Isolation, Identification, and Culture characteristics of a Genotype Ⅱ Goose Astrovirus
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摘要:
目的 明确鹅星状病毒（Goose astrovirus, GAstV）的致病性和体外培养特性。 方法 对广东某鹅场表现为内脏和关节痛风的发病鹅组织样品进行病原学检测、病毒分离鉴定、病毒基因序列测定与分析、病毒培养特性及致病性研究。 结果 从中分离到1株鹅星状病毒，命名为GD2208株；ORF2基因核苷酸序列比对分析显示，该毒株与基因Ⅱ型鹅星状病毒（GAstV-Ⅱ）参考株同源性最高，为97.4%～99.1%；进化树分析显示该毒株与GAstV-Ⅱ参考株处于同一进化分支。动物回归试验显示，以该毒株感染3日龄雏鹅后致死率为20%，感染鹅临床症状、剖检病变及病理组织学变化与自然发病鹅相似。禽胚及细胞适应性研究结果显示，该毒株能在鹅胚中稳定增殖，但盲传4代后仍无法适应SPF鸡胚和番鸭胚；病毒在GEK和LMH细胞中均能稳定增殖且可见明显细胞病变，在DF-1上可稳定繁殖，但盲传至8代后仍未见细胞病变。 结论 本研究自表现痛风症状雏鹅中分离到1株GAstV-Ⅱ毒株，该毒株感染3 日龄雏鹅可发生典型痛风症状，对鹅胚、GEK、LMH和DF-1细胞均有一定适应性，以上结果为进一步开展GAstV的致病机制、疫苗和药物研发奠定了基础。 Abstract:Objective To clarify the pathogenicity and culture characteristics of Goose astrovirus(GAstV). Methods Disease diagnosis and pathogen isolation, identification, gene sequencing, culture characteristics , and pathogenicity on a goose at a farm in Guangdong showing visceral and gout symptoms were conducted. Results The Goose astrovirus (GAstV) isolated from the tissue sample was named GD2208. It showed the highest homology of 97.4%–99.1% with the reference strain GAstV-Ⅱ based on the ORF2 gene nucleotide sequence. They belonged to the same evolutionary branch on the evolutionary tree. In an animal regression test, the chicks inoculated with GD2208 displayed similar clinical symptoms as well as the pathological and histopathological changes observed on the naturally infected 3-day-old goose and had a 20% mortality rate. The isolate could proliferate in goose embryos, but unable to adapt to SPF chicken or Muscovy duck embryos after 4 generations of blind transmission. It could also grow in GEK and LMH cells with the apparent cytopathic effects or in DF-1 cells without the effects after 8 generations of blind transmission. Conclusion A GAstV-Ⅱ strain isolated from a diseased goose when inoculated in healthy chicks produced similar symptoms on the birds, and it could grow in goose embryos, GEK, LMH, and DF-1 cells. -
Key words:
- Goose astrovirus /
- isolation /
- identification /
- cultural characteristics
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图 1 临床样品的（RT-）PCR检测
M：DL2000 Marker；1：鹅星状病毒阳性对照；2：阴性对照；3：心脏；4：肝脏；5：脾脏；6：胰腺；7：肾脏；8～14：AIV、GCV、GPV、ARV、GHPV、TMUV、FAdV。
Figure 1. RT-PCR detection on clinical samples
M: DL2000 marker; 1: GAstV positive control; 2: negative control; 3: heart; 4: liver; 5: spleen; 6: pancreas; 7: kidney; 8–14: AIV, GCV, GPV, ARV, GHPV, TMUV, and FAdV, respectively.
图 2 分离病毒的鹅胚
P：死亡鹅胚，C：正常鹅胚。
Figure 2. Healthy and GAstV-infected goose embryos
P: infected goose embryo; C: healthy goose embryo.
图 3 第三代（F3）鹅胚尿囊液RT-PCR检测
M：DL2000 Marker；1：鹅星状病毒阳性对照；2：阴性对照；3：第三代鹅胚尿囊液。
Figure 3. RT-PCR detection on embryo allantoic fluid of F3 goose
M: DL2000 marker; 1: GAstV positive control; 2: negative control; 3: embryo allantoic fluid of 3rd generation goose.
图 4 分离病毒核酸的PCR扩增结果
M：DNA 2000 Marker；1：阴性对照；2：ORF2-2；3：ORF2-1。
Figure 4. PCR amplification of nucleic acids of GAstV
M: DNA 2000 marker; 1: negative control; 2: ORF2-2; 3: ORF2-1.
图 7 F4代禽胚尿囊液RT-PCR检测结果
M：DL2000 Marker；1：GAstV阳性对照；2：阴性对照；3～5：鹅胚尿囊液；6～8：番鸭胚尿囊液；9～11：SPF鸡胚尿囊液。
Figure 7. RT-PCR results on avian embryo allantoic fluid of F4 goose
M: DL2000 marker; 1: GAstV positive control; 2: negative control; 3–5: goose embryo allantoic fluid; 6–8: Muscovy duck embryo allantoic fluid; 9–11: SPF chicken embryo allantoic fluid.
图 8 GAstV接种鹅胚F1～F4代的病毒滴度
Figure 8. Viral titers of F1–F4 GAstV-inoculated goose embryos
图 9 鹅星状病毒接种GEK、LMH细胞
A：病毒感染LMH细胞（200×）； B：LMH正常细胞（200×）；C：病毒感染GEK细胞（200×）；D：GEK正常细胞（200×）。
Figure 9. GEK and LMH cells inoculated with GAstV
A: virus infected LMH cells (200×); B: normal LMH cells (200×); C: virus infected GEK cells (200×); D: normal GEK cells (200×).
图 10 病毒接种不同细胞后F2、F4、F6、F8代的病毒滴度
Figure 10. Viral titers of F2, F4, F6, and F8 GAstV-inoculated cells
图 11 人工感染鹅临床症状及剖检病变
A：感染鹅，精神萎靡、蹲伏、腹泻、嗜睡、聚集；B：感染鹅，腿部关节肿胀、无法站立和行走；C：心脏；D：脾脏；E：肾脏；F：腿部关节。
Figure 11. Clinical symptoms and autopsy lesions of GAstV-infected geese
A: infected geese showing depression, crouching, diarrhea, lethargy, and aggregating; B: infected geese with swollen leg joints, inability to stand and walk; C: heart; D: spleen; E: kidney; F: leg joints.
图 12 动物回归试验主要器官的病理组织切片（HE）
A：心脏（200×）； B：肝脏（200×）；C：脾脏（200×）；D：肺脏（200×）；E：肾脏（200×）；F：腿部关节（200×）。
Figure 12. Pathological tissue sections (HE) of organs from animal regression test
A: Heart (200×); B: liver (200×); C: spleen (200×); D: lungs (200×); E: kidney (200×); F: leg joints (200×).
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