上海地区鸽Ⅰ型副黏病毒流行株HN基因遗传进化和生物信息学分析

doi:10.16431/j.cnki.1671-7236.2025.11.032

摘要/Abstract

摘要： 【目的】了解上海地区鸽Ⅰ型副黏病毒(PPMV-1)流行株HN基因遗传进化情况，探讨PPMV-1免疫失败的原因，为研发PPMV-1新型疫苗和抗病毒药物提供技术支持。【方法】采集临床送检疑似PPMV-1感染鸽样品(脑、气管、肺脏、肝脏、胰腺和脾脏)，应用RT-PCR方法进行PPMV-1鉴定，阳性样品进行HN基因测序和遗传进化分析，并通过生物信息学在线软件对HN蛋白结构功能进行分析。【结果】送检鸽鉴定为PPMV-1阳性，其HN基因全长为2 002 bp，编码区全长1 716 bp，编码571个氨基酸，与PPMV-1国内分离株Pi/SH/CH/0617/2013核苷酸相似性最高，为99.8%，与新城疫(NDV)疫苗株La Sota和Mukteswar核苷酸相似性分别只有83.6%和85.5%。遗传进化分析显示，流行株属于NDV Class Ⅱ群Ⅵ.2.1.1.2.2亚型，与NDV国内分离株Pi/SH/CH/0617/2013和pigeon/Ningxia/2068/2016同处一个分支，与La Sota和Mukteswar处于不同分支。HN蛋白为亲水性蛋白，分子质量约为62.71 ku，理论等电点(pI)为7.88，半衰期为30 h，不稳定系数为30.44，脂肪系数为85.48；该蛋白主要分布在线粒体和细胞质中，无信号肽，含有1个跨膜结构、5个潜在的N-糖基化位点、105个O-糖基化位点、13个半胱氨酸残基和75个磷酸化位点。HN蛋白二级结构、三级结构预测以无规则卷曲占比最高，其次为β-折叠和α-螺旋；B细胞抗原表位预测显示，HN蛋白含有10个抗原表位(氨基酸长度≥7)。【结论】上海地区PPMV-1流行株属于NDV强毒株的Class Ⅱ群Ⅵ.2.1.1.2.2亚型，其HN蛋白具有较好的抗原表位，可作为PPMV-1疫苗研制和抗病毒治疗的靶蛋白。本研究结果为PPMV-1 HN基因遗传进化、蛋白表达、新型疫苗研发和抗病毒药物的筛选等提供理论依据。

关键词: 鸽Ⅰ型副黏病毒(PPMV-1); HN基因; 遗传进化分析; 生物信息学分析

Abstract: 【Objective】 The purpose of this study was to understand the genetic evolution of the HN gene of Pigeon paramyxovirus type Ⅰ (PPMV-1) epidemic strain in Shanghai,explore the reasons for PPMV-1 immune failure,and provide technical support for the development of new PPMV-1 vaccines and antiviral drugs. 【Method】 Samples (brain,trachea,lung,liver,pancreas and spleen) were collected from suspected PPMV-1 infected pigeons.PPMV-1 identification was performed on the sampless using RT-PCR.Positive samples were subjected to HN gene sequencing and genetic evolution analysis,and the structure and function of HN protein were analyzed using bioinformatics online software. 【Result】 The diseased pigeon was identified as PPMV-1 positive,with a HN gene length of 2 002 bp and a coding region of 1 716 bp,encoding 571 amino acids.It had the highest nucleotide similarity of 99.8% with the domestic isolate Pi/SH/CH/0617/2013 of PPMV-1,and only 83.6% and 85.5% nucleotide similarity with the Newcastle disease (NDV) vaccine strains La Sota and Mukteswar,respectively.Genetic evolution analysis showed that the prevalent strain belonged to NDV Class Ⅱ group Ⅵ.2.1.1.2.2 subtype,which was on the same branch as the domestic isolates Pi/SH/CH/0617/2013 and pigeon/Ningxia/2068/2016,but on different branches from La Sota and Mukteswar.HN protein was hydrophilic,the theoretical molecular weight,isoelectric point (pI),estimated half-life,instability index and fatty index of HN protein was about 62.71 ku,7.88,30 h,30.44 and 85.48,respectively.HN protein was mainly distributed in the mitochondria and cytoplasm,without signal peptides.It contained 1 transmembrane structure,5 potential N-glycosylation sites,105 O-glycosylation sites,13 cysteine residues and 75 phosphorylation sites.The prediction of secondary and tertiary structures of HN protein showed that random coil had the highest proportion,followed by beta sheet and alpha helix.The prediction of B cell antigenic epitopes showed that HN protein contained 10 antigenic epitopes (amino acid length ≥7). 【Conclusion】 PPMV-1 epidemic strain in Shanghai belonged to Class Ⅱ group Ⅵ.2.1.1.2.2 subtype of the NDV virulent strain.HN protein of the epidemic strain had a good antigenic epitope and could be used as a target protein for PPMV-1 vaccine development and antiviral therapy.The results provided theoretical basis for the genetic evolution of PPMV-1 HN gene,protein expression,development of novel vaccines,and screening of antiviral drugs.

