OBJECTIVES: Direct genotyping of adenovirus or enterovirus from clinical material using polymerase chain reaction (PCR) followed by Sanger sequencing is often difficult due to the presence of multiple virus types in a sample, or due to varying efficacy of PCR amplifying the capsid gene on the background of foreign nucleic acids. Here we present a simple protocol for virus genotyping using massive parallel amplicon sequencing. METHODS: The protocol utilized a set of 16 tailed degenerate primers flanking the seventh hypervariable region of the adenovirus hexon gene and 9 tailed degenerate primers targeted to the proximal portion of the enterovirus VP1 gene. Subsequent addition of dual indices enabled simultaneous sequencing of 384 different samples on an Illumina MiSeq instrument. Downstream bioinformatic analysis was based on remapping to a set of references representative of the presently known repertoire of virus types. RESULTS: After validation with known virus types, the sequencing method was applied on 301 adenovirus-positive samples and 350 enterovirus-positive samples from a longitudinally collected series of stools from 83 children aged 3 to 36 months. We detected 7 different adenovirus types and 27 different enterovirus types. There were 37 (6.2%) samples containing more than one genotype of the same viral genus. At least one dual infection was experienced by 23 of 83 (28%) of the children observed over the 3 years' observation period. CONCLUSIONS: Amplicon sequencing with a multiplex set of degenerate primers seems to be a rapid and reliable technical solution for genotyping of large collections of samples where simultaneous infections with multiple strains can be expected.
- MeSH
- Adenoviridae klasifikace genetika izolace a purifikace MeSH
- adenovirové infekce virologie MeSH
- DNA primery genetika MeSH
- enterovirové infekce virologie MeSH
- Enterovirus klasifikace genetika izolace a purifikace MeSH
- genotyp * MeSH
- genotypizační techniky metody MeSH
- kojenec MeSH
- lidé MeSH
- longitudinální studie MeSH
- předškolní dítě MeSH
- sekvenční analýza DNA metody MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Norsko MeSH
The aim of this work was to express the recombinant hexon protein of the hemorrhagic enteritis virus, to establish the diagnostic value of this protein for serological detection of antibodies in turkey serum samples and to assess seroprevalence of the infection in the Czech Republic. The N' terminal part of the hexon protein was expressed in a bacterial expression system and used as an antigen in an ELISA test for the detection of hemorrhagic enteritis virus specific antibodies in turkey sera. Validation of the test was performed by comparison with a commercially available ELISA test. Serological reactivity was assessed on a panel of 126 turkey sera by a newly developed ELISA test. Serum samples were taken from turkey farms with the history of hemorrhagic enteritis virus infection, from farms with animals free of infection, and from turkey farms following vaccination. Both ELISA kits gave identical results (100 %) with the tested sera. ELISA based on the recombinant hexon protein thus proved useful and cheaper for detection of antibodies in turkey flocks infected with the hemorrhagic enteritis virus.
- MeSH
- Adenoviridae klasifikace genetika imunologie MeSH
- antigeny virové genetika imunologie MeSH
- ELISA MeSH
- exprese genu * MeSH
- krocani MeSH
- protilátky virové krev imunologie MeSH
- rekombinantní proteiny genetika imunologie izolace a purifikace MeSH
- specificita protilátek imunologie MeSH
- virové plášťové proteiny genetika imunologie izolace a purifikace MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Adenovirus type 5 (Ad5) gene therapy vectors require protection against antibodies, complement proteins and blood cells if they are to be delivered intravenously to treat metastatic disease. Such protection can be achieved by chemically modifying Ad5 with polymers based on hydrophilic HPMA. Here, such polymers were designed to include side chains bearing reactive carbonyl thiazolidine-2-thione groups (TTs) to covalently modify available amino groups of the lysine residues in the Ad5 capsid. Furthermore, the inclusion of side chains bearing positively charged quaternary ammonium groups (QAs) was designed to improve electrostatic interaction of the polymers with negatively charged Ad5 hexon protein. Finally, to enable triggered uncoating and reactivation of the Ad5, either the TTs or both the TTs and the QAs were linked to polymer backbone via reductively degradable disulfide bonds. SDS-PAGE demonstrated that these polymers covalently modified Ad5 capsid proteins in a reduction reversible manner. In infection studies, polymers containing QAs prevented binding of coagulation factor X to Ad5. Furthermore, the antibody and complement mediated binding of Ad5 to erythrocytes was reduced by such polymers (>95% without polymer, 25% following coating). These data indicate that coating Ad5 therapeutics with such polymers will improve blood circulation half-life and deposition at disease sites.
- MeSH
- Adenoviridae fyziologie genetika klasifikace MeSH
- adenovirové infekce prevence a kontrola MeSH
- aminy chemie MeSH
- barvení stříbrem MeSH
- erytrocyty metabolismus MeSH
- faktor IX metabolismus MeSH
- genetická terapie MeSH
- genetické vektory MeSH
- imunologické faktory metabolismus MeSH
- komplement metabolismus MeSH
- lidé MeSH
- luciferasy metabolismus MeSH
- molekulární struktura MeSH
- molekulová hmotnost MeSH
- nádorové buněčné linie MeSH
- polymery chemická syntéza chemie MeSH
- protilátky metabolismus MeSH
- statická elektřina MeSH
- vazba proteinů MeSH
- virové plášťové proteiny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
