孙莹, 张兵, 李岭, 黄小洁, 侯力丹, 刘丹, 李启红, 李俊平, 王乐元, 李慧姣, 杨承槐^,中国兽医药品监察所,北京 100081

Construction of a Recombinant Duck Enteritis Virus Expressing Hemagglutinin of H9N2 Avian Influenza Virus

SUN Ying, ZHANG Bing, LI Ling, HUANG XiaoJie, HOU LiDan, LIU Dan, LI QiHong, LI JunPing, WANG LeYuan, LI HuiJiao, YANG ChengHuai^,China Institute of Veterinary Drug Control, Beijing 100081

通讯作者: 杨承槐,Tel：010-61255386;E-mail：ychenghuai@163.com

孙莹和张兵为同等贡献作者
责任编辑: 林鉴非
收稿日期:2019-04-23接受日期:2019-07-22网络出版日期:2019-12-01

基金资助:

国家重点研发计划.2017YFD0500800 北京市自然科学基金项目.6162025

Received:2019-04-23Accepted:2019-07-22Online:2019-12-01
作者简介 About authors
孙莹,E-mail：sunyinggoodluck@163.com

张兵,E-mail：zhangbing06@163.com












摘要
【背景】H9亚型禽流感病毒（AIV）存在宿主范围扩大、毒力增强的趋势,并为其他亚型AIV重排提供基因,给养禽业和公共卫生造成极大威胁。水禽不仅是流感病毒的宿主,更是其天然储存库,在禽流感病毒的传播和变异中发挥着重要作用。因此有效控制水禽感染对养禽业健康发展、公共卫生安全具有重要意义。鸭肠炎病毒（DEV）属于疱疹病毒,能感染鸭、鹅等雁形目禽类,可引起产蛋下降及高死亡率。DEV基因组大,免疫原性好,具有开发成活疫苗载体的潜力。【目的】构建缺失gE基因、表达H9亚型AIV HA基因的重组病毒rDEV-△gE-HA,探讨重组病毒rDEV-△gE-HA作为防治DEV-AIV的二联重组活载体疫苗的可行性。【方法】以H9N2亚型禽流感病毒HA基因作为靶基因,构建含有HA基因表达盒的转移载体pT-gE-HA,将其与携带绿色荧光蛋白标记的重组rDEV-△gE-GFP共转染CEF细胞后,进行蚀斑筛选、纯化表达HA基因的重组病毒rDEV-△gE-HA;利用PCR、基因测序鉴定重组病毒;在CEF中连续传代重组病毒20次,测定外源基因传代稳定性。以10 ³ TCID₅₀免疫易感鸭,分析重组病毒rDEV-ΔgE-HA对致死性DEV强毒攻毒保护效果;将不同剂量（10 ³-10 ⁶TCID₅₀）rDEV-△gE-HA免疫鸭,免疫后14、21、28 d分别采集血清,测定H9血凝抑制（HI）抗体,并在免疫后28 d,以10 ⁸EID₅₀的剂量静脉注射H9N2 AIV（A/duck/GD/08）,攻毒后2 d,采集喉拭子,进行病毒分离试验。【结果】将构建的转移质粒载体pT-gE-HA与rDEV-△gE-GFP共转染CEF细胞,经过3轮蚀斑筛选,获得纯化的重组病毒rDEV-△gE-HA。PCR鉴定及基因测序结果显示,HA基因成功地插入到DEV基因组中,替换了绿色荧光蛋白。重组病毒在CEF中至少能稳定传代20代。重组病毒rDEV-ΔgE-HA以10 ³ TCID₅₀免疫易感鸭,能抵抗致死性DEV强毒攻击。重组病毒rDEV-ΔgE-HA免疫易感鸭后14 d,各剂量免疫组均能检测到H9 HI抗体效价;免疫后21日,各组抗体效价水平略有上升,10 ³TCID₅₀剂量免疫组HI抗体效价达到1:2 ⁴,而10 ⁴-10 ⁶TCID50剂量免疫组HI抗体效价在1:2 ^2.4-1:2 ³。免疫鸭后28 d,用H9N2 AIV进行攻毒,10 ³、10 ⁴、10 ⁶TCID₅₀免疫组均未从喉拭子分离到病毒H9N2,说明能完全保护,阻止喉头排毒,而10 ⁵TCID₅₀免疫组保护率为80%（4/5）,1/5病毒分离阳性。【结论】成功构建了稳定表达H9亚型AIV HA基因的重组DEV,该重组病毒保留了亲本毒的免疫原性,能抵抗致死性DEV强毒的攻击;免疫鸭后能诱导产生AIV HI抗体,尽管HI抗体滴度不高,但至少80%免疫鸭能阻止排毒。该研究为研制DEV-H9亚型AIV二联重组活载体疫苗奠定了基础。
关键词： 鸭肠炎病毒;H9亚型禽流感病毒;HA;重组病毒

