Abstract
More than 100 different enterovirus (EV) genotypes infect humans and contribute to substantial morbidity. However, current methods for characterisation of full-length genomes are based on Sanger sequencing of short genomic regions, which are labour-intensive and do not enable comprehensive characterisation of viral populations. Here, we describe a simple and sensitive protocol for the amplification and sequencing of near full-length genomes of human EV species using next generation sequencing. EV genomes were amplified from 89% of samples tested, with Ct values ranging between 15.7 and 39.3. These samples included 7 EV-A genotypes (CVA2, 5–7, 10, 16 and EV71), 19 EV-B genotypes (CVA9, CVB1-6, ECHO3, 4, 6, 7, 9, 11, 16, 18, 25, 29, 30, and EV69), 3 EV-C genotypes (CVA19 and PV2, 3) and 1 EV-D genotype (EV70). We characterised 70 EVs from 58 clinical stool samples and eight reference strains, with a minimum of 100X depth. We found evidence of co-infection in four clinical specimens, each containing two distinct EV genotypes (CVB3/ECHO7, CVB3/ECHO18 and ECHO9/30). Characterisation of the complete genome provided conclusive genotyping of EVs, which can be applied to investigate the intra-host virus evolution of EVs, and allows further identification and investigation of EV outbreaks.
Highlights
Enteroviruses (EVs) infect respiratory and gastrointestinal mucosa and are mostly transmitted through the faecal-oral route
Recombination in the 5′UTR region rescued defective mutations introduced to pathogenic circulating vaccine-derived poliovirus in vitro, with recombination between the cVDPV and the 5′UTR of EV-A/B species resulting in reduced viral replication and virulence, whereas recombination with the 5′UTR of EV-C/D species resulted in increased viral fitness[20]
Takara LA was the only polymerase other than KlenTaq LA to successfully amplify near full-length genome products of ~8 kb, and this enzyme was used in further testing of the method using four stool specimens from the NSW Health Pathology East virology diagnostic laboratory, which were previously confirmed as EV-positive using diagnostic qPCR
Summary
Enteroviruses (EVs) infect respiratory and gastrointestinal mucosa and are mostly transmitted through the faecal-oral route. Terminal deletions in the 5′UTR of up to 36 nucleotides which affect the domain I stem-loop are associated with an increased likelihood of viral persistence, and significantly reduced viral replication and prevent cytopathicity[21] This naturally-occurring deletion has been observed in virus derived from human hearts affected by EV-induced myocarditis and in the murine pancreas[22]. Detailed characterisation of EV infections may aid in the identification of EV strains or mutations that associate with adverse clinical outcomes, such as severe neurological complications or T1D. To this end, we describe a simple method for amplifying and sequencing near full-length EV genomes from human clinical specimens
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
