鸭甲肝病毒基因3型弱毒株与其返强毒株的生物学特性比较

doi:10.16431/j.cnki.1671-7236.2025.09.026

摘要/Abstract

摘要： 【目的】探究鸭甲肝病毒基因3型(Duck hepatitis A virus genotype 3,DHAV-3)弱毒株毒力返强的机制，通过雏鸭试验和基因组序列分析对DHAV-3弱毒株与其返强毒株的生物学特性进行比较。【方法】用DHAV-3分离株YDF的第140代鸡胚化弱毒株Y140及其第4代雏鸭传代毒株Y140/4R接种1日龄北京鸭，观察临床症状、病理变化、发病率和死亡率，用实时荧光定量PCR检测雏鸭肝脏、肾脏和血清中的病毒RNA水平。对Y140株及其第1～4代雏鸭传代病毒基因组序列进行测序分析，探究与毒力返强有关的分子机制。【结果】接种Y140株的雏鸭均未出现临床症状和病理变化，接种Y140/4R株的雏鸭出现鸭病毒性肝炎的特征性临床症状和大体病变，死亡率为40%。与Y140株相比，Y140/4R株感染后3 d存活鸭肝脏和肾脏中病毒RNA水平显著升高(P＜0.05)，感染后2～3 d死亡鸭肝脏和肾脏中病毒RNA水平极显著升高(P＜0.01)。感染后2～4 d，Y140/4R株产生的病毒血症水平显著高于Y140株(P＜0.05)。基因组测序结果显示，从第2代开始，2C、3D蛋白和3'-非翻译区分别发生I149V、V236I和A25G突变；从第3代开始，VP1蛋白发生E202D突变。【结论】雏鸭传代可驱动DHAV-3弱毒株基因组发生突变，导致毒力变异株出现，使病毒在靶器官的复制能力以及形成病毒血症的能力显著提高。

关键词: 鸭甲肝病毒基因3型(DHAV-3); 弱毒株; 毒力返强; 复制; 病毒血症

Abstract: 【Objective】 The experiment was to explore the mechanism of virulence reversion of Duck hepatitis A virus genotype 3 (DHAV-3) attenuated strain.The biological characteristics of the DHAV-3 attenuated strain and its revertant strain were compared through ducklings experiments and genomic sequence analysis. 【Method】 One-day-old Pekin ducklings were inoculated with DHAV-3 attenuated strain Y140,derived by 140 passages of isolate YDF in chicken embryos,and its revertant strain Y140/4R,derived by 4 passages in duckings,and monitored for clinical signs,gross lesions,morbidity and mortality.Viral RNA levels in liver,kidney and serum of ducklings were measured by Real-time quantitative PCR.Sequencing and analysis were conducted on the genomic sequences of the Y140 strain and the first to fourth passage viruses of Y140 to explore the molecular mechanisms related to virulence reactivation. 【Result】 Ducklings inoculated with Y140 strain showed no clinical symptoms or pathological changes.Ducklings inoculated with Y140/4R strain presented the characteristic clinical symptoms and gross lesions of duck viral hepatitis,and the mortality was 40%.Compared with Y140 strain,the viral RNA levels in liver and kidney of surviving ducks 3 days after infection with Y140/4R strain were significantly increased (P＜0.05),and the viral RNA levels in liver and kidney of dead ducks 2-3 days after infection were extremely significantly increased (P＜0.01).2-4 days after infection,the viremia level produced by Y140/4R strain was significantly higher than that of Y140 strain (P＜0.05). The genomic sequencing results showed that starting from the second generation,mutations I149V,V236I and A25G occurred in 2C protein,3D protein and 3'-UTR,respectively.Starting from the third generation,the E202D mutation occurred in VP1 protein. 【Conclusion】 Passage of duckings could drive mutations in the genome of DHAV-3 attenuated strain，leading to the emergence of virulence variants and significantly enhancing the replication ability of virus in target organs and the ability to form viremia.