Abstract
【Background】The H9N2 avian influenza virus (AIV) pathogenicity and transmissibility have recently showed an increasing trend. Moreover, it donates partial or even whole cassette of internal genes to generate novel reassortants, which is serious threat to poultry industry and public health. Waterfowls are considered as the natural host and reservoirs of AIVs and play an important role in the spread and mutation of AIV. Therefore, successful control of the spread of H9N2 in waterfowls contributes significantly to poultry industry and public health. Duck enteritis virus (DEV) taxonomically belongs to family Herpesviridae and infects ducks, geese, and swans, which results in high mortality and decreased egg production in domestic and wild waterfowl. DEV may be a promising candidate viral vector for aquatic poultry vaccination because it has a large genome and good immunogenicity. 【Objective】 In this study, we constructed a recombinant DEV expressing the hemagglutinin (HA) gene of a H9N2 virus that was inserted into the deleted viral gE gene, and then its characterization to explore the feasibility of the recombinant DEV as a live vectored vaccine was studied.【Method】 The HA gene of H9N2 was cloned to construct the transfer vector pT-gE-HA. Plasmid pT-gE-HA and rDEV-△gE-GFP were co-transfected into CEF cells. After plaque-purification, we obtained a pure recombinant virus which expressed H9N2 AIV HA protein, and named as rDEV-△gE-HA; PCR and sequencing assay were used to identify the recombinant virus. The recombinant virus was passaged in primary CEF 20 times to evaluate the genetic stability of the foreign gene in the recombinant virus. Ducks were inoculated with 10 ³EID₅₀ rDEV-△gE-HA, then challenged with lethal DEV. Ducks were vaccinated intramuscularly with 10 ³-10 ⁶ TCID₅₀ of rDEV-△gE-HA. At 14, 21, and 28 days post-vaccination (d.p.v.), sera were obtained from all ducks to monitor HI antibody against H9N2 AIV. At 28 d.p.v. all ducks were challenged with 10 ⁸ EID₅₀ H9N2 (A/duck/GD/08) by intravenous injection. Oropharyngeal swabs were collected from H9N2 virus challenged ducks to detect viral shedding.【Result】The recombinant expression vector pT-gE-HA was constructed and transfected with rDEV-△gE-GFP in chicken embryo fibroblasts (CEF). After 3 rounds of plaque-purification, the purified rDEV-△gE-HA was obtained. The results of the PCR and sequencing indicated that the HA expression cassette had already successfully been inserted into the DEV. The HA gene were stably maintained after the recombinant was passaged 20 times in CEF. Ducks inoculated with 10 ³ TCID₅₀ of rDEV-△gE-HA were protected against lethal DEV. HI antibody was detected in all vaccinated ducks at 14 d.p.v. and slightly increased at 21 d.p.v.. Challenge with H9N2 at 28 d.p.v., ducks inoculated with 10 ³, 10 ⁴ and 10 ⁶TCID₅₀ were completely protected from challenge, as evidenced by the finding that no virus was recovered from collected oropharyngeal swabs, while 80% ducks (4/5) inoculated with 10 ⁵TCID₅₀ were protected.【Conclusion】In this research, we successfully constructed a stable recombinant DEV expressing the HA of H9N2 AIV. The recombinant DEV remained the protective efficacy of the parental virus against lethal DEV parental virus. Moreover, it could induce HI antibody in ducks and protect no less 80% ducks against H9N2 AIV challenge, although the titer of HI antibody was not too high. This study laid a foundation for developing bivalent vaccine controlling DEV and AIV infection.
Keywords：duck enteritis virus;H9 subtype AIV;HA;recombinant virus


