Enrichment next-generation sequencing (NGS) provides a more cost-efficient and sensitive method for detecting and sequencing novel coronaviruses from wild bat populations, according to a study reported in mSphere, an open-access journal from the American Society for Microbiology.
With the appearance of the new zoonotic Wuhan coronavirus, the importance of monitoring the likelihood of new virus risks in wildlife reservoirs has been heightened. Bats in particular have been found to be the most common reservoir of coronaviruses, including being a probable source or mixing vessel for two previous modern epidemic coronaviruses: SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome).
“We should be alert and vigilant with the knowledge that bat CoVs [coronaviruses] are likely to cause another disease outbreak, not only because of their prevalence but also because the high frequency of recombination between viruses may lead to the generation of viruses with changes in virulence,” according to, of the Wuhan (China) Institute of Virology, and colleagues.
“We previously provided serological evidence that [HKU8-related] CoV had jumped over from bats to camels and recombined with MERS-CoV, alerting other researchers that the CoV species could be dangerous. ... Genome-level comparison is needed to monitor the risk of alterations in species tropism and pathogenesis,” according to study authors. They performed a study to develop a more effective and cost efficient method for detecting and sequencing novel coronaviruses in the bat population.
The taxonomy of coronaviruses is particularly complex and may be too narrowly defined, given the high level of genetic plasticity found. There are four genera (Alpha-, Beta-, Gamma-, and Deltacoronavirus) consisting of 38 unique species in the CoV subfamily Orthocoronavirinae, and the number is increasing. Viral taxomists rely on the open reading frame 1b (ORF1b) gene for classification, but viruses in the same species may show great diversity in regions outside ORF1b, confounding the species designation. In particular, bat CoVs classed as the same species can differ significantly in terms of receptor usage or virus-host interaction, as observed in bat SARS-related CoVs, according to the researchers.
The researchers obtained RNA from previous bat CoV surveillance projects, which used bat rectal swabs. Libraries for NGS were constructed from total RNA and processed to generate RNA fragments larger than 300 nucleotides. Following first- and second-strand cDNA synthesis, double-stranded cDNA was purified and the library was amplified by polymerase chain reaction (PCR) technology.
Targeted CoV genome enrichment was achieved using 4,303 customized biotinylated 120-mer baits. These baits were designed from 90 representative CoV genomes, and in silico analysis determined that these baits should target the known CoV species tested. These baits were added and hybridized to the libraries. To capture virus-specific library fragments, streptavidin magnetic beads (which bind to biotin) were added to the hybridization reaction mixture. The beads were then washed to remove unbound DNA. The postcapture virus-specific library fragments were then amplified using a subsequent round of PCR.
The enrichment NGS were retrospectively complemented with unbiased NGS and/or additional Sanger sequencing to obtain full-length genomes. The study showed that enrichment NGS not only decreased the amount of data requiring analysis but produced full-length genome coverage in both laboratory and clinical samples.
Using this technology, the researchers “effectively reduced sequencing costs by increasing the sensitivity of detection. We discovered nine full genomes of bat CoVs in this study and revealed great genetic diversity for eight of them.” In addition, they noted that using standard targeted PCR, which is common practice for many surveillance studies, would not have discovered this diversity.
“We should be alert and vigilant with the knowledge that bat CoVs are likely to cause another disease outbreak, not only because of their prevalence but also because the high frequency of recombination between viruses may lead to the generation of viruses with changes in virulence,” according to the researchers.
“We have provided a cost-effective methodology for bat CoV surveillance. The high genetic diversity observed in our newly sequenced samples suggests further work is needed to characterize these bat CoVs prior to or in the early stages of spillover to humans,” the authors concluded.
This study was supported by the Chinese government. The authors reported that they had no conflicts.
Viral genome data for new CoVs from this study are available in GenBank under accession numbersto .
SOURCE: Li B et al. mSphere 2020 Jan 29;5:e00807-19.