H9N2型禽流感病毒不同传播方式的分子机制

H 9N 2亚型禽流感病毒属正黏病毒科流感病毒属A型流感病毒。虽表现低致病性,但由于传播广泛、能造成免疫抑制并使宿主易发生继发感染,其危害不容忽视[1]。反向遗传技术是近几年快速发展的一项新方法,是根据病毒基因组及其复制的特点,建立操作系统,从克隆的cDNA产生病毒的过程[2     

Amino acid substitutions in the neuraminidase protein of an H9N2 avian influenza virus affect its airborne transmission in chickens

Cases of H9N2 avian influenza virus (AIV) in poultry are increasing throughout many Eurasian countries, and co-infections with other pathogens have resulted in high morbidity and mortality in poultry. Few studies have investigated the genetic factors of virus airborne transmission which determine the scope of this epidemic. In this study, we used specific-pathogen-free chickens housed in isolators to investigate the airborne transmissibility of five recombinant H9N2 AIV rescued by reverse genetic technology. The results show that airborne transmission of A/Chicken/Shandong/01/2008 (SD01) virus was related to the neuraminidase (NA) gene, and four amino acid mutations (D368E, S370L, E313K and G381D) within the head region of the SD01 NA, reduced virus replication in the respiratory tract of chickens, reduced virus NA activity, and resulted in a loss of airborne transmission ability in chickens. Similarly, reverse mutations of these four amino acids in the NA protein of r01/NASS virus, conferred an airborne transmission ability to the recombinant virus. We conclude that these four NA residues may be significant genetic markers for evaluating potential disease outbreak of H9N2 AIV, and propose that immediate attention should be paid to the airborne transmission of this virus.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0142-3) contains supplementary material, which is available to authorized users.     

1株H9N2亚型重组禽流感病毒的生物学特性

为研制高效特异的H9N2亚型AI疫苗,试验对利用反向基因操作技术构建的1株表面基因由A/chicken/Shanghai/10/01(H9N2)(简称SH10)提供、内部基因由12质粒系统提供的重组病毒SH/PR8的生物学特性进行了研究.结果表明,与亲本毒相比重组病毒的毒力有所下降,重组病毒在鸡胚上生长的适应性能更好,更适于作为新型疫苗株使用.     

H9N2亚型禽流感病毒流行病学及其疫苗的研究进展

禽流感(avian influenza AI)是由A型流感病毒引起的一种高度接触性传染病。H9N2是A型流感病毒的1个亚型,其谱系复杂,流行范围广,已经成为我国AI的主要亚型。尽管我国自1998年就开始进行H9N2亚型禽流感病毒(AIV)的防疫,但目前其疫苗株与流行株的抗原性已经出现了较大的差异。带毒野禽的迁徙不仅使H9N2AIV防控难度加大,而且使AI的流行不断复杂化;活禽交易市场为AIV重排以及跨种传播提供了有利的条件。此外,H9N2AIV感染谱在不断扩大,不仅感染哺乳动物,甚至已经感染了人群;因此,有必要重新认识H9N2AIV的兽医学和公共卫生学意义。现就H9N2AIV近年在我国的流行情况及其疫苗的研究进展作一综述。     

不同H9N2亚型禽流感病毒株致病力的比较试验

参照NDV评价毒力的方法对1998—2008年间收集的25株H9N2AIV的致病性进行了比较研究。结果显示,25株H9N2AIV不同毒株间致病性有较大差异,大致分为3类：致病性偏强、致病性中等和致病性较弱。从中选出致病力有差异的8个毒株进行了1日龄雏鸡脑内接种指数（ICPI）、6周龄鸡静脉接种指数（IVPI）以及8周龄SPF鸡人工感染排毒试验,结果证明大部分毒株ICPI和IVPI基本上为0,排毒散毒的高峰期在攻毒后第5天到第6天。在25个毒株中,3#和12#表现出了比较高的致病性,不仅其EID50值最高（分别为10-8.8/0.2mL和10-8.8/0.2mL）、ELD50值最高（分别为10-7.9/0.2mL和10-8.7/0.2mL）,而且鸡胚平均死亡时间也最短（分别为66.5,69h）。3#、12#还出现了1日龄雏鸡脑内接种指数（分别为0.238,0.437）和6周鸡静脉内接种指数（分别为0.34,0.51）。在8周龄SPF鸡人工感染排毒试验中3#和12#毒株的排毒量大,排毒时间也明显长于其他毒株（第2～9天）。以上试验结果表明我国H9N2AIV不同毒株致病性有明显差异,呈多态性,致病性偏强的毒株造成鸡群较高的死亡率。     

