Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (3): 613-626.doi: 10.3864/j.issn.0578-1752.2024.03.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Molecular Epidemic Characteristics and Genetic Variation Analysis of Porcine Circovirus 3 in Some Pig Farms in Central China from 2020 to 2022

LI BOTIAN1(), LI CHUNQI1(), LIU GUOPING1(), XIE JUN2(), ZENG PAN1, ZHAO RUNZE1, LI TONG1, PEI JIE2, GUO LIWEI1, WU RUI3, TAN LEI1   

  1. 1 College of Animal Science and Technology, Yangtze University, Jingzhou 434020 Hubei
    2 Hubei Provincial Center for Animal Disease Control and Prevention, Wuhan 430000
    3 College of Veterinary Medcine, Sichuan Agricultural University, Chengdu 610000
  • Received:2023-10-27 Accepted:2023-12-25 Online:2024-02-01 Published:2024-02-05

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Abstract:

【Objective】 The epidemiological and genetic variation characteristics of porcine circovirus 3 in some parts of central China were investigated through a systematic experimental protocol, which provided the data of basis for the research of PCV3 vaccine。【Methods】 A total of 3 500 clinical samples collected from 15 large-scale pig farms in central China (Hubei, Hunan and Henan) were detected by real-time PCR, and the relationship between different pig herds, different tissues and organs, different amount of detoxification and the characteristics of corresponding epidemiological symptoms was analyzed. The whole genome of PCV3 for some positive samples was amplified, sequenced and analyzed.【Result】 50.86% (1 780/3 500) of the tested samples were positive for PCV3 nucleic acid, pigs at different ages were susceptible to PCV3, and the positive rates of PCV3 were higher in nursing pigs, suckling piglets and growing finishing, which were 70.44% (498/707), 67.19% (596/887) and 41.75% (177/424), respectively. The positive rates of PCV3 in lymph nodes, lung, and postpartum placenta samples were 67.05% (59/88), 63.79% (74/116), and 49.11% (55/112), while the positive rates of PCV3 in blood and colostrum samples were 56.09% (502/895) and 44.20% (278/629), respectively, and PCV3 was detected in nasal swabs and saliva. The positive rates of PCV3 was high in the pig with symptoms of reproductive disorder, which were 44.43% (399/898), 36.43% (431/1183), and 28.04% (233/831), respectively. The length of the whole genome sequence of the 24 PCV3 was 2 000 nt, and the homology of the whole genome nucleotide sequence among the 24 sample strains was 98.4%-100%, and the homology of the whole genome with the reference strain PCV3 at home and abroad was 97.4%-99.5%. Genetic evolution analysis showed that 23 strains of PCV3 belonged to the PCV3b subtype and 1 strain belonged to the PCV3a subtype. The results of Rep amino acid sequence alignment showed that PCV3-L2, L23 and L14 strains had unique variation sites in (N124I), (A183E) and (V244I), respectively. The results of Cap protein amino acid sequence alignment showed that PCV3-L15, L21, L3 and L19 strains had unique variation sites in (T45P), (R2K), (R14K) and (F7L), respectively.【Conclusion】 PCV3 can infect pigs at different ages and is distributed in different tissues and organs. PCV3 can infect pigs through vertical transmission (such as colostrum and semen) and horizontal transmission (such as oral and nasal secretions and saliva). PCV3 infection may be closely related to reproductive disorders, respiratory disorders, multiple organ inflammation and arthritis in pigs. In addition, both PCV3a and PCV3b subtypes were prevalent in large-scale pig farms in the surveyed area, and the PCV3b subtype was the dominant strain. In this study, genome-wide amino acid sequence analysis revealed that some unique variation sites were located in Cap protein, which may lead to changes in the immunogenicity conferred by Cap protein. In this study, the genetic evolution of the whole gene sequence of PCV3 is elaborated, aming to lay a foundation for future research on PCV3 vaccines.

Key words: porcine circovirus 3, viral load, whole genome amplification, genetic evolution analysis

Table 1

Information of primers"

引物名称 Primer name 引物序列（5'-3'）Sequence (5'-3') PCR 产物大小 Size of PCR product (bp)
PCV3-1IF TAGTATTACCCGGCACCTCGG 816
PCV3-1IR GCACCCTCCCCATTATAACCA
PCV3-2IF CGGCTGCGCGGGAATTGAAGCTGT 491
PCV3-2IR GGTAAACGCCCTCGCATGTGGGGA
PCV3-3IF TGTCGCCCCCACCGAAGCATG 836
PCV3-3IR GCCCGGCACCAAAATGAGACA
PCV3F CACCCAGGACAAAGCCTCTT 89
PCV3R AAGCAGTGCTCCCCATTGAA

Table 2

Information of PCV3 reference strains"

