Detection and Characterization of Swine Origin Influenza A(H1N1) Pandemic 2009 Viruses in Humans following Zoonotic Transmission

Abstract

Human-to-swine transmission of seasonal influenza viruses has led to sustained human-like influenza viruses circulating in the U.S. swine population. While some reverse zoonotic-origin viruses adapt and become enzootic in swine, nascent reverse zoonoses may result in virus detections that are difficult to classify as "swine-origin" or "human-origin" due to the genetic similarity of circulating viruses. This is the case for human-origin influenza A(H1N1) pandemic 2009 (pdm09) viruses detected in pigs following numerous reverse zoonosis events since the 2009 pandemic. We report the identification of two human infections with A(H1N1)pdm09 viruses originating from swine hosts and classify them as "swine-origin" variant influenza viruses based on phylogenetic analysis and sequence comparison methods. Phylogenetic analyses of viral genomes from two cases revealed these viruses were reassortants containing A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) genes with genetic combinations derived from the triple reassortant internal gene cassette. Follow-up investigations determined that one individual had direct exposure to swine in the week preceding illness onset, while another did not report swine exposure. The swine-origin A(H1N1) variant cases were resolved by full genome sequence comparison of the variant viruses to swine influenza genomes. However, if reassortment does not result in the acquisition of swine-associated genes and swine virus genomic sequences are not available from the exposure source, future cases may not be discernible. We have developed a pipeline that performs maximum likelihood analyses, a k-mer-based set difference algorithm, and random forest algorithms to identify swine-associated sequences in the hemagglutinin gene to differentiate between human-origin and swine-origin A(H1N1)pdm09 viruses.IMPORTANCE Influenza virus infects a wide range of hosts, resulting in illnesses that vary from asymptomatic cases to severe pneumonia and death. Viral transfer can occur between human and nonhuman hosts, resulting in human and nonhuman origin viruses circulating in novel hosts. In this work, we have identified the first case of a swine-origin influenza A(H1N1)pdm09 virus resulting in a human infection. This shows that these viruses not only circulate in swine hosts, but are continuing to evolve and distinguish themselves from previously circulating human-origin influenza viruses. The development of techniques for distinguishing human-origin and swine-origin viruses are necessary for the continued surveillance of influenza viruses. We show that unique genetic signatures can differentiate circulating swine-associated strains from circulating human-associated strains of influenza A(H1N1)pdm09, and these signatures can be used to enhance surveillance of swine-origin influenza.

Keywords: A(H1N1)pdm09; influenza; pandemic; random forest; swine-origin; zoonotic transmission.

FIG 1

Evolutionary relationships of the hemagglutinin (A) and neuraminidase (B) nucleotide sequences. The evolutionary history was inferred using the neighbor-joining method (28). (A and B) The optimal trees with the sums of branch length equaling 1.65521810 (A) and 2.16241627 (B). The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches (29). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method (30) and are in the units of the number of base substitutions per site. The analysis involved 203 (A) and 209 (B) nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 1,795 (A) and 1,489 (B) positions in the final data set. Evolutionary analyses were conducted in MEGA7 (19).

FIG 1

Evolutionary relationships of the hemagglutinin (A) and neuraminidase (B) nucleotide sequences. The evolutionary history was inferred using the neighbor-joining method (28). (A and B) The optimal trees with the sums of branch length equaling 1.65521810 (A) and 2.16241627 (B). The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches (29). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method (30) and are in the units of the number of base substitutions per site. The analysis involved 203 (A) and 209 (B) nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 1,795 (A) and 1,489 (B) positions in the final data set. Evolutionary analyses were conducted in MEGA7 (19).

FIG 2

Genome constellations identified in A(H1N1) variant viruses.

FIG 3

Random forest tree output. Positions represented in the random forest tree represent the human-associated nucleotide. The green colored rectangles represent the fraction of strains in the collection that are swine associated. The blue colored rectangles represent the human-associated endpoint for the categorization of human-associated strains. Percentage values represent the fraction of the data set represented by in that endpoint. (i.e., 49% represents that human-associated strains make up ∼50% of the data set).