Oral abstracts

Journal of Clinical Virology 22 (2001) 149– 166 www.elsevier.com/locate/jcv

Abstracts

Oral abstracts

Viral zoonosen Antigenic Reactivity of baculovirus-expressed ebola virus VP35 and nucleoproteins JAN GROEN1, B. van den HOOGEN1, C. MAAS1, A. FOOKS2, C. GLEGG2, V. DEUBEL3, A. OSTERHAUS1 1 Institute of Virology, Erasmus Medical Center Rottedam, The Netherlands; 2Centre for Applied Microbiology and Research (CAMR), Porton Down, UK; 3Centre de Recherche Me´rieux Pasteur a` Lyon, France

Background: ebola virus (EBO) is a member of the Filo6iridae family and classified as a biosafety level 4 virus. This classification makes the preparation of diagnostic assays difficult and complicated. Objecti6e: in order to study the immunological reactivity against the EBO individual proteins and evaluate their usefulness for EBO diagnosis, the genes coding for the viral structural proteins were expressed using recombinant baculoviruses. Materials and methods: the fulllength EBO nucleocapsid (N) of the Gabon strain and the VP35 protein of the EBO Zaire strain were expressed as a his-tagged recombinant proteins. The antigenic reactivity and specificity of the recombinant N (r-N) protein and recombinant VP 35 (r-VP35) of EBO were determined by Westernblotting (WB) and enzyme-linked immunosorbent assay (EIA) using EBO virus specific monoclonal antibodies (MCA). The results obtained with the r-N and r-VP35 EIA’s were and compared with the results obtained in an indirect immunofluorescence assay (IFA) based on whole EBO. Serum samples from eight experimentally infected non-human primates, 110 EBO negative primate serum samples and 110 serum samples from health blood donors were used. Results: the EBO specific MCA’s reacted both in WB and EIA specifically with the homologous protein. Considering the EBO IFA as ‘gold standard’, EIA’s based on the r-N protein Gabon and r-VP35 Zaire correctly identified 7/8 of the positive primate serum samples. An EIA based on a mixture of both proteins resulted in 100% positivity (8/8). Both the primate negative serum

samples (n =110) and the serum samples from healthy blood donors tested negative in all the EIA’s coated with the respective recombinant EBO proteins. Conclusions: our data showed a good correlation between the baculovirus-expressed EBO antigens with the IFA based on whole virus. Therefore, EIA’s based on EBO recombinant proteins are valuable tools for diagnostic and epidemiological studies.

TBE in Estonia VEERA VASILENKO1, I. GOLOVLJOVA1, K. KUTSAR2, A. JO0 GISTE2, A, . LUNDKVIST3, A. PLYUSNIN4 1 Institute of Experimental and Clinical Medicine, Tallinn, Estonia; 2Health Protection Inspectorate, Tallinn, Estonia; 3 Swedish Institute for Infectious Disease Control, Stockholm, Sweden; 4Haartman Institute, Department of Virology, University of Helsinki, Finland

The TBE morbidity in Estonia was increased from (185), 12.8 per 100,000 inhabitants in 1999 to (272), 18.9 per 100,000 in 2000. The establishment and maintenance of natural TBE virus foci is complex function of several factors such as climate, vegetation, animal reservoirs, amplifying host animals, TBE virus factors and other (Hagelund, 2000). Taking into account this postulate TBE virus foci in Estonia are characterized by, (1) Existence of four TBEV high endemic areas, in South-Western, Eastern, Northern and Southern parts of Estonia, in spite of fact that TBE cases were registrated all over the territory of the country. (2) These TBEV high endemic areas differ between themselves by landscape, by type of forest (mixed forest with prevalence of firs or pines), by number of TBE cases diagnosed every year, by TBE virus vector (only I. ricinus or two species of ticks are TBE virus vector). (3) TBE virus was isolated from six species of small mammals which are TBE virus animal reservoir. Some of these species may be amplifying host animals. It is important that the two species of six (C. glareolus and A. agraius) are distributed all over the territory of Estonia. (4) Long observa-

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tion (1976 – 2000) of TBE morbidity indicated that every year the greatest number of TBE cases were diagnosed in South-Western part of Estonia. (5) TBE virus infectivity rate of ticks was studied by RT-RCR during 1999 –2000. The preliminary data indicated that the percentage of infected ticks is low. (6) Preliminary genetic study of TBE virus strains isolated from ticks and TBE patients showed that TBEV strains isolated in Estonia belong into two different TBEV serotypes (western and eastern serotype).

Correlation between kinetics of dengue virus specific serum immunoglobulin classes and subclasses and clinical outcome of infection PENELOPI KORAKA1, C. SUHARTI2, E. van GORP3, C. HACK4, J. GROEN1, and A. OSTERHAUS1 1 Institute of Virology, Erasmus Medical Centre Rotterdam, The Netherlands; 2University of Diponegoro, Semarang, Indonesia; 3Slotervaart Hospital, Amsterdam, The Netherlands; 4 Academic Hospital Free University, Amsterdam, The Netherlands

The kinetics of dengue virus specific serum immunoglobulin classes (IgM and IgA) and subclasses (IgG 1 –4) were studied in patients suffering from dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Serum samples from non-dengue febrile patients were included as controls. IgM, IgG 1 and IgG three serum antibodies were the predominant immunoglobulins throughout the course of illness in all three patient groups and their levels proved not to be associated with disease severity. In contrast, IgA antibodies were significantly higher in the acute phase in DSS patients compared with DF patients (P B0.05). The levels of IgG 1 differed significantly between patients with DF and those with DHF and DSS (PB 0.05). A significant difference was also found in IgG three levels between DF patients and DHF patients (PB 0.05) but not between DF patients and DSS patients. Finally, levels of IgG four antibodies did differ significantly between DF patients and DSS patients (PB 0.05). Collectively, these data show that increased levels of specific IgA, IgG 1, IgG 3 and IgG 4 serum antibodies are risk markers for the development of DHF and DSS and that their measurement may provide valuable guidance for therapeutic intervention.

Interferon-induced MxA protein inhibits la crosse virus replication by sequestering the viral nucleoprotein into highly ordered complexes GEORG KOCHS1, CHRISTIAN JANZEN1, HEINZ HOHENBERG2 and OTTO HALLER1 1 Department of Virology, University of Freiburg, Freiburg; 2 Heinrich-Pette-Institut for Experimental Virology and Immunology, Hamburg, Germany Bunyaviruses replicate in the cytoplasm of infected cells. New viral particles are formed by budding of viral ribonu-

cleoprotein complexes into the Golgi apparatus. The interferon-induced human MxA protein has been shown to inhibit bunyavirus replication by an as yet unknown mechanism. Here we demonstrate that MxA binds to the nucleoprotein of La Crosse 6irus (LACV) and prevents its accumulation in the Golgi compartment. In infected cells, MxA colocalized with the nucleoprotein of LACV in large and highly ordered complexes. Electron microscopy revealed that these complexes consisted of dense fibrillary structures and had a predominantly perinuclear localization. A similar redistribution of viral nucleoproteins by MxA was detected with other bunyaviruses known to be MxA-sensitive, such as Bunyamwera 6irus and Rift Valley fe6er 6irus. MxA(E645R), a mutant of MxA that has no antiviral activity against LACV, did not lead to the formation of aberrant nucleoprotein complexes. Wild-type MxA but not MxA(E645R) was able to bind to LACV nucleoprotein in coimmunoprecipitation assays, indicating that the carboxy-terminal effector domain of MxA is involved in nucleoprotein recognition. These results demonstrate a novel mechanism of interferon action whereby an essential viral component is trapped in the cytoplasm of infected cells and becomes unavailable for the generation of new virus particles.

Chlamydiae in the etiology of chronic human diseases chalmydia Viral persistence and antigen expression in coxsackievirus-induced acute and chronic murine myocarditis LISA BROWN, S. ILLAVIA, S. HIYAT, J. BANATVALA and PETER MUIR Department of Infection, King’s College London, St Thomas’ Hospital Campus, Lambeth Palace Road, London, UK

Group B coxsackieviruses (CVB) cause acute and chronic myocarditis and dilated cardiomyopathy, which are important causes of heart failure requiring cardiac transplantation. CVB may damage myocardium directly through killing or functional impairment of infected myocytes, or indirectly by triggering cytotoxic immune responses. Since viral infectivity is rapidly cleared from the heart, such immune responses are thought to target sequestered myocardial autoantigens which either cross-react with viral epitopes (molecular mimicry), or are released following virus-induced tissue damage, resulting in stimulation of autoreactive T cells (bystander activation). Although residual viral RNA persists beyond acute infection, it’s ongoing biological activity and role in pathogenesis has not so far been determined, and is considered by some to represent either a transient, terminal stage of infection, or a molecular fossil, indicative of a earlier infective process but of no ongoing consequence. Here we define a new paradigm of CVB persistence in which recurrence of chronic myocarditis 12 months after CVB3 infection is associated

Abstracts with persistence of viral RNA and reappearance of viral antigen in myocardium. The temporal association of viral antigen expression with disease recurrence, and its localization within myocardial lesions implicates viral persistence in chronic disease, and suggests that viral antigen is a target of inflammatory responses in myocarditis. These insights provide a rationale for new therapeutic strategies for viral heart disease and may also be relevant to other persistent infections caused by positive sense RNA viruses, and diseases believed to result from post-infectious autoimmunity.

Humoral and cellular immune response in viral infections Antibodies to human papillomavirus type 16 (HPV-16) in oral fluid (OF) from women with cervical neoplasia D. MARAIS1, JENNY BEST3, R. ROSE4, P. KEATING2, R. SOETERS2, L. DENNY2, C DeHAECK2, J. NEVIN1, P. KAY1, J-A. PASSMORE1 and A-L. WILLIAMSON1 1 Department of Medical Microbiology University of Cape Town, South Africa; 2Obstetrics and Gynecology, University of Cape Town, South Africa; 3Department of Infection, GKT, St Thomas’ Hospital, London, UK; 4Departments of Medicine, Microbiology and Immunology, University of Rochester, New York, USA

Purpose: human papillomavirus type 16 (HPV-16) is the most common type of papillomavirus found in precancer and cancer of the cervix (cervical neoplasia). The relationship between HPV-16 antibodies in oral fluid (OF) and serum, and detection of cervical HPV-16 DNA in women with cervical neoplasia was investigated. Methods: OF from 81 women and serum from 84 women with cervical neoplasia were tested for IgG and IgA antibodies to HPV-16 virus-like particles (VLP-16) by enzyme immunoassay (EIA). OF was obtained from 36 controls, who were laboratory staff. HPV-16 DNA was detected in cervical cells by polymerase chain reaction with HPV-16-specific primers. Results: anti-VLP-16 IgA antibodies were detected in OF from 44 of 81 (54.3%) women with cervical neoplasia, but in only 3 of 36 (8%) controls (P B0.0001), IgA antibody levels were significantly higher in patients with cervical neoplasia than in controls (P B0.001). Anti-VLP-16 IgG was detected in OF from 26 of 73 (43.9%) patients and 4 of 30 (13.3%) controls (P= 0.023). IgA antibodies were detected more frequently in OF (44/81, 54.3%) than in serum (28/83, 33.7%), but IgG antibodies were detected more frequently in serum (55/84, 65.5%) than in OF (26/73, 35.6%). There were no significant differences in the prevalence of IgA and IgG antibodies in OF or serum among HPV-16 DNA positive and negative women. Conclusions: OF antibody testing could provide a useful non-invasive screening method for HPV-16 infection and cervical neoplasia, particularly in developing countries. The OF method would also be useful for estimating mucosal antibody responses to candidate HPV vaccines.

