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Canine Influenza Virus A(H3N2) Clade with Antigenic Variation, China, 2016–2017

During 2012–2017, we collected throat swabs from dogs in China to characterize canine influenza virus (CIV) A(H3N2) isolates. A new antigenically and genetically distinct CIV H3N2 clade possessing mutations associated with mammalian adaptation emerged in 2016 and replaced previously circulating strains. This clade probably poses a risk for zoonotic infection.


The Study
During October 2012-July 2017, we collected 399 throat swabs from dogs with respiratory symptoms in pet hospitals and kennels in China to monitor for CIV H3N2 epidemics and virus evolution. We amplified the matrix gene by realtime reverse transcription PCR using Influenza A Virus V8 Rapid Real-Time RT-PCR Detection Kit (Beijing Anheal Laboratories Co. Ltd., http://anheal.company.weiku.com) and isolated and identified virus isolates using methods previously described (6). Of 399 samples, 54 (13.5%) contained CIV H3N2 isolates. Of these 54 isolates, 43 were from Beijing, 6 from Nanjing, 3 from Shanghai, and 2 from Xi'an.
To characterize the evolution of CIV H3N2, we sequenced the full genome of the 54 isolates (GenBank accession nos. MK212398-829) and performed genetic analyses using available sequences of related viruses from GenBank and the GISAID database (https://www.gisaid.org). Phylogenetic analysis of worldwide CIV H3N2 isolates indicated that each genome segment of the H3N2 isolates after 2016 formed a separate clade, distinct from other isolates from China, which grouped with isolates from South Korea and the United States (Figure 1; Appendix Figure, https://wwwnc.cdc. gov/EID/article/25/1/17-1878-App1.pdf). Each genome segment of the 41 H3N2 CIVs isolated after 2016 shared high nucleotide sequence identities (99.62% ± 0.09% to 99.88% ± 0.10%). Among these isolates, the time to most recent common ancestor computed by molecular clock analysis (10,11) was similar for each genome segment; all ancestors dated back to early to mid-2016 (Appendix Figure). Therefore, the introduction of this CIV H3N2 clade into China most likely occurred in 2016 as either a single event or multiple events involving genetically similar viruses. This clade was more closely related to earlier H3N2 CIVs than the ancestral H3N2 avian influenza viruses from South Korea (Figure 1), and viruses of this clade could have originated from H3N2 CIVs circulating in South Korea or the United States.
We then investigated the molecular characteristics of these viruses. Although all the CIV H3N2 isolates from this clade still possessed 226Q and 228G (which confer specificity to cell entry receptors in birds) in hemagglutinin, they also possessed the 4 amino acid substitutions 251R and 590S in polymerase basic 2 and 146S and 242I in hemagglutinin, which have frequently been identified in human influenza viruses. Of note, 251R and 590S in polymerase basic 2 are known determinants of adaptation to growth in mammals ( Antigenic analysis with ferret antiserum against representative viruses of different clades demonstrated a diversity of reaction patterns that generally corresponded with phylogenetic relationships ( Table 2). H3N2 CIVs isolated during 2016-2017 reacted well with antiserum against viruses of the same lineage and less well with antiserum against viruses of other lineages. Numeric analysis of these hemagglutinin inhibition (HI) titers with AntigenMap (http://sysbio.cvm.msstate.edu/software/AntigenMap) revealed that H3N2 CIVs isolated after 2016 had a distinguishable antigenic reaction pattern ( Figure 2).
The CIV H3N2-positive dogs in this study generally had only respiratory symptoms and recovered within 10 days. However, the virus spread rapidly. Among dogs in a cohort, 1 displayed mild disease (cough, runny nose, lethargy) soon after being introduced into a kennel. Within  In 2017, the percentage of dogs treated at the Veterinary Teaching Hospital of China Agricultural University that were seropositive for CIV H3N2 was 6.3%, higher than the percentage during 2012-2013 (3.5%) (14). The wide prevalence and increased seropositivity of H3N2 variants suggest the lineage that emerged in 2016 might possess greater infectivity in dogs than earlier viruses, which might have resulted in clade replacement. The possibility of stochastic events leading to the disappearance of the previous clade should not be excluded. Considering that preadaptation of influenza A(H1N1)pdm09 virus genes to mammalian hosts through prior circulation for several decades in swine might have contributed to the emergence of viruses containing these genes in humans, the potential adaptation of this CIV H3N2 clade to mammals and its public health threat should be further evaluated.
Because dog competitions and trade involving different countries are frequent and the surveillance of CIV is limited, further studies should focus on determining whether viruses of this CIV H3N2 lineage are prevalent in other countries. Global active surveillance to monitor the spread of these viruses among dogs should also be enhanced. Such efforts could prevent further CIV spread and adaptation and will be critical for identifying public health threats that could emerge at the animal-human interface.  Table 2. One unit (cell) represents a 2-fold change in HI titer.

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