Pathogenicity of an avian influenza virus serotype H9N2 in chickens

A low pathogenic avian influenza virus (AIV) serotype H9N2 affected many commercial flocks in the Middle East in late 1990s and early 2000s. Due to the varying pathogenicity ofAIV H9N2 reported in previous studies, this study was carried out to determine the pathogenicity of a Jordanian isolate of H9N2 in broiler and specific-pathogen-free (SPF) chickens. Mild tracheal rales were observed in the broilers but not in the SPF birds starting 3 days postinfection (DPI) and until the end of the experiment at 16 DPI. Infected chickens had gross and histologic changes limited to the respiratory system (sinuses, trachea, lungs, and air sacs) characterized by congestion and lymphoplasmacytic inflammation. However, the lesions in the broiler chickens were more severe than those in the SPF chicks. Furthermore, the virus caused significant (P = 0.004) reduction (230 g) in average body weight of the infected broiler group compared with the uninfected broiler group. Both broiler and SPF-infected groups seroconverted, and they had a geometric mean titer of 2(8.2) and 2(9.3), respectively, on the hemagglutination inhibition test at 16 DPI. Cloacal virus shedding was not detected by 9 DPI and 15 DPI in broiler and SPF-infected groups, respectively. This study demonstrated the pathogenic nature of the local Jordanian H9N2 isolate and the variation from what it has been reported in other countries of the region. Regional effort should be directed to start an eradication program of this disease because of its pathogenicity for chickens, wide distribution, and possible interference with surveillance for H5N1 serotype.     

H9N2亚型鸡源禽流感病毒分离与鉴定

禽流感 (AI)又名真性鸡瘟或欧洲鸡瘟 ,是由正粘病毒科 A型流感病毒引起的一种传染病 ,是国际兽医局规定的 A类烈性传染病。鸡、火鸡、鸭和鹌鹑等家禽及其他野禽均可感染。我们从新乡市某蛋鸡场分离鉴定 1株低致病力的 H9N2亚型的禽流感病毒毒株 ,现报告如下。1　材料与方法1.1　材料　病料来自河南职业技术师范学院禽病研究所接诊检验的病、死鸡。禽流感 A型琼扩抗原、标准阳性血清以及抗 HA、NA分型血清购自中国农科院哈尔滨兽医研究所 ;抗新城疫 (ND)血清和抗减蛋综合征 (EDS- 76 )血清由本院禽病研究所提供。 SPF鸡胚和雏鸡购自…     

Genetic characterization of H9N2 avian influenza virus previously unrecognized in Korea

In this study, we describe the isolation and characterization of previously unreported Y280-lineage H9N2 viruses from two live bird markets in Korea in June 2020. Genetic analysis revealed that they were distinct from previous H9N2 viruses circulating in Korea and had highest homology to A/chicken/Shandong/1844/2019(H9N2) viruses. Their genetic constellation showed they belonged to genotype S, which is the predominant genotype in China since 2010, where genotype S viruses have infected humans and acted as internal gene donors to H5 and H7 zoonotic influenza viruses. Active surveillance and control measures need to be enhanced to protect the poultry industry and public health.     

H9N2亚型流感病毒光动力失活的超微结构

使用H9N2亚型流感病毒作为系统模型,用不同浓度(2,4 μmol/L)的光敏剂经激光照射(12 J/cm2,20 min),使病毒颗粒失活后,使用透射电子显微镜(TEM)观察病毒粒子形态,并对病毒的感染性进行测定.结果显示,低浓度光敏剂处理组的病毒粒子膜结构虽然保持完整性,但表面糖蛋白缺失,同时丧失了对MDCK细胞的...     

H9N2亚型禽流感病毒神经氨酸酶基因的克隆及表达

根据已知H9N2亚型禽流感病毒神经氨酸酶(NA)基因序列设计,合成克隆引物。自H9N2亚型病毒感染的鸡胚尿囊液中提取总RNA,反转录后采用高可信度DNA聚合酶(PyobestTMDNAPolymerase)扩增NA基因,采用Invitrogen定向表达系统(ChampionTMpETdirectionalTOPOexpressionsystem)进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组NA,分子量约54·7ku。经免疫印迹及ELISA分析重组NA的免疫反应性和免疫动物分析其免疫原性,结果表明:重组NA能与H9N2亚型病毒抗血清发生特异性结合,且其免疫动物后能诱导机体产生特异性抗体,具有良好的抗原性。     

