Molecular detection and phylogenetic analysis of porcine circovirus type 3 in Tibetan pigs on the Qinghai-Tibet Plateau of China

We collected samples from the Tibetan pig farms located in the Sichuan, Gansu and Qinghai provinces, representing different areas on the Qinghai-Tibet Plateau. A total of 498 intestinal tissue samples from 56 farms were collected between June 2018 and December 2019. The collected samples included 316 diarrheic and 182 healthy animals. The PCV3 positivity rate was evaluated in Tibetan pigs with clinical signs of acute diarrhoea from 29 farms. Healthy control samples were collected in a randomized manner from 27 farms, based on 10% of the number of Tibetan in stock, and used to evaluate the latent PCV3 infection rate in healthy animals. All the sampled Tibetan pigs were at the weaner and grower stage, and were between 4 and 12 weeks old; no further categorization was performed on the basis of age. Intestinal tissues were stored in liquid nitrogen for DNA extraction. This study was granted approval from the veterinarians of the Animal Care and Use Committee at Gansu Agricultural University, China.

The intestinal tissues from Tibetan pigs were homogenized to powder with liquid nitrogen, and diluted threefold with phosphate‐buffered saline (PBS) in volume. All samples were centrifuged at 1800 × g for 10 min at 4 °C, and the supernatants were collected and transferred into a 1.5 mL tube. DNA extraction from supernatants was performed using a commercial DNA extraction kit (Omega, Norcross, GA, USA) according to the manufacturer’s instructions. The extracted sample DNA was stored at − 20 °C until being used for PCV3 detection. A pair of specific primers for PCV3 detection was designed based on the conserved sequence in the cap genes (Table 1, Fig. 1), and the expected size of the amplification product was 415 bp. The amplification reactions consisted of 1.0 μL of sample DNA, 1.0 μL of each primer at a concentration of 2.5 μM, 12.5 μL of 2 × PCR mix, and 9.5 μL of nuclease-free water added up to a final volume of 25 μL. The thermocycling program used was as follows: predenaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 58 °C for 25 s and 72 °C for 30 s, with a final extension for 10 min at 72 °C (Bio-Rad, USA). The PCV3 positivity rates in different provinces were calculated and marked on a map of China based on the electrophoresis results of the PCR products.

The PCV3 strains PCV3/CN/Xinjiang-11/2018 and PCV3-China/GD-ZQ-1/2017 (MK284236 and MG897485) were chosen from GenBank, and the conserved regions of the PCV3 genes were analyzed with MEGA 7.1 software to design specific primers to sequence entire Cap genes in PCV3 strains, which were then used to confirm the subtypes of PCV3 in the positive samples. The references for the amplification reactions and PCR parameters are given in the methods in section of DNA extraction and PCV3 detection. The PCR products were purified and cloned using the pMD19-T vector (TaKaRa, Dalian, China) for sequencing (Sangon Biotech, Shanghai, China). Twenty different nucleotide sequences of Cap proteins were obtained from the PCR products and analyzed using the MegAlign program. Twenty-one different subtypes representative of PCV3 strains that occurred in different countries in recent years were chosen, and phylogenetic analysis of the entire Cap nucleotide sequences was performed using the neighbour‐joining method with 1000 bootstrap replicates in MEGA 7.2 software. To compare the number of mutations in the amino acid sequences of the entire Cap protein among the three PCV3 subtypes, different PCV3 strains and one reference strain with highest homology were chosen. Prediction of the amino acid sequence from nucleotide sequences translation was performed to compare the point mutations using MEGA and edited in Microsoft Excel, version 2016.

Based on the phylogenetic analysis of the Cap nucleotide sequences, the DNAs from twenty positive samples with different Cap nucleotide sequences were subjected to PCR with three pairs of overlapping primers (Table 1, Fig. 1) to obtain the complete genome sequence of PCV3. Primers were designed based on conserved regions of the PCV3 strains PCV3/CN/Xinjiang-11/2018 and PCV3-China/GD-ZQ-1/2017 (MK284236 and MG897485). All the PCR assays were performed according to the methods described in section of DNA extraction and PCV3 detection. Three sequence fragments from one positive DNA sample were assembled and annotated using the EditSeq program of the DNASTAR software and aligned to obtain the final sequences of the PCV3 genomes. Twenty complete PCV3 genomic sequences in this study were used in phylogenetic analyses and sequence alignments with 22 other reference strains obtained in GenBank. Phylogenetic analysis of PCV3 whole-genome was performed using the neighbour‐joining method with 1000 bootstrap replicates in MEGA 7.2 software.

In the present study, a total of 498 intestinal tissue samples from 56 Tibetan pig farms were collected from three different provinces on the Qinghai-Tibet Plateau of China. The collected samples were then subjected to PCV3 detection by using conventional PCR. The results showed that positivity rate for molecular detection of PCV3 in Tibetan pigs was 17.68% (88/498) at the sample level and 25.00% (14/56) at the farm level. Detailed information on 88 samples that were positive for PCV3 is shown in Fig. 2 and Table 2. The PCV3 positivity rate at the sample level was 21.25% (44/207) in Gansu, 16.88% (27/160) in Qinghai and 12.98% (17/131) in Sichuan. The PCV3 positivity rate at the farm level were 25.00% (6/24) in Gansu, 18.75% (3/16) in Qinghai and 31.25% (5/16) in Sichuan. The prevalence of PCV3 in samples from pigs with diarrhoea was significantly higher than that in samples from healthy pigs (sample levels: 23.42% vs. 7.69%; farmer levels: 27.59% vs. 2.22%). The PCV3 positivity rate in Gansu was found to be significantly higher than that in Sichuan and Qinghai provinces (Table 2).