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IL-12 and IL-18 are important in innate defense against genital HSV-2 infection but are not required for the development of acquired IFN-g-mediated protective immunity ALI HARANDI1, B. SVENNERHOLM2, J. HOLMGREN1 and K. ERIKSSON1 1 Department of Medical Microbiology and Immunology, Go¨ teborg University, Sweden; 2Department of Clinical Virology, Go¨ teborg University, Sweden

Vaccination of mice with an attenuated strain of HSV-2 (TK−) elicits strong protective immunity to subsequent challenge with a virulent strain of HSV-2. We employed this model to assess the requirement of T helper 1-modulating cytokines in the control of primary and secondary genital HSV-2 infections. We found that IFN- is required for both innate control of the primary infection and for the induction of protective immunity to the challenge infection. Following challenge infection, an accelerated genital HSV-2 replication was observed in both naı¨ve and vaccinated IFN- − / − mice, and vaccinated IFN- − / − animals were unable to survive challenge infection. IL-18 and, to a lesser extent, IL-12 were shown to contribute to the innate control of primary infection. There were significantly elevated levels of shed virus in IL-18 − / − mice and neurological complications appeared significantly earlier in both IL-12 and IL-18 deficient animals. However, vaccinated IL-12 − / − and IL-18 − / − mice could control genital viral replication and remained healthy following a secondary infection. Vaccinated IL-12 − / − and IL18 − / − mice, as opposed to vaccinated IFN- − / − mice, were also able to mount specific IFN- and DTH responses. These results show that IFN- is required for both innate and acquired protection against genital HSV-2 infection whereas IL-12 and IL-18 are implicated in the innate defense but can be circumvented in development of the acquired protective immunity.

Enterovirus infections and the risk of Type I diabetes associated autoantibodies ¨ NNROT, KIMMO SALMINEN, K. SADEHARJU, M. LO M. KNIP, J. ILONEN, O. SIMELL, S. KORHONEN, T. SIMELL and H. HYO8 TY Juvenile Diabetes Research Foundation Centre for the Prevention of Type I diabetes in Finland; Universities of Turku, Tampere and Oulu, Finland We have analysed the role of enterovirus infections in the pathogenesis of Type 1 diabetes in a prospective birth cohort study (the Finnish Diabetes Prediction and Prevention Study). In this study all newborns are first screened for diabetes associated HLA-DQB1 alleles and those with high risk alleles are invited to follow-up. Enterovirus infections were diagnosed by serology and RT-PCR from serum samples taken during the follow-up of 41 case children who developed Type 1 diabetes associated autoantibodies or clinical

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diabetes and 196 control children matched for the time of birth, gender and HLA-DQB1 alleles. Enterovirus infections were more frequent in case children than in control children, 24% (59/248) versus. 16% (183/1114) of the follow-up sample intervals indicated infection, respectively, (P =0.004). The average enterovirus antibody levels were also higher in case children than in control children (P= 0.003 for coxsackievirus B4 IgG). To study further possible causal association we analysed if there was any temporal association between the induction of autoantibodies and enterovirus infections. Enterovirus infections were particularly frequent during the 6 months period preceding the first detection of autoantibodies, 51% (21/41) of the case children compared with 28% (55/196) of the control children had infection at that time (odds ratio OR 3.0, 95% CI 1.4-6.4). Enterovirus RNA was found in 17% of the case children and 3% of the control children during this period (OR 7.7, 95% CI 2.1-29.2). This data confirm our earlier findings and support the role of enterovirus infection in the pathogenesis of Type 1 diabetes.

The viral kinetics and histopathological changes of coxsackievirus B3 (CVB3) and B4 (CVB4) infection following oral infection of mice ´ 1, A. PETROSHUBHADA BOPEGAMAGE1, M. STARA ´ 1 and P. MUIR2 VICOVA 1 Virology Department, Institute of Preventive and Clinical Medicine, Limbova´ 14, Bratislava, Slovak Republic; 2 Department of Infection, King’s College London, London, UK

Murine models have been widely used to study CVB pathogenesis. However, in most models infection is initiated by intraperitoneal challenge. Since enteroviruses normally infect via the gastrointestinal tract, and since the interaction of virus with gut-associated lymphoid tissue may influence the natural history of infection, we studied the kinetics of viral replication and organ pathology following oral challenge. Immunocompetent Swiss Albino mice (strain ICR) were given high doses of CVB3 (Nancy) or CVB4 (JVB) by oral gavage, and controls were given PBS. Oral challenge led to acute viral replication in heart, pancreas, thymus, spleen, small and large intestines, with higher viral titres observed in CVB3-infected mice. Infectious virus was cleared by 28 days post infection in all organs accept spleen. Infection was accompanied by histological evidence of myocarditis but no changes of the exocrine and endocrine pancreas, although there was a transient hyperglycemia in CVB3-infected mice. The spleens contained activated enlarged Malphigian bodies with apparent lymphocytic periarterial sheaths. Activated Peyer’s patches were observed in the early phase of infection. Diffuse severe degenerative and inflammatory changes of the epithelial lining of the gut and destruction of the villi in apical parts with cell debris in the intestinal lumen in the late phase of infection were also evident. Our results provide evidence that the oral route of infection provides an excellent model for studying early events

in CVB infections, and their influence on subsequent pathogenesis. Acknowledgement: We thank Dr E. Mitrova´ , National Reference Center, Slow Virus Neuroinfections, Bratislava, Slovakia for the histological studies.

Hepatitis Identification of a new YMDD variant of Hepatitis B virus during lamivudine therapy SUZAN PAS1, R. de MAN2, A. OSTERHAUS1 and H. NIESTERS1 1 Department of Virology University Hospital Rotterdam, Rotterdam, The Netherlands; 2Gastroenterology, University Hospital Rotterdam, Rotterdam, The Netherlands

We describe twin sisters both chronically infected with HBV and treated with Lamivudine. Serum HBV DNA levels were routinely quantified and the polymerase region of the virus was sequenced indicated by a HBV DNA increase. Seventy-six weeks after initiation of therapy, a new variant at codon 550 in the YMDD region was identified in one patient; in the sister a YIDD variant was found at week 86. We designed molecular beacons for the quantitative detection of both wild type and variant virus. Together with RFLP analysis, the dynamics of the HBV variant population in these patients was further studied. RFLP analysis detected the presence of the new variant at week 72, with the molecular beacons at week 76. Samples from the new variant were cloned and sequenced at different time points. The new variant was already present at week 68 in 5% of the clones, this number increased to 27.5 and 28% in week 72 and 76, respectively. Segregation with the L526M mutation was found. In, week 85, all of the clones harboured the new variant in the YMDD region. The dynamics of both wild type and variant population was quantified using molecular beacons. Both patients were followed after end of therapy for 21 weeks and both RFLP analysis and molecular beacons detected equal amounts of variant DNA asides the wild type HBV DNA.

Core promoter deletion variants in Yemeni blood donors TALAL SALLAM1 and C. TONG2 Department of Microbiology, Sana’a University, Sana’a, Republic of Yemen; 2Department of Infection, King’s College London, London, UK 1

Background: genetic variations in the Core Promoter region of Hepatitis B virus (HBV) were reported to be associated with negative HBeAg phenotype and disease severity. This study investigates the presence of core promoter variations in asymptomatic Yemini blood donors, correlating variations with HBeAg phenotype and viral load. Method: nested PCR was used to amplify the surface and core promoter/precore regions of HBV in 107 HBsAg positive Yemini blood donors. PCR products were sequenced directly or after cloning. HBeAg and

Abstracts viral load were measured when HBV DNA was detectable. Result: 42/107 (39.3%) of HBsAg positive blood donors had detectable HBV DNA. Sequencing of the surface gene indicated that all were genotype D (ayw2 or ayw3). Of the 27 core promoter/precore sequences analysed, only 2 were wild type. The commonest substitutions were T at 1762 and A at 1764; 2 had 8bp deletions (1766 –1773) and 7 had 12bp deletions (1746– 1757) plus the 8 bp deletions. One donor had mixed infection with substitutions as well as deletion variants. Premature stop mutations in codon 28 were found in 12, but never in sequences with deletions. Deletion variants were all HBeAg negative with viral load B1000 copies/ml with the exception of the donor with mixed infections. Conculsions: the combination of 12 and 8 bp deletions is novel. Although this creates a new binding site for the hepatocyte-specific transcription factor HNF1, it also truncates the X open reading frame. The overall effect seems to be a negative HBeAg phenotype and a low level of viraemia.

Phylogenetic analysis of recent Nigerian hepatitis B isolates support endemicity of genotype E in West Africa MICK MULDERS, S. ODEMUYIWA and C. MULLER Laboratoire National de Sante´ , Dept. of Immunology, Luxembourg

Serum samples were collected from 20 acute and chronic hepatitis patients in Ibadan, a highly endemic region of Nigeria. All sera tested positive in a HBsAg ELISA and were sequenced across the complete pre-S/S gene. Only a single isolate contained a mutation with the ‘a’ determinant of the surface protein (T131N). In the pre-S2 region of the five isolates, three to seven amino acids were deleted suggesting that immune escape mutations earlier associated only with chronic HBV infection may be observed also in acute disease. Phylogenetic analysis of the complete pre-S2/S (large S) gene S (831 nt) demonstrated that all the viruses belonged to genotype E. No isolates of genotype E have been found in any other region of the world, including the Americas, despite the forced migration of slaves from West Africa. This suggests that the virus emerged not before the late 19th century in the West-Africa population. It could also explain the relatively low diversity of viruses of this genotype.

