Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (24): 4978-4989.doi: 10.3864/j.issn.0578-1752.2024.24.012

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Isolation, Identification and Genetic Variation of a Three-Lineage Strain Recombined Porcine Reproductive and Respiratory Syndrome Virus

LI XinLei1(), SUN JiuYing1, YANG Cheng1, CHENG Ning1, WANG KaiYue1, WANG HuanHuan2, CHENG XueJiao3, ZHAO Jian3, SUN YingFeng1()   

  1. 1 College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384
    2 Tianjin Nongken Kangjia Ecological Breeding Co., Ltd, Tianjin 300384
    3 Tianjin Zhongsheng Challenge Biotechnology Co., Ltd, Tianjin 300380
  • Received:2024-05-06 Accepted:2024-10-28 Online:2024-12-16 Published:2024-12-23
  • Contact: SUN YingFeng

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Abstract:

【Objective】This study aimed to provide basic data for prevention and control of swine diseases in pig farms through analysis about molecular characterization of PRRSV isolated from the lungs of diseased piglets in pig farm in Tianjin. 【Method】Lung tissues of diseased piglets were collected, and PRRSV nucleic acid positive samples were inoculated into porcine alveolar macrophages (PAMs) for virus isolation and identification. The virus was purified by limited dilution method and identified by indirect immunofluorescence assay (IFA). Then, it was segmented amplification using overlapping primers of RT-PCR, and whole genome sequence was obtained by cloning. The nucleotide and amino acid sequences of N1 strain were analyzed for homology, genetic evolution, and recombination events using biological analysis software. 【Result】The PRRSV strain, named N1, was successfully isolated with the whole genome length of 15016 bp, excluding polyA tail. The homology analysis based on whole genome sequences showed that the homologous of N1 strain was 85.0%-90.0% to other lineage PRRSV strains, with the highest homology of 90.0% to lineage 8.3, the representative strain of JXA1, and the N1 isolate shared 93.7% identity with lineage 3 (QYYZ-like) in the ORF2a, ORF2b, and 3'UTR regions, and shared 96.2% identity with in the ORF5-ORF7 regions with lineage 1.8 (NADC30-like), while the rest of the regions had a high degree of homology with sublineage 8.3 (JXA1-like). It had similar 131 (111+1+19aa) discontinuous amino acid deletion pattern with lineage 1.8 (NADC30-like) in Nsp2-coding region. The virulence sites and major neutralizing antigenic epitopes of GP5 protein were identical to lineage 1 (NADC34-like, NADC30-like), but three N-glycosylation sites locating at N33, N44 and N51 were mutated to a certain extent compared with the other representative strains. These potential glycosylation sites were likely to contribute to the differences in viral virulence and led to the occurrence of viral immune escape. According to the genetic phylogenetic tree constructed based on ORF5 and whole genome sequences, the genetic evolution tree based on the ORF5 gene showed that N1 strain was closer to lineage 1.8 (NADC30-like) and belonged to the same branch, but N1 strain was in the same branch as lineage 8.3 (JXA1-like) strain based on whole genome genetic evolution tree, suggesting the possibility of this strain recombination. Recombination events of the N1 strain were analyzed by Simplot and RDP4.0 softwares, and the results of the recombination analyses showed that the N1 was a multiple recombinant virus from NADC30-, JXA1-, and QYYZ-Like strains with 13 recombination events and recombinant fragments located at A(1-421 nt), B(422-1 735 nt), C(736-3 598 nt), D(3 599-4 439 nt), E(4 440-5 762 nt), F(5 763-6 451 nt), G(6 502-7 226 nt), H(7 227-8 295 nt), I(8 296-9 312 nt), J(9 313-11 536 nt), K(11 537-12 854 nt), L(12 855-13 859 nt), M(13 860- 15 016 nt) with recombinant breakpoints in Nsp1, Nsp2, Nsp3, Nsp4, Nsp7, Nsp9, Nsp12, ORF3, and ORF5. 【Conclusion】The PRRSV N1 strain isolated in this experiment was a three-lineage recombinant strain of PRRSV with the sublineage 8.3 (JXA1-like) strain as its main parent, it exhibited extensive and complex recombination patterns between sublineage 1.8, lineage 3, and sublineage 8.3, and the results provided the reference for the comprehensive prevention and control of porcine reproductive and respiratory syndrome in Tianjin area.

