A letter to the NEJM, published today: Sequencing-Based Detection of Avian Influenza A(H5N1) Virus in Wastewater in Ten Cities. Click or tap through for the citations and a chart.
Highly pathogenic avian influenza viruses are virulent members of the H5 and H7 subtypes of influenza A virus, cause systemic disease in avian species, and incur substantial costs to agricultural production because of the need for mass culling of infected animals. Since 1955, most avian influenza outbreaks have been caused by three lineages; one of them (a lineage that infected poultry) gave rise to the 2.3.4.4b clade, which has recently become widespread.
On March 25, 2024, H5N1 2.3.4.4b was detected in dairy cattle herds in Texas concomitantly with herds in Michigan and Kansas. The first case in humans in 2024 was detected shortly thereafter in Texas (on March 28, 2024) in a person with exposure to symptomatic cattle. As of July 28, 2024, H5N1 had been detected in 14 human infections in 3 U.S. states (4 cattle-associated and 10 poultry-associated), 171 dairy herds across 13 states, and 94 poultry flocks across 26 states.
Another group has reported the presence of influenza virus with an H5 hemagglutinin gene in wastewater beginning in March 2024 and has subsequently identified H5 influenza virus at 25 sites across 9 states (https://data.wastewaterscan.org/).
Since May 2022, the Texas Epidemic Public Health Institute has been using hybrid-capture sequencing to test weekly wastewater samples in mostly urban areas in cities throughout Texas (Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org) and has detected over 400 human and animal viruses to date, several of which (e.g., SARS-CoV-2, influenza virus, and mpox virus) correlate with clinical case data.
Seasonal influenza virus serotypes H3N2 and H1N1 are routinely detected in Texas Epidemic Public Health Institute wastewater samples, and levels in wastewater have corresponded to clinical caseloads from May 2022 through the beginning of March 2024. Until that point, serotype H5N1 had not been detected (0 of 1337 wastewater samples).
However, in samples from March 4 through July 15, 2024, H5N1 was detected in 10 of 10 cities, 22 of 23 sites, and 100 of 399 samples (Figure 1 and Fig. S1). The abundance of H5N1 sequences identified has not correlated with influenza-related hospitalizations, which declined in Texas during the spring of 2024 (Figure 1B).
Wastewater sequences were identified as H5N1 through the use of competitive mapping to an influenza genome database. Sequences were then manually validated and documented by three independent genomics researchers. Within the set of validated H5N1 sequencing reads, there are alignments to all eight genome segments, including the PB2, HA (hemagglutinin), and NA (neuraminidase) genes, which cover more than 85% of the genome (Figure 1A). All sequencing reads best match H5N1 genomes from birds and mammals (including those identified in humans) collected since 2023 and are assigned to the 2.3.4.4b clade.
Of importance, a glutamic acid, instead of lysine, in position 627 of PB2 suggests that this virus has not extensively adapted to humans (see Table S2, which shows all unique single-nucleotide polymorphisms).
The widespread detection of influenza A(H5N1) virus in wastewater from 10 U.S. cities is troubling. Although the exact origin of the signal is currently unknown, the lack of clinical burden along with genomic information suggests multiple animal sources.
Wastewater monitoring should be considered as a sentinel surveillance tool that augments our detection of evolutionary adaptations of concern. To aid efforts to identify the source and promote efforts to mitigate the next flu pandemic, we suggest expanded, agnostic sequencing of wastewater, livestock and their products, exposed agricultural workers (including fecal and urine samples), and migrating birds along major flyways.
