Characterisation of Newly Discovered Human Polyomaviruses WU and KI

Seweryn Bialasiewicz (2010). Characterisation of Newly Discovered Human Polyomaviruses WU and KI PhD Thesis, School of Chemistry & Molecular Bioscience, The University of Queensland.

       
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Author Seweryn Bialasiewicz
Thesis Title Characterisation of Newly Discovered Human Polyomaviruses WU and KI
School, Centre or Institute School of Chemistry & Molecular Bioscience
Institution The University of Queensland
Publication date 2010-11
Thesis type PhD Thesis
Total pages 237
Total colour pages 16
Total black and white pages 221
Subjects 06 Biological Sciences
Abstract/Summary Background: Until recently, only two polyomaviruses, JC virus (JCV) and BK virus (BKV), were known to commonly infect humans. In 2007, two new human polyomaviruses, KI polyomavirus (KIV), and WU polyomavirus (WUV) were discovered in respiratory samples of children suffering from acute respiratory tract disease. Due to their relative novelty, many details about KIV and WUV’s viral infectious cycles, pathobiology, and epidemiology could only be assumed by parallel comparison with other polyomaviruses. Thus, further studies were needed to elucidate the epidemiology, and pathogenesis of KIV and WUV. Methods: Epidemiology: Nearly 3000 respiratory samples collected over an entire year were screened for WUV and KIV by PCR, along with 8 other common respiratory viruses. An encapsidation assay was used to determine if whole viral particles were present in the respiratory tract of patients. Detection methods: Two KIV and six WUV real-time PCR (rtPCR) assays were designed and evaluated against a panel of other respiratory and blood pathogens, and clinical samples to establish sensitivity and specificity. Plasmid controls were created to establish analytical sensitivity. Tissue tropism: Collections of blood, urine, upper and lower respiratory tract secretions, faeces, and cerebrospinal fluid from both immunocompetent and immunocompromised adults and children were screened for WUV, KIV, JCV and BKV, and detection rates were compared. Representative WUV and KIV isolates had their VP1 and non-coding control regions (NCCR) sequenced, and analysed for changes respective to their site of detection. Transcription factor binding sites (TFBS) were predicted and compared to those of JCV and BKV. Global WUV diversity and genotyping: Forty nine WUV isolates collected across four continents had their genomes fully sequenced and analysed for levels of diversity, existence of genotypes, impact on predicted protein functional sites and association with disease or patient demographics. A genotyping scheme was also evaluated for fidelity against the whole-genome phylogenetic tree. Results: KIV and WUV showed a 2.6% and 4.5% average annual prevalence, respectively, in the study population. Predominance of WUV (93%) and KIV (78.6%) detections were in patients under the age of five. There was no significant seasonal variation of KIV, or WUV. The level of co-infection of KIV or WUV with other respiratory viruses was 74.7% and 79.7%, respectively. Respiratory symptoms were indistinguishable from those of other common respiratory infections. The encapsidation assay demonstrated that WUV DNA was fully protected from digestion in a respiratory sample with high WUV load. Seven rtPCR assays did not cross react with unrelated organisms, and all could reliably detect 10 copies of genomic DNA equivalents per reaction, which was more 10x more sensitive than conventional methods. Compared to the conventional PCR assays, clinical sensitivity was 100% in one of the KI and two of the WUV rtPCR assays. Clinical specificity was higher than 95% in four of the assays; the calculations suffered from poor sensitivity in the gold standard however. WUV and KIV were predominantly detected in upper and lower respiratory tract specimens, and faeces from paediatric patients. JCV and BKV were primarily detected in blood, urine and faeces from adult patients. WUV and KIV NCCR/VP1 sequence similarity ranged from 99.5-100% and 97.5-100%, respectively. No substantial difference was noted between the TFBS of JCV, BKV and WUV and KIV. Three main WUV genotypes and five subtypes were identified, provisionally termed Ia, Ib, Ic, II, IIIa and IIIb. Overall nucleotide variation was low (0-1.2%), however individual protein sequence variation between subtypes was as high as 3.0%, with VP1 being the most variable gene, indicating a possibility of multiple serotypes. A general geographical association was also noted. The discriminatory power of the previous VP2 fragment typing method was found to be limited and a new larger genotyping region within the VP1 was proposed. Conclusions: Both of the newly-discovered human polyomaviruses are frequently present in the respiratory samples of children suffering from upper and lower respiratory tract disease. The detection peaks for both WUV and KIV indicates an early age of primary exposure followed by reactivation in older age and under immunosuppression, similar to that seen in JCV and BKV. Clinical symptoms were indistinguishable from other respiratory infections, and the high rate of co-infections confounded any direct association with disease, however evidence of active WUV replication was observed. Multiple real-time PCR assays were developed which were sensitive and specific for use in the detection of WUV and KIV. Both novel viruses have a more restricted tissue tropism range in comparison to JCV and BKV, but VP1/NCCR sequences alone do not appear to offer suggestions as to why. The first extensive genomic survey of global WUV diversity revealed multiple genotypes which were more associated with geo-ethnicity rather than clinical disease. Overall, this series of studies has contributed substantial knowledge to the epidemiology and pathobiology of WUV and KIV, and has created validated tools to facilitate further studies into both of these viruses.
Keyword novel
polyomavirus
paediatric
WU polyomavirus
KI polyomavirus
respiratory disease
PCR
tissue tropism
genotyping
Additional Notes Colour pages: 31, 37, 69, 87, 93, 95-97, 118, 123, 148 Colour AND Landscape pages: 68, 145, 146, 149, 159 Landscape pages: 66, 137-140, 150, 151, 156

 
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Created: Mon, 22 Nov 2010, 14:53:55 EST by Mr Seweryn Bialasiewicz on behalf of Library - Information Access Service