First national external quality assessment scheme for avian influenza A virus (H5N1)

Abstracts: Oral Presentations

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Virology of avian influenza in relation to wild birds

R.A.M. Fouchier*, V.J. Munster, J. Keawcharoen, A.D.M.E. Osterhaus, T. Kuiken. Department of Virology,

Erasmus Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands The outbreak of highly pathogenic avian influenza of the H5N1 subtype in Asia, which has subsequently spread to Russia, the Middle East, Europe, and Africa, has put increased focus on the role of wild birds in the persistence of influenza viruses. The ecology, epidemiology, genetics, and evolution of pathogens cannot be fully understood without taking into account the ecology of their hosts. Here, we review our current knowledge on global patterns of LPAI influenza virus infections in wild birds, discuss these patterns in the context of host ecology and in particular birds' behaviour, and identify some important gaps in our current knowledge. In addition, we will discuss potentially important differences between LPAI influenza viruses and HPAI H5N1 viruses in wild birds and mammals, in particular with respect to pathogenesis, virus secretion, and hostrange.

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Epidemiology and potential clinical associations of human bocavirus

K. Templeton 1 *, A. Manning 2, V. Russell 2, K. Eastick 1, G. Leadbetter 1, N. Hallam 1, R Simmonds 2. 1Royal Infirmary

Hospital, Edinburgh; 2University of Edinburgh, Edinbrugh, UK Background and Aims: Human Bocavirus (HBoV) is a newly discovered human parvovirus; HBoV was first detected in respiratory samples with a potential role in human respiratory disease. This study compared frequencies, epidemiology and clinical backgrounds of HboV infections with those of other respiratory virus infections among diagnostic samples referred to the Specialist Virology Laboratory (SVL) in Edinburgh. Methods: Samples were coded and anonymised. Subject information was obtained from the SVL respiratory sample archive. Samples were screened by nested PCR for HBoV, and real-time PCR for respiratory syncytial virus (RSV), adenoviruses, influenza and parainfluenza viruses. Results: HBoV infection was detected in 47 from 574 study subjects (9.2%), ranking below RSV (15.7%) and adenovirus (10.3%) in prevalence. HBoV showed peak incidences among young children (6-24 months) in mid-winter months (December, January). HBoV was specifically associated with lower respiratory tract infections (LRTI) in children. HBoV infections were more prevalent in individuals infected with other respiratory viruses (17%), mainly adenovirus or RSV. HBoV infections were primarily seen in children less than 5 years (>90%) but also in immunosupressed adults. Conclusions and Discussion: HBoV was frequently detected and is a potential respiratory pathogen of LRTI, with a prevalence and epidemiology comparable to RSV. It is principally detected in children less than 5 years. Differentiating HBoV from RSV and other respiratory viruses may be clinically important in the future.

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Genotype analysis of measles outbreaks in the WHO European region 2005-2006

M. Mulders 1 *, G. Lipskaya 1 , J. Spika 1 , WHO European Regional Measles/Rubella Laboratory Network. 1World Health Organization,

European Regional Office, Copenhagen, Denmark The European Region of the World Health Organization has adopted a goal for measles and rubella elimination and control of congenital rubella by 2010. To achieve this goal high immunization coverage and in-depth surveillance are key strategies. Of the 52 Member States of the WHO European, 27 had a measles incidence of 1:1,000,000 or less. As of June 2006, only 17 countries reported this low incidence, and 9 reporting an incidence of more than 1:100,000. The increase in incidence was due to the occurrence of outbreaks of various nature in many of the Member States. A long-lasting outbreak started fall 2004 in Romania affecting well over 10,000 people and caused by a D4 virus, was also linked to smaller outbreaks in Portugal and Hesse, Germany in

$13 2005. Another devastating outbreak in Ukraine, which started in fall 2005, has affected well over 40,000 people and was caused by a single virus variant belonging to the D6 genotype. Genetically identical viruses were found in Germany (>1000 cases in Northern Rhine Westphalia), but also in Russian Federation, Belarus, Spain, Estonia, Latvia, Poland. The 3rd important genotype circulating in Europe was B3, a genetically highly diverse genotype extensively found in sub-Saharan Africa. Different variants of the B3 virus were found in Denmark, Sweden, Germany, UK, Spain. Although there is a marked improvement in the vaccination coverage and decrease in incidence for measles in the European Region, measles continues to have a severe public health impact throughout the entire Region. Increased vigilance and commitment at all levels is needed to ensure the Regional goals of measles and rubella elimination are met by 2010.