重组禽流感病毒灭活疫苗(H9N2亚型,Re-9株)对2010年～2011年流行病毒株免疫效力的研究

为评价H9N2亚型重组禽流感病毒(AIV)灭活疫苗Re-9株的免疫效力,本研究选取2010年～2011年分离的5株H9N2亚型AIV进行攻毒保护实验。这5个分离株均为类A/CK/Beijing/1/94病毒株,其HA和NA基因与Re-9株及其亲本株的同源性介于90.6%～97.5%和88.1%～98.7%之间,其抗原相关值在66.67%～100%之间。将Re-9株为种毒制备灭活疫苗,免疫4周龄SPF雏鸡后,3周时平均HI抗体效价达9.5 log2;在免疫后3周以2×107EID50的剂量攻击亲本株和5株流行病毒株,攻毒后采集3 d、5 d、7 d的拭子,免疫组拭子样品病毒检测均为阴性;对照组3 d、5 d拭子病毒检测的阳性率均在80%以上,7 d的阳性率在60%以下。以上结果表明灭活疫苗Re-9株抗原针对性强,而且具有良好的免疫效力,能够抵御近年来分离的H9N2亚型AIV,是理想的H9N2亚型禽流感疫苗株。     

H9N2禽流感病毒HA2基因重组杆状病毒的表达

从pMD18-THA阳性质粒扩增了H9N2亚型禽流感病毒的HA2基因，将扩增到的HA2基因克隆至昆虫杆状病毒转移栽体pBlueBacHis2A中。将其与杆状病毒共转染于Sf9昆虫细胞，经蚀斑筛选纯化重组杆状病毒，用其感染Sf9昆虫细胞，并优化表达条件。SDS-PAGE和Western-blotting分析表明，表达产物的分子质量约为27ku。Dot-ELISA分析表明，表达的HA2融合蛋白可与鸡抗HgN2亚型血清发生特异性反应，而与H5和H7亚型抗血清间无交叉反应。     

H9N2禽流感病毒分离株NS基因同源性分析

经RT-PCR扩增了三株国内H9N2亚型禽流感病毒(ATV)分离株的非结构(NS)蛋白基因,并把扩增的基因片段克隆到pGEM-T载体中测序,获得了NS1和NS2蛋白的完整编码序列.经与GenBank中发表的核苷酸序列比较表明,这三株病毒的NS基因之间的同源性为96%～98%;与1994年以来香港、韩国H9N2亚型分离株NS基因的同源性均在90%以上;其NSI蛋白的C-末端都缺失13个氨基酸,不同于香港分离株;在AIV NS基因系统发育进化树中,三者都处于等位基因A类的亚洲禽-猪群分枝.     

Isolation of avian influenza virus (H9N2) from emu in China

This is the first reported isolation of avian influenza virus (AIV) from emu in China. An outbreak of AIV infection occurred at an emu farm that housed 40 four-month-old birds. Various degrees of haemorrhage were discovered in the tissues of affected emus. Cell degeneration and necrosis were observed microscopically. Electron microscopy revealed round or oval virions with a diameter of 80 nm to 120 nm, surrounded by an envelope with spikes. The virus was classified as low pathogenic AIV (LPAIV), according to OIE standards. It was named A/Emu/HeNen/14/2004(H9N2)(Emu/HN/2004). The HA gene (1683bp) was amplified by RT-PCR and it was compared with other animal H9N2 AIV sequences in GenBank, the US National Institutes of Health genetic sequence database. The results suggested that Emu/HN/2004 may have come from an avian influenza virus (H9N2) from Southern China.     

一株鸡源H9N2亚型禽流感病毒的分离鉴定

从福州市某活禽市场采集的鸡泄殖腔棉拭子样品中分离出1株病毒,经血凝抑制试验(HI)和聚合酶链反应(PCR)方法鉴定为H9N2亚型禽流感病毒。在GenBank基因库中对该病毒株的3个基因片段的测序结果进行BLAST比对分析表明,3个基因片段均属于H9N2亚型禽流感病毒的基因。HA基因遗传进化分析表明,该病毒分离株与代表株DK/HK/Y280/97处于同一分支,与上海的鸡源分离株A/chicken/Shanghai/06/2015(H9N2)同源性最高,同源性为99.5%。     

Infectivity and transmissibility of H9N2 avian influenza virus in chickens and wild terrestrial birds

