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.