Recombinant expression of human bocavirus capsid proteins

Abstracts: Posters children (0-16 yrs from Edouard Herriot hospital, Lyon, France) was done to evaluate this assay. Among these 89 swabs, real-time NASBA detected 10 (11.2%) samples for Influenza A and 2 (2.2%) samples for Influenza B. Comparatively, by cell culture only 5 (5.6%) samples were identified as Influenza A and none as Influenza B. Conclusions: With the real-time NucliSens | system, a sensitive detection of Influenza A and Influenza B viruses in a single reaction can be done within 3 hours. It provides a valuable alternative to cell culture method for the clinical management of patients with Influenza infections.

~ W H O European Regional Measles/Rubella Laboratory Network - integration of activities M. Mulders 1 , E. Gavrilin 1 , D. Featherstone2, G. Lipskaya 3. 1World Health Organization - European Regional Office, Denmark, 2World Health Organization- Headquarters, Switzerland

Background and Aims: The World Health Organization European Regional Office has adopted the goal to eliminate measles and rubella and to prevent congenital rubella before 2010. To achieve these objectives, key strategies have been developed to meet the elimination targets. These not only include achieving high immunization coverage, but also strengthening surveillance by in-depth epidemiological investigation and laboratory confirmation of suspect cases. Methods: To ensure a reliable laboratory confirmation and differentiation of suspect measles and rubella cases, the Office coordinates a laboratory network involving 50 national reference laboratories from 48 of the 52 Member States of the European Region. The network has been built upon the experience obtained with the WHO Polio Labnet, while about half of the institutions involved fulfill polio and measles duties. The objectives of this laboratory network are to provide complete and reliable laboratory data to assist the Measles/Rubella Program at each stage of its implementation. Its main tasks are monitoring and verifying disease incidence rates and virus transmission, monitoring the susceptibility profile of the population, and investigating adverse events following vaccination. To ensure the data are reliable, standard operational procedures have been introduced throughout the laboratory network, and standard reference materials and reagents are being used. WHO coordinates a Quality Assurance mechanism including annual accreditation review (by correspondence or on-site) and annual proficiency testing. WHO has also developed an online tool for reporting epidemiological and virological data, called CISID (http://data.euro.who.int/CISID). Results: All 50 national as well as all 20 sub-national Measles/Rubella Laboratories have passed the annual proficiency testing, and all laboratories were accredited, albeit some only provisionally. In terms of timeliness of completeness of aggregate laboratory data, there is a marked difference between countries from Western Europe and Eastern Europe including Commonwealth of Independent States, where the lack of commitment from Western European is dramatic. Conclusions and Discussion: Overall, the objectives of the WHO/EURO Measles/Rubella Labnet are met. However, particularly Western European countries need to increase their involvement and commitment to the Labnet.

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DNA sequencing and phylogenetic analysis of L1 and LCR regions of human papillomavirus type 16 from Turkish patients

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Method: Fifteen cervical smear and 17 larynx cancer samples found to be positive for HPV16 DNA were included in this study. HPV DNA was identified by a real time PCR (Heliosis | Metis Biotechnology, Turkey). Following amplification of L1 and LCR regions, PCR products were directly sequenced (Visible Genetics, Canada). The L1 and LCR sequences of HPV type 16 available in GenBANK and representing main HPV genotypes were compared with 32 sequences handled in this study. Phylogenetic analyzis was done by MEGA 3 programme. Results: L1 region DNA sequences were clustered in a separate group with 2 European strains apart from the American and African isolates, and both isolates from cervical smear and larynx cancer showed a heteregenous distribution. Phylogenetic analyzis of LCR sequences showed that Turkish isolates were clustered in 2 different groups apart from American, African and European groups. LCR region phylogenetic analysis also showed that 12 of 15 isolates derived from cervical smears were clustered in just one group. Conclusion: HPV type 16 strains isolated from Turkish patients show a different clustering pattern in terms of L1 and LCR sequences, when compared to available sequences in gene banks from other regions of the world. Strain or subtype difference of HPV 16 may play a role in tissue tropism and pathogenecity of the virus. 1 ~

Detection and genetic characterisation of circulating measles virus in Ireland

A. Conway, J. Hassan, J. Connell. National Virus Reference

Laboratory, Dublin, Ireland This study investigates the epidemiology and genetic characterisation of measles outbreaks in Ireland since 2003. The study objective was achieved by (1) the collating of data generated from measles IgM detection in oral fluid specimens from patients with suspected measles infection. (2) The examination of this data in conjunction with patient demographics to determine the prevalence of measles infection in the population tested and (3) the genetic characterisation of the circulating measles strain. RNA isolated from a representative number of confirmed positive measles IgM samples were subjected to RT-PCR. Subsequently, specimens were genotyped and the resulting sequence was aligned using phylogenetic analysis. To date, within this study 515 specimens have been tested for measles IgM. Of these samples, 169 (32.8%) were confirmed positive for measles IgM. A number of specimens was selected from each of the Irish health board areas with confirmed measles cases using a 10% rule; ie: the total numbers of samples positive/equivocal for measles IgM was established per area and 10% of that number was used as the number of samples required for further investigation for that area. RT-PCR testing on RNA isolated from these specimens has, to date, identified strains D2 and D7 of the measles virus. Poor vaccine uptake in Ireland has ensured the incidence of measles in Ireland remains significantly higher than other European countries, with resultant sporadic outbreaks. Continued circulation of the D2 measles strain in Ireland was observed in the population tested, and this study has yielded the first documented identification of the D7 measles strain in Ireland.