Genetic changes in avian influenza viruses influence their infectivity, virulence and transmission. Recently we identified a novel genotype of H9N2 viruses in widespread circulation in poultry in Pakistan that contained polymerases (PB2, PB1 and PA) and non-structural (NS) gene segments identical to highly pathogenic H7N3 viruses. Here, we investigated the potential of these viruses to cause disease and assessed the transmission capability of the virus within and between poultry and wild terrestrial avian species. Groups of broilers, layers, jungle fowl, quail, sparrows or crows were infected with a representative strain (A/chicken/UDL-01/08) of this H9N2 virus and then mixed with naïve birds of the same breed or species, or different species to examine transmission. With the exception of crows, all directly inoculated and contact birds showed clinical signs, varying in severity with quail showing the most pronounced clinical signs. Virus shedding was detected in all infected birds, with quail showing the greatest levels of virus secretion, but only very low levels of virus were found in directly infected crow samples. Efficient virus intra-species transmission was observed within each group with the exception of crows in which no evidence of transmission was seen. Interspecies transmission was examined between chickens and sparrows and vice versa and efficient transmission was seen in either direction. These results highlight the ease of spread of this group of H9N2 viruses between domesticated poultry and sparrows and show that sparrows need to be considered as a high risk species for transmitting H9N2 viruses between premises.     

H9N2亚型禽流感病毒对鸡输卵管致病性的研究

为了研究H9N2亚型禽流感病毒(H9N2 AIV)对鸡输卵管的致病性,本研究首先采用凝集素亲和组化染色法检验了流感病毒受体SAα-2,3Gal在鸡输卵管上的分布;然后用H9N2 AIV人工感染产蛋母鸡,制作输卵管的病理组织切片观察其病理变化;最后,采用胶原酶Ⅰ灌注消化法培养鸡输卵管膨大部的原代上皮细胞,接毒H9N2 AIV后观察细胞病变.试验结果表明,鸡输卵管除漏斗部没发现SAα-2,3Gal受体,其余部分均呈强阳性分布;攻毒后病理组织切片观察,输卵管膨大部上皮细胞绒毛脱落,部分细胞坏死、脱落,组织间隙变大,子宫内部组织内部充血出血,其他部分变化不明显;原代细胞接毒72 h后,细胞病变不明显,但是上清中HA滴度可以达到3log2.从以上三方面可以看出,鸡输卵管上皮细胞膜上存在禽流感病毒受体,H9N2 AIV可以引起侵害鸡输卵管,并引起部分输卵管病变,而且H9N2 AIV可以感染鸡输卵管膨大部原代上皮细胞,H9N2 AIV有可能通过输卵管感染母鸡.     

表达H9N2亚型禽流感病毒HA蛋白重组乳酸杆菌的构建

H9N2亚型禽流感病毒的血凝素(HA)基因经树突状细胞靶向肽(DCpep)与异亮氨酸拉链(GCN4)基因修饰后,插入到穿梭表达载体p SIP409中,将质粒电转化至乳酸杆菌内。结果表明,该重组乳酸杆菌经诱导后,能够表达大小约为63 000且具有反应原性的蛋白,为进一步探讨重组乳酸杆菌在激发黏膜免疫反应过程中,DC调控黏膜免疫应答的机制及研制抗禽流感口服疫苗奠定基础。     

H9N2亚型禽流感病毒细胞高产株的拯救

为构建能够在MDCK细胞中高水平复制的H9N2亚型禽流感病毒(AIV)疫苗株,本研究在对病毒生长特性及HA基因遗传进化分析的基础上,筛选出一株细胞高度适应的国内流行株A/chicken/Shanghai/11/2011(H9N2) (SH11),并通过反向遗传操作技术拯救出全部基因均来自亲本株的AIV rSH11株.生物学试验结果表明rSH11在鸡胚半数感染量(EID50)、组织培养半数感染量(TCID50)、遗传稳定性等方面均与亲本株保持一致,rSH11经MDCK细胞连续传5代后血凝价稳定在1∶1024,表明该病毒株具有细胞高度增殖的特性.采用rSH11株制备油乳剂灭活苗免疫4周龄SPF鸡,免疫一周即可以检测出HI抗体,免疫3周后HI抗体平均效价高于1∶700,表明其具有良好的免疫原性.rSH11疫苗对不同的H9N2病毒株能够产生良好的免疫保护作用,显著抑制免疫鸡排毒.H9N2亚型AIV细胞高产株的拯救为该亚型AIV细胞苗的研制奠定了基础.