序号
Number		 毒株名称
Strain name		 登录号
Accession number		 地区
Location		 基因型
Gene type		 长度
Length (bp)
1 PCV3/KU-1605 KY996341.1 韩国South Korea b 2000
2 PCV3-BR/RS/8 MF079254.1 巴西Brazil b 2000
3 PCV3-Shandong-01 MH107161.1 中国China b 2000
4 PCV3-China/JL17-42 MG870095.1 中国China b 2000
5 PCV3/CN/Chongqing-148 KY075991.1 中国China b 2000
6 PCV3/CN/Chongqing-147 KY075990.1 中国China b 2000
7 PCV3/CN/Qinghai/2016 MG372486.1 中国China b 2000
8 PCV3/CH/HB/HD/2017 MH101645.1 中国China b 2000
9 PCV3 strain 2164 KX458235.1 美国America b 2000
10 PCV3-AH-201706 MG868946.1 中国China b 2000
11 PCV3-US/MO2015 KX778720.1 美国America a 2000
12 CN/Liaoning-2017 MH177453.1 中国China a 2000
13 PCV3/KU-1601 KY996337.1 韩国South Korea a 2000
14 PCV3-BR/RS/6 MF079253.1 巴西Brazil a 2000
15 PCV3-BJ-1 MF318453.1 中国China a 2000
16 Hainan/2017 MG253684.1 中国China a 2000
17 CN/FuJian-1215-2016 KY924474.1 中国China a 2000
18 CN/FuJian-820-2016 KY924473.1 中国China a 2000
19 CN/FuJian-318-2017 KY924475.1 中国China a 2000
20 PCV3-Chian/GX2016-2 MF155642.1 中国China a 2000
21 CN/Fujian-12/2016 KY075987.1 中国China a 2000

Table 3

Information of Positive sample"

不同猪场 Different pig farm PCV3阳性数(份) Number of positives 样本数(份) Number of samples PCV3阳性率 Positive rate
湖北
Hubei		 武汉Wuhan 169 330 51.21%
襄阳Xiangyang 107 228 46.93%
荆州Jingzhou 97 199 48.74%
恩施Enshi 148 265 55.85%
鄂州Ezhou 107 212 50.47%
随州Suizhou 252 398 63.32%
湖南
Hunan		 常德Changde 203 320 63.44%
湘潭Xiangtan 116 286 40.56%
益阳 Yiyang 50 104 48.08%
湘潭Xiangtan 91 157 57.96%
永州Yongzhou 121 241 50.21%
河南
Henan		 信阳Xinyang 112 193 58.03%
兰考县Lankao 96 192 50.00%
开封Kaifeng 101 185 54.59%
新乡Xinxiang 106 190 55.79%
总计 Total 1876 3500 53.60%

Fig. 1

Viral load map A: Standard curve; B: viral load map of different stages; C: viral load map of different parts; D: Virus load map of different samples; E: viral load map of different symptoms"

Table 4

Information of Positive sample"

不同阶段 Different stages PCV3阳性数(份) Number of positives 样本数(份) Number of samples PCV3阳性率 Positive rate
头胎母猪 Firstborn sow 114 366 31.15%
经产母猪Multiparous sows 243 675 36.00%
哺乳仔猪 Sucking pig 596 887 67.19%
保育猪 Nursery pig 498 707 70.44%
生长育肥猪 Growing-finishing pig 177 424 41.75%
种公猪 Boar 103 441 23.36%
总计 Total 1780 3500 50.86%

Table 5

Information of Positive sample"

不同器官 Uneven organs 样品数(份) Number of samples PCV3阳性数(份) Number of positives PCV3阳性率 Positive rate
心脏 Heart 130 29 22.31%
肝脏 Liver 124 53 42.74%
脾脏 Spleen 84 20 23.81%
肺脏 Lungs 116 74 63.79%
肾脏 Kidney 195 77 39.49%
淋巴结 Lymph node 88 59 67.05%
胃 Stomach 100 36 36.00%
产后胎盘 Placenta 112 55 49.11%
总计 Total 949 403 22.31%

Table 6

Information of Positive sample"

样品类型 Sample type 样品数(份) Number of samples PCV3阳性数(份) Number of positives PCV3阳性率 Positive rate
血液 Blood 895 502 56.09%
精液 Semen 658 206 31.31%
初乳 Colostrum 629 278 44.20%
外阴拭子 Vulvar swab 287 108 37.63%
鼻拭子 Nasal swab 431 112 25.99%
唾液 Saliva 400 93 23.25%
总计 Total 3300 1299 39.36%

Table 7

Information of Positive sample"

症状 Symptoms 样品数(份) Number of samples PCV3阳性数(份) Number of positives PCV3阳性率 Positive rate
不吃 Skip 400 119 29.75%
少吃 Eat less 327 170 51.99%
未吃完 Not finished 456 142 31.14%
咳 Cough 831 233 28.04%
跛腿 Limp 102 25 24.51%
流产 Abortion 477 198 41.51%
早产 Premature birth 272 126 46.32%
产木乃伊Mummification 149 75 50.34%
腹泻 Diarrhea 83 4 4.82%
小猪发红 Sow flush 68 22 32.35%
死亡 Death 42 7 16.67%
总计 Total 3207 1121 34.95%

Fig. 2

PCV3 Establishment results of fluorescence quantitative method A: PCV3 qPCR amplification curve; B: Specific detection results; C: melting curve; D: Standard Curve"

Fig. 3

PCR amplification results of PCV3 positive sample M: DL2000 DNA Marker; 1: PCR product of P1 primer (816 bp); 2: PCR product of P2 primer (419 bp); 3: PCR product of P3 primer (836 bp); -: Negative control"

Fig. 4

PCV3 whole-genome, ORF1 and ORF2 genetic evolution trees A: ●The genome sequences of PCV3 obtained in this study; ◇The genome sequences of PCV3 GenkBank outside China; B: ●The sequences of PCV3 ORF1 obtained in this study; ◇The sequences of PCV3 ORF1 GenkBank outside China; C: ●The sequences of PCV3 ORF2 obtained in this study; ◇The sequences of PCV3 ORF2 GenkBank outside China"

Fig. 5

Amino acid sequence alignment of Rep protein between 24 PCV3 strains and 6 reference strain"

Fig. 6

Amino acid sequence alignment of Cap protein between 24 PCV3 strains and 6 reference strain"

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