MATERIALS AND METHODS
To detect porcine circovirus-like agents in dogs and cats, we collected
199 clinical serum samples from dogs (n=158) and cats (n=41) with
clinical signs of respiratory tract or digestive tract disease from
veterinary hospitals in the cities of Nanjing (Jiangsu province, China)
and Shijiazhuang (Hebei province, China) between January 2018 and
December 2019. Samples were stored at −80°C before analysis. DNA
extraction and PCR amplification were performed as previously described
(Wen et al., 2019). Briefly, total DNA from 200 µL of serum was
extracted using a DNA kit (Tiandz, Inc., China) according to the
manufacturer’s instructions. Two sets of back-to-back PCR primers,
described previously, were designed to amplify the complete genome
sequences. PCR was carried out using the Mix (green) (TsingKe Biotech
Co., Ltd. Beijing, China) following manufacturer’s instructions. After
an initial denaturation at 98 °C for 2 min, the PCR conditions were 40
cycles at 98 °C for 15 s, 62 °C for 15 s, and 72 °C for 10 s, followed
by a final extension at 72 °C for 5 min. The PCR products were purified
using a Gel Extraction Kit (AXYGEN, China) according to the
manufacturer’s instructions, then cloned into the pMD18T vector (TaKaRa,
Dalian, China), and sequenced. In addition, all samples were screened
for PCV2, PCV3, and PCV4 by conventional PCR using reference primers
(Wen et al., 2012; Palinski et al., 2017; Zhang et al., 2019).
The resultant sequences were analyzed using DNAStar software version
7.10 (Lasergene).The complete genome sequences of porcine
circovirus-like agents in this study and reference strains were aligned
using the Clustal X program of DNAStar to determine nucleotide sequence
similarities. Based on complete genome sequences, a phylogenetic tree
was constructed using the neighbor-joining method in MEGA7.0
(http://www.megasoftware.net), using p -distances with 1,000
bootstrap replicates. We used 12 representative P1 strains for
phylogenetic analysis, including 2 cattle-origin P1 strains (GenBank
KY462783, KY462784), 2 rabbit-origin P1 strains (GenBank KY462785,
KY462786), 2 goat-origin P1 strains (GenBank KY462787, KY462788), and 6
pig-origin P1 strains [GenBank EF514716 (the prototype sequence),
JN040279, KU243695, KU323639 (the prototype elongated ORF1 sequence),
KU324511, and KU356939]. In addition, we also used all other related
porcine circovirus-like agents available on GenBank for analysis.