5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>followed by EV A (25%). The distribution <strong>of</strong> types in VIRO Typenedwas comparable to regular surveillance. VIRO typened also registered thedetection <strong>of</strong> 3 oral poliovirus (OPV) vaccine strains (2 OPV2 and 1 OPV3).Clinical data were only available in 20% 30% <strong>of</strong> patients. Meningitis wassignificantly more found among EV B strains, while Hand Foot Mouthdisease is frequently registered for EV A strains. Analysis <strong>of</strong> the sequencedata showed the circulation <strong>of</strong> specific strains.Conclusions: The VIRO Typened concept generates information on circulatingEVs comparable to regular surveillance. With Typened clinicaldata could be linked to specific EV species. Complete submission <strong>of</strong> clinicaldata in the future should provide more in depth analysis <strong>of</strong> typespecific illnesses. The sequence data allowed the analysis <strong>of</strong> strain specificsurveillance rather than the current type specific surveillance enablinganalysis <strong>of</strong> immune divergent strains.REF 499Molecular epidemiology and clinical association <strong>of</strong> enterovirus andparechovirus types in neonates and young infants in SpainMaria CABRERIZO 1 , Carmen MUÑOZ ALMAGRO 2 , Diana ROULA 2 ,Esther PEREZ 2 , Maria Pilar ROMERO 3 , Ines MARTÍNEZ RIENDA 4 ,Antonio MORENO DOCON 5 , Almudena OTERO 1 , GloriaTRALLERO 11 Instituto de Salud Carlos III, National Centre for Microbiology, Madrid,SPAIN; 2 Hospital Sant Joan de Déu, Barcelona, SPAIN; 3 Hospital dela Paz, Madrid, SPAIN; 4 Hospital de Cruces, Bilbao, SPAIN; 5 HospitalVirgen de la Arrixaca, Murcia, SPAINEnteroviruses (EV) and parechoviruses (HPeV) are the main viral causes<strong>of</strong> neonatal sepsis and meningitis. The relative frequencies <strong>of</strong> specific EVand HPeV types in infections affecting children under 1y were determinedovera3ysurveillance period in Spain, and compared with those observedin patients over 1y. Positive samples included were 461 EV and 24HPeV from children under 1y, and 587 EV from patients over 1y. Thus,79 and 88% <strong>of</strong> the EV and HPeV, respectively, could be directly typed bysequencing <strong>of</strong> VP1. All HPeV were type 3, exclusively in infants up to7m, whereas 27 different EV types were identified in children under 1yr.CV B3, CV B4, E 11, E 18 and E 25 were statistically more frequent ininfants than in patients over 1y, while E 6, E 30 and E 13 were prevalentin patients over 1y (p=0.02). Incidence <strong>of</strong> CV B and EV A in infants werealso statistically higher (p=0.006). Regarding clinical outcome in childrenunder 1y, frequency detection <strong>of</strong> predominant EV types as E 11 and CVB4, was similar in meningo encephalitis and febrile syndromes. Only E 6and HPeV 3 caused more meningo encephalitis and neonatal sepsis thanfever (p=0.001). All myocarditis cases (7) were caused by CV B, whileCV A are associated with HFMD (p=0.001). In neonatal sepsis (11 cases),different viruses were detected, including HPeV and CV B. In conclusion,HPeV 3, E 11 and CV B types were prevalent in neonatal and young infantinfections in Spain during 2010-2012, while not others that frequently circulatedin the same period. Overall, those types presented with greaterdisease severity.Methods: A multiplex <strong>of</strong> Real Time PCRs (RT PCR) was developed andvalidated on the VP1 gene to differentiate the 4 genotypes <strong>of</strong> BKV. 150BKV positive samples (17 plasma, 133 urine) were tested with these specificassays. Of these 150 samples, 50 were additionally confirmed bysequencing the 1630 1956 nucleotide fragment. Results: For every genotype,a 100% specific and internally controlled assay was developed. Theprecision <strong>of</strong> the 4 RT PCRs remained within 1SD and the limit <strong>of</strong> detectionwas log 3 copies/ml. Of the 150 BKV positive samples, 105 (70%)were genotype I, 8 (5.3%) genotype II, 19 (12.7%) genotype IV and 3(2%) genotype I+IV. In 15 (10%) samples genotyping was not successfuldue to a low viral load. By sequence analysis the genotypes <strong>of</strong> 46 <strong>of</strong> the50 and 2 <strong>of</strong> the 3 samples with the double infection could be confirmed.Conclusions: This study describes a new multiplex RT PCR for detection<strong>of</strong> the genotypes <strong>of</strong> BKV. It proved to be a rapid, cheap and sensitive genotypingtool compared to sequencing, and can detect double infections withdifferent BK genotypes. This