response of the differentiating epithelial cells (Clara) of the bronchi and bronchioles for expelling virus. Newborn and adult mice were intranasally inoculated with Py, the lungs were obtained starting two days post infection, and formalin fixed, paraffin embedded sections were made for evaluation. In situ hybridization, immunohistochemistry, and hematoxylin and eosin staining were performed to analyze viral replication, gene expression of viral and host proteins, and histopathological observations, respectively. A clear hyperplasia of the Clara cells, as evidenced by the presence of Clara cell secretory protein, began a few days after infection and continued for 48–72 hours resulting in the eventual sloughing of multiple layers of cells prior to lymphocytic infiltration. The sloughing eliminates apical cells positive for Py DNA and underlying cells expressing Py early proteins leaving a normal bronchiolar epithelium virtually free of detectable virus. A similar phenomenon has been reported for Sendai virus, a distinctly different virus than Py, and appears to be ubiquitous, though rarely analyzed, in other lung infections of viruses tropic for bronchiolar epithelium.

 

 

O-050

 

 The Hepatitis C virus core protein has a unique region that directs maturation and intracellular targetting of the protein 

JOHN McLAUCHLAN and R. Graham Hope

MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK

 

The HCV core protein forms the capsid shell of virions and has been implicated in modulating a number of key cellular processes. Our aim is to characterise fundamental properties of the protein and identify sequences that influence its behaviour. From sequence comparisons, we postulate that core consists of three distinct domains: an N-terminal domain that has a high content of basic amino acids; a region of about 55 amino acids that is absent in sequences from related flavi- and pestiviruses and is therefore unique; finally, a transmembrane segment, containing the signal peptide for glycoprotein El. This transmembrane segment is removed from the capsid protein of flaviviruses by a viral protease while, for HCV, processing to the mature form of core is performed by a cellular protease. Our data indicate that a hydrophobic domain that resides within the unique domain of HCV core is necessary for efficient production of mature protein and also is critical for protein stability. Hence, the need for a viral protease during maturation of flavivirus capsid proteins is substituted in HCV core protein by sequences that direct processing solely by the cellular machinery. The unique domain also functions to target core to intracellular lipid droplets and has sequence similarity to oleosin proteins that locate to equivalent structures in plant cells. The role of the unique sequences in HCV will be discussed in the light of recent data on the relationship between efficient virus production and coordinate processing during maturation of flavivirus capsid proteins.

 

 

O-051

 

 The non structural nss protein of rift valley fever Phlebovirus utilizes the microtubule network for its transport to the nucleus 

MICHÈLE BOULOY¹, F.Z. Yadani¹, A. Kohl¹, P. Gounon¹ and P. Denoulet^{1,2 

 1}Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex, France

 ²College de France, Paris, France

 

Rift valley fever virus (RVFV) is an arthropod-borne virus of the Bunyaviridae family (genus Phlebovirus) which sporadically causes widespread epizootics/epidemics throughout Africa and is responsible for hemorrhagic fevers. Among members of the family and Phlebovirus genus, the RVFV is unique as its nonstructural protein NSs forms filamentous structures in the nuclei of infected cells. The presence of viral components in the nucleus is unexpected since replication of RVFV as well as all the members of the family occurs only in the cytoplasm.

To investigate the mechanism of filament formation, we expressed the NSs protein via recombinant Semliki Forest replicon and demonstrated that the protein alone is able to form nuclear filaments and that the 10–17 carboxy-terminal amino acids play an essential role for the formation of filaments but not for nuclear localization.

This domain contains two serine residues, which are phosphorylated. However, phosphorylation did not seem to play an important role for the properties of the protein.

Using the yeast two-hybrid system, we found that NSs interacts specifically with beta-tubulin. Existence of this interaction in infected cells was confirmed by biochemical analyses and immunofluorescence assays: NSs copurified and colocalized with microtubules and appeared to function as a microtubule-associated protein. The domain of NSs interacting with beta-tubulin was mapped within the 15–18 N-terminal amino acids.

By expressing NSs mutants in which this domain was deleted, we found that the mutated protein remained in the cytoplasm and demonstrated that interaction of NSs with microtubules was essential for the transport of the protein to the nucleus.

 

 

O-052

 

 Characterisation of the immunnmodulative influence of respiratory syncytial virus matrixprotein M2 through interaxtion with the nfkappab complex 

KERSTIN REIMERS, Chrisitan Roder and Hermann Werchau

Medical Microbiology and Virology, Ruhr-University Bochum, MA 6/30, University Street 150, Bochum 44780, Germany

 

Respiratory syncytial virus (RSV) infection results in a persistent activation of nuclear factor kappa B in lung epithelial cells. Expression of the RSV proteins in A549 cells followed by detection of activated FB through electrophoretic mobility shift assays (EMSA) showed that the matrixprotein M2 is able to activate NF kappa B in contrast to the other RSV core proteins. These results were confirmed by a reporter gene construct consisting of a luciferase gene regulated by NF kappa B enhancer elements. Interestingly, N-terminal deletion mutants of M2 were not able to induce NF kappa B response. Immunoprecipitation followed by Western blotting revealed that M2 is interacting directly with the NF kappa B complex. Confocal laser microscopy as well as cellular subfractionation showed that M2 is in part translocated to the nucleus time-consistently with the activation of NF kappa B. These findings suggest that M2 besides its function as antitermination factor in viral transcription participates in the modulation of the immune system caused by RSV through interaction with the NF kappa B complex.

 

 

O-053

 

 Measles virus encephalitis among adult patients admitted with encephalitis in a Dutch hospital 

JAN GROEN, Y.A. Nur, H. Kruining, C. Copra, P. Koraka and A.D.M.E. Osterhaus

Institute of Virology, University Hospital Rotterdam, Dr Molewaterplein 40, Rotterdam 3015 GD, The Netherlands

 

 Background: Clinical features associated with many viral infections of the CNS are non specific and remain often undiagnosed. 53 patients admitted to our hospital with clinical manifestation of encephalitis followed by neurological deterioration of unknown origin were included in this study. Combined evaluation of clinical data, results of diagnostic serological tests and polymerase chain reactions (PCR) carried out on cerebrospinal fluids (CSF) and broncho alveolar lavages (BAL) may shed light on the aetiology of these cases of encephalitis.

 Aim: To identify undiagnosed cases of measles with CNS involvement.

 Methods: A retrospective analysis was performed on 53 adult patients admitted to our hospital with encephalitis followed by neurologic deterioration of unknown origin during 1998 (mean age=48 at sampling date). Serum and CSF samples were tested for the presence of IgM antibodies against tick-borne encephalitis virus (TBEV), dengue virus (DV) and measles virus (MV).

 Results: Clinical features reported were fever, headache, facial weakness, altered mental status seizures, impaired hearing, motor disturbances, limb paralysis and respiratory distress. IgM serum and CSF antibodies to MV were found in 2 out of 53 patients and in one patient borderline positive results was found. MV infection was confirmed in the borderline IgM positive patient, by showing MV–RNA by RT–PCR in a broncho alveolar lavage. No evidence of TBEV or dengue virus infections was found.

 Conclusion: The identification of three cases of measles encephalitis among 53 adult patients with encephalitis of unknown origin indicates that this condition tends to be overlooked with the continuing elimination of MV in developed countries.

 

 

O-054

 

 Human herpesvirus-6 infection in children with acute febrile illness and seizures 

GEORGE KAMKAMIDZE¹, Maia Butsashvili^1,2, Nino Gochitashvili¹, Lela Abashidze¹, Tengiz Tsertsvadze^{1 

 1}Georgian AIDS and Clinical Immunology Research Center, 16 Al. Kazbegi Avenue, Tblisi 380060, Georgia

 ²Fogarty International Center

 

Human herpesvirus-6 (HHV-6) is one of the recently discovered herpesviruses. Primary HHV-6 infection is causative agent of roseola infantum or exanthem subitum and is frequently associated with nonspecific febrile illness in children under the age of 3 years. The goal of our study was the investigation of HHV-6 infection in children with acute febrile illness complicated by seizures.

Twenty-two children from 6 to 24 months of age with acute febrile illness and seizures were enrolled in the study. Blood samples were obtained at the moment of the first examination in the emergency department and 10 days thereafter. Anti-HHV-6 IgG and IgM antibodies were evaluated by indirect immunofluorescent assay. HHV-6 DNA was detected by nested PCR assay in peripheral blood mononuclear cells. 15 children of the same age without acute illness were studied as controls.

Primary HHV-6 infection has been found in 6 out of 22 patients from the study group (3 cases: IgM+ and PCR+ in both samples, 1 patient: PCR+ in both, IgG+ in the second sample, 2 cases: IgM−, IgG− and PCR+ in both samples). In this group past infection was found in 7 patients (6 cases: IgG+ and PCR+ in both samples, 1 child: IgG+ an PCR− in both samples). Nine children showed no infection (IgG−, IgM−, PCR−). No primary HHV-6 infection was found in the control group while the past infection was documented in 8 out of 15 children. Primary HHV-6 infection is associated with acute febrile illness and seizures in the early childhood.

 

 

O-055

 Transmission of hepatitis C virus in a pediatric oncology ward 

ANTJE KNÖLL¹, Magdalena Helmig², Ove Peters² and Wolfgang Jilg^{3 

 1}Institute of Medical Microbiology and Hygiene, University of Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg 93053, Germany

 ²Childrens Hospital St. Hedwig, Steinmetzstrasse, 93049 Regensburg

 ³Institute of Medical Microbiology and Hygiene, Regensburg

 

Hospital-related Hepatitis C Virus (HCV) infections continue to occur despite the introduction of blood donor screening. The evaluation of this problem is difficult due to the frequently silent course of infection, the lack of prospective studies and the scarce data about patient to patient transmission in settings other than hemodialysis units.

