Multiple Incursions and Recurrent Epidemic Fade-Out of H3N2 Canine Influenza A Virus in the United States

Abstract

Avian-origin H3N2 canine influenza virus (CIV) transferred to dogs in Asia around 2005, becoming enzootic throughout China and South Korea before reaching the United States in early 2015. To understand the posttransfer evolution and epidemiology of this virus, particularly the cause of recent and ongoing increases in incidence in the United States, we performed an integrated analysis of whole-genome sequence data from 64 newly sequenced viruses and comprehensive surveillance data. This revealed that the circulation of H3N2 CIV within the United States is typified by recurrent epidemic burst-fade-out dynamics driven by multiple introductions of virus from Asia. Although all major viral lineages displayed similar rates of genomic sequence evolution, H3N2 CIV consistently exhibited proportionally more nonsynonymous substitutions per site than those in avian reservoir viruses, which is indicative of a large-scale change in selection pressures. Despite these genotypic differences, we found no evidence of adaptive evolution or increased viral transmission, with epidemiological models indicating a basic reproductive number, R₀, of between 1 and 1.5 across nearly all U.S. outbreaks, consistent with maintained but heterogeneous circulation. We propose that CIV's mode of viral circulation may have resulted in evolutionary cul-de-sacs, in which there is little opportunity for the selection of the more transmissible H3N2 CIV phenotypes necessary to enable circulation through a general dog population characterized by widespread contact heterogeneity. CIV must therefore rely on metapopulations of high host density (such as animal shelters and kennels) within the greater dog population and reintroduction from other populations or face complete epidemic extinction.IMPORTANCE The relatively recent appearance of influenza A virus (IAV) epidemics in dogs expands our understanding of IAV host range and ecology, providing useful and relevant models for understanding critical factors involved in viral emergence. Here we integrate viral whole-genome sequence analysis and comprehensive surveillance data to examine the evolution of the emerging avian-origin H3N2 canine influenza virus (CIV), particularly the factors driving ongoing circulation and recent increases in incidence of the virus within the United States. Our results provide a detailed understanding of how H3N2 CIV achieves sustained circulation within the United States despite widespread host contact heterogeneity and recurrent epidemic fade-out. Moreover, our findings suggest that the types and intensities of selection pressures an emerging virus experiences are highly dependent on host population structure and ecology and may inhibit an emerging virus from acquiring sustained epidemic or pandemic circulation.

Keywords: canine influenza; emerging virus; influenza; virus evolution; virus host adaptation.

FIG 1

Evolutionary relationships of H3N2 CIV sequences. Black branches on the phylogenies represent early emergent viruses isolated in Asia between 2006 and 2007, while branches leading to enzootic viruses isolated from different geographic regions are color coded as follows: green, China; blue, South Korea; and red, United States. Red triangles show viruses of Chinese origin that formed a transient and locally contained outbreak in Los Angeles, CA, in March 2017. Horizontal branch lengths are drawn to scale (nucleotide substitutions per site). (A) Individual segment phylogenies for all complete coding regions from all H3N2 CIV sequences available. Each segment tree is rooted on the closest related avian IAV sequence in the database. Identical sequences were collapsed into single branches for purposes of clarity. (B) Phylogeny of concatenated major reading frames from each genome segment for all nonreassortant viruses circulating in Asia and the United States (see inset). Bootstrap values for key nodes are indicated.

FIG 2

Clock-like nature of H3N2 CIV evolution. Regression of root-to-tip genetic divergence against sampling date was performed based on a phylogeny of concatenated major coding regions of H3N2 CIV sequences from Asia and the United States. Dotted ovals demark the approximate ranges of the MW2015, MW2016, and SE2017 U.S. phylogenetic clades.

FIG 3

Mean dN/dS values for major coding regions of H3N2 CIV and H3N2 AIV. All H3N2 AIV sequences available in the database were included. Only full genomes of nonreassorted CIVs were analyzed. NS1 contains a portion of the NS2 alternative reading frame. It should be noted that estimates for the shorter segments (M1 and NS1) show variance, especially among U.S. viruses that have circulated for a short period and accumulated only a small number of nucleotide substitutions.

FIG 4

Amino acid diversity among H3N2 CIVs in the United States. Clade consensus-level segregating amino acid positions across major coding regions in H3N2 CIV that differentiate various virus groups of interest in this study are shown. Numbering for each major reading frame starts at 1 on the first amino acid, with the exception of HA, which is numbered according to the standard H3 numbering scheme. See Table 1 for more details.

FIG 5

Spatiotemporal incidence of H3N2 CIV infection in the United States. Samples included those collected throughout the United States between March 2015, soon after the first introduction of virus, and September 2017. Blue marks indicate samples that were tested for CIV but were negative, while red marks indicate samples that tested positive for H3N2 CIV. Yellow bars on the incidence-by-date histogram (lower panel) indicate periods of elevated incidence. An animated version of this map is included in Movie S1 in the supplemental material.

FIG 6

Phylodynamics analysis of major outbreaks occurring during the circulation of H3N2 CIV in the United States. (A) Spatiotemporal incidence of the virus and sequencing coverage of the U.S. epidemic. Black ticks represent samples testing positive for CIV by qRT-PCR, gray ticks represent negative tests, and triangles represent sequenced viruses, with all samples separated by collection date (horizontal axis) and ZIP code (vertical axis). Epidemiologically defined outbreak clusters are demarked by colored boxes, and sequenced virus markers are colored accordingly. (B) Phylogenetic structure of U.S. epidemiological outbreaks based on concatenated segment major reading frames (as in Fig. 1B, inset). Branch tips (triangles) are colored by epidemiologically defined outbreaks as in panel A. Open node circles indicate bootstrap proportions of >98%.

FIG 7

Transmission rates among different U.S. outbreaks. (A) R₀ estimates for the different U.S. outbreaks based on epidemiological analysis. Estimates were performed using a gamma-distributed serial interval for both short (mean = 3.5 days; SD = 1.5 days; solid dots) and long (mean = 7 days; SD = 6 days; open dots) assumed serial interval periods. Error bars represent 95% credible intervals. (B) Estimates of the variation in the rate of transmission over the course of each outbreak.