Recovering influenza genomes from wild bird habitats for outbreak prevention and pandemic preparedness

Abstract

Purpose: Avian influenza viruses (AIVs) cause costly outbreaks in commercial poultry flocks and dangerous zoonotic infections in humans that threaten future pandemics and new types of seasonal flu. AIVs naturally circulate in wild waterfowl, so surveillance programs currently focus on testing individual birds and culturing viruses for identification by molecular methods. Unfortunately, these programs rarely detect dangerous strains before they cause outbreaks. We have developed an alternative, culture-free method for characterizing AIVs using targeted genomic sequencing. In addition to traditional swab specimens, this new approach can be applied to environmental specimens from wild bird habitats. Virus-laden feces from numerous bird species accumulates in wetlands sediment, so these specimens may enable extensive identification of locally circulating AIVs. Methods & Materials: Sediment specimens and bird swabs were collected from wetlands in British Columbia, Canada between September 2016 and April 2018. Total RNA was extracted from each sample and screened for AIV genomic material by qPCR. 230 qPCR-positive samples (184 sediments and 46 swabs) were prepared into metagenomic sequencing libraries using a custom protocol. Hemagglutinin (HA) and neuraminidase (NA) genome segments were enriched in these libraries using 3,600 custom hybridization probes then sequenced on an Illumina HiSeq. Genomic sequencing results were analyzed with custom bioinformatic pipelines to identify AIV sequencing reads and characterize their HA and NA subtypes. Results: AIV genomic material was weakly detected by qPCR in these sediment and swab specimens (Ct value median: 35.0, IQR: 32.3 - 37.6) yet, using our custom targeted genomic sequencing method, AIV genomic sequences were recovered from 107 sediment specimens (58%) and 25 swab specimens (54%). 13 of 16 avian HA subtypes were detected in sediment while 7 were detected in swabs. All 9 avian NA subtypes were detected in sediment while 6 were detected in swabs. Conclusion: Targeted genomic sequencing allows culture-independent AIV detection and subtyping in both swab and sediment specimens with a sensitivity comparable to or exceeding that of traditional culture and PCR-based methods. A broad diversity of HA and NA subtypes were recovered in sediment specimens, demonstrating the utility of environmental sampling for AIV surveillance.