HBV Vaccination coverage in HCW ROBERT VRANCKX1, P. JACQUES2 and G. MOENS2 1 Institute of Public Health, Belgium; 2IDEWE Occupational Health Services, Belgium

Introduction: Hepatitis B virus (HBV) infection is an occupational hazard for health care workers (HCW) who are exposed to human blood. The HBV vaccination policy for HCW started in Belgium in 1984. Studies with plasma-derived and DNA-recombinant HVB vaccines indicate that the vac-

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cines are effective and safe. An immune response level (antiHBs)\10 IU/l gives at least a 7 years’ protection. An update of the vaccination coverage and the rates of seroconversion and seroprotection among HWC can give an insight into the actual status and can encourage further development of vaccination programs. Material and methods: a sample of 5064 HCW, working in 22 hospitals were tested for antibodies to hepatitis B surface antigen (anti-HBs). A blood sample was taken as part of the regular occupational medical examination. The presence of anti-HBs was determined by radioimmunoassay (RIA). Anti-HBs titers were expressed in IU/l, using the WHO reference preparation (first International Reference Preparation for HBIG, lot 26.01.77). We considered those who had a positive anti-HBs test as seroconverted (SC) and those who had an anti-HBs titer \ 10 IU/l as seroprotected (SP). The cut-off for anti-HBs positivity used in this calculation of seroconversion rates was 2 IU/l. Vaccination results: studied population (N1 =5064). Anti-HBs positive without vaccination (N2 = 293). Population eligible for vaccination (N3 =5064 − 293 = 4771). Number vaccinated (N4 = 4049). Seroconverted with or without vaccination (N5 =4151). Seroconverted after vaccination (N6 =4151 −293 =3858). Vaccination coverage (4049/4771=84.9%). Non- or low-responders (N7 =4049 − 3858 =191, 191/4049=4.7%). Among high-risk professions, such as nurses, care and laboratory workers 94.79% (CI 93.98 – 95.60) were vaccinated, whereas, the vaccination coverage for the other professions was 69.26 (CI67.16 – 71.36). Results on risk assessment: out of the 1015 non-vaccinated persons, 293 showed a positive anti-HBs test and 277 were also SP. Among these 293 positives, 161 (54.95%) declared they had a earlier hepatitis infection that was serologically proven to be HBV. Of the remaining 132 positives, 89 (70.45%) had earlier undergone surgery and/or transfusion. Among these 1015 non-vaccinated persons, 373 were nurses, care or laboratory workers. 219 of these (59.03%CI 54.03 – 64.03) had a positive anti-HBs test. This contrasts with the results for workers in other sectors, where 74 persons (11.49%; CI 9.03 – 13.95) showed an anti-HBs positivity. This seems to provide additional proof of the higher exposure rate of this group of HC. Conclusions: in our sample, which can be considered as representative, high vaccination, seroconversion and seroprotection rates have been achieved, at least for those employees who are at higher risk. The same conclusion can be made if we consider the hospital departments, which carry a higher risk of blood-borne infections.

Inhibition of hepatitis C virus replicons by treatment with interferon-alpha or interferon-gamma M. FRESE1, T. PIETSCHMANN2, N. KRIEGER2, V. LOHMANN2, D. MORADPOUR3, R. BARTENSCHLAGER2 and OTTO HALLER1 1 Department of Virology, University of Freiburg, Germany; 2 Institute of Virology, University of Mainz, Germany; 3 Department of Internal Medicine II, University Hospital Freiburg, Germany

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Hepatitis C virus (HCV) persists in the majority of infected individuals, and is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Chronic hepatitis C is currently treated with interferon (IFN)- or with a combination of IFN- and ribavirin. The availability of an HCV replicon system (Lohmann et al. Science 285:110 –113, 1999) allowed us to investigate the effects of IFNs on genuine HCV replication in cultured cells. We show here that IFN- inhibits subgenomic HCV RNA replication in HuH-7 human hepatoma cells. Immunofluorescence, Western blot, and Northern blot analysis revealed that both HCV protein and replicon RNA levels were reduced after treatment with IFN-. In further experiments, we could demonstrate that IFN-, but not TNF-, has a comparable antiviral effect. Finally, we investigated whether MxA plays a role in the inhibition of HCV. The human MxA protein is an IFN-induced GTPase that is antivirally active against various RNA viruses. However, HCV RNA replication was not affected in transfected HuH-7 cells that transiently overexpressed MxA. Moreover, a dominant-negative mutant of MxA did not interfere with the antiviral activity of IFN- against HCV RNA replication. Taken together, these results demonstrate that IFN- inhibits HCV replicons via an MxA-independent pathway.

Risk factors for perinatal transmission of hepatitis C virus CHRISTOPH STEININGER, M. KUNDI, G. JATZKO, A. LISCHKA, H. KISS and H. HOLZMANN Institute of Virology, University of Vienna, Austria

128 HCV-infected pregnant women with detectable HCVspecific antibodies and a positive result in an HCV-specific RT-PCR or immunoblot, were investigated for the following risk factors for perinatal HCV transmission, HCV viral load, co-infection with HIV, and mode and course of delivery. These were assessed by questionnaires and review of delivery reports. Sera of the newborns were tested by HCV-specific RT-PCR immediately after birth and after =1 month for identifying a perinatal HCV infection. The risk factors were evaluated in a logistic regression analysis. The HCV status after birth was available for 101 children. In 75 of these children follow-up for a final diagnosis of transmission was done, and 9 of these children had become infected. Transmission occurred only in mothers with detectable viremia, and, except for one child of a HIV-coinfected mother, all of the HCV-infected infants had been delivered vaginally. In vaginal deliveries, the virus load was clearly higher in mothers whose children became infected than in those with non-infected children (8.1 × 105 vs. 1.6 × 104, PB 0.01). Analysis of birth parameters revealed a significantly increased risk for perineal laceration (OR =8.2, 95%-CI [1.77–38.18]) and for a reduction in umbilical cord blood pH (OR= 4.1, 95%-CI [1.11 –15.07]). Perinatal HCV infection appears to depend on the level of maternal viremia and on direct exposure of the child to maternal blood during vaginal delivery, if associated with perineal rupture and infantile asphyxia. Therefore, elective caesarian delivery before membrane rupture should be reconsidered as a protective measure.

Molecular epidemiology of a transcontinental shellfishborne outbreak of hepatitis A ´ NCHEZ, A. BOSCH and ROSA PINTO ´ G. SA Enteric Virus Group, Department of Microbiology, University of Barcelona

An outbreak of hepatitis A, affecting 183 people occurred in Valencia (Spain), during the late fall – early winter season. Epidemiological evidence pointed to an association of the outbreak with consumption of coquina clams (Donax sp), imported frozen from Peru. Shellfish were analysed for the presence of hepatitis A virus (HAV), and the molecular epidemiology of sequences from sera samples from patients has been investigated. Analysis of the VP1/2A junction region revealed that all isolates belonged to genotype IB. The complete capsid sequence has been elucidated from 18 patient samples. The mutant frequency was figured to be around 6 × 10 − 3. As expected, most changes corresponded to silent mutations, although three of them induced aminoacid substitutions, some of them on the capsid surface. One of these mutations was located in the immunodominant site and resulted in a loss of recognition by monoclonal antibody K34C8.

Common source outbreak of hepatitis A in an endemic area confirmed by limited sequencing within the VP1 region PATRICIA AURAUZ-RUIZ1, L. SUNDQVIST1, Z. ´ 2, L. TAYLOR2, K. VISONA ´ 2, H. NORDER1 and GARCIA L. MAGNIUS1 1 Swedish Institue for Infectious Disease Control, Stockholm, Sweden; 2Louisiana State University, International Center for Medical Research and Training, San Jose´ , Costa Rica

Hepatitis A virus (HAV) isolates from sera of 70 cases in two outbreaks in Costa Rica and of 216 sporadic cases in Central America were compared by phylogenetic analyses within the VP1 region. The outbreaks affected in all 531cases, in 1992 – 93 and 1999, respectively, and were presumed water borne. In the first of these outbreaks HAV RNA could be detected in 70% of the cases sampled during six weeks after onset of jaundice. All isolates belonged to subtype 1A. Background isolates from Costa Rica and El Salvador tended to form separate subclusters in the phylogenetic tree and were mostly unrelated to subtype 1A strains from other parts of the world. Based on their amino acid sequences four HAV strains, all related to CR326 sampled in Costa Rica 1960, were found to have circulated in the area during the last three decades. However, on the basis of nucleotide variability the isolated from the outbreaks could be distinguished from the strains from sporadic cases and sequence analysis could confirm the epidemiological homogeneity of both the outbreaks. Thus, limited sequencing within the VP1 region proved useful to identify outbreaks of hepatitis A in a highly endemic area, where most strains were local and only one subtype was prevalent.

Abstracts Screening of BMT patients for HGV before and after transplantation reveal no transfusion-related transmission LAURDIS CHRISTENSEN1, L. JENSEN2, J.O. BOCK1 and C. HEILMANN2 1 Department of Clinical Microbiology, Rigshospitalet, Denmark; 2Department of Pediatrics, Rigshospitalet, Denmark

A total of 60 patients consecutively admitted to the national unit in Denmark for bone marrow transplantation (BMT) were screened before and after transplantation for the presence of hepatitis G virus (HGV) by a PCR technique directed against the 5’ untranslated region. Eighteen patients were found positive for HGV before transplantation and 24 patients were found positive after transplantation. The over-all prevalence was 43.6% (26/60), and a total of 16 patients were found positive both before and after bone marrow transplantation. Sequencing of the PCR products revealed that the virus strain was identical before and after transplantation for all these 16 patients in spite of high numbers of transfusions of erythrocytes and thrombocytes between the two screenings. An apparent correlation between transfusion frequency and prevalence of HGV was found to be confounding with time. We conclude that the prevalence of detectable levels of HGV in this patient population is due to the disease progression in particular the immune suppression of the individuals. We furthermore suggest that the predominant route of transmission of HGV is a vertical one possibly taking place at the early stages of fetal development.

Treatment and prevention Comparison of genotypic HIV-drug resistance interpretation by virtual phenotype (Virco) and Beta Test (Stanford) ELISABETH PUCHHAMMER-STO8 CKL and F. HEINZ Institute of Virology, University of Vienna, Austria

The quality of genotypic HIV-resistance testing depends strongly on the interpretation of the mutations identified in the sequence of the pol region of HIV strains. In the present study, two resistance interpretation systems were compared, the virtual phenotype (Virco), and the more rule-based Beta Test (Stanford, Robert Shafer). So far HIV sequences obtained by capillary sequencing from 87 samples of HIV-infected persons under antiretroviral therapy were subjected to both interpretation models. The concordance of the interpretation results was highest for resistance against lamivudine (98.9%) and Nelfinavir (96.6%). Discordant results were obtained especially for ddC (44.8%, 39/87 samples), and ddI (41.4%). In 99.5% of all discordant results (209/210) the Beta Test predicted resistance (low, intermediate, or high), while the virtual phenotype revealed sensitivity. 96.8% of all samples judged as lowly resistant, and 41.5% of those intermediately resistant by the Beta Test were sensitive in the virtual phenotype interpretation. Highly discordant results (highly resistant versus sensitive)

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were obtained in 17 cases, ten of them involving ddC-resistance. Analysis of the underlying mutations showed that in all of these 10 samples mutations at codon 74 or at codon 69 were present in the RT gene (both accepted as significant resistance mutations for ddC in the literature), but did not lead to a resistant virtual phenotype above the given cutoff provided by the Virco system. The current data already show that there are discrepancies between the two interpretation systems, and that HIV resistance interpretation will require further investigation.