Key words: PRRSV, isolation and identification, recombination, genetic evolution analysis

Table 1

Genome-wide sequence information of the PRRSV reference strain"

名称
Name		 登录号
Gene ID		 国别
Country		 年份
Year		 名称
Name		 登录号
Gene ID		 国别
Country		 年份
Year
APRRS GQ330474.2 China 2009 Prime Pac DQ779791.1 USA 2006
NADC34 MF326985 USA 2014 HK15 KF287142.1 China 2013
1483 KP998403.1 China 2015 QYYZ JQ308798.1 China 2011
NADC30 JN654459.1 USA 2008 SH1211 KF678434.1 China 2013
HNhx KX766379.1 China 2016 RespPRRS MLV AF066183.4 USA 2005
Q94-136 KP998421.1 China 2015 CH-1a AY032626.1 China 1996
MN184C EF488739.1 USA 2007 VR2332 U87392.3 USA 1992
GD-KP KU978619.1 China 2015 JXA1 EF112445.1 China 2006
TJ EU860248.1 China 2006

Fig. 1

Clinical samples RT-PCR amplification M: NormalRun 250 bp-I DNA ladder; 1: Positive material for disease; 2: Negative control"

Fig. 2

Isolation and identification of PRRSV strain N1 A: Normal PAMs cells; B: Cytopathy of PAMs; C: IFA assays for normal PAMs cells; D: IFA assay isolate N1 infected PAMs cells"

Fig. 3

N1 genome full-length segmented amplification M: NormalRun 250bp-I DNA ladder; 1-14: Amplification products of each fragment of the N1 genome"

Table 2

Nucleotide homology of the ORF region of isolate N1 with representative PRRSV strains"

项目
Project		 NADC34
Sublineage1.5		 NADC30
Sublineage1.8		 SH1211
Lineage2		 QYYZ
Lineage3		 VR2332
Lineage5		 ARRS
Lineage7		 JXA1
Sublineage8.3		 CH-1a
Sublineage8.1
全长 Total length 83.7% 87.1% 88.7% 85.0% 86.9% 86.1% 90.0% 88.8%
5′UTR 91.1% 89.5% 89.4% 91.1% 91.1% 89.5% 91.1% 91.1%
ORF1a 80.3% 85.7% 85.9% 81.4% 84.5% 83.5% 87.3% 86.0%
ORF1b 86.2% 88.6% 91.9% 88.2% 89.2% 89.0% 94.2% 92.3%
ORF2a 85.6% 84.8% 90.1% 89.0% 88.6% 85.7% 87.8% 88.5%
ORF2b 86.0% 90.5% 92.8% 93.7% 91.5% 90.1% 91.0% 91.4%
ORF3 82.6% 81.6% 95.5% 89.5% 88.5% 87.3% 97.4% 94.4%
ORF4 85.5% 84.9% 96.6% 94.4% 89.4% 90.9% 96.8% 95.9%
ORF5 86.9% 90.0% 85.1% 84.2% 84.9% 85.9% 85.1% 86.4%
ORF6 92.0% 94.1% 88.6% 87.6% 89.0% 89.1% 88.8% 87.8%
ORF7 93.8% 96.2% 94.9% 86.8% 92.7% 93.0% 90.1% 90.6%
3′UTR 92.1% 93.9% 92.7% 90.1% 94.7% 94.1% 89.5% 91.4%

Table 3

Amino acid homology of isolate N1 with representative PRRSV strains"

