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3rd International Conference on Clinical Microbiology and Microbial Genomics

Genetic variability of HCMV strains isolated from Polish pregnant women, their fetuses and newborns. W. Wujcicka 1 , M. Rycel 1 , B. Zawilińska 3 , E. Paradowska 4 , P. Suski 4 , Z. Gaj 1 , J. Wilczyński 1,2 , Z. Leśnikowski 4 , D. Nowakowska 1,2.

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3rd International Conference on Clinical Microbiology and Microbial Genomics

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  1. Genetic variability of HCMV strains isolated from Polish pregnant women, their fetuses and newborns W. Wujcicka1, M. Rycel1, B. Zawilińska3, E. Paradowska4, P. Suski4, Z. Gaj1, J. Wilczyński1,2, Z. Leśnikowski4, D. Nowakowska1,2 1 Department of Fetal-Maternal Medicine and Gynecology, Polish Mother's Memorial Hospital Research Institute, Lodz, Poland; 2 Department of Fetal-Maternal Medicine and Gynecology, IIIrd Chair of Gynecology and Obstetrics, Medical University of Lodz; 3 Department of Virology, Jagiellonian University Medical College, Cracow, Poland; 4 Laboratory of Molecular Virology and Biological Chemistry, Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland 3rd International Conference on ClinicalMicrobiology and MicrobialGenomics Valencia, SpainSeptember 24th-26th 2014

  2. Seroprevalence of HCMV infections Prevalence from 40% to 100%varyingbetweencontinents and countries The most common etiologic agent of intrauterine infections in fetuses (0.2% to 2.2% of all live births) and a major cause of infection induced hearing loss and mental retardation http://www.abbottdiagnostics.com.au/Your_H ealth/Infectious_Diseases/CMv/ Hyde TB et al. (2010) Rev Med. Virol.20: 311-326

  3. Geneticvariability of HCMV Approximately 20 HCMV ORFs encoding viral envelope glycoproteins B (gB, UL55), H (gH, UL75) and N (gN, UL73), and chemokines and chemokine receptors, as well as TNF-α receptor (pUL144, UL144) Puchhammer-Stöckl E and Görzer I. (2011)FutureVirol.6(2): 259-271

  4. HCMV glycoprotein B (gB) • The major HCMV envelope protein, important for viral replication in vivo and in vitro as well as host cell entry, cell-to-cell virus transmission, and fusion of infected cells • Thesiteat position 460cleaved by cellular endoprotease • The region between 448 and 481 codonsincluding the area of the highest genetic variability of UL55 Pignatelli S et al. (2004) RevMedVirol. 14(6):383-410

  5. HCMV UL55genotypesincongenitalcytomegaly The gB1 genotype of HCMV most commonly observed in congenital cytomegalyworldwide Single gB1 genotype of HCMV identifiedincongenitalinfectionsfrom Southern Hungary Co-infectionswithvarious HCMV gBstrainsobservedininfantsfromthe U.S. and China

  6. Aims of study: • Determination of HCMV UL55genotypesin pregnant Polish women, their fetuses and newborns • Estimation of the impact of viralgenotypes on both the transplacental transmission of the virus and disease outcome in the offsprings

  7. Materials and Methods: Collection of clinicalspecimens

  8. Patients’ classification for moleculartesting • Clinicalsymptomsobservedinpregnantwomen and theirfetuses: • Flu-likesymptomsinmothers • Ultrasound markers in fetuses: • ventriculomegaly, hydrocephaly and fetal • hydrops as well as intrauterine growth • restriction, ascites, pericardialeffusion, cardiomegaly and the presence of hyperechogenic foci in different organs like • the fetal brain, liver and pancreas Serologicalscreening: Anti-CMV IgG and IgM tests(Diasorin/Biomedica, Italy)based on Chemiluminescence Immunoassay (CLIA) between 2009 and 2011 years Testsbased on an Enzyme-Linked Fluorescence Assay (ELFA)between 2012 and 2013 Determination of IgGavidity Preparation of DNA to furthermolecularstudies: NucleicacidextractionwithQIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and storageat -20oC

  9. HCMV DNA detection and quantification Real-time Q PCR assay for UL55 gene fragment of length 150 bpswith forward and reverse primers and TaqMan probe of thefollowingsequences:5’-GAGGACAACGAAATCCTGTTGGGCA-3’, 5’-TCGACGGTGGAGATACTGCTGAGG-3’, and5’-6-FAM-CAATCATGCGTTTGAAGAGGTAGTCCA-TAMRA-3’ Absolute quantification analysisusingstandard curves for serial 10-fold HCMV DNA dilutions from 105 to 1 viral copy

  10. Genotyping of HCMV strains • Real-time PCR assay to amplify DNA fragments of lengths between 72 and 79 bp, dependent on HCMV UL55 genotype • Single reactionsperformed in two separate tubes for gB1 and gB3 as well as gB2 and gB4 genotypes • Serial dilutions of HCMV reference strains Towne (gB1; ATCC: VR-977) and AD-169 (gB2; ATCC: VR-538) usedincalibration curves

  11. Results: HCMV load and UL55genotypeinpregnantwomen

  12. HCMV load, UL55genotype and cytomegalyoutcomeinfetuses and newborns

  13. Maternalvs. fetal and neonatalinfectionswith HCMV

  14. Maternalvs. fetal and neonatalinfectionswith HCMV Significantcorrelationbetween genotypes of HCMV identified in the pregnant women and theircongenitallyinfectedoffsprings

  15. Conclusions • Infections with single and multiple HCMV strains occur in pregnant women, as well as in their fetuses and neonates, both with the asymptomatic and symptomatic infections. • HCMV infections, identified in mothers, seem to be associated with the viral genotypes in their children.

  16. ThankYou for Yourattention!

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