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本文引用格式
孙莹, 张兵, 李岭, 黄小洁, 侯力丹, 刘丹, 李启红, 李俊平, 王乐元, 李慧姣, 杨承槐. 表达H9亚型禽流感病毒HA基因重组鸭肠炎病毒的构建[J]. 中国农业科学, 2019, 52(23): 4398-4405 doi:10.3864/j.issn.0578-1752.2019.23.020
SUN Ying, ZHANG Bing, LI Ling, HUANG XiaoJie, HOU LiDan, LIU Dan, LI QiHong, LI JunPing, WANG LeYuan, LI HuiJiao, YANG ChengHuai. Construction of a Recombinant Duck Enteritis Virus Expressing Hemagglutinin of H9N2 Avian Influenza Virus[J]. Scientia Acricultura Sinica, 2019, 52(23): 4398-4405 doi:10.3864/j.issn.0578-1752.2019.23.020

0 引言

【研究意义】禽流感是由A型流感病毒（avian influenza virus,AIV）引起的禽类的急性、烈性、接触性传染病,分为高致病性禽流感和低致病性禽流感^[1]（http://www.oie.int）。H9亚型禽流感可导致家禽发病、混合感染、免疫抑制^[2,3],还为H5N1、H7N9、H10N8和H5N6等亚型禽流感重排提供内部基因^[4,5,6,7,8],对公共卫生造成威胁^[9]。水禽作为流感病毒最复杂的生态系统,在H9亚型禽流感病毒传播和变异中发挥着重要作用^[10,11,12]。因此,水禽免疫是H9亚型禽流感防制的关键环节。目前H9N2禽流感油乳剂灭活疫苗对于水禽存在着抗体水平不高、免疫效果不确实等缺点,因此急需一种高效、安全、适用于水禽的新型疫苗。鸭病毒性肠炎,也称为鸭瘟,是由鸭肠炎病毒（duck enteritis virus,DEV）引起的鸭、鹅等多种雁形目禽类感染的一种急性、热性、接触性传染病^[13]。鸭病毒性 肠炎引起产蛋下降及高死亡率,造成巨大的经济失,是目前对水禽养殖业危害最严重的疫病之一^[13]。本研究探索以DEV为载体表达H9亚型AIV抗原的新型活载体疫苗的可行性,可以同时防治鸭、鹅等水禽的DEV和AIV的感染,具有很好的发展前景。【前人研究进展】DEV属于疱疹病毒科、甲型疱疹病毒亚科、马立克病毒属、鸭疱疹病毒1型^[14] (http://www. ictvonline.org)。DEV基因组大,158—162k^{[15,16,17,18,19,20]},免疫后3日即可产生保护力,安全性好,只感染雁形目禽类,对其他家畜和人不致病,可用作基因工程疫苗活载体。近年来,以DEV为载体表达外源基因进行了探索研究,如表达H5亚型AIV HA基因^{[21,22,23,24]}、H9亚型HA基因^[25]、1和3型鸭肝炎病毒VP1蛋白^[26]、小鹅瘟病毒VP2蛋白^[27,28]、鸭坦布苏病毒E和PrM蛋白^[29,30]、鸡传染性支气管炎病毒S、N、S1蛋白^[31]。动物试验结果令人鼓舞,表达H5N1亚型AIV HA基因的重组DEV,以10⁶ PFU免疫鸭一次,3 d后即可抵抗致死性H5N1病毒,另有研究报道,以10⁵ PFU免疫鸭一次,即可抵抗致死性H5N1病毒^[24]。表达H9亚型AIV HA的重组病毒DEV,以10³ EID₅₀免疫鸭后能产生高水平的HI抗体（1:256）,并能完全阻止H9N2亚型AIV排毒^[25]。同时表达1、3型鸭肝炎病毒VP1蛋白的重组DEV,免疫鸭2周后即可检测到中和抗体,4周后中和抗体达峰值1:32;免疫后3 d即可抵抗1、3型鸭肝炎病毒的攻击^[26]。这些研究结果表明,DEV作为活疫苗载体潜力巨大。【本研究切入点】外源基因的插入位点对其表达影响较大,插入位点不同,抗体水平、免疫效果也明显差异。【拟解决的关键问题】寻找稳定、高效表达外源基因的插入位点,为研制DEV-AIV基因重组活载体疫苗奠定了基础。

1 材料与方法

1.1 试验时间、地点

本试验于2014年6月至2016年7月在中国兽医药品监察所完成。

1.2 试验材料

1.2.1 毒株和细胞 DEV细胞适应毒、DEV强毒株和H9N2禽流感（A/duck/GD/08）由中国兽医药品监察所保存;SPF鸡胚由北京梅里亚维通实验动物技术有限公司提供,按常规方法制备鸡胚成纤维细胞（CEF）。