method will be <strong>of</strong> value to obtain insight inthe relation <strong>of</strong> BKV genotype with nephropathy and hemorrhagic cystitis.REF 501New method targeting VP2 capsid gene for direct enterovirus genotypingin clinical specimensWafa IBRAHIM 1,2,3 , Nabila BOUKHADRA 1 , PhilippeBERTHELOT 1,2 , Dorsaf NASRI ZOGHLAMI 3 , Shabir OMAR 1 ,Thomas BOURLET 1,2 , Bruno POZZETTO 1,2 , Sylvie PILLET 1,21 University Hospital <strong>of</strong> Saint Etienne, Saint Etienne, FRANCE; 2 Faculty<strong>of</strong> Medicine <strong>of</strong> Saint Etienne, University <strong>of</strong> Lyon, Saint Etienne, FRANCE;3 Faculty <strong>of</strong> Pharmacy <strong>of</strong> Monastir, Monastir, TUNISIATyping <strong>of</strong> human enterovirus (HEV) by sequencing the viral capsid (VP) 1coding region is the gold standard for HEV typing, even in the absence <strong>of</strong>cell culture <strong>of</strong> the specimen thanks to the CODEHOP strategy. We describean alternative method based on the same strategy but in the VP2 codingregion. By testing 10 fold dilutions <strong>of</strong> reference and clinical strains, thenew test was shown to be at least as sensitive as the VP1 method, except forpoliovirus 1. A total <strong>of</strong> 98 specimens [48 cerebrospinal fluids (CSF) and50 peripheral specimens] taken from patients <strong>of</strong> the University Hospital <strong>of</strong>Saint Etienne and found positive for HEV RNA by routine techniques wereanalyzed by the VP1 and VP2 methods. The concordance between bothtyping techniques was <strong>of</strong> 70.41% (P
5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>with particular ethnic group and frequently used as a genetic marker forhuman migration in both prehistoric and modern times. The aims <strong>of</strong> thisstudy were to determine the frequency <strong>of</strong> JCV urinary shedding and genotypedistribution. Material and methods: Urine samples collected from107 healthy individuals were tested for presence <strong>of</strong> JCV DNA by PCR.A semi nested PCR was performed for amplification <strong>of</strong> 495 bp fragmentwitin VP1coding region and amplified fragments were directly sequenced.The genotypes <strong>of</strong> JCV isolates were determined by comparison to prototypesequences <strong>of</strong> the known genotypes in BioEdit s<strong>of</strong>tware. Results: JCVDNA was detected in 31.7% <strong>of</strong> healthy individuals. Males had a higherexcretion rate than did the females (62% vs. 38%) and difference wasstatistically significant. In Serbian population genotype 1 was most prevalent41.2%, followed by genotype 4 in 31.4% and genotype 2 in 26.4%.A new variant <strong>of</strong> subtype 1A with a nucleotide substitution (C>G) at aposition 1940 was found in two samples. Conclusion:Considering geographicalposition and knowing that distribution <strong>of</strong> JCV genotypes mayreflect migration patterns, it is not surprising that the most prevalent genotypesin Serbia are 1 and 4 (<strong>European</strong> types) followed by genotype 2B, 2Cand 2D (Eurasian types).REF 503HIV 1 epidemic in Portuguese injecting drug users may be evolvinginto a unique molecular epidemiological patternJoão PIEDADE 1,2 , Carina SOUSA 1 , Sandra VIDEIRA E CASTRO 1 ,Elizabeth PÁDUA 3 , Ricardo PARREIRA 1,2 , Aida ESTEVES 1,21 Grupo de Virologia, Unidade de Microbiologia Médica, Instituto deHigiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, POR-TUGAL; 2 Unidade de Parasitologia e Microbiologia Médicas (UPMM),Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa,Lisbon, PORTUGAL; 3 Lab. Nac. Referência IST VIH/SIDA e Hepatites Be C, Dep. de Doenças Infecciosas, Instituto Nacional de Saúde Dr. RicardoJorge, Lisbon, PORTUGALHIV 1 is characterised by a high degree <strong>of</strong> genetic diversity. HIV 1 geneticvariants are classified in 4 phylogenetic groups (M P) and group M issubdivided into 9 subtypes (A–D, F–H, J, K). Fast genetic recombinationgives rise to mosaic viruses, some <strong>of</strong> which gain epidemic proportions (circulatingrecombinant forms, CRF). According to their coreceptor (CCR5or CXCR4), HIV 1 variants can also be classified as R5, X4 or dual/mixedtropic. The aims <strong>of</strong> this study were to assess the genetic diversity <strong>of</strong> protease(PR), reverse transcriptase (RT), integrase (IN) and C2V3C3 codingsequences and to estimate the frequency <strong>of</strong> coreceptor usage in HIV 1strains circulating among 61 intravenous drug users from the Greater Lisbon.Viral RNA was amplified by RT nested PCR to originate PR, RT, INand C2V3C3 amplicons <strong>of</strong> 460, 650, 906 and 565 bp, respectively. 