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Recombinant expression of human bocavirus capsid proteins

K. Kantola, S. Aranko, K. Hedman, M. S6derlund-Venermo. G. Bozdayi 1 , B. Dogan 1 , S. Tuncer 2, A.R. Turkyilmaz 3, A. Biri 5, Y. Kemaloglu 4, S. Rota 1 . 1Gazi University, School of Medicine,

Department of Microbiology and Clinic Microbiology, Besevler, Ankara; 2Metis Biotechnology Ltd., Ostim, Ankara; 3Ankara University, Institute of Hepatology, Cebeci, Ankara; 4Gazi University, School of Medicine, Department of ENT, Besevler, Ankara; 5Gazi University, School of Medicine, Department of Gynecology and Obstetrics, Besevler, Ankara, Turkey

Purpose: Aim of this study is to sequence the L1 and LCR region of HPV type 16 isolated from 2 different Turkish patient groups; patients with larynx cancer and cervical smear with HPV infection, and to analyze these sequences phylogenetically.

Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital Laboratory, Finland

Background: A new human-pathogenic parvovirus, human bocavirus, has been identified and cloned from respiratory tract samples. According to recent studies this virus may be associated with acute respiratory illness in small children. Sequence analysis of the HBoV genomic DNA showed the virus to belong to the genus Bocavirus, subfamily Parvovirinae, family Parvoviridae. The aim of this study is to express the previously uncharacterized putative structural proteins of this virus for immunological studies. Methods: The VPl-unique and VP2 regions of human bocavirus were amplified with PCR from a near-full-length HBoV genomic

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

clone (kindly donated by Dr. T. Allander; Genbank accession no. DQ000496), and were cloned into E. coli and baculovirus expression vectors. Results and Discussion: SDS-PAGE and immunoblotting showed that the VPlu-GST fusion protein in E. coli was expressed in full length. The protein was affinity purified on glutathione beads. Similar recombinant proteins were also successfully expressed in High Five insect cells, predominantly in insoluble form. The expression of VP2 is ongoing. The expressed human bocavirus proteins will be used in immunoblot and ELISA studies for detection of antibodies in human patients (diagnosis), and for generation of antibodies (research).

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Detection of enteric adenovirus in childhood gastroenteritis with real-time PCR

X.L. Pang, M. Slatnik, J.K. Preiksaitis, B. Lee. Provincial Laboratory

for Public Health, Edmonton, Alberta, Canada Introduction: Enteric adenovirus (Ad40/41) is an important cause of gastroenteritis. We report the enhanced surveillance of Ad40/41 in childhood gastroenteritis with a real-time TaqMan PCR assay (TaqMan-PCR) as compared to electron microscopy (EM). Methods: Primers and probe were selected from the Ad41 hexon gene to detect both Ad 40 and Ad 41. Specificity of the assay was evaluated using 31 non-enteric adenovirus strains. Available stool samples from children <7 years submitted to Provincial Laboratory for viral studies from January 2003 to April 2004 were included in the study. Results: The TaqMan-PCR did not cross-react with adenovirus serotypes 1 4 , 10, 19 and 30. Eighty-four percent (1267/1509) of the specimens were tested by the assay. After removing 97 duplicate specimens (<15 days apart) that had the same results, 238 of the 1170 individual cases tested positive for Ad40/41. The monthly detection rate for Ad40/41 ranged from 2.4% to 77.8% in all 16 months with no clear seasonality. Ninety-one percent (1069/1170) of the cases were also tested by EM. TaqMan-PCR detected significantly more Ad40/41 as compared to EM, 215 (20.1%) versus 27 (2.5%) respectively, (p < 0.001, McNemar test). Twenty-five of the 27 cases tested positive for adenovirus by EM were positive by TaqMan-PCR demonstrating high sensitivity and specificity of the assay. Conclusions: With our highly sensitive and specific TaqManPCR, we found an unusually high prevalence of Ad40/41 in children diarrhea in Northern Alberta. Further studies to correlate quantitative virus with clinical symptoms and the natural history of diarrhea in children are underway. I ~