Key words: Duck hepatitis A virus genotype 3 (DHAV-3); attenuated strain; reversion to virulence; replication; viremia

中图分类号:

S834

胡晓阳, 黄晶晶, 彭铎, 张大丙. 鸭甲肝病毒基因3型弱毒株与其返强毒株的生物学特性比较[J]. 中国畜牧兽医, 2025, 52(9): 4284-4290.

HU Xiaoyang, HUANG Jingjing, PENG Duo, ZHANG Dabing. Comparison of Biological Properties of Duck Hepatitis A Virus Genotype 3 Attenuated Strain and Its Reversion Strain[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(9): 4284-4290.

参考文献

[1] DAVID E S.Diseases of Poultry (14th Ed)[M].New Jersey:John Wiley & Sons，Incorporated,2020.
[2] LEVINE P P，FABRICANT J.A Hitherto-undescribed virus disease of ducks in North America[J].The Cornell Veterinarian,1950,40(1):71-86.
[3] TSENG C H,KNOWLES N J,TSAI H J.Molecular analysis of Duck hepatitis virus type 1 indicates that it should be assigned to a new genus[J].Virus Research，2007,123(2):190-203.
[4] TSENG C H,TSAI H J.Molecular characterization of a new serotype of Duck hepatitis virus[J].Virus Research,2007,126(1-2):19-31.
[5] KIM M C,KWON Y K,JOH S J,et al.Recent Korean isolates of Duck hepatitis virus reveal the presence of a new geno- and serotype when compared to Duck hepatitis virus type 1 type strains[J].Archives of Virology,2007,152(11):2059-2072.
[6] WANG L Y,PAN M,FU Y,et al.Classification of Duck hepatitis virus into three genotypes based on molecular evolutionary analysis[J].Virus Genes,2008,37:52-59.
[7] FU Y,PAN M,WANG X Y,et al.Molecular detection and typing of Duck hepatitis A virus directly from clinical specimens[J].Veterinary Microbiology,2008,131(3-4):247-257.
[8] ASPLIN F D.Duck hepatitis:Vaccination against two serological types[J].The Veterinary Record,1965,77(50):1529-1530.
[9] HAIDER S A,CALNEK B W.In vitro isolation,propagation,and characterization of Duck hepatitis virus type Ⅲ[J].Avian Diseases,1979,23(3):715-729.
[10] GOUGH R E,BORLAND E D,KEYMER I F,et al.An outbreak of Duck hepatitis type Ⅱ in commercial ducks[J].Avian Pathology,1985,14(2):227-236.
[11] GOUGH R E,COLLINS M S,BORLAND E,et al.Astrovirus-like particles associated with hepatitis in ducklings[J].The Veterinary Record,1984,114(11):279.
[12] TODD D,SMYTH V J,BALL N W,et al.Identification of Chicken enterovirus-like viruses,Duck hepatitis virus type 2 and Duck hepatitis virus type 3 as Astroviruses[J].Avian Pathology,2009,38:21-30.
[13] FU Y,PAN M,WANG X Y,et al.Complete sequence of a Duck astrovirus associated with fatal hepatitis in ducklings[J].The Journal of General Virology,2009,90(5):1104-1108.
[14] LIU N,WANG F M,SHI J J,et al.Molecular characterization of a Duck hepatitis virus 3-like Astrovirus[J].Veterinary Microbiology,2014,170(1-2):39-47.
[15] DOAN H T,LE X T,DO R T,et al.Molecular genotyping of Duck hepatitis A viruses (DHAV) in Vietnam[J]. Journal of Infection in Developing Countries,2016,10(9):988-995.
[16] HASSAN T I R,EID A A M,GHANEM I A I,et al.First report of Duck hepatitis A virus 3 from duckling flocks of Egypt[J].Avian Diseases,2020,64(3):269-276.