1.2.2 试验动物 4周龄易感麻鸭（DEV中和抗体和H9亚型禽流感HI抗体均<1:4）购自北京昌平某鸭场。

1.2.3 质粒和菌株 重组质粒PT-gE-GFP-gpt、表达绿色荧光蛋白的重组病毒rDEV-△gE-GFP-gpt由中国兽医药品监察所病毒制品检测室构建保存;pMD18T载体购自大连TaKaRa公司,DH5α受体菌购自天根生化科技（北京）有限公司。

1.2.4 主要试剂 Ex Taq DNA 聚合酶、限制性内切酶、T4 DNA 连接酶、T4 DNA 聚合酶购自大连TaKaRa公司;胶回收试剂盒等均购自天根生化科技（北京）有限公司;胎牛血清、M199培养液购自Hyclone公司;OPTI-MEM培养液购自Gibco公司;lipofectamine2000购自Invitrogen公司;无内毒素高纯质粒提取试剂盒Endo-free Plasmid Mini Kit II购自Omega公司。

1.2.5 引物试验所用PCR引物见表1,由上海Invitrogen生物公司合成。

Table 1
表1
表1目的基因的扩增引物及鉴定引物
Table 1Primers for amplification and identification of target gene

引物名称 Primer name	序列（5′-3′） Sequence (5′-3′)
H9Nhe I-F	CAGCTAGC CGCCACCATGGAGACAGTATCACTA ATAACTA
H9BHI-R	CGGGATCCTTATATACATATGTTGCATCTGC
Re-JD-F	TCAGGATGTAACGCTGGAG
Re-JD-R	GGCATCGCAGTCGGTTTT

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1.3 试验方法

1.3.1 重组质粒的构建 构建策略见图1。以H9N2亚型AIV RNA反转录的cDNA为模板扩增HA基因,经Nhe I和BamH I双酶切克隆到质粒pT-gE-GFP- gpt,获得重组质粒pT-gE-HA。

图1

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图1重组病毒的构建示意图

a：DEV基因组示意图,包括UL, IRS, US和TRS区域;b：GFP基因表达盒插入到DEV gE基因内,获得rDEV-△gE-GFP-gpt;c：质粒pT-gE-HA与rDEV-△gE-GFP-gpt进行同源重组,获得重组病毒rDEV-△gE-HA
Fig. 1Construction of recombinant DEV

a: Map of the DEV genome, which consists of long (UL) and short (US) unique regions with inverted repeat sequences (IRS,TRS) flanking the US region; b: Construction of rDEV-△gE-GFP-gpt. An expression cassette encoding GFP was inserted in DEV genome; c: Plasmid pT-gE-HA was used to generate rDEV-△gE-HA by recombination with rDEV-△gE-GFP-gpt


1.3.2 重组病毒的制备、纯化及鉴定 重组质粒pT-gE-HA与表达绿色荧光蛋白的重组病毒rDEV-△gE-GFP-gpt共转染CEF,筛选、纯化按文献[32]方法进行。用鉴定引物Re-JD-F、Re-JD-R进行PCR扩增,PCR产物送上海Invitrogen生物公司测序。

1.3.3 重组病毒的稳定性 将重组病毒接种CEF（moi=0.01）,待80%细胞产生病变后,冻融3次,再接种CEF,如此连续传代20次。用引物Re-JD-F、Re-JD-R进行PCR鉴定外源基因HA是否稳定存在。

1.3.4 动物试验

1.3.4.1 DEV免疫原性 将15只4周龄易感麻鸭分成3组,5只/组,分别肌肉注射10³ TCID₅₀ rDEV-ΔgE- HA、DEV亲本毒或PBS作为对照。每组单独隔离饲养。在免疫后14 d,腿部肌肉注射接种DEV强毒（CVCC AV1221）,每只10³MLD。观察14 d,每天记录发病死亡情况。

1.3.4.2 H9免疫原性 将30只4周龄易感麻鸭分成6组,5只/组,其中4组分别以10³—10⁶TCID₅₀的剂量肌肉注射rDEV-ΔgE-HA,1组肌肉注射10³TCID₅₀ DEV亲本毒,另1组注射PBS作为对照,1 mL/只,免疫后第2、3、4周分别采集血清,测定H9血凝抑制抗体,免疫后28 d,以10⁸EID₅₀ 剂量静脉注射H9N2 AIV（A/duck/ GD/08）,攻毒后2 d,采集喉拭子,进行病毒分离试验。