We report the transmission of HCV to nine pediatric patients of an oncology ward. The index case was a two-year-old child who had been hospitalized for intravenous feeding since birth. In October 1996, he developed clinical hepatitis, and HCV infection was confirmed by positive HGV–PCR and seroconversion to anti-HCV. The source of infection could not be traced.

All precautionary hygienic measures to prevent HCV transmission were taken, and the patient remained hospitalized. During the following months, eight additional children who had been treated at overlapping periods became infected with HCV. Sequencing of three HCV genomic regions (5′NTR, NS3, hypervariable region of E3/NS1) showed very high homology (genotype 1b). The mode of virus transmission could not be established by screening of family members, medical staff, and blood products. We assume that involuntary non-observance of precautionary measures was responsible for this outbreak.

Meanwhile, the index patient has received a combined liver and small bowel transplantation. He remains HCV–PCR negative in serum and lives at home. Four of the other patients are HCV–RNA positive, two are HCV–RNA negative, and two have died from their oncologic disease.

 

 

O-056

 

 Detection of enteroviral sequences in frozen spinal cord samples of Japanese ALS patients 

BRUNO LINA², Pierric Giraud¹, Frédérik Beaulieux², Seiitsu Ono³, N. Shimizu³ and Guy Chazot^{1 

 1}Service de Neurologie B Hôpital Neurologique Pierre Wertheimer, 59, Bvd. Pinel, Lyon cedex 03, France

 ²Centre National de Référence pour la Grippe, Laboratoire de Virologie, Unité de Virologie Médicale, EA 3085, Domaine Rockefeller, 69373, Lyon cedex 08, France

 ³Department of Neurology, Teikyo Uni School of Medicine, Ichihara Hospital, Anesaki, lchiahra Japan

 

The possible association of persistent enterovirus infection with the development of amyotrophic lateral sclerosis (ALS) was suggested again when we detected and located enteroviral sequences in the spinal cord of 15/17 (88.3%) of ALS cases compared to 1/29 (3.4%) of control cases using Reverse Transcriptase Polymerase Chain Reaction (RT–PCR) and direct RT in situ PCR (RT–IS–PCR) in formalin-fixed samples, respectively. These results had to be confirmed in a second study. This confirming second study was carried out with frozen spinal cord samples collected from 19 Japanese patients. The samples were blinded in Japan and subsequently sent on dry ice to the French Lab. Using an adapted procedure, total RNA was extracted from the samples and the extracts were processed for enterovirus detection by RT-PCR in the 5′ non coding region. Among the 19 samples, five were ALS cases and 14 were non-ALS controls. Among the five ALS cases, three were positive for enterovirus detection (3/5; 60%) compared to 2/14 (14%) in the controls.

The PCR products obtained from both controls and patients are going to be sequenced.

Even if the results of this second study were not as dramatic as those of the previous study, it nonambiguously confirms that enteroviral sequences can be detected in the spinal cord samples from ALS patients. There is a link that has to be elucidated between the presence of these sequences and the development of ALS.

 

 

O-057

 

 Herpes neonatorum: a molecular diagnosis 

 

CAROLINE SWANINK, Willem J.G. Melchers, Michael A. Gaytant and Jochem M.D. Galama

University Medical Center St. Radboud, Dept. of Medical Microbiology, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

 

A female neonate was delivered by caesarean section at 35+6 weeks of gestation because of fetal distress. Apgar scores were 5/5/6 after 1/5/10 minutes respectively. The mother had premature rupture of membranes and signs of infection (fever and leukocytosis). Immediately after birth bacterial cultures were taken and the child received amoxycillin and cefotaxim. On day 4 she was transferred to the NICU of our hospital because she had persistent convulsions. A cerebrospinal tap was performed and a nasopharyngeal swab and rectal swab was taken for both bacterial and viral culture. Antibiotic therapy was changed to amoxycillin 200 mg/kg/day, ceftazidim 150 mg/kg/day and aciclovir 30 mg/kg/day on suspicion of a meningoencephalitis. The cerebrospinal fluid revealed 11 leukocytes/ml, normal protein and normal glucose level. E lectro-encephalog ram did not show abnormal temporal activity. All bacterial and viral cultures were negative. Unfortunately there was no cerebrospinal fluid (CSF) for viral diagnostics. Three days later a serum sample and spinal tap were taken for viral diagnostics. Polymerase chain reaction (PCR) on both serum and CSF were positive for herpes simplex virus (HSV). Despite continuation of aciclovir, she died on day 12. Viral cultures from brain, lungs, liver, spleen and throat remained negative. PCR on brain, liver, serum and throat swab were all positive for HSV thus confirming the diagnosis herpes neonatorum.

 Conclusion: Prompt treatment with aciclovir on suspicion of herpes neonatorum may hamper the diagnosis using conventional virus culture. PCR is an important diagnostic tool in herpes neonatorum.

 

 

O-058

 

 Influenza B virus in seals 

ALBERT OSTERHAUS, G.F. Rimmelzwaan, B.E.E. Martina, T.M. Bestebroer and R.A.M. Fouchier

Dept. of Virology, Erasmus University Rotterdam, Dr. Molewaterplein 50, 3000 DR Rotterdam, The Netherlands

 

Influenza B virus is a human pathogen whose origin and possible reservoir in nature are not known. An influenza B virus was isolated from a naturally infected harbor seal (Phoca vitulina) and was found to be infectious to seal kidney cells in vitro. Sequence analyses and serology indicated that influenza virus B/Seal/Netherlands/l/99 is closely related to strains that circulated in humans 4 to 5 years earlier. Retrospective analyses of sera collected from 971 seals showed a prevalence of antibodies to influenza B virus in 2 percent of the animals after 1995 and in none before 1995. This animal reservoir, harboring influenza B viruses that have circulated in the past, may pose a direct threat to humans.

 

 

O-059

 

 Rabies 

PAUL-PIERRE PASTORET¹ and Bernard Brochier^{2 

 1}University of Liège, Faculty of Veterinary Medicine, Immulogie–Vaccinologie, Boulevard de Colonster Bât.b B, Liège 4000, Belgium

 ²Pasteur Institute, Brussels, Belgium

 

Rabies has been known for centuries as a disease of humans and of domestic and wild animals. For many years rabies virus was thought to be unique, but the existence of several distinct rabies-related viruses is now recognized. Of special interest are recent lyssavirus isolates from bats such as fruit bats (flying foxes). The presence of a pseudogene between the G and L cistrons Implies that rabies virus is evolutionarily intermediate in the Rhabdoviridae family; apart from that, the gene order is similar to that of the Vesiculovirus genus. Glycoprotein can, alone, elicit a protective immunity as has been shown by using a vaccinia virus recombinant containing the rabies in Europe and North America emphasis is nowadays placed on the vaccination of the wildlife rabies reservoir to control rabies from the start.

Wildlife such as red fox (Vulpes vulpes) can be vaccinated by the oral route using baits. Efficacious when orally administered, the recombinant vaccinia-rabies virus vaccine which is stable in the environment and perfectly safe for target and non target animals has been extensively used in continental Europe (Belgium, France, Grand-duchy of Luxembourg) together with attenuated strains of rabies virus

Fox vaccination against rabies did provoke a dramatic decrease of rabies incidence in both wild and domestic animals and consequently a similar decrease of post-exposure treatments in humans.

Elimination of rabies from large areas in Continental Europe did contribute to change in quarantine regulation against rabies in United Kingdom and therefore improve the free movements of pets within the European Union.

 

 

O-060

 

 Menangle virus, a zoonotic member of the family Paramyxoviridae infecting fruit bats and associated with reproductive disease in pigs 

Peter D. Kirkland¹, Robert J. Love², Anthony D. Ross¹, Rodney J. Davis¹, Anne B. Gleeson¹ and ADRIAN PHILBEY^{3 

 1}New South Wales Agriculture, Elizabeth Macarthur Agricultural Institute, Camden, Australia

 ²Department of Animal Health, University of Sydney, Camden Australia

 ³Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, EH26 0PZ, UK

 

Menangle virus is a member of the family Paramyxoviridae isolated from stillborn piglets in Australia in 1997 1 The affected piggery experienced reproductive disease and stillborn piglets frequently had skeleta deformities and degeneration of the brain and spinal cord. Inclusion bodies were observed in neurones. The virus produces vacuolation, syncytia and focal lysis in cultured cells and is non-haemagglutinating. It appears to be a member of the genus Rubulavirus. Neutralising antibodies against Menangle virus were detected in sera from sows that produced affected litters, as well as a high proportion of other pigs at the affected piggery and two associated piggeries. No antibodies were detected in samples collected prior to the onset of reproductive disease. Neutralising antibodies were also detected in sera from two humans, both piggery workers, exposed to infected pigs and in sera from three species of fruit bats (Pteropus spp) in Australia. A colony of grey-headed fruit bats (P. poliocephalus) roosts near the affected piggery during summer and is thought to be the source of infection for pigs. Menangle virus was eradicated from the three infected piggeries and there has been no evidence of infection in any other piggeries in Australia.