A viral sensitivity assay for influenza using human fibroblasts and in situ ELISA CAMILLA KOLMSKOG and A. LINDE Department of Virology, Swedish Institute for Infectious Disease Control, Solna, Sweden

Aim: to establish a viral sensitivity assay (VSA) for influenza in a human fibroblast cell line (HS 27) with in situ ELISA in microplates. Materials and methods: to establish the method HS cells in 96 well microplates were inoculated with 15TCID50 of influenza B/Beijing/1/87P3-1, zanamivir sensitive strain and influenza B/Beijing/1/87P3-12, zanamivir resistant strain. Different amounts (0.01 – 150 g/ml) of neuraminidase inhibitor (zanamivir or oseltamivir) were added to reduce virus growth. After 3 days, the cells were fixated in the plates and antigen expression was measured in an in-situ ELISA with mabs against the influenza nucleocapsid antigen. IC50 for each virus and antiviral was calculated, based on 50% reduction of the absorbance in the antigen ELISA. Results: when zanamivir was used in the assay IC50 for the resistant strain was 100 g/ml and 0.1 g/ml for the sensitive. B/Beijing/1/87[P3-12] was also resistant against oseltamivir. IC50 for the resistant strain was 100 g/ml and 1 g/ml for the sensitive. Discussion and Conclusion: in situ ELISA is a rapid and objective method for evaluation of growth inhibition. The use of HS cells is preferable to Madin – Darby canine kidney (MDCK) cells since these expresses the human sialic acid receptor and give no background in the in situ ELISA. The results are comparable to those obtained in the plaque reduction assay. Examination on clinical isolates will be presented.

Anti-herpes drugs and epstein-barr virus (EBV): effect on cerebrospinal fluid EBV DNA load in patients with brain aids related lymphomas SIMONA BOSSOLASCO1, K. FALK2, A. BESTETTI1, M. VIGANO1, A. LAZZARIN1, A. LINDE2 and P. CINQUE1 1 Clinic of Infectious Diseases, San Raffaele Hospital, Milan, Italy; 2Department of Virology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden

Background and aims: the effect of anti-herpes drugs on EBV DNA load in CSF was analysed in patients with brain AIDS related lymphoma (ARL), observed between 1994 and

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Abstracts

2000. Patients and methods: EBV DNA was quantified by real time PCR in CSF samples drawn from 39 patients with brain ARL at the time of neuroradiological presentation (25 with primary central nervous system lymphoma, 13 with systemic lymphoma and brain localization). At the time of sampling, 15/39 patients were receiving maintenance or induction antiherpes drugs treatment for concomitant CMV infection (ganciclovir in 11, foscarnet in one, both drugs in two) or maintenance therapy for recurrent herpes zozter (acyclovir in one). Serial CSF samples were drawn from 11 patients, five of whom received antiherpes treatment after brain lymphoma onset. Results: EBV DNA was detected in 20/24 anti-herpes drug untreated (83%) and in 10/15 treated patients (67%). A significantly higher EBV DNA load was found in CSF from untreated compared with treated patients (median 110,000 vs. 3000 copies/ml, P =0.01). Following onset of brain lymphoma, EBV DNA decrease was significantly higher in CSF samples from treated than untreated patients (median 1.00 vs. 0.00 log copies/ml, P= 0.010). Conclusions: antiviral drugs might affect EBV DNA load in CSF. The absence or low amounts of EBV DNA in CSF from patients receiving antiherpes drugs might be misleading towards a diagnosis of brain lymphoma. The potential effect on EBV replication in CSF in earlier stages of brain lymphoma might require investigation.

Measles elimination: is there a time factor? CLAUDE MULLER and J. MOSSONG Department of Immunology, Laboratoire National de Sante´ , Luxembourg and Centre de Recherche Public-Sante´ , Luxembourg

There is increasing evidence that individuals that are fully protected against measles disease can become infected and develop an asymptomatic secondary immune response (SIR). In vaccinated populations the percentage of susceptibles to SIR may be as much as 5 –10 times higher than among late convalescents. On one occasion virus has been isolated from such a healthy contact person and such individuals would be the most likely to support viral transmission in the absence of disease, but this is difficult to demonstrate. The accelerated waning of immunity in vaccines compared with persons with natural protection is likely to further increase the proportion of individuals that may contribute to MV circulation. Our mathematical model predicts a worse case scenario where even a greatly reduced transmission potential with a basic reproduction number of 1.24 would undermine control efforts based on a single– dose vaccination strategy. There is a clear need to experimentally define the transmission capacity of vaccinated individuals during measles outbreaks and even in the absence of clinical measles. In our studies in Nigeria, waning of maternal antibodies seems to be more rapid in low-income developing countries than earlier estimated even in children born to mothers with natural immunity. Children born to vaccinated mothers will loose passive protection even sooner

and their role in viral transmission is likely to increase. Our observations suggest that eradication may be more easily achieved as long as a large proportion of the world population is still protected by wild-type virus-induced immunity. Do we have to rush not to miss an opportunity?

Molecular epidemiology of measles in the UK 1992 to 2001 D. BROWN, BERNARD COHEN, R. HUNJAN, L. JIN, M. RAMSAY and J. WHITE Central Public Health Laboratory and Communicable Disease Surveillance Centre, Colindale, London NW9 5HT, UK

Enhanced surveillance of measles was introduced in the United Kingdom (UK) in 1994 to monitor the outcome of a mass vaccination campaign implemented to prevent a measles epidemic. The surveillance programme uses measurement of measles-IgM in oral fluid to provide laboratory confirmation of cases notified by physicians, the ‘non-invasive’ nature of the test resulting in a high level of compliance. Oral fluid samples collected in the acute phase are also adequate specimens for RT-PCR detection of measles virus and sequencing RT-PCR products provides a means of genotyping circulating virus strains. Phylogenetic analyses of N, M and H gene sequences from RT-PCR products generated similar lineages and prior to 1994 revealed the co-circulation of mainly 2 indigenous strains, D6 and C2 (Jin et al, J Gen Virol 1997, 78, 1287 – 1294). Following the 1994 vaccination campaign, the circulation of these strains was interrupted. After a period of very low incidence in 1995, a wide diversity of strains has been detected, often associated with separately imported measles cases. The imported genotypes (and number of cases) between 1995 and 1999 were A (1), C2 (3), D2 (2), D4 (7), D5 (2), D6 (5), D (8) and G2 (1). Importations were usually associated with sporadic or small clusters of cases in unvaccinated adults but in 1997 an outbreak of 150 cases due to genotype D6 was confirmed principally in children from 1 to 15 years in a community who refuse vaccination. Molecular epidemiological studies of measles in the UK, utilising the compliance advantages of oral fluid testing, have confirmed the interruption of widespread indigenous transmission and demonstrated the importation of multiple genotypes of different geographical origin.

Rapid and recent spread of new genotypes of measles virus in Germany SABINE SANTIBANEZ, H. HENGEL and A. TISCHER Robert Koch Institute, Berlin, Germany

The WHO has set the goal to eliminate measles from the European Region by the year 2007. To estimate the epidemiological situation of measles in Germany, a nationwide sentinel, that includes laboratory surveillance, has been established. It facilitates a continuous and an areawide monitoring of circu-

Abstracts lating measles viruses. A total of 50 virus samples were selected from the 151 PCR positive measles cases collected during the year 2000. They were characterized by nucleotide sequence analysis of the variable part of the N gene, and in some cases, of the H gene. Phylogenetic analysis, using the classification scheme recommended by WHO in 1998, revealed the appearance of three recognized genotypes (B3, C2 and D6) and one earlier undescribed genotype, which we propose to be D8. Surprisingly, it is the predominant genotype in Germany since 55% of cases were associated with D8. It is widely distributed over all parts of the country whereas the typical European genotypes of the nineties, C2 and D6, are still circulating in the south of Germany. In addition, two cases from the central region of Germany were identified as genotype B3, which was detected in 1997/98 in sub-Saharan Africa. This is the first report on the appearance of a clade B virus in Europe, most likely as a result of a new virus importation. Altogether, the data suggest a rapid qualitative change of measles virus circulation in Germany compared with the situation in the nineties.

Food and water-borne viral infections The use of molecular techniques for monitoring enteric virus contamination of shellfish harvesting areas ´ and D. LEES KATHLEEN HENSHILWOOD, W. DORE The Centre for Environment, Fisheries and Aquaculture Science, Weymouth Laboratory, The Nothe, Weymouth, Dorset, UK

Filter feeding bivalve shellfish concentrate viral pathogens from sewage polluted growing waters. Conventional bacterial indicators have failed to prevent shellfish-associated outbreaks of viral illness. Viruses of principle concern are Norwalk-like virus (NLV) causing gastro-enteritis and hepatitis A virus. This study describes the application of a recently developed nested RT-PCR for detection of NLVs in oysters (Crassostrea gigas) from five harvesting areas subject to varying degrees of pollution. The results showed a clear correlation between the degree of pollution and NLV occurrence. NLV prevalence in each site was consistent with known associations with gastroenteritis outbreaks. Prevalence and diversity of the NLV strains present was determined by sequencing RT-PCR positive amplicons. A variety of NLV strains of both Genogroup I and II were seen, with many samples containing a mixture of up to three different strains. This common occurrence of strain mixtures may have implications for the role of shellfish as vectors of NLV disease. NLV monitoring of harvesting areas was utilised as an investigative tool following several oyster-associated outbreaks. Oysters from a site implicated in an outbreak were shown to be contaminated with NLVs despite compliance with bacterial standards. NLV contamination persisted in oysters for at least two month at this site. This and

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other data demonstrated the potential contribution of NLV monitoring for improved public health controls. This study demonstrates the feasibility of applying molecular methods for the monitoring of NLV contamination in shellfish. The results also raise interesting questions regarding the role of shellfish as vectors for the transmission of NLVs.

Detection of norwalk-like viruses and other human enteric viruses in shellfish SOIZICK le GUYADER1, F. LOISY1, F. NEILL2, S. PARNAUDEAU1, M. POMMEPUY1, R.L. ATMAR2 1 IFREMER, Microbiology Laboratory, Nantes, France; 2 Molecular Virology, Baylor college of Medicine, Houston, TX, USA.

Sewage pollution can contaminate shellfish growing waters with various enteric viruses of human origin and molluscan shellfish have been implicated in gastro-enteritis or hepatitis A outbreaks. Current methods are now sensitive enough to detect viruses in environmental samples and studies were undertaken to evaluate viral contamination in different sites over a period of several years. Samples with no bacterial contamination are rarely contaminated by viruses or phages, whereas, these occasionally containing bacteria showed a low positivity (for example 23% for Norwalk-like viruses and 19% for enteroviruses). Samples collected in areas routinely impacted by human sewage were more contaminated (35% for Norwalklike viruses and 45% for enteroviruses). An increase of the contamination was observed during late winter-early spring months which can be related to the release of viral particles during the winter gastroenteritis peak. Finally, depuration of naturally contaminated oysters were done in semi-professional tanks. Parameters such as temperature/aeration/flow circulation were monitored and viruses (human and phages) elimination was followed. Results show a diversity among replicates, but at least 10 days seems to be necessary in the monitored to eliminate both viruses and phages. The development of methods (simplification or quantitative PCR) will allow to carried out collaborative studies in shellfish growing areas with producer and epidemiologist. This may improve the quality of shellfish and will prevent in the future gastro-enteritis outbreaks.