Development and evaluation of a real time NASBA for the detection of noroviruses

S. Corden, J. Watkins, C. Moore, D. Westmoreland. NPHS

Microbiology Cardiff, UK Background and Aims: Noroviruses are a leading cause of outbreaks of viral gastroenteritis both in hospital and the community. Outbreaks within the healthcare setting can have a significant health and economic impact as a direct result of ward closures and cancellation of operations. Due to the lack of suitable tissue culture systems electron microscopy (EM) was the gold standard for the detection of noroviruses. However, with the advent of ELISA and advances in molecular diagnostics EM has been superseded. The aim of the study was to develop and evaluate a real time NASBA for the rapid routine detection of noroviruses. Methods: A real time NASBA assay was developed using primers and probes, which amplified and detected a region within the ORF-1/ORF-2 overlap of the norovirus genome. The primers and probes were genogroup specific. The assay was evaluated using known norovirus positive and negative clinical material and comparing the results to end point NASBA, which has been used routinely in Cardiff since 2002. Results: The results of the analysis of 96 clinical samples showed that the sensitivity and specificity of the real time NASBA were 80% and 91% respectively when compared to end point NASBA. The positive and negative predictive values of the real time NASBA were 97% and 57% respectively.

Abstracts, 9th ESCV Annual Meeting

Discussion: The reason for the low NPV is believed to be sequence variation within the target gene. Therefore false negative results were generated. The results obtained highlight the difficulty in developing molecular methods to detect highly genetically diverse viral pathogens. 1 ~

Arthritis and arthralgia three years after Sindbis virus infection

S. Kurkela 1,2, T. Helve 3, A. Vaheri 1,2, O. Vapalahti 1,2. 1Haartman

Institute, University of Helsinki, 2Department of Virology, Helsinki University Central Hospital Laboratory, 3Department of Rheumatology, Helsinki University Central Hospital, Finland Background: Similar to many other alphaviruses, Sindbis virus (SINV) is a mosquito-borne causative agent of a clinical disease that manifests with rash and joint symptoms. It has been suggested that in many of these infections joint symptoms might persist for years. SINV infection is clinically relevant especially in Finland, where hundreds or even thousands of serodiagnosed cases occur during outbreaks that take place every seven years. Persistence of joint symptoms in SINV infection was approached in the present study. Methods: A prospective cohort of 49 patients was physically examined three years after verified SINV infection to reveal persistent joint or other symptoms. We carefully interviewed the patients to exclude factors that might have biased the prevalence of persistent joint symptoms in previous studies. Results: Arthritis, determined as swelling and tenderness, was diagnosed in 2/49 of the patients. Pain in palpation and in movement of a joint was found in 14.3% of the patients in the rheumatologic examination, whereas additional 10.2% complained of arthralgia in the interview; a total of 24.5% of the patients had such joint symptoms that were attributable to the SINV infection three years earlier. Small and peripheral joints were commonly affected. Conclusions: The high frequency of persistent arthralgia caused by SINV infection was in agreement with previous studies. Although evident in two patients, persistent arthritis was not a typical manifestation. Considering the high seroprevalence of SINV in Finland, it can be estimated that persistent symptoms of SINV infection may have considerable public health implications. 1 ~

Focus of Siberian subtype TBEV in Kokkola Archipelago, Western Finland

A.E. J~i~iskel~iinen 1, T. Tikkakoski 2, N.Y. Uzc~tegui 1 , A. Vaheri 1,3, O. Vapalahti 1,3,4. 1Department of Virology, Haartman Institute,

RO. Box 21 (Haartmaninkatu 3), FIN-O0014 University of Helsinki, Helsinki, Finland, 2Department of Radiology, Keski-Pohjanmaa Central Hospital, Mariankatu 16-20, FIN-67200 Kokkola, Finland, 3Department of Virology, Helsinki University Hospital Laboratory (HUSLAB), P.O. Box 400, FIN-O0029 HUS, Helsinki, Finland, 4Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine, RO. Box 66 (Agnes SjSbergin katu 2), FIN-O0014 University of Helsinki, Helsinki, Finland Background: Tick-borne encephalitis (TBE) is a relatively severe CNS infection endemic from Central and Eastern Europe through Siberia to Far East Asia. Three subtypes of the TBE virus are known: the European subtype, carried by the tick species Ixodes ricinus, and the Siberian and Far Eastern subtypes, both carried by I. persulcatus. The boundary between the distribution areas of the two tick species lies at the Russian side of the Finnish-Russian border, and both tick species overlap in Eastern Europe. Finland is situated at the northernmost limit of the TBE-endemic area in Europe. Here, TBE is endemic in ,&land and a few other locations in southern Finland, and, interestingly, in an isolated focus in the archipelago of Kokkola. Until now, only I. ricinus ticks and the European subtype of TBEV have been found in Western Europe. Results and Discussion: Out of 1181 ticks collected from Kokkola archipelago in 2004, 1.1% were positive for TBEV-RNA by RT-PCR. Based on a 205-nt amplicon from the NS5 gene, the local TBEV strains unexpectedly clustered together with the Siberian subtype strains of TBEV. Therefore we studied the tick species and identified the ticks as I. persulcatus. We isolated 11 TBEV strains