2 结果

2.1 重组病毒的制备、纯化及鉴定

转移载体pT-gE-HA与DEV共转染CEF细胞后,经过3代筛选,所有的蚀斑都无绿色荧光,获得纯化的重组病毒rDEV-ΔgE-HA。经测序证实,插入的HA基因大小和位置均正确。

2.2 重组病毒的传代稳定性

重组病毒经过传代20次,对重组病毒的HA基因进行PCR鉴定,各代次重组病毒的PCR扩增片段大小均为2 500 bp（图2）,证实HA基因在重组病毒中稳定存在。

图2

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图2重组病毒的传代稳定性检测

M：DL15000。数字代表重组病毒的代次
Fig. 2Identification of stability of the recombinant DEV

M: DL15000 DNA Marker. The numbers show the passages of the recombinant viruses

2.3 动物试验

重组病毒rDEV-ΔgE-HA及其亲本毒分别以10³ TCID₅₀免疫易感鸭,均能抵抗致死性DEV强毒攻击,在14 d观察期内未出现任何临床症状,而对照组鸭攻毒后3 d均表现出精神萎靡、食欲不振等症状并在6 d内死亡。表明HA基因的插入并不影响亲本毒免疫原性,能100%抵抗DEV强毒攻击（图3）。

图3

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图3重组病毒对DEV的攻毒保护效果

Fig. 3Efficacy of the recombinant virus against lethal DEV challenge



以不同剂量rDEV-△gE-HA（10³—10⁶TCID₅₀）免疫鸭,在免疫后14、21和28 d分别采血,测定HI抗体效价。在免疫后14 d,各组HI抗体效价水平均在1:2²左右;免疫后21 d,各组抗体效价水平略有上升,10³TCID₅₀剂量免疫组HI抗体效价达到1:2⁴,而10⁴—10⁶TCID₅₀剂量免疫组HI抗体效价在1:2^2.4—1:2³;免疫后28 d,各组HI抗体效价均略有降低,在1:2²—1:2³（图4）。

图4

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图4重组病毒诱导产生的HI抗体

Fig. 4HI antibody induced by the recombinant virus in ducks



不同剂量免疫鸭后28 d,用H9N2禽流感病毒（A/duck/GD/08）进行攻毒。10³、10⁴、10⁶TCID₅₀免疫组均未从喉拭子分离到病毒H9N2,说明能完全保护,阻止喉头排毒;然而,10⁵TCID₅₀免疫组部分保护率为80%（4/5）,1/5病毒分离阳性。

这些数据表明,重组病毒rDEV-△gE-HA能诱导产生HI抗体,对H9N2亚型禽流感病毒具有一定的保护。

3 讨论

在中国,自从1992年首次报道,H9N2亚型AIV存在宿主范围扩大、毒力增强的趋势,对养禽业造成巨大威胁^[33];此外,H9N2亚型AIV不仅能直接感染人类,还为其他亚型流感病毒发生重排提供内部基因,对公共健康存在巨大风险^[9]。鸭被认为是禽流感病毒的主要自然储存库,因此控制H9N2禽流感病毒在鸭群内的感染与传播对保护其他动物和公共健康具有重要意义。本研究将水禽源H9N2亚型禽流感病毒HA基因插入到鸭肠炎病毒中,成功地构建了表达H9亚型禽流感病毒HA基因的重组鸭肠炎病毒rDEV-ΔgE-HA。

以不同剂量rDEV-△gE-HA（10³—10⁶TCID₅₀）免疫鸭后,均能产生HI抗体,但HI抗体滴度较低（1:2²—1:2⁴）,且HI抗体滴度与免疫剂量无明显相关性。下一步将尝试加强免疫,是否能提高HI抗体滴度。在另一研究中,笔者将相同的HA基因表达盒插入到DEV UL2基因内,获得重组病毒rDEV-△UL2-HA,该重组病毒免疫鸭后14 d,HI抗体滴度为1:2⁴,免疫后28 d,HI抗体滴度上升到1:2⁸,抗体滴度显著高于本研究结果^[25]。因此,HA基因的插入位点对HI抗体滴度具有重要影响。在疱疹病毒中,如EHV-1^[34]、EHV-4^[35]、HSV-1^[36]、PRV^[37]、BHV-1^[38]、FHV-1^[39],糖蛋白gE和gI形成异构体,有助于病毒在细胞间扩散,这两个糖蛋白缺失后细胞间扩散的能力减弱。DEV gI/gE基因缺失后,蚀斑明显减小,病毒在细胞间扩散能力减弱^[17],这是否与HI抗体滴度较低有关,有待进一步研究。

