Materials and methods
From January to May 2018, a total of 80 nasal swabs from sick dogs with respiratory disease were collected in veterinary hospitals in Guangdong Province in southern China. The presence of CIV RNA/antigen was detected using a commercial RT-qPCR kit (Anheal, China) or an immunochromatographic strip kit (RapiGEN, Korea). The positive nasal swab samples were processed for virus isolation in SPF eggs as reported in a previous study. A hemagglutination (HA) test was performed to determine the viral titer.
Total RNA was extracted from the viral stocks using TRIzol (Takara, Japan) following the manufacturer’s instructions and then reverse transcribed into cDNA using the Uni12 primer (AGCAAAGCAGG). The viral genome was obtained by PCR using universal primers targeting IAV. DNA fragments with the expected size by 1% agarose gel electrophoresis were purified, cloned into pCloneEZ-blunt (CloneSmarter, USA) and sequenced (BGI, China).
The raw sequencing data were assembled, processed using SeqMan 7.1.0 and then aligned with other H3N2 CIV strains from China, the USA, and Korea using BioEdit 5.0.7.0. Their genomic sequences in eight segments were compared at the nucleotide (nt) and amino acid (aa) level. A phylogenetic tree was established with MEGA 6.0 using the neighbor-joining method based on the bootstrap values of 1,000 replicates.