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Identification of a fourth human parechovirus serotype

K. Benschop 1 *, J. Schinkel 1 , M. Luken 4, R van den Broek 4, M. Beersma 5, N. Menelik6, H. van Eijk 1, H. Zaaijer 1, C. VandenBroucke-Grauls 3, M. Beld 1 , K. Wolthers 1,2. 1Cfinical

Virology and 2Human Retrovirology of the Department of 3Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands, 4primagen, Amsterdam, The Netherlands, 5Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands, 6Department of Pediatrics, Boven IJ Ziekenhuis, Amsterdam, The Netherlands. Introduction: Since the recent discovery of a novel human parechovirus (HPeV) serotype in Japan, three different serotypes are recognized within the genus of HPeVs (formerly echovirus22 and 23). Infections with HPeVs are commonly associated with mild gastrointestinal and respiratory symptoms in young children, but more severe conditions, such as flaccid paralysis and neonatal sepsis, have also been described. While genotyping HPeV isolates from our laboratory, we identified a deviant HPeV isolate (K251176-02) from a stool specimen from a neonate with high fever. Method: The complete genome sequence was determined by combinations of consensus primers to generate partially overlapping PCR fragments, which were subsequently sequenced. Extensive phylogenetic analysis was carried out on the complete genome sequence. For serotyping, neutralisation assays were carried out with antisera directed against the known serotypes 1-3. Results: Phylogenetic analysis of the complete genome showed that K251176-02 was most related to the HPeV2 prototype CT86-6760. However, the genetic distance is considerable (0.327) and comparable with the distances to other HPeV prototypes. A SimPIot analysis showed K251176-02 to be most similar to HPeV2 CT86-6760 in the highly variable P1 region and to HPeV3 in the more conserved P2-P3 region, suggesting that K251176-02 originated from an early recombination event. Furthermore, K251176-02 could not be neutralized by antisera directed against HPeV1-3. Conclusion: K251176-02 represents a new genotype based on phylogenetic analysis. As K251176-02 could not be neutralized with antibodies against the known HPeV serotypes, the newly identified HPeV should be classified as a fourth HPeV serotype. 1~

First national external quality assessment scheme for avian influenza A virus (H5N1)

H. Zeichhardt 1,2 *, B. Schweiger 3, V. Lindig 1,4, H.-P. Grunert 1,4.

1Charit~ University Medicine Berlin, Campus Benjamin Franklin, Institute of Virology, Berlin; 21NSTAND (Society for Promotion of Quality Assurance in Medical Laboratories), Duesseldorf,, 3Robert Koch-lnstltut, National Reference Center for Influenza, Berlin; 4Gesellschaft fuer Biotechnologische Diagnostik, Berlin, Germany External quality assessment schemes (EQASs) for influenza virus detection were implemented in Germany in 2000. These EQASs survey the laboratory proficiency as well as the robustness and quality of diagnostic tests. These EQASs are organized by INSTAND and authorized by the German Medical Association and three scientific societies. The EQAS cover the detection of human influenza viruses (homogenates of cells infected with influenza A subtype H1N1 or

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Journal of Clinical Virology 2006, Vol 36 (suppl 2)

H3N2 viruses or influenza B virus or mock infected cells). These viruses represent circulating influenza virus strains covered by influenza vaccines as proposed by WHO. In March 2006 a sample with avian influenza A virus H5N1 (allantoic fluid, chemically inactivated) was included. 170 laboratories from Germany and 7 other countries participated in this EQAS. Antigen detection: The majority of the laboratories reported correct results (95.7-100%). The sample containing avian influenza A virus H5N1 revealed difficulties for antigen detection and type differentiation. The rapid test of one manufacturer led to false negative results in 15/28 laboratories (only 46.4% correct results). The influenza A virus specific ELISA of another manufacturer was capable to detect H5N1 virus, however, led to false positive results indicating influenza B virus. Genome detection: The influenza viruses were well detected by type specific and non-differentiating PCRs (92-100% correct results). Specificity problems reflected by false positive results occurred with the negative sample. 98.6% of all subtyping results for the H5N1 sample were correct (68/69 analyses). 3 of 19 laboratories subtyped the H3N2 sample wrongly as H5.

Posters

Abstracts, 9th ESCV Annual Meeting Viral detection in patient and control samples was performed by means of PCR whereas long-term BM cultures (LTBMCs) were used for functional experiments. Results: HSV-1 DNA was detected by PCR in BM cells of 22.2% of CIN patients and 16.6% of the controls (p>0.05). EBV was found in 11.1% of CIN patients and 8.3% of the controls (p > 0.05). Interestingly, CMV was detected in BM cells of 55.5% of CIN patients whereas it was not present in the control group (p = 0.006). Further investigation for the identification of CMV genome in CD3+, CD14+ and CD34+ BM cells, representing T-lymphocytes, monocytes/macrophages and progenitor cells respectively, and also in stromal cells, revealed the viral DNA only in the latter cell type. Infection of LTBMCs with CMV AD169 strain resulted in viral latency at the stromal cells, whereas CMV could establish a lytic infection at the stromal cells in long-term cultures but in the absence of non adherence bone marrow cells. The number and the clonogenic potential of non adherent cells in CMV-infected LTBMCs were lower compared to the mock-infected cultures. Conclusion: CMV is frequently detected in BM stromal cells of patients with CIN. Based on the functional assays we postulate that the CMV latent infection of BM microenvironment cells may affect the growth of haemopoietic progenitor cells and may, therefore, have a role in the pathogenesis of neutropenia in the affected subjects.

