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
Adenoviruses are a widespread cause of diverse human infections with recently confirmed zoonotic roots in African great apes. We focused on savanna-dwelling chimpanzees in the Issa Valley (Tanzania), which differ from those from forested sites in many aspects of behavior and ecology. PCR targeting the DNA polymerase gene detected AdV in 36.7% (69/188) of fecal samples. We detected five groups of strains belonging to the species Human mastadenovirus E and two distinct groups within the species Human mastadenovirus C based on partial hexon sequence. All detected AdVs from the Issa Valley are related to those from nearby Mahale and Gombe National Parks, suggesting chimpanzee movements and pathogen transmission.
- MeSH
- Adenoviridae genetika izolace a purifikace MeSH
- adenovirové infekce epidemiologie veterinární virologie MeSH
- DNA-dependentní DNA-polymerasy genetika MeSH
- feces virologie MeSH
- fylogeneze MeSH
- nemoci lidoopů epidemiologie virologie MeSH
- Pan troglodytes virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Tanzanie epidemiologie MeSH
- MeSH
- adenovirové infekce diagnóza farmakoterapie virologie MeSH
- coxsackie virózy diagnóza farmakoterapie virologie MeSH
- enterovirové infekce diagnóza farmakoterapie virologie MeSH
- infekce dýchací soustavy diagnóza klasifikace virologie MeSH
- infekce viry z čeledi Coronaviridae diagnóza farmakoterapie virologie MeSH
- infekce viry z čeledi Orthomyxoviridae diagnóza klasifikace virologie MeSH
- nachlazení klasifikace virologie MeSH
- Publikační typ
- přehledy MeSH
