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Genetic variability of HCMV strains isolated from Polish pregnant women, their fetuses and newborns W. Wujcicka1, M. Rycel1, B. Zawiliska3, E. Paradowska4, P. Suski4, Z. Gaj1, J. Wilczyski1,2, Z. Lenikowski4, D. Nowakowska1,2 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 1 3rd International Conference on Clinical Microbiology and Microbial Genomics Valencia, Spain September 24th-26th 2014 Seroprevalence of HCMV infections Prevalence from 40% to 100% varying between continents and countries The most common etiologic agent of intrauterine infection s 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 Genetic variability 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- receptor (pUL144, UL144) Puchhammer-Stckl E and Grzer I. (2011) Future Virol. 6(2): 259-271
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 The site at position 460 cleaved by cellular endoprotease The region between 448 and 481 codons including the area of the highest genetic variability of UL55 Pignatelli S et al. (2004) Rev Med Virol. 14(6):383-410 HCMV UL55 genotypes in congenital cytomegaly The gB1 genotype of HCMV most commonly observed in congenital cytomegaly worldwide Single gB1 genotype of HCMV identified in congenital infections from Southern Hungary Co-infections with various HCMV gB strains observed in infants from the U.S. and China Aims of study: Determination of HCMV UL55 genotypes in pregnant Polish women, their fetuses and newborns Estimation of the impact of viral genotypes on both the transplacental transmission of the virus and disease outcome in the offsprings Materials and Methods: Collection of clinical specimens Patients classification for molecular testing Serological screening: Anti-CMV IgG and IgM tests (Diasorin/Biomedica, Italy) based on Chemiluminescence Immunoassay (CLIA) between 2009 and 2011 years
Tests based on an Enzyme-Linked Fluorescence Assay (ELFA) between 2012 and 2013 Determination of IgG avidity Clinical symptoms observed in pregnant women and their fetuses: Flu-like symptoms in mothers Ultrasound markers in fetuses: ventriculomegaly, hydrocephaly and fetal hydrops as well as intrauterine growth restriction, ascites, pericardial effusion, cardiomegaly and the presence of hyperechogenic foci in different organs like the fetal brain, liver and pancreas Preparation of DNA to further molecular studies: Nucleic acid extraction with QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and storage at -20oC HCMV DNA detection and quantification Real-time Q PCR assay for UL55 gene fragment of length 150 bps with forward and reverse primers and TaqMan probe of the following sequences: 5-GAGGACAACGAAATCCTGTTGGGCA-3, 5-TCGACGGTGGAGATACTGCTGAGG-3, and 5-6-FAM-CAATCATGCGTTTGAAGAGGTAGTCCA-TAMRA-3 Absolute quantification analysis using standard curves for serial 10-fold HCMV DNA dilutions from 105 to 1 viral copy 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 reactions performed 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) used in calibration curves
Results: HCMV load and UL55 genotype in pregnant women Patient No. 1. 2. Clinical specimen HCMV load(*) UL55 genotype PBMC 1.23 x 102 gB3 plasma 1.31 x 103 gB2 PBMC 51 3. PBMC 46 gB2 4. urine 1.5 x 103 gB2 5. plasma 5.59 x 102 gB1-gB2 6. PBMC 5.82 x 102 gB1-gB2 urine 8.17 x 103
3 7. whole blood 1.08 x 10 gB1 urine 4.28 x 102 gB1-gB2 8. plasma 1.81 x 102 gB2 urine 1.32 x 103 3 2 plasma 1.98 x 10 whole blood3 1.66 x 102 gB2-gB3 urine3 7.16 x 103 9. plasma 1.36 x 102 whole blood 9.67 x 102 gB2 urine 8.74 x 102 10. whole blood 4.88 x 102 gB2 urine 1.14 x 103 2
11. whole blood 6.90 x 10 gB2 urine 2.35 x 102 12. whole blood 2.78 x 102 gB2 urine 2.41 x 104 3 13. whole blood 1.14 x 10 gB2 urine 8.05 x 103 14. PBMC 28 whole blood 1.28 x 102 gB2 urine 1.49 x 102 15. whole blood 1.30 x 102 gB2 urine 4.10 x 102 16. whole blood 1.59 x 102 gB2 urine
2.48 x 102 2 17. urine 1.51 x 10 gB4 18. urine 5.35 x 102 gB4 (*) - HCMV load per 1 mL of study fluid (blood, plasma, urine) or 5 x 105 cells (PBMC) HCMV load, UL55 genotype and cytomegaly outcome in fetuses and newborns Patient No. Clinical specimen HCMV load(*) amniotic fluid cells plasma urine amniotic fluid cells ascitic fluid brain kidney 4.32 x 106 6.15 x 103 9.33 x 105 8.2 x 104/1 x 105 cells 1.54 x 102 52.6*** 42.4***
3. amniotic fluid cells 1.86 x 105/1.4 x 105 cells gB1-gB2 Asymptomatic 4. whole blood plasma 4.49 x 10 2.11 x 103 gB1-gB2 Asymptomatic 5. whole blood 6.61 x 103 gB1-gB2 Asymptomatic 6. whole blood 1.55 x 102
gB1-gB2 Asymptomatic 7. amniotic fluid cells ascitic fluid brain kidney liver 2.98 x 104/5 x 104 cells 6.66 x 103 1.40 x 103*** 3.31 x 102*** 3.18 x 103*** gB2 Symptomatic (death) 8. amniotic fluid and whole blood of child 1.34 x 103 gB2 Symptomatic 9. amniotic fluid
2.19 x 102 gB2 Symptomatic 10. amniotic fluid 6.36 x 102 gB2 Asymptomatic 11. amniotic fluid 1.47 x 103 gB2 Asymptomatic 1.** 2.** 2 12. UL55 genotype gB2
Outcome Symptomatic gB1-gB2 gB2-gB3 gB3 Symptomatic (death) brain 7.82 x 103*** Symptomatic kidney 4.11 x 103*** gB1 (death) 3 liver 7.01 x 10 *** (*) - HCMV load per 1 mL of study fluid; ** - HCMV loads identified in fetal and neonatal body fluids that were included in our previous study  *** - HCMV load per one cut section of paraffin-embedded tissue Maternal vs. fetal and neonatal infections with HCMV HCMV UL55 genotype (No. tested (%)**) Study group Total No. tested* Single gB1
gB2 gB3 gB4 All single infections Multiple gB1-gB2 gB2-gB3 All multiple infections Pregnant women 18 0 (0) 11 (61.1) 1 (5.55) 2 (11.1) 14 (77.8) 3 (16.7) 1 (5.55) 4 (22.2)
1 (16.66) 2 (33.3) Asymptomatic fetuses and newborns 6 0 (0) 2 (33.3) 0 (0) 0 (0) 2 (33.3) 4 (66.7) 0 (0) 4 (66.7) * No. tested number of tested patients; ** % - part of all the patients included in each study group. Maternal vs. fetal and neonatal infections with HCMV Significant correlation between genotypes of HCMV identified in the pregnant women and their congenitally infected offsprings Maternal genotype gB2 gB1-gB2 gB2-gB3
Total 2 = 9; P 0.050 Fetal and neonatal neonatal genotype gB2 gB1-gB2 4 0 1 5 0 4 0 4 Total 4 4 1 9 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. Thank You for Your attention!
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