Multi-drug resistance is increasing at alarming rates. The efficacy of phage therapy, treating bacterial infections with bacteriophages alone or in combination with traditional antibiotics, has been demonstrated in emergency cases in the United States and in other countries, however remains to be approved for wide-spread use in the US. One limiting factor is a lack of guidelines for assessing the genomic safety of phage candidates. We present the phage characterization workflow used by our team to generate data for submitting phages to the Federal Drug Administration (FDA) for authorized use. Essential analysis checkpoints and warnings are detailed for obtaining high-quality genomes, excluding undesirable candidates, rigorously assessing a phage genome for safety and evaluating sequencing contamination. This workflow has been developed in accordance with community standards for high-throughput sequencing of viral genomes as well as principles for ideal phages used for therapy. The feasibility and utility of the pipeline is demonstrated on two new phage genomes that meet all safety criteria. We propose these guidelines as a minimum standard for phages being submitted to the FDA for review as investigational new drug candidates.
Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Yúnnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Yúnnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term.
The virosphere is largely unexplored and the majority of viruses are yet to be represented in public sequence databases. Bats are rich reservoirs of viruses, including several zoonoses. In this study, high throughput sequencing (HTS) of viral RNA extracted from swabs of four body sites per bat per timepoint is used to characterize the virome through a longitudinal study of a captive colony of fruit nectar bats, species Eonycteris spelaea in Singapore. Through unbiased shotgun and target enrichment sequencing, we identify both known and previously unknown viruses of zoonotic relevance and define the population persistence and temporal patterns of viruses from families that have the capacity to jump the species barrier. To our knowledge, this is the first study that combines probe-based viral enrichment with HTS to create a viral profile from multiple swab sites on individual bats and their cohort. This work demonstrates temporal patterns of the lesser dawn bat virome, including several novel viruses. Given the known risk for bat–human zoonoses, a more complete understanding of the viral dynamics in South-eastern Asian bats has significant implications for disease prevention and control. The findings of this study will be of interest to U.S. Department of Defense personnel stationed in the Asia-Pacific region and regional public health laboratories engaged in emerging infectious disease surveillance efforts.
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