Hospital-based assessment of human astrovirus infections in the US FERENC JAKAB1, J.E. WALTER1, E. PARADA RICART1, M. STAAT2, P. AZIMI3, T. BERKE1, D. MATSON1 and D. MITCHELL1 1 Center for Pediatric Research, Children’s Hospital of The King’s Daughters, Eastern Virginia Medical School, Norfolk, VA; 2Children’s Hospital Medical Center, Infectious Disease Department, Cincinnati, OH; 3Children’s Hospital Oakland, Infectious Disease Section, Oakland, CA

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Purpose: this prospective study determined the frequency and clinical characteristics of human astrovirus (HAstV) infections among hospitalized children in Oakland, Norfolk and Cincinnati during a 2-year time period. Methods: children hospitalized November 1997 –October 1999, 2 weeks –5 years of age, with diarrhea (D), vomiting (V), and/or fever (F) were evaluated. Questionnaires were administered and stool samples collected from 1856 children with 1910 admissions. HAstV was detected in stool samples by RT-PCR using a primers from a non-structural region (Mon348/ Mon340). Clinical data, co-infection with rotavirus (RV) or human calicivirus (HuCV), seasonality, and ages were analyzed. Results: HAstV was detected in 7% (N =131) of hospitalized children, of whom 29% (N= 38) had co-infection with RV and 5% (N = 6) with HuCV. HAstV infection was more common in Norfolk (10.2%) than in Cincinnati (5.5%) (P B 0.05). More hospitalizations from HAstV infections (73%) occurred from January to June and were most frequent among children B2 years of age (85%). Diarrhea (69%) occurred more commonly than V (47%) (PB 0.01). Fever was present in 61% and all three symptoms (D, V, F) in 17% of children. In contrast, HAstV/RV co-infections resulted in all symptoms in 66% (P B0.0001). HAstV infection without diarrhea occurred in 31% of children, but co-infection with RV without diarrhea occurred only in 5% (PB 0.001). Conclusions: HAstV was detected in 7% of children hospitalized for D, V, and/or F and 1/3 of those children were co-infected with RV. Hospitalized children infected with HAstV may have only F or V as presenting symptoms.

Molecular epidemiology of astrovirus infection in Barcelona, Spain ´ S. GUIX, S. CABALLERO, C. VILLENA, R.M. PINTO and ALBERT BOSCH Enteric Virus Group, Department of Microbiology, University of Barcelona, Spain

A 3-year study, involving 2347 gastroenteritis samples, was conducted to determine the prevalence, time-distribution and medical significance of human astrovirus infection in Barcelona, Spain. The overall incidence of astrovirus was found to be 4.9%. Mixed infections with other enteric agents were detected in 17.2% of all astrovirus positive samples. During the 3-year period, the highest astrovirus incidence was reported in the winter months, although infections also occurred in summer. The peak detection rate was observed in children between 2 and 4 years of age, although astroviruses were also found in 7% of the assayed adult samples. Overall, HAstV-1 was the most prevalent type, followed by HAstV-4, HAstV-3, HAstV-8 and HAstV-2. HAstV-5, HAstV-6 and HAstV-7 were not detected during these 3 years. From our serotype data in each age group, we observed that HAstV-1, HAstV2 and HAstV-3 affected mostly children younger than

3 years of age, while HAstV-4 and HAstV-8 had a greater impact in older children. Genetic variability was analyzed between astroviruses isolated in Barcelona and other strains isolated in other parts of the world. A fourth lineage was described for HAstV-1, most likely due to the large number of assayed samples, which may also explain the high level of genetic variability observed in the astrovirus isolates.

Respiratory viral infections Antigenic and genetic heterogeneity among recent swine influenza A (H1N1) virus isolates, indicating farm-restricted virus circulation JAN de JONG1, P. HEINEN2, W. LOEFFEN3, A. van NIEUWSTADT2, R. FOUCHIER1, G. RIMMELZWAAN1, E. CLAAS1, T. BESTEBROER1, K. BIJLSMA4, C. VERWEIJ4, A. OSTERHAUS1 and T. KIMMAN4 1 National Influenza Centre, Department of Virology, Erasmus University Rotterdam, Dr Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands; 2Department of Mammalian Virology, ID-Lelystad, Lelystad, the Netherlands; 3Animal Health Service, Boxtel, the Netherlands; 4Research Laboratory of Infectious Diseases, National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands

In order to explore the occurrence of antigenic and genetic drift in swine influenza A (H1N1) viruses we examined 26 virus isolates from outbreaks of respiratory disease among finishing pigs in the Netherlands in the 1995/1996 season and compared them with reference strains from earlier outbreaks using antigenic and molecular methods. In contrast to swine H3N2 influenza viruses (de Jong et al., Vaccine 1999; 17: 1321 – 1328), we did not observe a significant antigenic drift in swine H1N1 influenza viruses isolated from the late 1980s up to 1996 inclusive. We did, however, detect a marked antigenic and genetic heterogeneity in haemagglutination inhibition reactivity as well as in nucleotide sequences of the HA1 gene among the 26 swine H1N1 influenza virus strains isolated during our 1995/1996 surveillance. Interestingly, the observed antigenic and molecular variants were not randomly distributed over the farms. This finding indicates independent introductions of different swine H1N1 influenza virus variants at the various farms of the study and points to the existence of a marked difference between the epidemiologies of human and swine influenza viruses. Possibly there is an endemic circulation of swine H1N1 influenza viruses on each of the concerning farms. The observed heterogeneity indicates the necessity of regular monitoring of the antigenic reactivity of influenza viruses from swine and suggests that a replacement of the H1N1 component of the swine influenza vaccine may be justified.

Abstracts Antigenic and genetic characterization of swine influenza A (H1N1) viruses isolated from pneumonia patients in The Netherlands G. RIMMELZWAAN1, JAN de JONG1, T. BESTEBROER1, A.M. van LOON2, E. CLAAS1, R. FOUCHIER1 and A. OSTERHAUS1 1 National Influenza Centre, Department of Virology, Erasmus University Rotterdam, Rotterdam, The Netherlands; 2 Department of Virology, University Hospital Utrecht, The Netherlands

Since the late 1970s, avian-like influenza A (H1N1) viruses are circulating extensively among pigs in Europe. Infections of humans with classical swine influenza A (H1N1) viruses has been described. To our knowledge, however, only one report of human disease from possibly avian-like swine influenza A (H1N1) viruses has been published. In 1986, swine influenza A (H1N1) viruses were isolated from three individuals aged 3, 29 and 50 years, who lived in The Netherlands and Switzerland and suffered from illnesses ranging from minor upper respiratory disease to severe pneumonia (de Jong et al., Lancet 1986; ii: 1329 – 1330). In the summer of 1993, an influenza A (H1N1) virus was isolated from a 5-year-old girl living in The Netherlands and suffering from severe pneumonia. Both this virus and the virus from the pneumonia case in 1986 mentioned above proved to be antigenically and genetically similar to avian-like swine influenza A (H1N1) viruses from Europe. It is concluded that these viruses can infect humans and cause serious disease without the need for prior reassortment with human influenza viruses.

Diagnosis of influenza C virus infections by reverse transcription polymerase chain reaction and molecular evolution of influenza C in Finland M. HIRSILA8 1, 5, R. PYHA8 LA8 2, A. RUOHOLA3 and THEDI ZIEGLER4, 5 1 Department of Medical Biochemistry, University of Oulu, Finland; 2Influenza Laboratory, National Public Health Institute, Helsinki, Finland; 3Department of Pediatrics, Turku University Hospital, Finland; 4Department of Virology, University of Turku, Finland; 5Department of Medical Microbiology, University of Oulu, Finland

The diagnosis of influenza C infections is usually done by the isolation of the virus in embryonated hens’ eggs or by the demonstration of a significant antibody titer increase between acute and convalescent phase sera by the hemagglutination inhibition test (HAI). However, a successful recovery of influenza C in eggs requires more sophisticated inoculation and incubation techniques than those commonly used for the cultivation of influenza A and B. Therefore, even in laboratories that routinely used egg inoculation for the recovery of influenza viruses, influenza C usually remains undetected. Thus to date little is known about the clinical significance of this virus. We have used reverse transcription-polymerase chain reaction (RT-PCR) to

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determine the incidence of influenza C in young children and young adults with upper respiratory tract infections. Nasopharyngeal secretions were aspirated during the first 48 h after onset of symptoms. Influenza C-specific sequences were detected by two RT-PCR protocols, one specific for the hemagglutinin – esterase-fusion protein gene (HEF), and the other for the non-structural protein 1 gene (NS1), respectively. Influenza C-specific IgG antibodies were measured in paired serum samples collected from the adult individuals. The incidence of influenza C was approximately 3.5% in both groups. All influenza C-positive adults had also a significant antibody response. The HEF, NS1, and NP genes of selected influenza C-positive specimens were amplified by RT-PCR and the product was subjected to nucleotide sequencing. Data on the genetic relatedness of Finnish influenza C viruses to published sequences and on the molecular evolution will be presented.

Genetic clustering of 91 human rhinovirus prototype strains C. SAVOLAINEN, S. BLOMQVIST, M.N. MULDERS1 and TAPANI HOVI National Public Health Institute (KTL), Enterovirus Laboratory, Helsinki, Finland; 1Present address: Laboratoire National de Sante´ , Department of Immunology, Luxembourg Human rhinoviruses (HRV), common agents of mild respiratory infections, comprise 102 serotypes. The aim of this study was to investigate genetic relationships of HRV prototype strains and to probe the possibility to use genetic identification of a given HRV strain, instead of serotyping. Genomic sequences in the VP4/VP2 region were obtained from 86 different prototype strains. Phylogenetic analysis was carried out including the five published entire genome sequences and partial sequences of 61 recently isolated Finnish field strains. Seventy-one out of the 91 different HRV proto-type strains clustered in the HRV genetic group A (HRV1b group) and 19, including HRV14, in the group B. HRV 87 formed a group of its own. The interserotypic differences were generally similar to those between different enterovirus serotypes, but for four pairs of HRV serotypes less than 10%. The maximum variation in the genetic group A was 51% at the nucleotide level and 39% at the amino acid level, and in the genetic group B 36% and 19%, respectively. Judging from the observed interserotypic differences, the 61 Finnish field isolates might represent as many as 19 different serotypes. Preliminary analysis of a 450 nt motif in the VP3/VP1 region, which in the case of enteroviruses is more suitable for genetic identification of untyped strains than the VP4/2 region, suggested a similar preferential use of this region for HRV identification as well. However, larger numbers of field isolates need to be characterized in order to evaluate the feasibility of genetic typing of HRV strains.