项目
Project		 NADC34
Sublineage1.5		 NADC30
Sublineage1.8		 SH1211
Lineage2		 QYYZ
Lineage3		 VR2332
Lineage5		 ARRS
Lineage7		 JXA1
Sublineage8.3		 CH-1a
Sublineage8.1
Nsp1 82.7% 83.5% 92.9% 86.4% 86.8% 86.9% 95.6% 92.0%
Nsp2 74.3% 84.6% 81.4% 75.7% 80.9% 79.3% 83.5% 81.0%
Nsp3 85.7% 90.2% 83.5% 82.6% 86.1% 85.9% 83.6% 84.9%
Nsp4 84.7% 83.7% 89.3% 82.3% 86.7% 84.0% 90.5% 88.8%
Nsp5 81.8% 87.7% 84.8% 80.2% 85.0% 85.4% 85.9% 88.0%
Nsp6 88.5% 90.4% 86.5% 84.6% 86.5% 88.5% 86.5% 84.6%
Nsp7 81.8% 83.8% 91.8% 89.4% 86.8% 85.5% 92.5% 90.8%
Nsp8 92.1% 90.1% 98.7% 95.4% 96.0% 95.4% 99.3% 100.0%
Nsp9 87.3% 89.7% 91.2% 88.3% 89.2% 88.8% 92.6% 91.6%
Nsp10 85.7% 84.6% 95.5% 90.8% 90.0% 90.2% 99.1% 95.2%
Nsp11 85.7% 90.1% 91.6% 87.2% 89.4% 89.4% 95.0% 93.1%
Nsp12 82.8% 92.4% 85.8% 82.8% 86.0% 85.4% 86.0% 86.0%
GP2a 84.5% 84.1% 90.1% 87.8% 87.5% 84.8% 88.4% 87.7%
GP2b 84.7% 90.5% 93.0% 93.4% 90.9% 93.0% 89.7% 90.5%
GP3 82.4% 81.2% 95.5% 90.0% 88.4% 87.2% 97.6% 94.5%
GP4 84.8% 84.3% 94.4% 92.7% 88.4% 89.9% 95.3% 94.3%
GP5 86.7% 89.3% 85.0% 83.8% 84.4% 85.3% 84.9% 85.8%
M 87.7% 94.2% 88.9% 87.7% 89.1% 89.3% 89.1% 88.0%
N 92.9% 95.6% 94.8% 85.7% 92.1% 92.4% 89.4% 89.4%

Table 4

Comparison of key amino acid sites of N1 strain GP5 protein with representative PRRSV strains"

毒株
Strain		 毒力位点
Virulence locus		 毒株鉴别位点
Strain identification site		 主要中和抗原表位
Major neutralizing antigenic epitopes
13 151 137 37 38 39 40 41 42 43 44 45
N1 Q K S S H L Q L I Y N L
NADC34
Sublineage1.5		 Q K S S H L Q L I Y N L
NADC30
Sublineage1.8		 Q K S S H L Q L I Y N L
QYYZ
Lineage3		 Q K S S Y S Q L I Y N L
VR2332
Lineage5		 R R A S H L Q L I Y N L
JXA1
Sublineage8.3		 R R S S H I Q L I Y N L

Fig. 4

Analysis of GP5 N-glycosylation sites"

Fig. 5

Phylogenetic tree of isolated strain N1 based on whole genome sequence and ORF5 gene A: Phylogenetic tree based on whole genome sequence; B: Phylogenetic tree based on ORF5 gene"

Fig. 6

Recombination analysis of N1"

Table 5

Recombination region analysis of N1"

重组区域
Reorganization region		 重组位点(nt)
Recombination site		 主要亲本
Major parent		 次要亲本
Minor parent		 P value
RDP BootScan MaxChi Chimaera SiScan Phylpro
A 1-421 JXA1 QYYZ 1.095×10-20 2.080×10-19 3.278×10-9 1.373×10-10 3.970×10-7 2.220×10-15
C 1736-3598 JXA1 NADC30 2.788×10-53 4.086×10-29 2.750×10-22 5.663×10-24 2.572×10- 31 1.110×10-15
E 4440-5762 JXA1 NADC30 1.992×10-29 2.616×10-26 2.940×10-6 5.158×10-27 3.269×10-16 3.330×10-15
G 6502-7226 JXA1 NADC30 1.950×10-16 6.263×10-18 8.705×10-22 6.763×10-16 1.982×10-4 3.330×10-15
I 8296-9312 JXA1 NADC30 6.450×10-33 1.764×10-32 1.784×10-18 2.944×10-20 2.522×10-14 4.440×10-15
K 11537-12854 JXA1 NADC30 1.447×10-18 1.273×10-18 4.880×10-25 4.469×10-17 4.652×10-3 5.804×10-11
M 13860-15016 JXA1 NADC30 5.238×10-27 2.180×10-27 5.437×10-17 2.184×10-6 8.484×10-13 3.330×10-15
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