 

 

O-061

 

 European hantaviruses: diagnostics and epidemiology 

OLLI VAPALAHTI¹, A. Plyusnin^1,2, A. Lundkvist², H. Kallio-Kokko¹, M. Brummer-Korvenkontio¹, V. Koistinen¹, H. Henttonen³, A. Näjrvänen⁴ and A. Vaheri^{1 

 1}Department of Virology, Haartman Institute, University of Helsinki, PO Box 21, Helsinki SF-00014, Finland

 ²Karolinska Institute, Stockholm, Sweden

 ³Finnish Forest Research Istitute, Vantaa, Finland

 ⁴University of Kuopio, Kuopio, Finland

 

Only two hantaviruses, Puumala (PUU) and Dobrava (DOB) were seen to cause human disease in Europe according to reliable typing methods (focus-reduction neutralization assays using appropriate hantavirus serotypes or RT–PCR with subsequent sequencing). In addition, Tula virus can infect man but has not been associated with human disease. A phylogenic tree showing ancient cospeciation with the rodent host(s) and geographical clustering of individual hantavirus strains found in different parts of Europe will be presented.

We have developed sensitive and specific IgM and IgG ElAs based on baculovirus-expressed PUU, DOB and HTN nucleocapsid (N) proteins. Our data suggest that baculovirus-expressed N is antigenically indistinguishable from the native protein and that the whole N protein is more sensitive than a truncated N-terminal antigen. When studied by proteins expressed in alphavirus expression system, the early IgG response is towards N protein only and IgG antibodies to glycoproteins are detectable usually only at convalescence. Furthermore, although during the first days of the disease IgM, and often IgG antibodies to N are present, in rare cases (<2%) PUU IgM antibodies may be negative up to 5 days after onset of illness. For point-of care diagnostics, a sensitive, rapid (5 minutes) immunochromatography test for detection of PUU IgM antibodies based on baculovirus-expressed PUU N was developed

We detected 1989–1996 7000 PUU infections in Finland (957/year) resulting in an incidence of 19/100 000 (in hyperendemic regions 90/100 000) with <0.1% mortality%. In 1999, however, altogether 2300 cases were diagnosed. Women contracted the disease at 44 (mean), men at 40 years (male:female ratio 2:1). The PUU antibody prevalence for women entering the maternity clinics nationwide was 3%, but for the whole population endemic areas, 10–20%. Comparison of the incidence and prevalence data suggests that approximately 13% of PUU infections were diagnosed.

 

 

O-062

 

 Evaluation of two commercially available immunoassays for the detection of Hantavirus antibodies in serum samples 

PENELOPI KORAKA¹, T. Avsic-Zupanc², A.D.M.E Osterhaus¹ and J. Groen^{1 

 1}Laboratory for Exotic Viral Infections, Department of Virology, Erasmus Medical Center Rotterdam, Dr. Molenwaterplein 40 3015GD Rotterdam, The Netherlands

 ²Institute of Microbiology and Immunology, Medical Faculty, Zaloska 4, 1000 Ljubljana, Slovenia

 

 Background: Hantaviruses (HV) are members of the family Bunyaviridae and are known to cause a variety of diseases from haemorrhagic fever with renal syndrome (HFRS) to pulmonary syndrome. Several serotypes have been described from which at least five are pathogenic to humans. Each serotype has a different animal reservoir and geographical distribution. In the acute phase of the disease the clinical diagnosis may be confirmed by serology or by PCR.

 Objective: To evaluate two commercially available immunoassays using sera from HV suspected and non-HV patients: an enzyme immunoassay (EIA) developed by MRL Diagnostics, for the detection of immunoglobulins M (IgM) and G (IgG) against several HV serotypes and an indirect immunofluorescence assay (IFA) from Progen, based on slides coated with Hantaan virus (HNTV) and Puumala virus (PUUV), infected cells.

 Study design: A total of 145 serum samples were used for this study. The serum panel included serum samples from patients suspected of mild (n=91), severe (n=10) HFRS and patients with other viral infections (n=44)

 Results: The agreement between the MRL EIA and the Progen IFA for the detection of IgM and IgG serum antibodies ranged from 87% to 91%, respectively. In the non-HV group one out of 44 samples was positive by the Progen HNTV IgM IFA, none in the Progen PUUV IFA and two samples in the MRL IgM EIA, resulting in specificities of 98%, 100% and 95%, respectively. The sensitivities and specificities of the MRL ElAs compared to the Progen overall PUUV and HNTV IFAs were 90% and 91% for IgM respectively and 96% for IgG in both immunoassays

 Conclusions: The MRL EIA proved to be sensitive and specific assay for the serological diagnosis of mild and severe HFRS.

**NO ABSTRACT RECEIVED FOR NUMBER 63

 

 

**NO ABSTRACT RECEIVED FOR NUMBER 64

 

 

O-065

 

 Folding and quality control of viral glycoproteins in the endoplasmic reticulum 

MAURIZIO MOLINARI and Ari Helenius

Institute of Biochemistry, ETHZ, Zurich, Switzerland

 

Folding of proteins synthesized in the endoplasmic reticulum (ER) of living cells depends on the assistance of molecular chaperones and other factors present in the ER lumen. Interactions with ERresident proteins begin already as the growing nascent chain enters the ER lumen through the translocon complex, and continue until the mature protein is ready to leave the ER. The most commonly observed associations involve BiP, the major HSP70 homologue in the ER, the two lectin-like chaperones calnexin (Cnx) and calreticulin (Crt), and members of the thiol oxidoreductase superfamily such as protein disulfide isomerase (PDI) and ERp57. Together with glucosidases I and II and UDP-glucose: glycoprotein glucosyltransferase they promote proper folding, prevent premature oligomerization, inhibit degradation and support quality control of newly synthesized proteins in the ER.

When different proteins have been analyzed, it has become evident that some interact first with BiP and then with Cnx, some bind Cnx and Crt but do not associate with BiP. Others are sequentially assisted by BiP and GRP94. Formation of intramolecular disulfide bonds is in some cases catalyzed by PDI, in other cases by ERp57, or by both oxidoreductases acting together

Our results indicate that to understand the molecular basis for such differential chaperone selection it is important to analyze growing nascent chains in live cells, and to consider the kinetics of elongation and the precise timing of co-translational modifications such as signal peptide cleavage, N-linked glycosylation and oligosaccharide trimming

When analyzing the early events in folding of the Semliki forest virus El and p62 glycoproteins, and influenza hemagglutinin, we found that already during co-translational translocation of a glycoprotein a choice is made between distinct chaperone systems, one comprising BiP and possibly PDI, and the other Cnx, Crt and ERp57. The selection of chaperones depends on the location of N-linked glycans in the growing nascent chain. The chaperones compete for binding sites on the incoming polypeptide. Whichever binds first dominates during early stages of co- and post-translational folding. Its association with the nascent chain and the full-length glycoprotein delays or prevents binding of the other chaperone. Later in the folding process a transfer from one pathway to the other may however take place.

 

 

O-066

 

 Of course we need electron microscopy! 

C.R. MADELEY

Newcastle-upon-Tyne, UK

 

Most people recognise friends by their appearance, rather than by their voice, their footfalls or even their clothes. It is the same with viruses, though many diagnostic techniques detect either their traces rather than the virus particle itself. When classifying a virus, we only decide finally when we have seen what it looks like; no virus has ever been misclassified on morphology alone, a fact underlined by the IVTC (1). Clearly, then, there is a need to provide electron microscopic visualisation of viruses within any country providing a comprehensive diagnostic service to identify viruses which may invade, or circulate within its borders. Present trends restrict the availability of electron microscopes (EMs) to central reference laboratories, most of them remote from front-line diagnostic virology.

This is dire, for the recognition of a variety of viruses in diagnostic material is not a skill that can be taken up and put down at will-it needs regular practice and nurture. The microscopist needs the daily challenge of looking for virions in diagnostic preparations, not knowing what a specimen contains, nor even if it contains anything at all. ‘Standard’ preparations do not, and cannot, provide the same challenge. Such regular and comprehensive work, though, brings the side-effect that each test becomes cheaper the more it is used-the major expense is providing the instrument and the microscopist, the running expenses are trivial. For reasons that have more to do with novelty than science, diagnostic EMs are seriously under threat-we can only blame ourselves if we lose them by failing to argue strongly enough to retain them.

 

(1) Murphy FA, Fauquet CM, Bishop DHL, et al. Sixth report of the International Committee on Taxonomy of Viruses (ICTV): classification and nomenclature of viruses. Arch Virol Suppi 10. Vienna, New York: Springer. 1995

 

 

O-067

 

 Quality control in EM viral diagnosis 

STEFAN S. BIEL and Hans R. Gelderblom

Konsiliarlaboratorium für elektronenmikroskopische Erregerdiagnostik Robert Koch-Institut, Nordufer 20, D-1 3353 Berlin, Germany

 

Unlike other diagnostic techniques for infectious diseases, EM by its undirected, ‘open’ view is a direct and rapid ‘catch-all method’ detecting even agents not previously considered to be involved. After negative staining following a simple and rapid protocol, particle size, shape, and specific fine structure are assessed criteria which help to assign the observed virion to one of the 27 animal-specific virus families. The rapid morphological diagnosis is advantageous and in most cases fully sufficient for the clinician to guide the patient's treatment.

In order to keep a certain diagnostic standard in EM diagnosis, regular external quality control measures are essential. Hence we have started an External Quality Assessment Program on EM Viral Diagnosis (EQA-EMV) in 1994 comprising two EQA runs per year. At the present EQA-9 there are 84 participants from 25 countries registered, mainly from European countries. Information on the EQA program, workshops and laboratory courses can be obtained from the world wide web at http//www.electronmicroscopy.org.

The effectiveness of and need for this program becomes obvious looking at the still increasing numbers of participating laboratories and countries as well as the increasing rate of correct diagnostic results. Provided modern enrichment techniques and regular EQA measures are implemented, EM also in the future will play a useful and competitive role within the conventional and molecular-genetic diagnostic armament.

