Results and discussion
To sequence the genomes of CIV strains from southern China, 180 nasal swab samples were collected from veterinary hospitals. Among the tested nasal swabs collected from sick dogs with respiratory disease, 3 samples (3.75%) were positive for CIV RNA/antigen. After 3 passages in SPF eggs, allantoic fluids collected from eggs incubated with the positive samples tested positive in the HA test, with a titer of 27. After sequencing, three H3N2 subtype CIV strains (A/canine/China/Guangdong/1/2018, A/canine/China/Guangdong/2/2018, and A/canine/China/Guangdong/3/2018) were finally determined (Table 1). Their genomic sequences in eight segments have been deposited in the GenBank database (Nos. MK119981 - MK120004).
To further characterize the genome of H3N2 CIV in China, the nt and aa similarities of the genome sequences of H3N2 CIV strains in eight segments were calculated (Tables 2). The HA and NA genes of the H3N2 CIV isolates in this study showed genetic similarities of 99.4%-99.8% and 99.6%-99.9% among each other, respectively, and 96.1%-99.5% and 95.2%-99.8% with previously reported H3N2 CIV strains, respectively. HA and NA of the H3N2 CIV isolates in this study showed aa similarities of 96.1%-99.5% and 95.2%-99.8% among each other, respectively, and 95.7%-99.5% and 94.3%-99.8% with previously reported H3N2 CIV strains, respectively. In addition, all eight segments of these three field strains were more genetically similar to the recently determined strains from the USA than to Asian strains. All internal genes of the isolates in this study were most genetically similar to the H3N2 CIV strain A/canine/Georgia/89750.1/2017 from the USA.
The HA and NA aa sequences of the Chinese and USA isolates were aligned and compared (Table 3). The results indicated that the HA sequences of A/canine/China/Guangdong/1/2018, A/canine/China/Guangdong/2/2018, and A/canine/China/Guangdong/3/2018 contained five (V128I, N187S, A289S, K328S, N481S), four (V128I, N187S, A289S, K328S), and two (V128I, K328S) aa substitutions, respectively. In the analysis of the antigenic epitope sites, we found two (G162S, N204D) aa substitutions in the HA1 of H3N2 CIV isolates in this study which are same as USA isolates and different from other Chinese isolates(Nakajima et al. , 2003;Catonet al. , 1982).
When the NA aa sequences were aligned and compared, A/canine/China/Guangdong/1/2018, A/canine/China/Guangdong/2/2018, and A/canine/China/Guangdong/3/2018 contained two (V/I20A, N43K), two (V/I20A, N43K), and three (V/I20A, A/R30T, N43K) aa substitutions, respectively. In the analysis of the NA stalk region, we found that two (V50I, Y60H) aa substitutions in the NA of H3N2 CIV isolates in this study which are same as USA and Korea isolates and different from other Chinese isolates. And A/canine/China/Guangdong/1/2018 and A/canine/China/Guangdong/2/2018 have an(N43K) aa substitutions in NA stalk region. Furthermore, GD isolates in this study have three(G147S, R338K, E357D) aa substitutions in the NA antigenic epitope sites which are same as USA isolates and different from other Chinese isolates(Linet al. , 2012;Bunpapong et al. , 2014;Sun et al. , 2013).
To understand the genetic relationship between the three field isolates in this study and other CIV strains, we conducted phylogenetic analyses based on the HA and NA genes (Figure 1). The HA and NA genes of H3N2 CIV were clustered into three groups (groups A-C). The three groups included H3N2 CIV from China, Korea, and the USA. The strains in the present study were clustered in a new group (group D) and had were most closely related with the strains from the USA (group C) but were distant from the previously reported H3N2 CIV strain in China (group A).
Notably, all the H3N2 CIV strains in China before 2018 were located in group A. The CIV strains in group C from the USA were determined from 2015-2017. However, the molecular characteristics of the Chinese CIV strains circulating during the same period are unclear (the genomic sequences of only two strains were available online.). Accordingly, the origin of the CIV strains in group D remains unclear. Group D may be the result of continuous evolution of the field CIV strains in China. Alternatively, group D may be from the transboundary transmission of group C strains from the USA to China. A retrospective epidemiological investigation may help determine the true origin of group D.
In conclusion, in this study, we isolated and sequenced three emerging H3N2 CIV strains from China (A/canine/China/Guangdong/1/2018, A/canine/China/Guangdong/2/2018 and A/canine/China/Guangdong/3/2018), which are divergent from the reported sequences of Chinese H3N2 CIV strains but closely related with strains from the USA. This study will strengthen the understanding of the epidemiology and genetic diversity of H3N2 CIV.
The results of this study indicate that H3N2 CIV may have been transmitted from the USA to China, where it caused an epidemic in Chinese canines. These results suggest that we should strengthen monitoring of this disease at borders during the transportation of canines and continue to monitor CIV in China for a long period to observe the recombination of domestic and foreign-like strains. It is also important to monitor whether the coexistence presence of the two origin viruses of different origin has a greater impact on canines, which requires further research.