Human picornaviruses are predominant viruses in acute otitis media in young children JOHANNA NOKSO-KOIVISTO1, 2, R. RA8 TY1, S. BLOMQVIST1, A. PITKA8 RANTA2, S. VESA1, T. KILPI1 and T. HOVI1

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1

National Public Health Institute, Helsinki, Finland; Department of Otorhinolaryngology, University of Helsinki, Finland

2

Purpose of the study: to investigate the association of different virus infections with acute otitis media (AOM) in children less than two years of age. Methods: a total of 611 children participating in the pneumococcal vaccine trial (Finnish Otitis Media Vaccine Trial) were closely followed from 2 to 24 months of age. In the case of AOM nasopharyngeal aspirates (NPA) and middle ear fluids (MEF) were collected and analysed for nine common respiratory viruses by TR-FIA or RT-PCR methods. An AOM episode was defined to be a 30-day period starting with an AOM diagnosed by the study physician. An AOM-episode was designated to be virus positive, if one or more of the NPA and MEF specimens collected during the 30 day period was positive for a given virus. Results: out of the 611 study children, 473 (77%) children experienced 1566 AOM episodes (median 3, range 1 –16). Altogether 1016 (65%) AOM episodes were positive for at least one virus; 480 (31%) for rhinoviruses, 385 (25%) for enteroviruses and 142 (9%) for RSV. Other viruses (adenovirus, influenzaviruses A and B, parainfluenzaviruses 1, 2 and 3) were found in 153 (15%) episodes. Enteroviruses were present throughout the whole winter in contrast to the spring and fall peaks of occurrence of rhinoviruses. Conclusions: more than one half of AOMs in young children are associated with infections caused by the two major genera of human picornaviruses, rhinoviruses and enteroviruses.

Nucleic acid based diagnoses Diagnosing herpes virus infections by real time amplification and rapid culture HUBERT NIESTERS, J. GULDEMEESTER, A. OSTERHAUS and G. van DOORNUM Department of Virology, University Hospital Rotterdam, The Netherlands Purpose of the study: the aim of the study was the evaluate the use of real time detection methodology for HSV type 1 and 2, and VZV to replace rapid culture techniques in a clinical diagnostic setting. Methods: routine samples were collected from a clinic for sexually transmitted disease, as well samples from transplant patients. Nucleic acid were extracted on a MagnaPure LC isolation station, including a seal herpes virus as universal internal control. Primers for HSV type 1 and 2 were validated in the EU QCCA program. Preliminary results: a total of 366 swabs were tested to date, of which 145 were positive by either method. Eight double infections were detected using amplification techniques. A total of 63 samples were positive for HSV1 by both methods, of which two double infections with CMV and HSV2. Furthermore, five additional HSV1 were detected by amplification, one was only tissue culture positive. For HSV2, agreement was for 35 samples, 16

additional samples were detected by amplification techniques, all of them with viral load less than 5000 copies per ml viral transport medium. For VZV, six samples were in agreement, while five were only positive by amplification, one by culture techniques. These samples had a high viral load (above 10 million copies per ml). The clinical data of the patients with discrepant results were reviewed, and in all cases the patients could be considered as truly infected by the virus(es) detected. Conclusion: real time amplification techniques are suitable for the detection of human herpesvirus infection and are an alternative for viral culture as being more sensitive and quantitative. The speed for the generation of ‘negative’ results is an advantage.

Human papillomavirus testing in the colposcopy clinic NICOLA BRINK, R. JELLEY, P. WATTS, H. JALAL, C. PERRONS and R. TEDDER Departments of Virology and Obstetrics and Gynecology, University College London Hospitals and Royal Free and University College Medical School

Purpose of the study: to evaluate the use of HPV DNA detection to rationalise the management of patients attending an inner London colposcopy clinic with pre-malignant cervical disease. Patients and methods: the following clinical groups were included: mild or borderline smears on cervical cytology; persistent CIN 1 or mild dyskariosis, women undergoing local treatment of cervical pathology, atypical glandular cells on cervical cytology and women with complex medical problems. Samples were tested for HPV DNA at the initial colposcopy visit and approximately 7 months later. HPV DNA detection was done using the Digene Hybrid Capture II assay. Results were compared with cytology, histology and colposcopy. Results: paired samples were available from 243 women. The mean age was 37 years (range 20 – 70 years) and the mean time between samples 7 months (range 2 – 15 months). Results were as follows,

High risk HPV

Number of patients

‘Absent’ (not detected) ‘Acquired’ infection ‘Persistent’ infection ‘Resolution’ of infection

124 10 43 66

Thirty four women underwent local treatment of their cervical pathology, all of whom had HR-HPV DNA detected in their pre-treatment sample. 28 (82%) of this group had no detectable HPV DNA in their post-treatment sample. Conclusion: HPV DNA detection can be successfully integrated into the management of women attending a colposcopy clinic. It

Abstracts allows for a more rational and targeted treatment of pre-malignant cervical disease. The absence of detectable HPV DNA in the post-treatment sample provides additional confirmation of ‘cure’, but must be used in conjunction with the colposcopy, cytology and histology results. The detection of HR-HPV DNA also allows the identification of women at potential risk of disease progression.

HPV genotyping by sequence analysis and the line probe assay L. van DOORN1, C. BERNHARD KLETER1, VERMEULEN2, Y. ZOMERDIJK-NOOIJEN2, S. ULJEE2, W. QUINT1, G. FLEUREN2 and E. SCHUURING2, 3 1 Delft Diagnostic Laboratory, Delft, The Netherlands; 2 Department of Pathology, Leiden University Medical Center, The Netherlands; 3Department of Pathology, Academic Hospital Groningen, The Netherlands

Human papillomavirus (HPV) is the etiologic factor in the development of cervical cancer. Accurate detection and genotyping of HPV DNA is required for adequate patient management. The aim of the present study was to evaluate the INNO-LiPA HPV prototype research assay for detection and simultaneous identification of 25 different genotypes. A total of 194 cervical carcinoma specimens were collected and paraffin-embedded specimens were tested by PCR using four broad-spectrum PCR primer sets: SPF10 (L1 region, 65 bp), CPI/IIG (E1 region, 188 bp), GP5 + /6+ (L1 region, 150 bp) and MY09/11 (L1 region, 450 bp). SPF10 LiPA genotyping results were compared with direct sequence analysis of CPI/ IIG, GP5+/6 + and MY09/11 amplimers. HPV genotypes of sequenced fragments were assigned by BLAST search. The SPF10 PCR primer set was the most sensitive method, followed by the CPI/IIG, GP5+ /6+ and MY09/1 HPV primer sets. This may be explained by the amplimer size, the primer set generating the smallest product being the most sensitive. The SPF LiPA typing results were confirmed by sequence analysis of the CPI/IIG, GP5+ /6+ and MY09/11 amplimers. Only high-risk HPV genotypes were found, including HPV16 (43.6%), HPV 18 (18.9%), HPV 52 (8.9%), and HPV 33 (6.8%). Multiple genotypes were detected in 16% of the samples by the SPF10 LiPA system, whereas direct sequencing only revealed single genotypes. In conclusion, genotyping by the SPF10 LiPA system was confirmed by direct sequence analysis of PCR products. Therefore, the SPF10 LiPA is reliable, highly sensitive and offers a rapid tool for HPV genotyping studies.

Detection of multiple HPV genotypes by broad-spectrum SPF10 PCR and LiPA genotyping and confirmation by type-specific PCR BERNHARD KLETER1, L. van DOORN1, G. SCHOLTE2, J. LINDEMAN2 and W. QUINT1 1 Delft Diagnostic Laboratory, Delft, The Netherlands; 2 Slotervaart Hospital, Amsterdam, The Netherlands

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Human papillomavirus (HPV) infection of the cervix is associated with the development of cervical cancer. We have found that a considerable proportion of patients are infected by multiple HPV genotypes. This may implicate that a single lesion may contain multiple genotypes, or such biopsy specimens may represent multiple lesions, each containing a single HPV genotype. Among patients with cervical carcinoma, the prevalence of multiple genotypes is low, which is consistent with the hypothesis that cervical carcinomas are clonal. A total of 11 patients were selected, based on the presence of multiple HPV genotypes. A cervical scrape and a biopsy specimen taken on the same day were analyzed by the SPF10-LiPA genotyping system as well as individual type-specific PCRs for HPV 16, 18, 31, 33, 35, 39, 51, 52, 56, 58, 59, 66 and 68. The specimens contained 2 – 4 different genotypes. There was complete agreement between the SPF10-LiPA system and typespecific PCR in eight patients. In the remaining three patients, additional types were detected by type-specific PCRs in either the biopsy specimen or the scrape. However, if an additional type was detected in the biopsy by a type-specific PCR, the same genotype was detected by LiPA in the scrape, and vice versa. In conclusion, the SPF10-LiPA system is a reliable tool for identification of HPV genotypes in clinical samples. In some cases, the prevalence of multiple genotypes may be underestimated, due to competition between different genotypes when using broad-spectrum HPV-DNA PCR primers.

Development of a robust semi automated procedure for the molecular detection of high risk human papillomavirus in liquid based cytology samples KATIE CUSCHIER, L. SEAGAR, C. MOORE, and H. CUBIE Regional Clinical Virology Laboratory, Edinburgh

It is becoming increasingly accepted that for accurate and efficient reading of cytological preparations, liquid based cytology (LBC) is the way forward. Moreover, as only a small volume of the LBC sample is required for cytological preparation the residual material harbors enormous potential for ancillary molecular based clinical testing. Currently there is substantial justification for the implementation of high-risk human papillomavirus (HR-HPV) testing as an indicator of the development and maintenance of cervical intraepithelial neoplasia. Should HR-HPV testing become more widespread, the development of automated, efficient nucleic acid extraction techniques from LBC samples will be essential. We have successfully developed a robotic extraction procedure that is optimized for the extraction of HPV DNA from LBC samples. Our priority was to generate a system that allowed for high throughput, which did not compromise the sensitivity of the HPV test downstream. To assess the efficacy of the procedure we extracted from 200 LBC samples that had been selected for underlying cytological abnormality. HPV testing was performed by real-time PCR coupled with detailed melt analysis of amplified HPV product by utilisation of the ‘LightCycler’

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Abstracts

apparatus. Our results revealed that an extract from as little as 1 ml of the LBC sample was required for a reproducible HPV positive result. Robotic extraction of the LBC sample prior to real-time PCR ensured a rapid, robust assay for the assessment of HPV in clinical samples. Application of this technique has the potential enhance the predictive values of conventional cytology considerably.