[17] YEHIA N,ERFAN A M,OMAR S E,et al.Dual circulation of Duck hepatitis A virus genotypes 1 and 3 in Egypt[J].Avian Diseases,2021,65(1):1-9.
[18] KIM M C,KIM M J,KWON Y K,et al.Development of Duck hepatitis A virus type 3 vaccine and its use to protect ducklings against infections[J].Vaccine,2009,27(48):6688-6694.
[19] 李子恒.鸭甲肝病毒基因3型弱毒疫苗候选株毒力稳定性评估[D].北京:中国农业大学,2019. LI Z H.Assessment of virulence stability of Duck hepatitis A virus genotype 3 attenuated vaccine candidate[D].Beijing:China Agriculture University,2019.(in Chinese)
[20] 杨云川,刘锦琳,刘泓邑,等.DHAV-3适应鸡胚毒株的培育及特性测定[J].中国兽医学报,2023,43(1):41-48. YANG Y C,LIU J L,LIU H Y,et al.Development and characterization evaluation of chicken embryo-adapted strain of Duck hepatitis A virus serotype 3[J].Chinese Journal of Veterinary Science,2023,43(1):41-48.(in Chinese)
[21] WANG M H,LI Z H,LIU H C,et al.Effect of fetal calf serum on propagation of Duck hepatitis A virus genotype 3 in duck embryo fibroblast cells[J].BMC Veterinary Research,2019,15(1):153.
[22] 谢小雨.1株3型鸭甲肝病毒鸡胚化弱毒株的培育[D].北京:中国农业大学,2011. XIE X Y.Development of an attenuated Duck hepatitis A virus type 3 by passaged in SPF chicken embryos[D].Beijing:China Agriculture University,2011.(in Chinese)
[23] YANG W X,TERASAKI T,SHIROKI K,et al.Efficient delivery of circulating Poliovirus to the central nervous system independently of Poliovirus receptor[J].Virology,1997,229(2):421-428.
[24] KOTECHA A,WANG Q,DONG X,et al.Rules of engagement between αvβ6 integrin and Foot-and-mouth disease virus[J].Nature Communications，2017,8:15408.
[25] REN J,WANG X,HU Z,et al.Picornavirus uncoating intermediate captured in atomic detail[J].Nature Communications，2013,4:1929.
[26] WAHID R,MERCER L,GAST C,et al.Evaluating stability of attenuated Sabin and two novel type 2 oral Poliovirus vaccines in children[J].NPJ Vaccines，2022,7(1):19.
[27] EMERSON S U,HUANG Y K,NGUYEN H,et al.Identification of VP1/2A and 2C as virulence genes of Hepatitis A virus and demonstration of genetic instability of 2C[J].Journal of Virology，2002,76(17):8551-8559.
[28] CAINE E A,MONCLA L H,RONDEROS M D,et al.A single mutation in the VP1 of Enterovirus 71 is responsible for increased virulence and neurotropism in adult interferon-deficient mice[J].Journal of Virology，2016,90(19):8592-8604.
[29] EKANAYAKA P,LEE S Y,HERATH T U B,et al.Foot-and-mouth disease virus VP1 target the MAVS to inhibit type-Ⅰ interferon signaling and VP1 E83K mutation results in virus attenuation[J].PLoS Pathogens，2020,16(11):e1009057.
[30] GUAN H X,TIAN J,QIN B,et al.Crystal structure of 2C helicase from Enterovirus 71[J].Science Advances，2017,3(4):e1602573.
[31] WATKINS C L,KEMPF B J,BEAUCOURT S,et al.Picornaviral polymerase domain exchanges reveal a modular basis for distinct biochemical activities of viral RNA-dependent RNA polymerases[J].The Journal of Biological Chemistry,2020,295(31):10624-10637.
[32] STEIL B P,BARTON D J.Conversion of VPg into VPgpUpUOH before and during Poliovirus negative-strand RNA synthesis[J].Journal of Virology，2009,83(24):12660-12670.