Molecular analysis of adenoviruses from clinical samples

At present, 56 types of human adenovirus (HAdVs) have been identified and found to be associated with a variety of clinical features in the respiratory tract, eye, gastrointestinal tract, and other organs. In additions, HAdVs are able to establish persistent and latent infections in humans. Most of the work which has been carried out recently is related to adenovirus vectors and little has been done in other areas such as the nature and mechanisms of adenovirus persistence and latency in human tissues. Another area needing more investigation is the stability of the adenovirus genome which is useful for the development of adenovirus vectors and vaccines and for better understanding of adenovirus evolution especially with conflicting views about this issue.Recombination between two types of adenovirus can happen when the hexon epitope from one type and the fibre epitope from another type are found (intermediate strains). These recombinants can be detected by the conflicting results for serum neutralization (SN) and haemagglutination inhibition (HI) tests or by sequencing and phylogenetic analysis of the hexon and fibre regions of the adenovirus genome. The first part of this study is related to the stability and evolution of different adenovirus species. A total of 31 clinical isolates from AIDS patients previously typed in the hexon L2 region and the fibre knob region were analysed. These isolates were found to be from species D adenovirus (HAdV-D) and 28 of them had contradictory typing results in these two regions so they are clearly intermediate strains. Two isolates appear to be completely new and one isolate (Aids32) was typed as HAdV-D23 variant in both hexon L2 and fibre knob regions. Sequencing and phylogenetic analysis of the hexon L1, fibre shaft and penton regions of these adenoviruses revealed that no intragene recombination events occurred between the hexon L1 and L2 regions or between the fibre knob and fibre shaft regions. Sequencing and phylogenetic analysis of the penton showed that some of the intermediate strains had sequences from a third type of adenovirus in these regions. The penton analysis showed also that intragene recombination between penton HVR and RGD loop regions was common. New types of adenovirus were detected and sequential infection with different adenovirus variants was observed in some patients which indicates that the genome of HAdV-D from AIDS patients are not stable. Full genome sequencing and analysis was carried out for three isolates, two of them appeared to be new types of HAdV-D and the result of multi-recombination events and the third isolate appeared to be a variant of HAdV-D23.The stability of species B adenovirus (HAdV-B) was also analysed. A total of 96 isolates collected from the Manchester area typed previously by serum neutralization (SN) were analysed in five genome regions. Most of these isolates were HAdV-B3 and HAdV-B7 collected during a 15 months outbreak. The rest of the isolates were HAdV-B types 3 and 7 collected in different years following the outbreak in addition to other adenovirus types isolated from different years. The phylogenetic analysis results of all the isolates in the structural regions: hexon L2, penton and fibre knob were found to be consistent and no mismatches (hexon from one type and fiber from another type) were observed. Most of the isolates in the DNA polymerase and E1A regions had the same clustering patterns as the structural regions. However, one HAdV-B7 and one HAdV-B11 isolate changed their clustering patterns in the DNA polymerase region. In addition, HAdV-B16 isolates changed their clustering patterns in both DNA polymerase and E1A regions. The changes of the clustering patterns of some isolates is more likely related to natural variations rather than recombination which indicate that species B adenovirus genome is stable in general. The last part of this study is investigating adenovirus persistence and latency in human tissues. Tonsils and adenoids (106 right and left tonsils and 10 adenoids) were obtained from 57 patients who underwent routine tonsillectomies and/or adenoidectomies. Eighty four (72.41%) tonsils and adenoids samples were positive for HAdV by real-time PCR. The viral load was not the same in the right and left tonsils in most of the cases and ranged from 280 to more than 2.6 x 106 copies/107 cells. Seventy eight of 84 positive samples could be typed by sequencing of the hexon L1 region. Species C types were detected in 82% of the samples followed by species B (7.7%), HAdV-E4 (7.7%) and HAdV-F41 (2.56%). No DNA methylation was detected in the major late promoter (MLP) and E1A promoter regions of six tonsils and adenoids samples and two clinical isolates.