 

 

O-068

 

 Linking prophylactic and therapeutic intervention to molecular evolution of viruses 

L.S. CHRISTENSEN

Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark

 

Vaccination programs and therapeutic interventions involving contaminated blood products both have the potentials of significantly affecting the evolution of viruses, and studies on how evolution is affected by these measures in turn might significantly contribute to our understanding of the driving force and mechanisms of evolution. Studies on animal herpesviruses are reviewed which revealed clonal population structures of globally circulating strains on one hand and—on the other hand—cambrian explosion-like events taking place in day-to-day evolution of virus due to co-infections. Such explosion events were shown to take place as a result of the use of live vaccines and involve a transition stage of a fitness peak jump characterised by an extreme inherent diversity of virus strains. The question is addressed if such cambrian explosion-like events might explain the evolutionary patterns of parvovirus B19 and measles virus. In addition, the need and perspectives of establishing a European network for studies on molecular epidemiology and evolution is discussed in this context.

 

 

O-069

 

 A two-year cyclic pattern of RSV epidemics in Finland 1980–2000 

M. WARIS^1,2, O. Ruuskanen³, T. Heikkinen³ and P. Halonen^{2 

 1}Laboratory of Biophysics, University of Turku, Turku, Finland

 ²Department of Virology, University of Turku, Turku, Finland

 ³Department of Pediatrics, University of Turku, Turku, Finland

 

Over two decades, the Diagnostic Virology Unit in Turku has used antigen detection methods for the surveillance of RSV infections in Finland. Since 1987, the method has been essentially the same time-resolved fluoroimmunoassay (TR-FIA) with monoclonal antibodies. The diagnostic service has been backed up with culture and serology, allowing occasional evaluation of the immunoassay performance. During the past twenty years, RSV epidemic seasons in Finland have occurred in two-year cycles. They have started every second year with a minor spring outbreak followed by a major one the next winter. During each season, RSV has been circulating for more than 12 months. Monoclonal antibodies differentiating between A and B strain groups have been used to type clinical specimens and isolates. Group A or B strains predominated (>70%) in alternating cycles, except for 1991–92, when prevalence of group B strains was lower than expected and neither strain group predominated. Results of genotyping and limited nucleotide sequencing of G protein genes for group A strains from 1991–92 implicated emergence of a group A genotype that had been rare in previous years. In a clinical study performed in Turku University Hospital, RSV infections with group A strains were found to be more severe than those with group B strains, whereas the latter were more often associated with acute otitis media. Although hospitalised cases with laboratory verified re-infections were rare, findings in them suggest an age-dependent, strain group-specific protection. The two-year cyclic pattern of RSV infections seems to be a manifestation of the race between herd immunity and virus variation.

 

 

O-070

 

 Molecular epidemiology of the measles virus: what can we learn from it for monitoring vaccination strategies 

CLAUDE P. MULLER, Frank Hanses, Mick Mulders and Anh T. Truong

WHO Collaborative Center for Measles, Laboratoire National de Santé, PO Box 1102, L-1011 Luxembourg

 

Sequencing of wild-type isolates has proved to be an effective tool to monitor measles circulation and to confirm epidemiological links between cases. In the United States, molecular epidemiology has demonstrated interruption of virus transmission. In Europe measles incidence remains high and in some parts the disease is likely to be still endemic due to insufficient vaccination. Luxembourg experienced an outbreak with at least 110 cases in 1996, and cases continued to be reported throughout 1997 suggesting sustained virus transmission. We used molecular epidemiology to investigate this apparent endemicity. On the basis of their N gene sequences, the isolates were assigned to the typical European C2 and D6 genotypes. Sequence diversity within the temporally and spatially confined outbreak was 0.2% over the C-terminus. This was far lower than the diversity within each of the two co-circulating clusters of of the endemic B3 viruses that we observed in Nigeria. The nucleotide distance between the C2-viruses of the outbreak and the other C2 isolates was at least three or four times higher, suggesting an independent origin of the latter viruses. Similarly, the four D6 viruses found in Luxembourg were thought to be of at least two or three origins. Thus using intraoutbreak diversity as a measure we were able to differentiate between sporadic infections caused by endemic virus strains and a ‘pseudo-outbreak’ due to multiple unrelated imported cases. The approach described here may also useful to monitor the effectiveness of mass campaigns, where measles reporting is incomplete and the community is highly mobile as is the case in many developing countries.

 

 

O-071

 

 An aberrant genotype revealed in recombinant hepatitis B virus strains from Vietnam 

CHARLES HANNOUN^1,2, Heléne Norder, Magnus Lindh

 ¹Department of Clinical Virology, Göteborg University, Göteborg, Sweden

 ²Department of Virology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden

 

Seven genotypes of hepatitis B virus (HBV) have been described, but relatively few complete genomes from East Asia, where most of the world's HBV carriers live, have been studied. Five complete HBV genomes of Vietnamese origin were studied because on genotyping they produced atypical patterns. Their HBsAg sequences had markers for serotype adw. In phylogenetic tree analysis, two of the genomes clustered with genotype C, and three on a separate branch between genotypes A, B and C. In similarity plot and bootscanning analysis, these 3 strains showed signs of recombination, and phylogenetic tree analysis of two segments separately supported recombination between genotype C and a putative new genotype (or possibly a subgroup of genotype A): One segment (nt 1801-2865) was of genotype C origin, while the major part of the genome (nt 2866-1800) placed on a branch near genotype A. The findings encourage further study of genotypes and recombination in HBV from this geographic region.

 

 

O-072

 

 Molecular clock analysis of the three major lineages of the primate t-cell lymphotropic viruses 

SONIA Van DOOREN, Marco Salemi and Anne-Mieke Vandamme

Rega Institute for Medical Research, Minderbroedsstraat 10, B-3000 Leuven, Belgium

 

The molecular clock hypothesis was tested and statistically evaluated on full genome aligned sequences of the three major lineages of primate T-lymphotropic viruses (PTLVs): STLV-L, HTLVI/STLV-1 and HTLV-11/STLV-11. When first and second codon position of the coding regions were used, these analyses showed that the different PTLV lineages evolve at different rates. However, the clock hypothesis cannot be rejected for STLV-L and HTLV-I/STLV-I considering only the third codon position. Anthropologically documented migrations of the PTLV host were correlated with PTLV divergence times to estimate an evolutionary time scale for the PTI-Vs. According to our calculations this family of viruses originated more than one million years ago. We also estimated that HTLV-11 separated from STLV-11 (in Pygmy chimpanzees), not much earlier than 400 000 years ago. Thus, human and simian lineages of the primate T-lymphotropic viruses probably arose after speciation of their hosts through ancient interspecies transmissions. Finally, molecular clock analysis showed that HTLV-11 has two different evolutionary rates. The virus seems to evolve 50–250 times faster in injecting drug users (IDUs) than in endemic infected tribes. The dramatic increase of the rate of evolution among IDUs, that according to our analyses occurred just in the last one hundred years, might have important consequences for the future pathogenicity of this virus.

 

 

O-073

 

 Some effects of ignoring recombination in phylogenetic analyses 

MIKKEL H. SCHIERUP and Jotun Hein

Department of Ecology and Genetics, University of Aarhus, Aarhus, Denmark

 

Recombination appears to be common in population surveys of HIV and most other viruses. At the same time, phylogenetic trees are reconstructed from these data sets and used for evolutionary inferences. When recombination occurs, different parts of the sequence have different histories and a single phylogenetic tree does not exist. We quantified the amount of recombination needed before phylogenetic trees are affected by simulating sequences with recombination and inferring phylogenetic trees ignoring this recombination. We found that even very small amounts of recombination causes the molecular clock to disappear, thus questioning dating of events from virus phylogenies. Furthermore, a large amount of the rate heterogeneity inferred in viruses may be an artefact of ignoring recombination. Finally, ignoring recombination make the tree appear star-shaped which may be taken as evidence for exponential growth rather than recombination.

 

 

O-074

 

 The long lasting multiepitopic B- and T-cell responses induced in humans by an HIV-1 lipopeptide vaccine 

HANNE GAHERY-SEGARD¹, Gilles Pialoux², Suzanne Figueiredo¹, Hubert Poncelet², Helene Gras-Masse³, Jean-Paul Lévy¹ and Jean-Gérard Guillet^{1 

 1}INSERM, INSERM U445, Hopital Cochin, 27 Rue du Fauborg St. Jaques, Paris 75014, France

 ²Institut Pasteur, Paris, France

 ³CNRS URA1309, Institut Pasteur de Lille, Lille, France

 

We have developed an anti-HIV lipopeptide vaccine using several HIV-specific extent large peptides added with a lipid group. A mixture of six lipopeptides derived from regulatory or structural HIV-1 proteins (NEF, GAG, and ENV) was prepared. A phase I study was conducted to evaluate the immunogenicity and the tolerance of the vaccine in HIV-seronegative volunteers after three injections. A fourth injection of the lipopeptide vaccine was given to volunteers to follow the booster effect. Finally we evaluated the long lasting immune response over a period up to 2 years.

We showed for the first time that a lipopeptide vaccine containing different HIV-1 epitopes characterized in NEF, GAG and ENV viral proteins are able to induce a strong multiepitopic B and T cell responses in humans already after three injections (Gahéry-Ségard et al., J. Virol., 2000). The vaccinated subjects produced specific IgG antibodies that recognized the NEF and GAG proteins. Helper CD4+ T cell responses as well as specific cytotoxic CD8+ T cells were also obtained. These CD8+ T cells were able to recognize naturally processed viral proteins. Moreover, HIV-specific IFN-gamma secreting CD8+ T cells were detected, ex-vivo and confirmed after one stimulation. The B- and T-cell responses were extensively boost after the fourth injection. Finally a long lasting and polyepitopic CD4+ and CD8+ T cell responses were sustained over a period up to 2 years.