Standardisation of a competitive PCR assay for the detection of B19 virus contamination of plasma pools GIORGIO GALLINELLA1, E. MORETTI2, E. BUCCI2, M. MUSIANI1 and M. ZERBINI1 1 Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Italy; 2Kedrion Biopharmaceuticals, Bolognana, Lucca, Italy Screening of blood donations or plasma for the detection of B19 virus and removal of donations contaminated above a threshold value can limit considerably the input of B19 virus into production pools and thus the transmission of virus via blood or blood derivates. In our work we employed a standardised competitive PCR assay for the detection of B19 DNA in pooled plasma samples. The assay is based on the coamplification of the original B19 target and of an exogenous, internal competitor. Amplified products can be hybridised to probes specific for either the target or competitor sequences, and hybrids can be detected by means of an immunoenzimatic reaction. The efficiency of recovery of B19 virus and competitor plasmid following two different treatments of plasma samples (heat treatment of plasma prior to the amplification reaction or lysis of plasma following the Ampliscreen [Roche] protocol) was evaluated by competitive PCR titration. Coextraction of different amounts of B19 virus in the presence of competitor added as a working reagent at a single defined concentration enabled us to construct a viral titration curve, and then to discriminate positivity against a threshold value of viral load in the sample. Eighty plasma pools, consisting each of 960 donations, were analysed with both heat and Ampliscreen treatments for the presence of B19 virus and for viral contamination over a threshold value of 104 geq/ml. The assay developed proved to be suitable for large-scale screening of plasma pools and semi-quantitative evaluation of donations contaminated with B19 virus.

Second trimester congenital rubella infection: a case report JULIAN TANG1, N. BRINK1, E. AARONS1, E. JAUNAUIX1, L. HESKETH2, G. ENDERS3, S. STROBEL3 and G. SCHALASTA3 1 Departments of Virology and Obstetrics and Gynecology Royal Free and University College London Medical School; 2 Department of Virology, Public Health Laboratory, Preston; 3 Institute for Virology, Infectiology and Epidemiology e.V. G. Enders and Labor Professor Dr G. Enders and Partner, Stuttgart, Germany

Purpose: to describe the diagnosis and management of a case of second trimester congenital rubella infection. Case: a 30-year-old, nulliparous woman with a history of rubella immunisation in childhood, was found to be rubella IgG negative at booking. At 16 weeks gestation, she visited her general practitioner with a rash. A diagnosis of rubella was made by the detection of rubella-specific IgM together with the demonstration of a rubella IgG seroconversion. The sample taken at the time of the rash illness also had low avidity rubella IgG, providing further confirmation of recent rubella. The patient was counseled about the estimated risk of fetal damage between 13 and 16 weeks (about 10 – 15% risk of deafness) and offered the option of prenatal diagnosis. Prenatal diagnostic procedures were done at 19 and 23 weeks. Samples were tested using an in-house rubella reverse-transcriptase polymerase chain reaction (nested RT-PCR) (amniotic fluid and cord blood) and serology (rubella-specific IgM in fetal blood). Results3

Gestational age

Sample type

Results

19 weeks

Amniotic fluid

Rubella RNA not detected

22 weeks and 4 days

Amniotic fluid

Rubella RNA not detected Rubella RNA detected by nested RT-PCR (semiquantified) at 1000– 5000 genome copies/ml rubellaspecific IgM detected by 3 commercial test kits

Fetal cord blood

Conclusion: fetal infection was confirmed using nested RTPCR to detect rubella RNA and serology to detect rubella-specific IgM in a fetal blood sample. The combination of these two methodologies ensured a secure diagnosis of congenital infection, which would have been inadequate by sampling of amniotic fluid alone.

Evaluation of RT-PCR and virus culture in the detection of enteroviruses from cerebrospinal fluid samples TYTTI VUORINEN1, R. VAINIONPA8 A8 2 and T. HYYPIA8 2 1 Department of Virology, University of Turku; 2Haartman Institute, Department of Virology, University of Helsinki, Finland

Abstracts Enteroviruses are currently the most common course of viral meningitis. We have evaluated cerebrospinal fluid samples sent to the diagnostic laboratory at the Department of Virology, University of Turku between 1996 and 2000 by virus isolation and enterovirus RT-PCR test. Virus isolation was done in LLC-Mk2, A549, and CaCo cell cultures. Enterovirus genome was detected from the samples by an RTPCR assay, which amplifies all the enterovirus serotypes. Virus culture was performed from 1312 specimens and enteroviruses were isolated from 35 (3%) samples. The most common enterovirus typed by the neutralization assay was echovirus 30. Enterovirus RT-PCR test was done from 604 samples and it was positive in 82 (14%). Both virus isolation and RT-PCR were performed from 301 samples. Of those, 245 were negative with both assays and enterovirus was isolated from three samples, which remained negative by RTPCR. Enterovirus genome was detected in 53 cerebrospinal fluid samples, of which only 15 grew in cell culture. Our results show that RT-PCR test is the most powerful tool to detect enteroviruses from cerebrospinal fluid samples. Moreover, it is more rapid and the results can be obtained in one day when compared with the conventional virus isolation method. Therefore, enterovirus RT-PCR should be used as primary laboratory test in the diagnosis of enterovirus meningitis.

Follow-up study on the proficiency testing of molecular methods for the detection of enterovirus KARIN van VLIET2, P. MUIR1, J. ECHEVARRIA1, P. KLAPPER1, G. CLEATOR1 and A. van LOON2 1 For the European Union (EU) Concerted Action on Quality Control (QCCA) of Nucleic Acid Amplification in Diagnostic Virology; 2University Medical Center, Utrecht, The Netherlands

A multicenter quality assessment of nucleic acid amplification tests (NAT’s) for the detection of enteroviruses (EV) was conducted as part of the EU-QCCA program. Three different proficiency panels, consisting of heat-inactivated, freeze-dried enteroviruses, were distributed over a period of 3 years. A total of nine representative EV serotypes at various concentrations, a rhinovirus sample and ‘negative’ controls were used to test sensitivity and specificity of commercial assays and in-house methods. Panels and questionnaires were sent to a total of 109 laboratories in Europe, 41% of which participated two or three times. A total of 200 data sets were submitted. The seven different ‘strong-positive’ samples were detected by over 90% of the laboratories; the ‘positive’ samples by 70 – 90%; 49% and 16% of the participants correctly reported one or two ‘weak-positive’ samples, respectively. The average false –positivity rate was 2.8%. A scoring system was introduced to compare the performance of laboratories and methods employed. Overall, the performance was comparable between the proficiency panels; 67 –70% of the laboratories performed satisfactory. The sensitivities of

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the various NAT’s differed by a factor of 103 – 104. All types of methods proved capable of generating maximum scores. However, the nested PCR format was generally associated with a higher performance. Comparison of data obtained from 21 laboratories who examined all three panels showed an improvement in performance due to achievement of a higher sensitivity. In view of the unsatisfactory performance of one-third of the participants, ongoing proficiency testing remains necessary to assess, validate and improve the quality of molecular diagnostic investigations.

An European programme for external quality control of molecular diagnosis in infectious disease: past, present and future ANTON van LOON (Utrecht), J. REID (Glasgow), P. KLAPPER (Leeds), A. LINDE (Stockholm), B. VESTERGAARD (Copenhagen), J-M. ECHEVARRIA (Madrid), and G. CLEATOR (Manchester) For the EU Concerted Action for Quality Control of Nucleic Acid Amplification in Diagnostic Virology, Department of Virology, University Medical Center Utrecht, Utrecht, The Netherlands

The European Union Concerted Action for Quality Control of Nucleic Acid Amplification in Diagnostic Virology ( EU-QCCA) is now in its 3rd and final year. So far, a total of 14 proficiency panels have been distributed. These included panels for detection and quantitation of enteroviruses (3), HSV (3), CMV, HBV (2), HCV (2), HIV (2), and Chlamydia trachomatis. The number of participating laboratories ranged between 46 and 105, and increased with nearly every distribution. Laboratories were located in up to 20 countries per distribution. Whereas most participating laboratories used in-house methods for detection of enteroviruses and HSV, commercial assays were mainly used for detection and quantitation of HBV, HCV, HIV and Chlamydia trachomatis. Compared with studies in the 1990’s, the false – positivity rate had dropped markedly to levels between 1 and 3%, indicating that laboratories are better managing contamination risks. The sensitivity, particularly of in-house methods, varied considerably between laboratories (up to 1000-fold) but less so between types of method (b DNA, NASBA, (n)PCR, etc.). Since the EU programme is in its final year, various parties, including ESCV, have requested the EU-QCCA to continue its programme, and even develop it further. A plan has been made for continuation as a not-for-profit QC organisation, managed by professionals in the field, albeit under a different name: QCMD, Quality Control of Molecular Diagnostics. The scope of the programme will be extended to include non-viral pathogens as well as molecular typing. Also, more attention will be given to development of reference materials such as ‘run controls’. ESCV, ESCMID and the major molecular diagnostic companies are endorsing the programme. Further support will be sought from the EU, but the programme will only be viable when members of ESCV and ESCMID actively participate.

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Abstracts

Viral infections in the immunecompromised host Early diagnosis of lymphoproliferative disease with quantitative EBV PCR SANNA AALTO, E. JUVONEN, L. VOLIN, T. RUUTU, H. PIIPARINEN, P. MATTILA, J. TARKKANEN, S. KNUUTILA and K. HEDMAN Department of Virology, Department of Hematology, Department of Pathology, Department of Otorhinolaryngology and Department of Medical Genetics, Helsinki University Central Hospital, Finland

Introduction: Lymphoproliferative disease (PTLD) is a potentially fatal complication of organ transplantation. The incidence of PTLD ranges from 1 to 10%. In bone marrow transplant (BMT) recipients, rapid diagnosis of PTLD is mandatory. In this context, new PCR-based methods seem very promising. Objecti6e: to determine the suitability of quantitative EBV PCR (EBV qPCR) for early diagnosis of lymphoproliferative disease. Methods: a total of 103 serum samples drawn consequtively from 12 BMT recipients with fatal PTLD were studied with a TaqMan-based EBV qPCR. The post-mortem criteria for PTLD were strict; disseminated lymphocytic infiltrates in several organs with EBV antigens and/or RNA (EBER) detectable by in situ technology. For control, we studied 378 consecutive serum samples from 38 matched BMT recipients. A positive-control group were 16 patients with EBV mononucleosis (criteria: EBV IgM together with EBV IgG of low avidity). A negative-control group were 21 immunocompetent individuals with latent EBV infection. Results: all the 12 BMT recipients with fatal PTLD had EBV DNA in serum, with progressively rising copy numbers before death. The EBV DNA appeared 24 –152 (mean 72) days after BMT, i.e. earlier than clinical symptoms which appeared at 30– 173 (mean 77) days. The progression of PTLD was rapid: the mean time from first symptom to death was 21 days. In the 378 sera from the 38 asymptomatic BMT controls, EBV DNA occured only sporadically (3.7%). Nearly all (15/16) the patients with EBV mononucleosis had EBV DNA in serum, in contrast to none of the 21 immunocompetent individuals. Conclusion: EBV qPCR is a highly sensitive and gratifyingly specific method for early diagnosis of PTLD.

