Detection and characterization of H5N1 HPAIV in environmental samples from a dairy farm

Abstract

The recent expansion of HPAIV H5N1 infections in terrestrial mammals in the Americas, most recently including the outbreak in dairy cattle, emphasizes the critical need for better epidemiological monitoring of zoonotic diseases. In this work, we detected, isolated, and characterized the HPAIV H5N1 from environmental swab samples collected from a dairy farm in the state of Kansas, USA. Genomic sequencing of these samples uncovered two distinctive substitutions in the PB2 (E249G) and NS1 (R21Q) genes which are rare and absent in recent 2024 isolates of H5N1 circulating in the mammalian and avian species. Additionally, approximately 1.7% of the sequence reads indicated a PB2 (E627K) substitution, commonly associated with virus adaptation to mammalian hosts. Phylogenetic analyses of the PB2 and NS genes demonstrated more genetic identity between this environmental isolate and the 2024 human isolate (A/Texas/37/2024) of H5N1. Conversely, HA and NA gene analyses revealed a closer relationship between our isolate and those found in other dairy cattle with almost 100% identity, sharing a common phylogenetic subtree. These findings underscore the rapid evolutionary progression of HPAIV H5N1 among dairy cattle and reinforces the need for more epidemiological monitoring which can be done using environmental sampling.

Keywords: Bovine; Environmental; H5N1; Highly pathogenic avian influenza virus; Mammalian.

Fig. 1

Characterization of samples for H5N1 HPAIV using RT-qPCR assays. A The violin graph shows mean Ct values on x-axis, sample swab source on y-axis, grouped together with four different RT-qPCR assays for H5N1 HPAIV characterization and detection. B The bar graph shows standard-curve derived virus loads in log 10 of pfu/ml values on x-axis, sample swab source on y-axis

Fig. 2

Evolutionary analysis of PB2 and NS1 proteins by Maximum Likelihood method. The evolutionary history was inferred by the Maximum Likelihood method using 2024 mammalian PB2 (A) or NS1 (B) protein sequences and E249G substitution (shaded with green in A) or the R21Q substitution (shaded with purple in B) using the GISAID database. The yellow sequence is representing submitted consensus sequence from our study (A/dairy_cattle/Kansas/5/2024). Open red circle represents H5N1 sequences from mammalian host, filled red circle represents sequences from 2024 H5N1 dairy cattle isolates, and filled blue diamond represents sequences from avian H5N1 isolates

Fig. 3

Evolutionary analysis of HA genes by Maximum Likelihood method. The evolutionary history was inferred by the Maximum Likelihood method using top 100 blastn results from NCBI and recent H5N1 sequences from the GISAID database. The yellow sequence is representing the submitted consensus sequence from our study (A/dairy_cattle/Kansas/5/2024), the red text sequences represent the common subtree. Open red circle represents sequences from mammalian host, and filled red circle represents sequences from 2024 dairy cattle isolates

Fig. 4

Evolutionary analysis of NA genes by Maximum Likelihood method. The evolutionary history was inferred by the Maximum Likelihood method using top 100 blastn results from NCBI and recent H5N1 sequences from the GISAID database. The yellow sequence is representing submitted consensus sequence from our study (A/dairy_cattle/Kansas/5/2024), the red text sequences represent the common subtree. Open red circle represents sequences from mammalian host, and filled red circle represents sequences from 2024 dairy cattle isolates

Fig. 5

Evolutionary analysis of PB2 genes by Maximum Likelihood method. The evolutionary history was inferred by the Maximum Likelihood method using top 100 blastn results from NCBI and recent H5N1 sequences from the GISAID database. The yellow sequence is representing submitted consensus sequence from our study (A/dairy_cattle/Kansas/5/2024), the red text sequences representing the common subtree. Open red circle represents H5N1 sequences from mammalian host, filled red circle represents sequences from 2024 H5N1 dairy cattle isolates, and blue filled diamond represents sequences from avian H5N1 isolates

Fig. 6

Evolutionary analysis of NS genes by Maximum Likelihood method. The evolutionary history was inferred by the Maximum Likelihood method using top 100 blastn results from NCBI and recent H5N1 sequences from the GISAID database. The yellow sequence is representing submitted consensus sequence from our study (A/dairy_cattle/Kansas/5/2024), the red text sequences representing the common subtree. Open red circle represents sequences from mammalian host, and filled red circle represents sequences from 2024 dairy cattle isolates