To conclude, we believe that the development of an anti-HIV lipopeptide vaccine is a suitable method of inducing B and T cell multiepitopic responses in humans.

 

 

O-075

 

 Does vaccination against visna-maedi protect sheep against natural infection? 

MARGRÉT GUDNADÓTTIR

Institute for Virology, University of Iceland, Ármúil 1A, PO Box 8733, Reykjavík IS-128, Iceland

 

One twin lamb in each of eight pairs of twins was immunized with formalin inactivated whole visna virus, strain K 796, to which alun was added. The vaccinees developed antibodies against all major proteins of the virus as shown by Western Blots, and neutralizing antibodies (Ann. N.Y. Acad. Sci. 1994, Vol. 724, 140–147). After vaccination all the twins were housed with five heavily infected sheep in a closed sheep hut.

4 years later five unvaccinated twins had seroconverted. These five twin pairs were then slaughtered and tissue samples from spleens, hilar lymph nodes, lungs and chorioid plexuses collected for virus cultivation.

Virus was grown from all the five unvaccinated twins and two vaccinees, but three vaccinees were negative although every drop of tissue culture fluid from explants and cocultures of their organs were passed on for over 6 months.

Although there is only a small number of animals in this experiment, the results seem to indicate that it is possible to vaccinate against a slow lentivirus infection by conventionally prepared inactivated vaccine.

 

 

O-076

 

 Effect of amount, cellular location, and stability of an antigen on its capacity to induce a cytotoxic T cell response 

ANKE HUCKRIEDE, Barry-Lee Waarts, Marijke Holtrop, Derk-Jan de Groot, Jan Wilschut

Molecular Virology, University of Groningen, A. Deusinglaan 1, Groningen 9713 AV, The Netherlands

 

Activation of cytotoxic T lymphocytes (CTLs) requires presentation of antigenic peptides by MHC class l molecules on antigen-presenting cells. The peptides to be presented are generated by proteasomal degradation from antigens present in the cytosol. They are subsequently transported via the TAP transporter to the ER where they associate with nascent MHC class l molecules. Thus loaded MHC class l complexes are transported via the Golgi apparatus to the plasma membrane. When attempting to induce a CTL response by vaccination efficient presentation of the antigen in question is very important. Using influenza nucleoprotein (NP) as a model antigen we therefore investigated the effect of amount, location, and stability of an antigen on its presentation by MHC class l molecules and its ability to prime specific CTLs in vivo. Immunizations were performed using recombinant Semliki Forest virus (SFV) particles encoding NP. Transfection with recombinant SFV leads to very high expression of the transgene for 24–72 h whereafter the transfected cells die by apoptosis. Four different constructs were prepared: (1) SFV-NP: full-length NP, (2) SFV-enhNP: NP cloned behind a translational enhancer sequence derived from the SFV capsid protein sequence, (3) SFV-^∧nlsNP: NP missing the first 16 amino acids including the nuclear localisation signal (nls), and (4) SFV-ubNP: NP with a ubiquitin monomer fused to the N-terminus. Histochemical examination of transfected cells demonstrated that NP was located in the nucleus while ubNP was found mainly in the nucleus but also in the cytoplasm.?nls-NP was detected exclusively in the cytoplasm indicating that the first 16 amino acids are indeed essential for transport of NP to the nucleus. Biochemical analysis of transfected cells showed that all constructs were expressed to substantial levels. As expected, expression of enh-NP was 8–10× higher than that of the other constructs. Pulse-chase experiments revealed that NP and enh-NP were very stable with an estimated half-life of about 16 h. Despite the cytoplasmic localization (and thus excellent accessibility for proteasomal degradation) ^∧nls-NP showed the same half life as the other two constructs. In contrast, ubNP, which is targeted directly to the proteasomes via its ubiquitin moiety, appeared to be degraded much faster. Using the different constructs for immunisation of mice we are currently investigating whether the extent of the immune response (in terms of frequency of activated CTL precursors) is determined predominantly by the amount of antigen or by its degradation kinetics.

 

 

O-077

 

 Development of a hepatitis C virus vaccine: the benefice and limitations of DNA vaccination 

GENEVIÈVE INCHAUSPE, D. Boucreux, C. Brinster, A. Fournillier, N. Renard and O. Vidalin

INSERM U 271, 151 Cours Albert-Thomas, 69003 Lyon, France

 

While the study of the natural infection by hepatitis C virus (HCV) remains incomplete, increasing evidence support the concept that both humoral and cellular immune responses play a role in the control, possibly in the prevention, of the infection. These responses, except in an apparent minority of cases, are generally incompetent to eliminate the virus from an infected host. A successful vaccine should do more than simply mimic responses found in the natural infection but should rather induce optimized and more vigourous ones. We have explored different ways to formulate HCV vaccine-DNAs in order to reach this goal. Plasmids have been engineered to express different forms of the glycoproteins El and E2, in particular forms that will keep the ability to interact and produce noncovalent dimers presumed to exist on the surface of viral particles. Additional studies have addressed the issue of the role play by sugars in the immunogenicity of these glycoproteins. Different DNA-expression vectors as well as molecular targeting strategies have also been employed to evaluate the capacity of HCV-immunogens to induce either or both humoral and cellular immunity. Data have been gathered in BALB/c mice, mice transgenic for the human HLA A2.1, as well as in limited instances, in small primates.

 

 

O-078

 

 Developing safe vectors based on trans-complementation of defective TGEV by packaging cell lines 

JAVIER ORTEGO¹, I. Sola¹, D. Escors¹, H. Laudet² and L. Enjuanes^{1 

 1}Centro Nacional De Biotecnología (Csic), Department of Molecular and Cell Biology, Campus Universidad Autónoma, Madrid 28049, Spain

 ²Inra, Unite De Virologie Immunologie Moleculaires, France

 

A TGEV full-length infectious cDNA has been generated in our laboratory. This cDNA is being used to develop safe vectors for vaccination and gene therapy based on trans-complementation by a packaging cell line of defective TGEV genomes expressing heterologous genes. Packaging cell lines stably expressing individual TGEV structural proteins (E, M, N, and 7) were established by using the non-cytopathic Sindbis virus replicon expression vector pSINrep21 (Frolov, I., et al., 1996).

The cell lines were generated by transfection of these constructs in BHK-21 cells that constitutively expressed porcine aminopeptidase-N, the major receptor for TGEV (Delmas, B. et al. 1992). The expression of TGEV proteins was detected by Western blot and immunocitochemistry and could be maintained at least during ten passages in cell culture. The presence of the Sindbis virus replicon did not interfere the production of infective TGEV viral particles in these cell lines, as assessed by virus titration (108 p.f.u./ml). Knocked-out TGEV genomes are being generated from the cDNA in order to produce defective viral particles that can be complemented in trans by the packaging cell lines. To increase the cloning capacity of TGEV derived vectors, the non-essential genes 3a and 3b of TGEV were deleted. This modified cDNA produced infectious virus with titers comparable to that of the wild type TGEV cDNA. The green fluorescent protein (GFP) gene was introduced into this defective cDNA under the transcription regulatory sequences of the 3a gene, leading to high GFP expression levels. The generation of TGEV genomes deficient for the structural genes and the complementation with packaging cell lines to generate defective viral particles to develop safe vectors for vaccine development and gene therapy will be discussed.

 

 

O-079

 

 Highly sensitive SPF10 PCR and reverse hybridization line probe assay (LIPA) for detection and identification of HPV genotypes 

BERNHARD KLETER, A.C. Molijn, L.J. van Doorn and W.G.V. Quint

Delft Diagnostic Laboratory, R. de Graafweg 7, Delft 2625 AD, The Netherlands

 

Adequate detection and genotyping of HPV DNA is required to assess the role of HPV infections in cervical pathology. The aim of the present study was to evaluate the recently developed SPF10 primer set and a reverse hybridization assay (LiPA) for detection and identification of HPV genotypes (6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 70, and 74). This method was evaluated in a large number of clinical materials.

SPF10-PCR permitted HPV analysis in cervical scrapes (including ThinPrep samples) as well as freshly frozen biopsy specimens. The SPF10 primers are highly sensitive, detecting HPV DNA in about 23% of women with normal cytology, in approximately 50% of the ASCUS cases, in 98% of the cases with mild to severe dyskaryosis, and in 98–100% of patients with cervical carcinoma. Due to the small amplimer size, the SPF10 primers also detect HPV DNA in formalin-fixed, paraffin-embedded materials and in archival Pap smears.

LiPA results were compared with direct sequence analysis of SPF and My09/11 amplimers and type-specific PCR, and showed highly concordant results. Multiple HPV genotypes were more frequently detected by LiPA: in 12% of the cases with normal cytology or ASCUS, in 35% of the dyskaryotic cases, and in 4–13% of the carcinomas. Sequence analysis underestimates the prevalence of multiple genotypes.

The SPF-PCR, combined with the HPV-LiPA offers a highly sensitive and specific tool to investigate HPV infections and will facilitate further clinical and epidemiological studies.