Pre-emptive anti B-cell immunotherapy (PE) for epstein-barr virus (EBV) reactivation effectively reduces the incidence of EBV-lympho-proliferative disease (EBV-LPD) following T-cell depleted (TCD) allogeneic stem cell transplantation (ALLOSCT) HUBERT NIESTERS1, J. van ESSER1, B. van der HOLT1, E. MEIJER2, J. GRATAMA1, B. LO8 WENBERG1, L. VERDONCK2, J. CORNELISSEN1 and A. OSTERHAUS1 1 University Hospital Rotterdam; 2Utrecht Medical Center, The Netherlands In order to prevent EBV-LPD after TCD allo-SCT, we evaluate the efficacy of anti B-cell immunotherapy as PE-

therapy. Retrospectively, a plasma viral load of 1000 genome equivalents per ml (geq/ml) was associated with a positive predictive value (PV+) of 39% and chosen for PE by a single infusion of Rituximab. Forty-nine recipients of a TCD-alloSCT were included, 35 received stem cells from a family donor (sib) and 14 from a matched unrelated donor (MUD). Patients were monitored weekly for EBV-reactivation by quantitative PCR. Forty-three donor/recipient (D/R) combinations were EBV-seropositive, 3 were D +/R − and 3 D −/ R+ . EBV-reactivation occurred in 49% (24/49) of the patients. Twelve patients showed EBV-reactivation =1000 geq/ml and received PE (eight sib, four MUD) at a median EBV-DNA level of 3600 (range 300 – 86 300). Median time to EBV-DNA=1,000 geq/ml was 120 days (range 39 – 507), and median time to initiation of PE was 125 days (range 43 – 514) after SCT. Eleven patients showed complete clearance of EBV-DNA within a median of 6 days (range 1 – 46) after PE and did not develop EBV-LPD. One recipient of a MUDSCT developed EBV-LPD despite PE, but obtained complete remission (CR) after two infusions of Rituximab and donor lymphocyte infusion. Two recipients of a MUD-SCT presented with EBV-LPD before initiation of PE. These patients showed a CR on repeated Rituximab therapy and are alive to date. In conclusion, PE with a single infusion of Rituximab given at EBV-DNA =1000 geq/ml in recipients of a TCDallo-SCT is an effective strategy to decrease the incidence of EBV-LPD.

CMV infection is usually associated with concurrent HHV-6 and HHV-7 antigenemia in liver transplant patients IRMELI LAUTENSCHLAGER, M. LAPPALAINEN, K. LINNAVUORI, J. SUNI, and K. HO8 CKERSTEDT Departments of Virology and Surgery, Helsinki University Hospital, Helsinki, Finland

Human herpesvirus 6 and 7 (HHV-6, HHV-7) have been recently described in liver transplant patients. HHV-6 may cause fever, neurological disorders and hepatitis. Clinical significance of HHV-7 is less clear. HHV-6 and HHV-7 are closely related to cytomegalovirus (CMV) and interactions between the viruses have also been suggested. In this study, we investigated the post transplant HHV-6 and HHV-7 antigenemia in relation to CMV disease after liver transplantation. Consecutive 34 adult liver allograft recipients transplanted during 1999 – 2000 were included in the study. CMV infections were diagnosed by the frequent monitoring of pp65-antigenemia and by viral cultures. HHV-6 and HHV7 were demonstrated, by using immunoperoxidase staining and monoclonal antibodies against the virus specific antigens, in the mononuclear cells from the same blood specimens which were obtained for CMV pp65 monitoring. Altogether 322 blood specimens were analyzed. CMV disease was diagnosed in 12 (35%) patients during the first 3 months (first pp65 positive specimen mean 25 days, range 8 – 61 days) after transplantation. Concur-

Abstracts rent HHV-6 antigenemia was detected in 10/12 (mean 14 days, range 6 – 22 days) and HHV-7 antigenemia in 9/12 patients (mean 25 days, range 10 –89 days) after transplantation. HHV6 usually appeared slightly before CMV. All CMV infections were successfully treated with ganciclovir, and the CMV-antigenemia subsided. HHV-6 and HHV-7 antigenemia also responded to the antiviral treatment, but more slowly than CMV. In conclusion, CMV infection was usually associated with HHV-6 and HHV-7 antigenemia in liver transplant patients. The results support the suggestion that CMV, HHV-6 and HHV-7 may have interactions.

Prevalence of herpes group infections in early childhood in Finland JOHANNA AARNISALO, J. ILONEN, R. VAINIONPA8 A8 , I. VOLANEN and O. SIMELL Departments of Virology and Paediatrics, University of Turku, Finland

In this long-term prospective study we wanted to clarify the prevalence of cytomegalovirus (CMV), varicella-zoster-virus (VZV) and herpes simplex-virus (HSV) infections among young children in Finland and analyse the age of acquirement of the infections. Serum samples of 199 children born in 1989 and 1990 were collected at the age of 7 and 13 months and yearly thereafter until the age of 8 years. Two to eight samples were available from each child. A minimum of 106 samples were studied in each age group. IgG class antibodies to CMV, VZV, and HSV were measured using EIA. In the first sample at the age of 7 months the prevalence of CMV antibodies was 27% whereas only 3% of children had antibodies against HSV or VZV. The prevalence of seropositivity for CMV increased slowly up to 41% at the age of 8 years. The number of children acquiring varicella infection increased rapidly after 2 years age and at the age of 8 years 83% of children had antibodies against VZV. The proportion of children showing markers of HSV infection remained quite low throughout the observation period and at the age of 8 years 17% of children had antibodies to HSV. These data demonstrate that HSV infection is acquired later in life than before and the proportion of uninfected subjects is increasing. The proportion of cytomegalovirus infections during the perinatal period and early infancy remains relatively high, comprising a third of the children. Most children also experience an VZV infection during early childhood.

Aggravated anaemia in solid organ transplant recipients with low level parvovirus B19 DNA-anaemia PETER RAUTENBERG1, K. ZAHN1, F. FAENDRICH2, S. HIRT2, N. STUMPP3 and T. HARDER1 1 Institute Med. Microbiology and Virology; 2Deptartment of Surgery, University of Kiel, Germany; 3Medac, Incorp., Wedel, Germany

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Objecti6e: to investigate the prevalence and incidence of parvovirus B19 infections in solid organ transplant recipients (SOTR). Design: a prospective 10 month study was conducted on 63 SOTR (14 kidney, 32 heart, eight lung, nine liver). Patients were followed for at least 90 days post transplantation Material and methods: antibodies specific to VP-1 and VP-2 proteins were detected by IgG and IgM ELISA. DNA was determined by a seminested PCR targeting a VP1 gene fragment and a nested PCR specific for a NS1 gene region. The latter was also probed by a quantitative light cycler PCR (Harder et al. submitted). Results: a total of 53 out of 63 patients were IgG seropositive prior to transplantation. There was no IgM seroconversion, but four patients developed a significant IgG titre rise. Low level parvovirus B19 DNA-anemia (range 5 – 800 geq/ml) were detected in 18 of 63 patients before transplantation. In addition, eight seropositive patients developed DNA-aemia after transplantation. Posttransplant samples from renal, cardiac and hepatic recipients with DNAanemia showed reduced hemoglobin (94.5 vs. 105 g/l; P= 0.0054), erythrocytes (3.04 ×1012 vs. 3.3 × 1012/l; P=0.0016) and PCV (28 vs. 31%; P =0.0013). MCV, MHC, leukocyte and thrombocyte counts were not associated with DNAaemia. Donation of either blood, frozen plasma, albumin or thrombocytes ( \20, \25, \5, \6 units, respectively) was associated with \3 fold risk of low level DNA-aemia (95% CI 1.3 – 10.8). Low level DNA-aemia was not associated with a higher risk of organ rejection. Conclusion: while the impact of parvovirus B19 infection on erythrogenic differentiation is well known, the mechanisms triggering and supporting low level viral DNA-aemia and erythropoetic stress in SOTR await further elucidation.

Viral vaccines Molecular epidemiology of mumps virus and its transmission in the UK LI JIN1, J. WHITE2, S. BEARD1, R. HUNJAN1, P. LITTON1, B. COHEN1 and M. RAMSAY2 1 Enteric, Respiratory and Neurological Virus Laboratory, Central Public Health Laboratory; 2Immunisation Division, Communicable Disease Surveillance Centre, 61 Colindale Avenue, London

In the absence of an immunisation programme, epidemics of mumps occurred in the UK at three yearly intervals. Combined measles, mumps and rubella (MMR) vaccine was introduced in the UK in October 1988. The proportion of notified mumps cases confirmed by salivary IgM antibody tests increased from B 5% in 1995 to 38% in 2000. The majority of cases are now linked to secondary school outbreaks (age 13 – 18 years), a cohort too old to have routinely received MMR, although many received the measles-rubella (MR) vaccine in the school campaign in 1994. Of the cases where vaccination status is known, 47% had not received any MMR vaccine, 49% had received only one dose and 4% had received

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Abstracts dengue tetravalent attenuated vaccines have been submitted to clinical trials. JE chimeric vaccine with YF virus as vector and cell-derived inactivated vaccines are also at the level of clinical evaluation. In the area of measles the WHO priority is to develop new forms of current vaccine to facilitate mass immunization campaign in developing countries including nebulized aerosol, powder aerosol and Jet Injector technology. During the past 7 years, studies to ensure suitability of the transgenic mouse expressing human receptor for poliovirus for quality control of oral poliomyelitis vaccine (OPV) has been carried out by an international collaborative group under the aegis of WHO. Another cutting- edge quality in virto test for polio vaccine, called MAPREC assay, was also developed and evaluated in the WHO collaborative study. Recently, WHO has approved both of these tests for ensuring the quality control of OPV. The new diagnostic test was introduced in practical laboratories for rapid detection of polioviruses in clinical and environmental samples. The test is based on use of mouse cells expressing human receptor for poliovirus (L20B). The studies are in progress to develop standardized neutralization test for dengue viruses and international standards for human antibodies against JE and dengue viruses. Development of simpler assays for laboratory confirmation of measles infection is ongoing that is based on the detection of antimeasles IgM antibodies. Detailed information about the above-mentioned projects and some other ones will be presented.

two doses, emphasising the need to include a second dose of MMR in the schedule, which was introduced in 1996. Based on phylogenetic analysis, which was performed on the entire SH gene sequence (318 nucleotides), multiple mumps genotypes/strains were identified in the UK between 1995 and 2000 by direct sequencing of over 180 PCR amplicons from a variety of specimens. Continual surveillance has shown that some of the currently circulating genotypes were imported from abroad. Our studies show that monitoring geographical and temporal distribution of mumps genotypes in the UK, against a background of high herd immunity, is essential for mumps surveillance and contributes to programmes for control and elimination of mumps disease. WHO activity in development and introduction of new viral vaccines and related biologicals YURI PERVIKOV World Health Organization, Geneva

Development and introduction of new viral vaccines and biologicals for diagnosis of viral diseases and quality control of vaccines is important research priority of WHO. The main activity focuses on the following viral infections: measles, poliomyelitis, dengue, Japanese encephalitis (JE), rotaviruses, and respiratory syncytial virus. At present, two

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