The entire Cap genes of PCV3 strains in 88 positive samples were subjected to sequencing and used to confirm the subtypes of PCV3 in Tibetan pigs. Twenty different nucleotide sequences of Cap proteins were obtained from 88 PCV3-positive DNA samples. Sequence analysis identified 20 different viral strains, the Cap proteins of which shared 95.84–99.18% nucleotide identitiy (Table 3). They also shared 97.51–98.75% nucleotide identity with Chinese PCV3 reference strains, and 96.34–98.91% nucleotide identity with PCV3 reference strains from other countries. Phylogenetic analysis based on twenty nucleotide sequences of Cap proteins demonstrated that the 20 PCV3 strains formed three clades, including PCV3a (8/20, 40.00%), PCV3b (5/20, 25%) and PCV3c (7/20, 35.00%) (Figs. 2, 3 and Table 2). For PCV3 strains from the same geographic origin, the strains in the PCV3a subtype were divided into the same branches at three levels, while the strains in the PCV3b and PCV3c subtypes were divided into different branches at three levels. In all three provinces on the Qinghai-Tibet Plateau of China, the positivity rates of PCV3a and PCV3b at both the sample and farm levels in the samples from diarrheic Tibetan pigs were significantly higher than those in healthy samples. The positive rates of PCV3c in samples collected from pigs with diarrhoea at the sample level were significantly lower than those in healthy samples. From the 46 PCV3a-positive samples, eight different Cap genes were obtained, of which seven were from samples from pigs with diarrhoea, and one was from healthy a sample (CN/GS/4/2019/MN848593 (Table 2), located in one independent branch at three levels in the phylogenetic tree based on the Cap gene. Five different Cap genes were identified in 25 PCV3b-positive samples, of which four of them were from diarrheic animals and one from a healthy animal (CN/SC/4/2019/MN848601), which was also located in one independent branch. Seven different Cap genes were identified in 17 PCV3c-positive samples, of which three were from 8 diarrheic animals, and four were identified in 9 healthy samples (CN/QH/5/2019/MN848604, CN/GS/7/2019/MN848606, CN/GS/8/2019/MN848607, CN/GS/9/2019/MN848608), and these genes were divided into different branches at three levels.

To identify amino acid substitutions in the Cap proteins of PCV3 strains isolated from Tibetan pigs, twenty deduced amino acid sequences of each genotype were selected and aligned with the reference sequence of the same genotype prevalent in China and obtained from GenBank. Amino acid sequence analysis of Cap proteins showed that the 20 different viral strains shared 87.86–99.52% identity (Table 3). As shown in Fig. 4, nine amino acid mutations were found in Cap proteins of PCV3a, and eight amino acid mutations were found in Cap proteins of the PVC3b and PVC3c subtypes. The amino acid substitutions in the Cap proteins occurred primarily in the 1–100 amino acid regions in PVC3a subtype (amino acid residues 8, 23, 54, 65, 79, 99). Mutations were found at sites 113, 169 and 210, while no mutation was observed at the site 113 in the strain obtained from healthy samples. In the PVC3b subtype, eight amino acid mutations were found sites 28, 79, 94, 96, 129, 164, 193 and 212, two mutation sites were in Loop DE region, while no mutation was observed at the site 129 in the strain obtained from healthy samples. There were three amino acid substitutions (7, 10 and 26) located in the functional domains of NLS among the PVC3c subtype strains in Tibetan pigs compared with the reference sequence. Two mutations (118 and 128) were found in the loop EF domain, and the other amino acid mutations were found at amino acid positions 101, 183 and 186. No mutation was observed at site 118 in the strains obtained from healthy samples.

To compare the complete genome sequences of twenty PCV3 strains found in Tibetan pigs, three amplified fragments from twenty PCV3 strains were sequenced and sequences information was submitted to the GenBank database under the accession numbers MN848590-MN848609. The PCV3 sequences isolated in our present study were used to construct a phylogenetic tree with sequences from 22 reference strains, obtained from GenBank, by the neighbour-joining method (Fig. 5). The whole genome sequence similarity ranged from 94.64 to 99.82% among the 20 different viral strains, and from 93.12 to 98.65% compared with reference sequences obtained from GenBank (Table 3). The complete genome sequence revealed that these strains formed one branch in the phylogenetic tree, and there were three subclusters found in the first-order branches (Fig. 5). However, there was crossover among different subtypes in some subclusters, PCV3a, PCV3b and PCV3a were in one subcluster, and PCV3b and PCV3a were in another subcluster. Only one subtype was found in the tertiary branches of the evolutionary tree (Fig. 5).