Key words: Pigeon paramyxovirus type Ⅰ(PPMV-1); HN gene; phylogenetic analysis; bioinformatics analysis

中图分类号:

S852.65

刘健, 周锦萍, 徐卫兵, 白艺兰, 杨显超, 桂亚萍, 夏琦琦, 徐锋, 沈莉萍, 刘佩红, 王建. 上海地区鸽Ⅰ型副黏病毒流行株HN基因遗传进化和生物信息学分析[J]. 中国畜牧兽医, 2025, 52(11): 5371-5380.

LIU Jian, ZHOU Jinping, XU Weibing, BAI Yilan, YANG Xianchao, GUI Yaping, XIA Qiqi, XU Feng, SHEN Liping, LIU Peihong, WANG Jian. Phylogenetic and Bioinformatics Analysis of HN Gene of Pigeon Paramyxovirus Type Ⅰ Epidemic Strains in Shanghai[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5371-5380.

参考文献

[1] 李珮瑶,高晶萍,田勇,等.1株赛鸽新城疫强毒株的致病特性及分子特征分析[J].河南农业科学,2019,48(10):140-146.LI P Y,GAO J P,TIAN Y,et al.Pathogenicity analysis and molecular characterization of Newcastle disease virus isolated from racing pigeon[J].Journal of Henan Agricultural Sciences,2019,48(10):140-146.(in Chinese)
[2] 裴育,孙雅丽,赵烨,等.4株鸽新城疫病毒的基因组序列与致病性分析[J].病毒学报,2022,38(2):402-414.PEI Y,SUN Y L,ZHAO Y,et al.Genome sequencing and pathogenicity analyses of four isolates of the Newcastle disease virus in pigeons[J].Chinese Journal of Virology,2022,38(2):402-414.(in Chinese)
[3] ATHER S,WAJID A,BATOOL A,et al.Genomic and comparative clinico-pathological assessment of two Pakistani pigeon-derived Newcastle disease virus sub-genotypes ⅩⅪ.1.1 and ⅩⅪ.1.2 isolated in 2017[J].Comparative Immunology,Microbiology and Infectious Diseases,2023,94:101957.
[4] SHAN S H,BRUCE K,STEVENS V,et al.In vitro and in vivo characterization of a Pigeon paramyxovirus type 1 isolated from domestic pigeons in Victoria,Australia 2011[J].Viruses,2021,13(3):429.
[5] 云骜,张思远,成子强,等.6株鸽源新城疫病毒广东分离株F、HN基因的克隆及遗传进化分析[J].中国家禽,2024,46(2):106-112.YUN A,ZHANG S C,CHENG Z Q,et al.Cloning and phylogenetic analysis on F and HN genes of six pigeon Newcastle disease virus strains isolated from Guangdong province[J].China Poultry,2024,46(2):106-112.(in Chinese)
[6] 连聪,温肖会,吕殿红,等.我国新城疫分子流行病学研究进展[J].广东农业科学,2023,50(2):115-124.LIAN C,WEN X H,LYU D H,et al.Progress of molecular epidemiology of Newcastle disease in China[J].Guangdong Agricultural Sciences,2023,50(2):115-124.(in Chinese)
[7] ZHANG Y X,REN S F,YUAN X Y,et al.Biological characterization of isolated Pigeon paramyxovirus-1 and its pathogenicity analysis in SPF chickens[J].Veterinarski Arhiv,2023,93(1):65-70.
[8] CAO H W,LI D S,ZHANG H.Analysis of synonymous codon usage in Newcastle disease virus hemagglutinin-neuraminidase (HN) gene and fusion protein (F) gene[J].Virus Disease,2014,25(1):132-136.
[9] CHEN Y,GONG J,ZHAN T,et al.The role of dual mutations G347E and E349D of the Pigeon paramyxovirus type 1 hemagglutinin-neuraminidase protein in vitro and in vivo[J].Veterinary Sciences,2024,11(12):592.
[10] HE D,SUN L,LI C,et al.Anti-tumor effects of an Oncolytic adenovirus expressing hemagglutinin-neuraminidase of Newcastle disease virus in vitro and in vivo[J].Viruses,2014,6(2):856-874.
