Isolation, Identification, and S1 Gene Sequencing of Two Strains of Avian Infectious Bronchitis Virus
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摘要:
目的 了解福建地区鸡传染性支气管炎病毒（Infectious bronchitis virus，IBV）的流行及其S1基因变异情况。 方法 对2021年在福建地区鸡群中分离的2株病毒，通过鸡胚致病性试验、电子显微镜观察及RT-PCR方法进行鉴定。对2株分离株的S1基因进行克隆、测序并利用生物学软件进行分析。 结果 分离到的2株病毒均为IBV，分别命名为FJ-NP01和FJ-FZ01；FJ-NP01株和FJ-FZ01株的S1基因全长分别为1629 nt和1620 nt，编码543 aa和540 aa。FJ-FZ01株的 S1基因裂解位点为HRRRR，与基因型I分支所有参考毒株裂解位点一致；分离株FJ-NP01的 S1基因裂解位点为HRRKR，与已报道的基因型ⅣIBV分离毒株的裂解位点不同，与基因型Ⅵ参考毒株TC07-2的裂解位点一致。FJ-NP01株和FJ-FZ01株之间的核苷酸和氨基酸同源性较低，分别为83.2%和79.6%；与中国使用的Mass型常规疫苗H120和H52核苷酸和氨基酸序列同源性仅为75.7%～76.3%和77.1%～83.5%。FJ-NP01株是由基因型Ⅳ毒株CK CH GD LZ12-4与基因型I毒株L-1148在S1基因处发生重组而产生的新毒株，其核苷酸序列1438～1506 nt与推测的亲本毒株CK CH GD LZ12-4同源性达97%，其他S1基因核苷酸序列与推测的亲本毒株L-1148同源性达95.9%。 结论 以上结果表明福建地区IBV流行较为复杂，现有Mass型疫苗在福建省可能起不到良好的免疫保护作用。 -
关键词:
- 鸡传染性支气管炎病毒 /
- 分离 /
- 鉴定 /
- S1基因 /
- 序列分析
Abstract:Objective Epidemiology and genetic variations of the infectious bronchitis virus (IBV) in Fujian province were studied. Method Two strains of virus isolated from the diseased chickens in Fujian in 2021 were identified by chicken embryo pathogenicity test, electron microscope observation, and RT-PCR. S1 genes of the isolates were cloned, sequenced, and analyzed using biological software. Result The two IBV strains were code named FJ-NP01 and FJ-FZ01. The full length of S1 of FJ-NP01 was 1629 nt encoding 543 amino acids, and that of FJ-FZ01, 1620 nt encoding 540 amino acids. The S1 gene cleavage site of FJ-FZ01 was HRRRR, same as all reference strains of genotype I branch; while that of FJ-NP01 HRRKR differed from the reported site of IBV isolated from genotype Ⅳ but same as that of TC07-2 reference strain of genotype Ⅵ. The homology of nucleotide and amino acid between the two isolates was 83.2% and 79.6%, respectively, but merely 75.7%–76.3% and 77.1%–83.5% with the Mass-type conventional vaccines H120 and H52, respectively. Further analysis showed that FJ-NP01 was from a recombination event between CK CH GD LZ12-4 and L-1148, the homology of nucleotide acid between 1438–1506 nt of FJ-NP01 with CK CH GD LZ12-4 was 97%, and 95.9% between the other nucleotide acid of S1 gene with L-1148 . Conclusion It appeared that the IBV epidemic experienced in the province was complex in nature and that the existing Mass vaccines would not provide sufficient immune protection to deter the spread. -
Key words:
- Infectious bronchitis virus /
- isolation /
- identification /
- S1 genes /
- sequence analysis
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图 3 分离株的鉴定
M为2000 DNA Ladder，1为阳性对照，2为阴性对照，3为FJ-FZ01，4为FJ-NP01。
Figure 3. Identification of isolates
M: 2000 DNA ladder; 1: positive control; 2: negative control; 3: FJ-FZ01; 4: FJ-NP01.
图 4 2株分离株S1 基因RT-PCR扩增电泳图
M为2000 DNA Ladder，1为阳性对照，2为FJ-FZ01，3为FJ-NP01，4为阴性对照。
Figure 4. RT-PCR amplification electrophoresis of S1 of two isolates
M: 2000 DNA ladder; 1: positive control; 2: FJ-FZ01; 3: FJ-NP01; 4: negative control.
图 5 IBV分离株与参考毒株S1基因进化树
Figure 5. Phylogenetic tree constructed based on S1 from IBV isolates and reference strains
表 1 IBV参考毒株
Table 1. IBV reference strains
毒株 Virus 登录号 Locus 毒株 Virus 登录号 Locus Ma5
H120
LDT3
4/91
H52
28/86
M41
G13-1
Holte
UK/7/93
Italy-02
TW2575/98
3468/07
JP9758
TA03
GX-NN-6
TC07-2
PSH050513
QXIBVAY561713
FJ888351
AY702975
KF377577
EU817497
AY846750
DQ834384
L14069
L18988
Z83979
AJ457137
DQ646405
EU822336
AY296746
AY837465
JX291985
GQ265948
DQ160004
AF193423ZJ971
L-1148
A2
LX4
HB08
HN08
CK/CH/GD/HY09
CK/CH/HN/HN09
CKCHJSLYG1911-8
CKCHJSLYG1911-10
CKCHJSLYG1911-11
CK/CH/LGX/091109
CK/CH/LDL/07II
X
CK/CH/LCQ/08II
ck/CH/LGX/091110
CKCHJSLYG1912-1
CK CH GD LZ12-4AF352313
DQ431199
AY043312
AY338732
GQ265934
GQ265940
HQ018887
HQ018886
MW044574
MW044575
MW044583
KF411041
EU563940
FJ8298882
GQ258305
HM194643
MW044590
KC692277
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表 2 RDP 4. 101软件分析 IBV 分离株的 S1 基因遗传重组结果
Table 2. Recombinant results of S1 of IBV isolate analyzed by RDP 4. 101 software
重组
Recombinant破裂点
Break points主要亲本
Major parent次要亲本
Minor parentP值
P value毒株
Strain基因型
Genotype头端
Begin/nt末端
End/nt命名
Name基因型
Genotype同源性
Homology/%命名
Name基因型
Genotype同源性
Homology/%FJ-NP01 Ⅳ 1438 1506 CK CH GD LZ12-4 Ⅳ 97% L-1148 I 95.9 6.974×10−8
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