 

 

O-080

 

 Rapid identification of viral pathogens in cerebrospinal fluid by multiplex PCR followed by line probe assay 

PETER MUIR¹, Lisa J. Brown¹, Marjo van der Weide², Dianne N. Irish³, Nicholas W.S. Davies³, Robin S. Howard³, Mohammed K. Sharief^1,3, Richard O. Robinson^1,3, Geert Jannes² and Rudi Rossau^{2 

 1}Department of Infection, King's College London, St. Thomas’ Hospital Campus, Lambeth Place Road, London, SE1 7EH, UK

 ²Innogenetics NV, Gent, Belgium

 ³Department of Infection, Guy's & St. Thomas’ Hospital Trust, London, UK

 

We describe two mutliplex assays which allow detection of seven of the commonest viruses encountered in patients with suspected viral meningitis or encephalitis. One assay detects herpes simplex virus (HSV) types 1 and 2, varicella zoster virus (VZV), human herpesvirus type 6 (HHV6) and human enteroviruses, and a second detects cytomegalovirus (CMV) Epstein–Barr virus (EBV) and JC virus, as well as Toxoplasma gondii. Following a single round of multiplex PCR amplification, specific identification of viral or protozoan sequences is achieved by reverse hybridization to specific probes in a Line-Probe Assay (LiPA). This multiplex PCR-LiPA system was found capable of detecting 10–100 genome equivalents in 10 l of CSF when evaluated using external quality assessment panels for HSV, enterovirus and CMV. Upon evaluation of a panel of 577 CSFs submitted for diagnostic evaluation, a virus was identified in 86 samples (15%), and T. gondii was identified in one sample. Viruses were found more commonly in CSFs showing leukocytosis and/or elevated protein levels. Interestingly, however, none of eleven patients in whom HSV 1 was detected had CSF leukocytosis, whereas seven of eight patients with HSV 2 infection had leukocytosis. HSV 2 infection was frequently associated with aseptic meningitis in young women. Multiple herpesviruses were commonly detected in patients with HIV-related disease. Use of these assays could thus reduce the time and number of PCR assays required to confirm or exclude a viral diagnosis in patients with central nervous system disease. The LiPA detection format also achieves reliable and sensitive post-PCR viral identification.

 

 

O-081

 

 Real-time PCR to detect herpes viruses in samples from immunosupressed patients 

SUSAN NICOLL, A. Brass and H.A. Cubie

RCVL, City Hospital, Greenbank Drive, Edinburgh EH9 3ED, UK

 

 Purpose: Herpes viruses are an important cause of morbidity and mortality in patients immunocompromised due to transplantation or AIDS. As treatment is available it is important to develop a rapid screening system to identify patients at risk of clinical disease.

 Methods: Gene 29 from VZV, gpD of HSV1, gpG of HSV2 and MIE and gB genes of CMV were amplified using nested and real-time PCR. Systems were optimised using material obtained from cell culture and products were cloned into pGEM-T to ascertain the sensitivity of each system. DNA was extracted from 250 clinical samples and was used in both PCR systems. Unextracted samples were also tested. Results were compared with conventional serology or culture.

 Results: The sensitivity for VZV, HSV1 and HSV2 was bewteen 1 and 10 copies, while that of CMV was 10–100 copies. The sensitivity of the CMV system was increased by carrying out an initial 10 cycles of PCR using outer primers and then adding to the real-time PCR. 34 VZV, 34 HSVI, 32 HSV2 and 10 CMV positive results were obtained (one HSV2 was not detected by culture). All spiked CSF samples were detected, whether extracted or not. Turn-around time was less than 2 hours compared with a mean of 4.4 days for culture? immunofluorescence.

 Conclusions: Real-time PCR is a cost effective, sensitive and rapid means of detecting these viruses, with no need for extraction of most samples. This system will therefore be of benefit in screening immunocompromised patients.

 

 

O-082

 

 Diagnostic detection and quantification of pathogenic bunyaviridae by a TaqMan-RT-PCR approach 

MANFRED WEIDMANN and F.T. Hufert

Dept. of Virology, Inst. for Med. Microbiology and Hygiene University of Freiburg, Hermann-Herder-Str. 11, Freiburg 79104, Germany

 

The TaqMan-PCR technology offers the possibility of specific and highly sensitive one tube RT-PCR. There is no need for a second round nested PCR. Including all sequences available in the EMBL-databank the concept of this PCR-system has been applied to devise a set of primers to detect and distinguish pathogenic Bunyaviridae of the genera Bunyavirus, Hantavirus, Nairovirus and Phlebovirus. In a modular approach for Hantaviruses a first set of primers identifies pathogenic viruses at the genera level. Simultaneously this signal also gives a preliminary indication as to the identity of the putative species. The second set of primers is entirely species specific and able to identify pathogenic Hantaviruses without doubt. Apart from the pure diagnostical value this TaqMan-PCR scheme also offers the possibility to monitor disease development in infected patients by quantifying the viral load at continuos time points.

 

 

O-083

 

 Role of nested polymerase chain reaction for detection of respiratory pathogens in children with bronchiolitis 

GRACE ONG, P.V Coyle, D.E Wyatt, H.J. O'Neill and C. McCaughey

Regional Virus Laboratory, Royal Hospitals Trust, Grovesnor Road, Belfast BT12 6BN, Northen Ireland, UK

 

 Purpose: Influenzae A (H3N2) and Respiratory Syncytial Virus (RSV) co-circulated during the winter months of November 1999 & January 2000. Nested polymerase chain reaction (PCR) was compared with immunofluorescence (IF) for the detection of RSV serotype A and B in children with suspected bronchiolitis. Coinfection with Influenzae A (H3N2), Chlamydia spp. and enterovirus/Rhinovirus was also investigated using molecular techniques.

 Methods: A total of 50 nasopharyngeal secretions collected from babies admitted with bronchiolitis in the month of January 2000 comprising IF RSV positive (n=27) and RSV negative (n=23) specimens were tested for both RSV serotypes, Influenzae A (H3N2), Chlamydia spp. and enterovirus/Rhinovirus by nPCR. All specimens were also negative by IF for Influenzae A, Influenzae B, Adenovirus and Parainfluenza virus types 1, 2 and 3.

 Results: Twenty-six specimens were positive for RSV by both methods. Two secretions which were negative for RSV by IF were positive for RSV A and B respectively by nPCR (RSV A=20, RSV B=8). Influenzae A (H3N2) was detected in 3/50 specimens which were RSV negative by nPCR. Chlamydia spp. (identified as C. trachomatis) was present in one infant with RSV B and who gave a recent history of treated C. trachomatis conjunctivitis. Enterovirus was found in 11/50 secretions of which 3 had RSV or Influenzae A (RSV A=11, RSV B=1, H3N2=1) detected by nPCR and one had RSV by IF.

 Conclusions: nPCR proved more sensitive and specific than IF in detecting RSV and Influenzae A (H3N2). The presence of Influenzae A infection in secretions reported negative using conventional techniques highlights infection control issues in the cohorting of bronchiolitis patients in hospital. The demonstration of RSV and C. trachomatis infection addressed the contribution of the latter to bronchiolitis and the efficacy of treatment in eradicating Chlamydia carriage in this child. Further differentiation of enterovirus to genus level will be reported in the near future.

 

 

O-084

 

 The use of accurate measurement of HBV DNA in combination with the INNO-LiPA HBV resistance assay during lamivudine treatment: implications for disease management 

HUBERT G.M. NIESTERS¹, Suzan D. Pas¹, Leonieke Wolters², Annick van Nunen², Robert A. de Man², Edwin Fries¹ and Albert D.M.E. Osterhaus^{1 

 1}Department of Virology, University Hospital Rotterdam, Rotterdam, The Netherlands

 ²Department of Gastroenterology, University Hospital Rotterdam, Dr Molewayterplein 40, Rotterdam 3015 GD, The Netherlands

 

Lamivudine is a potent inhibitor of hepatitis B virus replication. Hepatitis B virus resistance to lamivudine has been described in patients under treatment with characteristic mutations in the so-called YMDD motif, combined with a mutation at position Leu528 in the polymerase gene. The appearance of this resistance is usually observed by an increase in viral HBV DNA load using hybridization assays with a limit of detection above 1 million copies per ml, combined with sequence analysis of PCR products. This resulted in the conclusion that the actuarial cumulative resistance incidence in our Rotterdam cohort was 39%. New generations of assays, both commercial (Digene Hybrid Capture 11) and in-house developed real time detection assays, can measure more accurately HBV DNA over a large dynamic range. The development of mutations linked to the resistance to lamivudine were monitored using the INNO-LiPA HBV DR resistance strip, which enabled to semi-quantitatively detect both wildtype and mutated viral sequences. In a retrospective analysis, we analyzed the occurrence of lamivudine resistance in relation to changes in viral load and increase of serum transaminases (ALT). Eleven patients were treated with 100 or 150 mg lamivudine per day for median 90 weeks (range 52–120 weeks) with median viral load of 2E9 copies per ml (range 1.08E8–7.75E10). Patients were monitored at least on a monthly basis. Reduction in viral load was median 4.42 log (range 2.7–8.91 Log), but always detectable by real time detection assays. Time from start therapy to appearance of first mutation detected by INNO-LiPA HBV DR was at week 42 (range 20–69), while increase in viral load was detected at week 45 (range 12–82). In 8 of these patients, a mixture of both YIDD and YVDD variants was observed at given timepoints. Reduction of ALT was observed in all patients with an average of 74.5% (range 37.7–91.8). An increase of ALT was observed at week 68 (range 49–107), which was on average 26 weeks later than the appearance of a mutation linked to resistance.

 Conclusion: A combination of accurate HBV DNA measurements and INNO-LiPA HBV DR resistance assays identifies HBV lamivudine resistance strains at the earliest timepoints. Elevations of ALT levels cannot be used for clinical patient management.

