Isolation of Novel Goose Astrovirus, FJ-NP
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摘要:
目的 确定福建某鹅场雏鹅以痛风为主要病征导致高死亡率的病原。 方法 2019年8月福建省南平市某鹅场暴发一种以痛风为主要特征的疾病，该病在5～20日龄的雏鹅中发病率高达40%，死亡率为80%，对该场采集的3份样品进行实验室检测和病原分离鉴定。 结果 PCR检测结果显示3份样品新型鹅星状病毒均为阳性；细菌分离结果显示，未分离到细菌，排除细菌感染引起；接种11日龄鹅胚未出现死亡，但胚体全身皮肤出现点状出血，肾脏、肺脏均有出血点，对收集尿囊液和组织样品进行PCR检测结果显示只有新型鹅星状病毒阳性，未检测到其他鹅常见病毒，将其命名为FJ-NP；并对分离株部分ORF2基因进行遗传进化分析，结果表明该分离株与安徽（GD AHAU2、AHAU3、AHAU5）、黑龙江（AstV-Goose-2018-HLJ01）和河南（AstV-HN02-Goose-1119-18、AstV-AH02-Goose-0715-18、AstV-HB02-Goose-0310-19、AstV-HN03-Goose-0402-19）株在同一进化分支上，与湖北、北京、福建株亲缘关系较远。 结论 成功分离到了1株新型鹅星状病毒，同时进行ORF2部分基因遗传进化分析，为福建省后续开展新型鹅星状病毒相关研究提供了素材。 Abstract:Objective To determine a previously unknown pathogen that caused an outbreak of highly lethal disease in a geese farm. Method In August 2019, a disease characterized by gout broke out in a goose farm in Nanping City, Fujian Province. Three clinical samples from the diseased geese, which showed symptoms of the infectious disease characterized by gout with an incidence rate as high as 40% that resulted in a mortality rate of 80% on 5- to 20-d-old geese, were collected and tested in laboratory for pathogen identification. Result The PCR results confirmed the existence of novel goose astrovirus in all three samples, but no bacteria were isolated from medium culture. Inoculating the virus into the 11-d-old goose embryos did not induce death but showed dotted hemorrhage on the body skin and hemorrhage on the kidneys and lungs. The allantoic fluid and tissue specimens collected for PCR were tested positive as novel goose astrovirus, not other viruses commonly known to infect geese. The isolated virus was named FJ-NP, and its partial ORF2 genes showed a close relationship with the strains found in Anhui (i.e., GD AHAU2, AHAU3, and AHAU5), in Heilongjiang (i.e., AstV-Goose-2018-HLJ01) and in Henan (i.e., AstV-HN02-Goose-1119-18, AstV-AH02-Goose-0715-18, AstV-HB02-Goose-0310-19, and AstV-HN03-Goose-0402-19) but far from the Hubei, Beijing or Fujian strains. Conclusion This study successfully isolated a strain of novel goose astrovirus, and performed a genetic evolution analysis on its partial ORF2 genes, which provided material for further research on novel goose astrovirus in Fujian Province. -
Key words:
- Gout /
- novel goose astrovirus /
- isolation
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图 1 临床样品中新型鹅星状病毒RT-PCR检测
注：M为2 000 DNA Ladder，1为阴性对照，2为阳性对照，3~5为临床样品。
Figure 1. RT-PCR detection of novel goose astrovirus in clinical samples
Note: M=2 000 DNA ladder; 1=Negative control; 2=Positive control; 3-5=Clinical samples.
图 2 死亡鹅胚体变化
注：A、C为试验组；B、D为对照组
Figure 2. Pathologic changes in goose embryo
Note: A and C are experimental groups; B and D, control groups.
图 3 分离株新型鹅星状病毒RT-PCR检测结果
注：M为2000 DNA Ladder，1为新型鹅星状病毒，2~7为禽流感病毒、新城疫病毒、禽呼肠孤病毒、鹅细小病毒、鹅多瘤病毒及鹅圆环病毒，8为空白对照。
Figure 3. RT-PCR result on isolated strain of novel goose astrovirus
Note: M=2000 DNA ladder; 1=Goose astrovirus; 2-7=Avian influence virus (AIV), Newcastle disease virus (NDV), Avian orthoreovirus (ARV), Aoose parovovirus (GPV), Goose polyoma virus (GPoV), and Goose circovirus (GoCV); 8=blank control.