[11] 熊晓妍,孙兴臣,李敏婕,等.赛鸽新城疫病毒南京分离株的生物学特性及其F和HN基因的分析[J].中国兽医科学,2017,47(11):1363-1370.XIONG X Y,SUN X C,LI M J,et al.Biologic characterization and F and HN gene analysis of racing pigeon Newcastle disease virus strain from Nanjing[J].Chinese Veterinary Science,2017,47(11):1363-1370.(in Chinese)
[12] DIMITROV M K,ABOLNIK C,AFONSO L C,et al.Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus[J].Infection,Genetics and Evolution,2019,74:103917.
[13] 李婷婷,陆冰洋,丁树荣,等.一株鸽源Ⅰ型副黏病毒的分离鉴定和F基因序列分析[J].黑龙江畜牧兽医,2021,24:51-56.LI T T,LU B Y,DING S R,et al.Isolation and identification of an isolate of pigeon Paramyxovirustype 1 and sequence analysis of F gene[J].Heilongjiang Animal Science and Veterinary Medicine,2021,24:51-56.(in Chinese)
[14] 岳楚昕,莫贞峰,李颖,等.新城疫病毒山东分离株ShD-5-06F和HN基因序列分析[J].动物医学进展,2008,29(8):5-9.YUE C X,MO Z F,LI Y,et al.Sequence and analyses on the F and HN gene of Newcastle disease virus ShD-5-06 isolated in Shandong[J].Progress in Veterinary Medicine,2008,29(8):5-9.(in Chinese)
[15] YU X H,LUO Y Y,WANG J J,et al.A molecular,epidemiological and pathogenicity analysis of Pigeon paramyxovirus type 1 viruses isolated from live bird markets in China in 2014-2021[J].Virus Research,2022,318:198846.
[16] MELANSON V R,IORIO R M.Amino acid substitutions in the F-specific domain in the stalk of the Newcastle disease virus HN protein modulate fusion and interfere with its interaction with the F protein[J].Journal of Virology,2004,78(23):13053-13061.
[17] 王雅静,谷长勤,张万坡,等.鸽源新城疫病毒的鉴定及HN基因遗传演化分析[J].畜牧与兽医,2017,49(6):106-110.WANG Y J,GU C Q,ZHANG W P,et al.Identification of Newcastle disease virus from pigeons and phylogenetic analysis of HN gene[J].Animal Husbandry & Veterinary Medicine,2017,49(6):106-110.(in Chinese)
[18] PANDA A,ELANKUMARAN S,KRISHNAMURTHY S,et al.Loss of N-linked glycosylation from the hemagglutinin-neuraminidase protein alters virulence of Newcastle disease virus[J].Journal of Virology,2004,78(10):4965-4975.
[19] 梁俊文.新城疫分离株P和HN基因分子进化与致病性的相关研究[D].济南:山东师范大学,2010.LIANG J W.Molecular evolution of P and HN genes in relation to pathogenicity of field Newcastle disease viruses[D].Jinan:Shandong Normal University,2010.(in Chinese)
[20] 刘晶雪,刘颖,迟苗苗,等.副粘病毒HN蛋白颈部与F相互作用区功能的研究[J].病毒学报,2018,34(3):318-327.LIU J X,LIU Y,CHI M M,et al.Study on functions of the fusion interaction region in the stalk domain of Paramyxovirus hemagglutinin-neuraminidase[J].Chinese Journal of Virology,2018,34(3):318-327.(in Chinese)
[21] SAYANTAN B,ALBERT S S,THEODORE S J,et al.Fusion activation through attachment protein stalk domains indicates a conserved core mechanism of Paramyxovirus entry into cells[J].Journal of Virology,2014,88(8):3925-3941.
[22] LIU B,JI Y H,LIN Z Q,et al.Two single amino acid substitutions in the intervening region of Newcastle disease virus HN protein attenuate viral replication and pathogenicity[J].Scientific Reports,2015,5(1):13038.
[23] 马吉强,刘建华.新城疫病毒昌吉株F基因的生物信息学特征分析[J].新疆农业科技,2019,4:14-18.MA J Q,LIU J H.Bioinformatics analysis of the F gene of the Changji strain of Newcastle disease virus[J].Xinjiang Agricultural Science and Technology,2019,4:14-18.(in Chinese)