158DNA sequences were analysed, from 49 samples successfully amplifiedfor, at least, one <strong>of</strong> the regions. Subtype classification was achieved byphylogenetic analysis with MEGA4 and recombinant analysis was carriedout by bootscanning. A significant degree <strong>of</strong> HIV 1 diversity was shown.This epidemic is dominated by B and G subtypes, and their recombinantforms, but other genetic forms (F1, CRF02 AG) were also found. On thewhole, non B subtypes were identified in 58.9% (93/158) <strong>of</strong> the sequences.It is also significant that 45.2% (19/42) <strong>of</strong> the concatenated sequences studiedwere derived from inter genotype recombinants. Finally, coreceptorusage prediction classified 30 V3 amino acid sequences as R5 and 5 as X4or dual/mixed tropic.REF 504The value <strong>of</strong> real time sequence based information in surveillance <strong>of</strong>healthcare associated viral infectionsJ.C. RAHAMAT LANGENDOEN 1 , D.S. LUIJT 2 , A.D. PRENGER 3 ,M.WAGELAAR 3 ,A.OTT 2 , H.G.M. NIESTERS 11 Department <strong>of</strong> Medical Microbiology, Division <strong>of</strong> Clinical <strong>Virology</strong>, University<strong>of</strong> Groningen, University Medical Center Groningen, Groningen,THE NETHERLANDS; 2 Laboratory for Infectious Diseases, Groningen,THE NETHERLANDS; 3 Municipal Health Service, Groningen, THENETHERLANDSObjectives: sequence based information can serve as a tool to definetransmission routes. As most laboratories have not incorporated sequenceanalysis in their daily routine, information is mostly available retrospectively.Reducing the time to obtain sequence based information shouldbenefit the understanding <strong>of</strong> transmission and guide the implementation<strong>of</strong> appropriate infection control measures. Methods: in August 2012, realtime sequencing is introduced at the UMCG, a large tertiary referral hospital.A set <strong>of</strong> viruses, particularly noro, rhino, parecho and enterovirus, ischaracterized immediately after detection. To gain insight in viral diversityoutside the UMCG, a regional network is set up in which a regionallaboratory and the municipal health service submit samples for typing. Incase <strong>of</strong> an outbreak <strong>of</strong> gastroenteritis or respiratory illness in healthcareassociated institutions a limited set <strong>of</strong> clinical and epidemiological data iscollected.Results: sequence analysis results were available less than a week afterdetection. Several clusters <strong>of</strong> identical viruses were identified, especiallywith norovirus, confirming clonal transmission. Real time sequencing alsoenabled us to rapidly detect pseudo outbreaks where several norovirusgenotypes were found, providing evidence for multiple introductions <strong>of</strong>different strains rather than an ongoing transmission. Conclusion: realtime sequence analysis contributes to the understanding <strong>of</strong> transmission<strong>of</strong> healthcare associated viral infections and enables us to focus infectioncontrol interventions adequately and timely.REF 505Frequency <strong>of</strong> distribution and variation <strong>of</strong> anal HPV genotypes andcorrelation with lifestyle and sexual behaviors among HIV infectedand non infected MSMs, compared to cervix samplesEszter UJHELYI, Csaba KOSA, Eszter SZABO, Edit BABARCZI, JanosSZLAVIK, Denes BANHEGYI, Istvan VALYI NAGYUnited Saint Istvan and Saint Laslo Hospital, Budapest, HUNGARYBackground: Anal cancer is one <strong>of</strong> the leading causes <strong>of</strong> death in nonAIDS defining cancers. Most <strong>of</strong> these cancers are associated with highrisk HPV (HR HPV) infection. No survey was made on anal HPV infectionin Hungarian MSM population before. We evaluated incidences<strong>of</strong> cytological abnormalities and different genotypes, known and suspectedrisk factors, compared cervical and anal pattern. Materials andMethods: After obtaining informed concern, cervical and anal cytobrushwere taken and HPV genotyping with PCR (Roche Linear Array HPVgenotype).Every patient were inquired and tested about their sexual behavior,socioeconomic factors, drug use, and other known and suspected riskfactors. Risk assessments on this cross sectional cohort study were madeby Chi squared and odds ratio were calculated.S260 Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013
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