图 4 FJ-NP 株ORF2基因PCR扩增图
注：M为5000 DNA Ladder，1为FJ-NP，
Figure 4. RT-PCR amplification of FJ-NP ORF2 genes
Note: M=5000 DNA ladder; 1=FJ-NP.
图 5 ORF2
部分基因核苷酸同源性比较 注：红色方框为分离株
Figure 5. Homology of partial ORF2 gene nucleotide
Note: Red box indicates isolated strain.
图 6 ORF2部分基因遗传进化分析
注：红色三角形为分离株
Figure 6. Phylogenetic tree analysis based on partial ORF2 gene
Note: Red triangle indicates isolated strain.
表 1 鹅常见病毒引物
Table 1. Primers for common goose viruses
引物名称
Primers引物序列
Sequences（5′-3′）退火温度
Annealing temperature片段长度
Product sizeGPV-F TGTGGCAGCATCTGAAATGGCAGAG 60 ℃ 640 bp GPV-R GAGGTTGTTGGAGTGACACAATG AIV-F GCCATCCTAGCAACGACTGT 55 ℃ 1275 bp AIV-R CCTGAAGTGCCACAAAATACAA ARV-F AAGGATCATGGACGGATTACG 55 ℃ 681 bp ARV-R AGTCGATTAGGTACGATGCCA NDV-F ACYTGCGGAGYGTGAAAGTC 57 ℃ 584 bp NDV-R GTCGCCAAGAGGAGTGAGCARAGT GPoV-F GAGGTTGTTGGAGTGACACAATG 55 ℃ 580 bp GPoV-R ACAAACCTGCAATTCCAGGGTTC GoCV-F AGGGATCCTGAGTCTCGA 55 ℃ 452 bp GoCV-R ACAACCACATCTGCCCATAT 
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表 2 新型鹅星状病毒参考毒株
Table 2. Reference strains for novel goose astrovirus
毒株 Virus 登录号 Locus 分离地 Isolate 时间 Time AstV-Goose-2018-HLJ01 MN175321 China Heilongjiang 2018 AstV-AH01-Goose-0512-18 MN307115 China Henan 2018 AstV-AH02-Goose-0715-18 MN307116 China Henan 2018 AstV-HB01-Goose-0123-19 MN307118 China Henan 2019 AstV-HB02-Goose-0310-19 MN307119 China Henan 2019 AstV-HBXG-Goose-2019 MN894548 China Hubei 2019 AstV-HN01-Goose-0103-18 MN307114 China Henan 2018 AstV-HN02-Goose-1119-18 MN307117 China Henan 2018 AstV-HN03-Goose-0402-19 MN307120 China Henan 2019 AstV-SDPY-Goose-1116-17 MH052598 China Shandong 2017 AAstV-Goose-CHN-2017-SD01 MF772821 China Shandong 2017 AstV-Goose-CXZ-18 MH807626 China Shandong 2018 AHAU1 MN428641 China Anhui 2018 GD AHAU2 MN428642 China Anhui 2018 AHAU3 MN428643 China Anhui 2018 AHAU4 MN428644 China Anhui 2018 AHAU5 MN428645 China Anhui 2018 AHHF MN099162 China Anhui 2018 AHCZ2 MG882764 China Beijing 2018 AHCZ4 MG882765 China Beijing 2018 JSCZ4 MG882766 China Beijing 2018 XX MN337323 China Beijing 2018 SDXT MN399857 China Shandong 2019 JSHA MK125058 China Jiangsu 2018 AstV-HNZZ MT323163 China Shandong 2019 AstV-SDTA MN809622 China Shandong 2019 GTF-04 MN068023 China Fujian 2018 GTF-07 MN068024 China Fujian 2018 AstV-SDLC MT323162 China Shandong 2019 AstV-JSXZ MT323161 China Shandong 2020 GD MG934571 China Guangdong 2017 HN1G KY807085 China Beijing 2014
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