 

 

O-085

 

 Implementation of a surveillance network of the herpes simplex virus resistance to antiviral drugs 

BRUNO LINA¹, D. Thouvenot ¹, C. Danve¹, F. Morfin¹, A. Boucher¹, I. Berlin², M. Aymard¹ And The Laboratories of Virology of Annecy, Bordeaux, Brest, Clermont-Ferrand, Grenoble, Marseille, Montpellier, Nancy, Nantes, Paris, Poitiers, Saint-Etienne, Toulouse and Army (Paris)

 ¹Laboratoire de Virologie, Domaine Rockfeller, 8 avenue Rockefeller 69373, Lyon cedex 08, France

 ²Laboratoire GlaxoWellcome, 100 route de Versailles, 78163 Marly-le-Roi, France

 

 Background: As the continuous increase in acyclovir (ACV) consumption could rise the prevalence of Herpes simplex virus (HSV) ACV-resistance, we established, with GlaxoWellcome laboratory collaboration, a pilot network of 15 hospital laboratories of virology for evaluating the prevalence of ACV-resistance.

 Methods: Samples were collected from 567 immunocompetent and from 514 immunocompromised patients. Herpes simplex viruses were isolated in MRC-5 or Vero cells, identified by immunofluorescence test as type 1 or 2 and sent to Lyon. There, the HSV ACV-susceptibility was tested using a simple colorimetric assay in microplates (2 HSV isolate doses tested with 5 and 10μM of ACV), read after 2 days incubation (37°C) at OD 540 nm.

 Results: Overall, 1268 HSV isolates were collected from 1081 patients. Twenty-seven patients (2, 5%) excreted ACV-resistant HSV, with a percentage ranging from 0, 35% in immunocompetents to 5, 22% in immunocompromised patients (haemopathies: 3%, organ transplants: 4, 3%, HIV infection: 6%, bone marrow transplants: 12%). Among the 25 immunocompromised patients excreting

ACV-resistant HSV, 21 previously received ACV treatment, and 15 exhibited clinical resistance (71, 5%). Out of the 470 immunocompromised patients excreting ACV-sensitive HSV, 84 previously received ACV treatment, and 6 exhibited clinical resistance (1.3%).

 Conclusion: ACV-resistance prevalence does not seem to increase. However, it remains a major concern for the management of HSV infections.

 

 

O-086

 

 Identification of mutations associated with resistance to BAY 38-4766—a novel non-nucleosidic inhibitor of cytomegalovirus replication 

IRIS BUEGER¹, S. Hallenberger¹, H.-D. Klenk² and H. Ruebsamen-Waigmann^{1 

 1}Bayer AG, Business Group Pharma, Antiinfective Research, Virology, Aprather Weg 18a, Wuppertal D-42096, Germany

 ²Institute of Virology, University Marburg, Robert-Koch-Str. 17, D-35037 Marburg, Germany

 

Human cytomegalovirus (HCMV), a beta-herpesvirus, causes sight- and life-threatening disease in immunocompromised patients. Treatment with currently marketed anti-HCMV drugs is associated with poor oral bioavailibility and dose-limiting toxicity. The clinical development compound BAY 38-4766 is a member of a new class of non-nucleosidic drugs which revealed inhibition of a late stage in the replicative cycle of CMV in kinetic studies.

In order to identify the viral target, resistant strains of HCMV and MCMV were generated by in vitro selection with increasing inhibitor concentrations. Sequence analysis of resistant HCMV strain AD169 and resistant MCMV Smith mapped mutations to orfs UL89 and UL104. Both proteins are thought to be essential for viral DNA cleavage and packaging of the genome into procapsids. Mutational results were experimentally confirmed by a functional DNA cleavage assay including Southern blot technique. In the case of wildtype MCMV, cleavage of viral DNA at the genome terminus correlated with different doses of inhibitor while viral DNA cleavage of resistant MCMV was unaffected. Recombinant MCMV mutants were generated by cotransfection of mouse embryo fibroblasts with combinatorial sets of overlapping MCMV DNA fragments. Drug susceptibility profiles of reconstituted virus populations were determined using a quantitative lacZ-based enzyme assay. The role of mutations of both, M89 and M104, in development of MCMV resistance to BAY 38-4766 are presented.

These findings strongly suggest, that BAY 38-4766 specifically inhibits cleavage of high-molecular-weight viral DNA concatemers to monomeric genome lengths performing a unique mechanism of action distinct from the marketed DNA replication inhibitors.

 

 

O-087

 

 Prevalence and patterns of antiretroviral resistance in treatment-naïve patients recently infected with HIV-1 

ANNA MARIA GERETTI¹, M. Smith², M. Donati², P. Easterbrook¹ and M. Zuckerman^{2 

 1}Academic Department of HIV Medicine, King's College Hospital, Weston Education Centre, Cutcombe Road, London SE5 9RT, UK

 ²Dept. of Virology, King's College Hospital, Guys, King's & St. Thomas’ School of Medicine, London, UK

On behalf of the South London Viral Resistance Collaborative Group

 

 Background: A high prevalence of drug-resistant HIV has been reported in treatment-naïve patients in North America. Data from European patients remain limited.

 Objective: To examine the distribution of HIV-1 subtypes and the prevalence of antiretroviral resistance in 26 treatment-naïve patients infected with HIV-1 after January 1995.

 Methods: 9 patients presented during seroconversion; 17 had an interval of <24 months between first positive and last negative HIV test. There were 18 Caucasian (83% homosexual males), 4 Caribbean and 2 African patients (4 females and 2 males with heterosexually acquired infection). Viral RNA from plasma collected at diagnosis was analysed by direct sequencing of RT and protease genes. Subtypes were determined by sequence analysis and peptide-EIA.

 Results: Non-B subtypes or intersubtype recombinants were found in 10 (38%) patients. A ZDV primary resistance mutation M41L was found in one homosexual Caucasian patient. The same patient also carried the secondary mutations T215C (RT) and L63P (protease). The two RT mutations persisted for >28 months while the patient was receiving d4T, ddl and nevirapine. The L63P mutation disappeared over time. Secondary mutations were detected in the RT gene of 2 other patients (K103R/I) and in the protease gene of 17 patients (L10V/I, K20R, M361, L63P, A71T, V771).

 Conclusions: In this heterogeneous South London cohort we found a high prevalence of non-B subtypes and intersubtype recombinants. There remains a low rate of transmission of primary resistance mutations. The M41L mutation, however, was persistent in the absence of ZDV pressure. Analysis of an expanded cohort is ongoing.

 

 

O-088

 

 Genotypic and phenotypic resistance testing in PCR negative HIV-1 patients on HAART 

ANNE SPIELHOFEN, B. Gröschel, J. Cinatl Jr. and H.W. Doerr

Institute for Medical Virology, Johann Wolfgang Goethe-University, Frankfurt/Main, Paul-Ehrlich Str. 40, 60596 Frankfurt, Germany

 

Objective: The introduction of ‘highly active antiretroviral therapy’ (HAART) made it possible to lower the viral load (VL) of most HIV-1 infected patients to below the detectable level of most commercial test kits, i.e. <50 RNA cop/ml. Despite HAART, however, it is still not possible to eradicate virus completely because of resting and latently infected memory T-cells that cannot be reached by therapy. The aim of this study was to determine whether single clones of latently infected memory cells obtained from patients with an undetectable viral load differed in resistance patterns and the occurrence of syncytium-inducing (SI) and non-SI (NSI) strains.

 Methods: 107 PBMCs from HIV-positive donors were CD8 depleted, CD4+CD45RO and CD4+CD45RA separated and cultivated in microtitre plate with immobilised anti CD3/CD28 antibodies. Six responders were tested who maintained VL <50 RNA cop/ml on HAART for more than one year. HIV strains were cultivated with a frequency of 0.3–0.6 per 1×10⁶ CD4 cells. Resistance was genotypically analysed by reverse transcriptase, protease and V3-loop gene sequencing and phenotypically by culture with PHA-PBMCs in the presence of antiretroviral drugs. Sl and NSI phenotype was determined using MT-2 assay system.

 Results: In four patients, both drug-sensitive and drug-resistant strains could be detected. The other two patients did not develop any drug resistance within the viral genome. Four patients showed both Sl and NSI strains, two patients had NSI strains only.

 Conclusions: The results demonstrate that in HAART responders (<50 cop/ml) heterogeneous virus strains occur in terms of drug sensitivity and SI/NSI. Further investigations should show which viral strains replicate when HAART fails.

 

 

O-089

 

 Molecular evolution of echovirus 30: limited range of genetic diversity associated with relative excess of amino acid substitutions 

TAPANI HOVI^1,2, Carita Savolainen² and Mick Mulders^{2,3 

 1}Department of Virology, National Public Health Institute (KTL), Mannerheimintie 166, Helsinki FIN-00300, Finland

 ²Enterovirus Laboratory, National Public Health Institute (KTL), Helsinki, Finland

 ³Present address: Laboratoire National de Santé, Department of Immunology, Luxembourg

 

Echovirus 30 (E30), a member of the human enterovirus-B species (family Picornaviridae), and a common cause of epidemic aseptic meningitis, shows a strikingly monotypic pattern of molecular evolution. The overall genetic divergence in the capsid coding region of the genomes of 131 independent field isolates is clearly less than that of the previously studied other enteroviruses. While several independent genetic lineages, or designated genotypes, within an enterovirus serotype typically cocirculate, a single genotype of E30 appears to have been almost exclusively prevalent during the last two decades in both Europe and Northern America. Within this genotype, strains isolated in the late 1970s through late 1980s cluster separately from those of the 1990s, and the strains causing the widespread epidemic in different parts of Europe during the second half of 1990s are all closely related. In spite of the limited range of genetic variation, the range of amino acid sequence differences in VP1 between the 131 analysed E30 strains is almost double compared to that of 107 coxsackievirus B4 strains showing wider genetic divergence. The variable amino acid positions cluster in regions known to be antigenic in other enteroviruses. It is possible that antigenic selection plays a role in the evolution of E30 but so far, there is no data supporting this hypothesis.