1 ~ ] HPV-16 E5 oncogene affects cellular pathways

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Proteomic profiling in echovirus 30 infected cells

K. Zimmermann 1 , H. Junker 2, S. Venz 2, A. Karger 3, R. Mentel 1

1Friedrich Loeffler Institute of Medical Microbiology, ErnstMoritz-Arndt-University Greifswald, Germany, 2Department of Medical Biochemistry and Molecularbiology, Ernst-Moritz-ArndtUniversity Greifswald, Germany, 3Institute of Molecular Biology, Friedrich-L oeffler-lnstitute Greifswald-lnsel Riems, Germany Human enteroviruses (EV) are one of the notable cause of meningitis in children. Despite the great advances in the knowledge of EV currently there is no licensed therapy effective in prevention or interrupting this virus infection. The elucidation of cell-virus interaction can help to find out potential targets for antiviral strategy. HeLa cells infected with echovirus 30 (E30), a circulating enterovirus strain associated with central nervous system disease, were analysed by two-dimensional gel electrophoresis followed by mass spectrometry. Notable changes in the protein expression pattern were observed already 4 hours after infection. Analysed proteins differed more than 1.5fold - 12 were down-regulated and 20 up-regulated after virus infection. Up to no a total of 32 proteins were identified. The majority of these belonged to the cytoskeleton, actin-associated network, metabolic enzymes, signal transduction and apoptosis. Our findings are preliminary data to understand the molecular basis of cellular response associated with to E30 infections. Further data and investigations are on the way.

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Cytomegalovirus as a potential cause in the pathogenesis of chronic idiopathic neutropenia

E. Dalpa 1 , E. Kourepini 1 , H. Papadaki 2, G.D. Eliopoulos 2, D.A. Spandidos 1 , G. Sourvinos 1 . 1Department of Virology and

2Haemopoiesis Research Laboratory of the University of Crete School of Medicine, Heraklion, Crete, Greece Background: Chronic idiopathic neutropenia (CIN) is a granulocytic disorder characterised by the prolonged, unexplained reduction in the number of circulating neutrophils. Increased apoptosis of bone marrow (BM) myeloid progenitor cells due to an inflammatory BM milieu has been implicated in the pathophysiology of the disease. The cause of these abnormalities, however, remains unknown. Aim: To evaluate the possible involvement of herpes virus superfamily members, namely HSV-1, HSV-2, CMV, EBV and VZV in the pathophysiology of CIN. Patients and Methods: BM cells, peripheral blood mononuclear cells and serum were examined from 14 CIN patients. Four patients with other types of neutropenia and 3 patients with myelodysplastic syndrome as well as five healthy donors, served as control group.

N. Kivi 1 , D. Greco 2, IR Auvinen 2, E. Auvinen 1 . 1University of Helsinki, Haartman Institute and HUSLAB, Department of Virology, Helsinki, Finland, 2Institute of Biotechnology, University of Helsinki, Helsinki, Finland

Background: Human papillomavirus type-16 E5 protein is an oncogenic protein, whose functions are much less studied than those of E6 and E7. To obtain large-scale information about the effects of E5 protein on cellular gene expression in epithelial cells, we examined cellular gene expression by cDNA microarray in a stable HPV-16 E5 expressing epithelial cell line at 24h post induction. Methods: The cDNA microarray analysis was made useing total RNA extracted from the cells. Background correction, normalization and analysis of variance were performed to the data. The mRNA expression levels were confirmed with real-time quantitative RT-PCR. The comparative threshold cycle method was used to determine fold changes of transcript present in E5 expressing cells compared to control cell samples. The effects at protein level were analyzed by immunoblotting. Results: In a screen of 16,000 annotated genes, approx. 200 genes were found to be statistically significantly altered due to E5 as expression compared to HaCaT-pMSG cells. 65 genes were studied by qRT-PCR and 14 of them also at the protein level. The protein levels of lamin A/C were found to be downregulated, protein kinase C-delta and phosphoinositide-3-kinase were found to be upregulated in immunoblotting. Conclusions: Several genes from important signaling pathways were found to be altered due to E5 protein expression. Among the genes of PIPK pathway, several genes were altered in microarray, and upregulation of PI3K at protein level was detected. E5 affects also protein de/phosphorylation and cell adhesion. Except for protein expression levels, the E5 protein may affect posttranslational modifications of cellular proteins.

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Immunological response to cytomegalovirus in congenitally infected neonates

J. Hassan 1 , S. Dooley 1 , W.W. Hall 2. 1National Virus Reference

Laboratory and 2Centre for Research into Infectious Diseases, University College Dublin, Belfield, Dublin 4, Ireland This study uses congenital CMV as an in vivo model to analyse the primary immune repertoire in virally infected neonates and their mothers. Ten pairs of matched neonates and their mothers were evaluated for specific IgM responses to 3 immunodominant CMV antigens; pp38 (pUL80a), pp52 (pUL44) and pp150 (pUL32). In contrast to conventional ELISA testing for CMV specific IgM which found 5 of the mothers and 4 of the neonates to be positive, western