 

 

O-090

 

 Molecular epidemiology of rabies and rabies-related viruses 

LORRAINE McELHINNEY, J. Smith, J.H.A. Bowen-Davies, P.R. Heaton, E.M. Black, C. Black, J.P. Lowings and A.R. Fooks

Rabies Research and Diagnostics Group, Department of Virology, Veterinary Laboratories Agency, Weybridge, Woodham Lane, New Haw, Surrey KT15 3NB, UK

 

The aim of this study was to develop rapid and highly sensitive molecular tools capable of identifying rabies virus isolates from zoonotic animals in the event of a rabies outbreak. Rabies and rabies-related viruses have been classified into six distinct genotypes with a proposed seventh genotype recently reported in Australia. Rabies viruses form multiple biotypes within each genotype. Many biotypes have been identified by the use of monoclonal antibody reactivity patterns but high-resolution differentiation has only recently become possible with the application of genetic sequencing of specific gene regions. Currently, we have sequenced the variable part of the N gene encoding the rabies virus nucleoprotein from more than 800 virus isolates obtained from around the world. These molecular studies have demonstrated genetically distinct groups of viruses, within the genotypes, related by host species and geographic distribution. The resultant database of virus sequences has the potential to form an important epidemiological tool that could contribute to the global disease control of rabies.

 

 

O-091

 

 Use of HBV surface antigen variability to confirm migration patterns of Pacific peoples 

ASHRAF BASUNI¹, Lesley-Anne Butterworth², Graham Cooksley², Stephen Locarnini³ and William F. Carman^{1 

 1}Institute of Virology, University of Glasgow, Church Street, Glascow G11 5JR, UK

 ²Clinical Research Centre, Royal Brisbane Hospital Foundation, Brisbane, Australia

 ³Research & Molecular Development, Victorian Infectious Diseases Reference Laboratory, North Melbourne 3051, Australia

 

There is evidence from history and mitochondrial DNA sequencing that the people of south east Asia migrated either eastwards into Micronesia or southwards into Indonesia and then on into Melanesia and Polynesia. HBV is classified into seven genotypes, A to G; genotypes A and D are widely distributed in old world, while genotypes B and C are confined to east Asia. Genotype E is mainly found in sub-Saharan Africa, genotype F in aboriginal population of the Americas and the recently discovered G in USA and France. There are nine subtypes, so more than one subtype can be found within a genotype. We have studied HBV surface antigen (HBsAg) variation from four different island groups: Kiribati in Micronesia, and Vanuatu, Fiji and Tonga (these three in Melanesia/Polynesia, listed from West to East relative to SE Asia). Samples were collected from unvaccinated children and adults in a number of villages on each island and tested for HBsAg. HBsAg positives were amplified by PCR and sequenced. The predominant sequence in Kiribati (genotype D; subtype ayw2) was different to that in Melanesia and Polynesia (genotype C; adrq-). However, there was a substantial proportion of both in Fiji, implying mixing because of population interchange over centuries. In keeping with the hypothesised migration from West to East (Vanuatu, Fiji, Tonga), specific variants were identified in each island that either increased, or decreased, from West to East. In some cases, such variants were not found in samples from Vanuatu, yet occurred in a majority of persons in Tonga, or vice versa. In addition, linkages of two amino acids were found that were specific for each island. We conclude that each population wave carried with it a specific subtype of HBV and that, within each subtype, only selected viral strains were carried on to the next island that then evolved independently. We believe that this is the first example of using viral strain to confirm other evidence for migration patterns.

 

 

O-092

 

 Divergence of hepatitis B virus-like infections in orang-utans 

ERNST VERSCHOOR¹, Kristin S. Warren^2,3, Susan Langenhuijzen¹, Heriyanto⁴, Ralph A. Swan², Jonathan L. Heeney^{1 

 1}Division of Molecular Virology, Biomedical Primate Research Centre, Lange Kleiweg 139, Rijswijk 2288 GJ, The Netherlands

 ²Division of Veterinary and Biomedical Sciences, Murdoch University, Western Australia

 ³Wanariset Orang-utan Reintroduction Centre, East Kalimantan, Indonesia

 ⁴Department of Animal Quarantine, East Kalimantan, Indonesia

 

A high prevalence of hepatitis B virus (HBV) infection was suspected in 195 ex-captive orang-utans due to sera that cross-reacted with human HBV antigens. Initially, it was assumed that such viral infections were contracted from humans during captivity. However, two wild-caught orang-utans were identified which were HBV surface antigen-positive, indicating that HBV or related viruses may be naturally circulating in wild orang-utan populations. Sequence analyses of 7 isolates revealed that orang-utans were infected with hepadnaviruses, but that these were clearly divergent from the known 6 human HBV genotypes and other nonhuman primate hepadnaviruses reported. Phylogenetic analyses of the sequences encoding the pre-S and small S proteins indicated that the various isolates of the orang-utan hepadnavirus (OHV) could be divided in two types, OHV-1 and -2. Two representative isolates (OU-Somad and OU-Papa) from the types of OHV were analyzed both molecularly and phylogenetically. OHV-1 and OHV-2 were highly similar in their the genome organisation to other primate hepadnaviruses. The complete genome sequences of both OHV types had an overall 5% sequence. Using a PCR-RFLP assay on sera of twenty-five OHV-positive Bornean orang-utans we found 19 OHV-1-infected individuals, while the remaining were infected with OHV-2. Phylogenetic analysis was performed using the full-length genomes of various hepadnaviruses. The tree topology showed a bifurcation, with one branch to the ape viruses and the other to the human genotypes A to E. These data suggest an ancient transmission event with a common ancestor virus and subsequent coevolution of this virus in different orang-utan populations.

 

 

O-093

 

 Prevalence and distribution of TT virus genotypes in german and ghanese individuals: identification of a new virus genotype from Ghana 

KATRIN LEITMEYER, S. Neifer and H. Meisel

Institute of Medical Virology, Charité, School of Medicine, Humboldt University Berlin, Schumannstr. 20/21, Berlin 10117, Germany

 

TT virus (TTV), a circular single-stranded DNA virus, recently found in the sera of Japanese patients with post-transfusion hepatitis of unknown origin (Okamoto et al., 1998) exhibits an unusual high degree of genetic diversity. Considerable divergence on the nucleotide and deduced amino acid level led to classification of currently at least 16 different genotypes (>30%). TTV is worldwide prevalent in humans, however with prevalence rates that vary significantly. In order to investigate the prevalence of TTV in two geographically distant countries, we have tested a total of 192 serum samples from German blood donors (n=30) and pregnant women (n=55), as well as 107 sera from Ghanese individuals by semi-nested polymerase chain reaction. To assess the genetic diversity of the TTV genome in more detail, a partial nucleotide sequence analysis within the ORF1 was performed and 222nt long fragments of 11 sequences were compared with representatives of all known genotypes. Twenty of 85 (23.5%) individuals from Germany and 95/107 (88.7%) from Ghana were found to be PCR-positive, indicating that TTV infection is common, respectively endemic in the areas studied. Phylogenetic analysis revealed that all but one sample belonged to genotypes already recognized among humans, however, with different distributions; four of five German samples grouped into universal genotypes such as genotype 1 and 2, found in all countries examined; one into genotype 3. The Ghanese samples grouped into genotype 2, 3 and 7. Most interesting, nucleotide and amino acid homologies of one sample from Ghana (Ghana33) to sequences of all known genotypes were as low as 31.2–58.4% and 22.2–58.5%, respectively, indicating the identification of a new variant representing a genotype not been reported before.

 

 

O-094

 

 Analysis of the origin of the African human T-cell lymphotropic virus type-1 (HTLV-1) subtypes 

SONIA Van DOOREN¹, M. Salemi¹, X. Pourrul², E. Delaporte², J. Desmyter¹ and A.-M. Vandamme^{1 

 1}Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, Leuven 3000, Belgium

 ²Institut de recherche pour le développement/SIDA Labo des Rétrovirus, Montpellier Cedex 1, France

 

To investigate the HTLV-I/STLV-1 interspecies transmissions, we phylogenetically analyzed the LTR and env sequences of HTLV-I and STLV-I strains. At least four of the five African HTLV-I subtypes cluster with STLV-I strains, more according to geographic origin rather than to host species. Pan troglodytes seems to be the simian reservoir for HTLV-1b. Mandrillus sphinx strains seem to be at the origin of both the HTLV-Id and HTLV-If subtype. HTLV-le clusters with Cercopithecus and Papio spp strains. HTLV-1a, however, has no simian counterpart so far. The Melanesian HTLV-Ic is related to Indonesian STLV-I strains and seems to have been brought to Melanesia and Australia along with the first human settlers.

To date the origin of the African HTLV-I/STLV-1 clades, we performed a molecular clock analysis on the combined LTR-env third codon position sequences, for which the molecular clock holds, using as starting date the anthropologically documented migration of Melanesian settlers from Indonesia to Melanesia and Australia, 50 000 years ago. We estimated that the virus spread in Africa around 25 000 years ago. The different African HTLV-I subtypes arose between 16 000 and 8000 ya for HTLV-1b, -1d and -1e, although less than 530 ya for HTLV-1f. Interspecies transmissions must have occurred in that time period, but are probably still ongoing.

The driving force for the African PTLV-I evolution was studied using the ML tree of the env gene reconstructing ancestral state characters. The calculated synonymous/non-synonymous substitution ratios provide evidence for a strong negative selective pressure, supporting purifying selection, irrespective of intra- or interspecies transmissions.