目前关于应用DEV作为载体表达各种免疫原性基因的报道已有很多,插入外源基因的靶位点不尽相同,免疫原性也有一定差别。将H5N1亚型AIV的HA基因分别插入DEV UL41基因内以及US7与US8基因之间,分别构建出重组病毒rDEV-△UL41-HA以及rDEV-△US78-HA,以10⁶ PFU的rDEV-△US78-HA免疫鸭2周后,大部分免疫鸭（5/6）能产生HI抗体,免疫3、4周后,所有免疫鸭均能产生HI抗体,最高滴度可达1﹕64左右,随后HI抗体滴度迅速下降;rDEV-△UL41-HA免疫效果比rDEV-△US78-HA略差^[22]。在另一研究中,将鹅源H5N1亚型AIV的HA基因分别插入到gI、gE基因内和US2基因内,2个重组病毒免疫鸭后第7天能检测到DEV抗体,抗体滴度无明显差异,但是比亲本毒低;然而未诱导产生AIV HI抗体,只能通过Western blotting可以检测到HA抗体^[23]。将H5N1亚型AIV的HA基因插入到DEV gC基因内构建的重组病毒rDEV-△gC-HA,通过IFA及蛋白免疫印记试验可检测到HA基因在感染细胞内高效表达^[21]。将H5N1 AIV 的HA基因插入gB和UL26基因之间,获得的重组病毒免疫1月龄SPF鸭,能够产生HI抗体,最高滴度可达1:64左右^[24]。在DEV US7和US8之间插入鸭坦布苏病毒的TE基因和PrM基因或单独插入TE基因,免疫鸭后2周能检测到鸭坦布苏病毒中和抗体,加强免疫后中和抗体升高约8倍,滴度高达1:128^[30]。在DEV SORF3与 US2连接区插入鸭坦布苏病毒的E基因,免疫鸭后2周能检测到坦布苏病毒中和抗体,免疫后4周中和抗体滴度达1:32左右^[29]。将传染性支气管炎病毒的N、S或S1基因插入DEV US10基因内,免疫后7 d即可产生体液和细胞免疫应答^[31]。

4 结论

在本研究中,成功地将H9亚型AIV HA基因插入到DEV gE基因内,获得了稳定表达HA基因的重组病毒rDEV-ΔgE-HA。该重组病毒保留了亲本毒的免疫原性,能抵抗致死性DEV强毒的攻击;免疫鸭后能诱导产生AIV HI抗体,尽管HI抗体滴度不高,但能部分阻止排毒。

参考文献 View Option 原文顺序
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[1] 于康震, 陈化兰 . 禽流感. 北京: 中国农业出版社, 2015.
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[2] SUN Y, LIU J . H9N2 influenza virus in China: A cause of concern. P
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The recent human infection with avian influenza virus revealed that H9N2 influenza virus is the gene donor for H7N9 and H10N8 viruses infecting humans. The crucial role of H9N2 viruses at the animal-human interface might be due to the wide host range, adaptation in both poultry and mammalian, and extensive gene reassortment. As the most prevalent subtype of influenza viruses in chickens in China, H9N2 also causes a great economic loss for the poultry industry, even under the long-term vaccination programs. The history, epidemiology, biological characteristics, and molecular determinants of H9N2 influenza virus are reviewed in this paper. The contribution of H9N2 genes, especially RNP genes, to the infection of humans needs to be investigated in the future.

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In the current study, we characterized H9N2 influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation during a 5 year period (1998-2002). Phylogenetic analysis of the 8 genes of 11 representative viruses showed that they all shared high similarity to that of the first isolate, A/Chicken/Shanghai/F/1998 (Ck/SH/F/98), and clustered to the same lineages. Furthermore, all 11 viruses had a 9 nt deletion between positions 206 and 214 of the neuraminidase gene. These genetic characteristics strongly suggest that these viruses are descendants of the first isolate. In addition, our study also showed that the H9N2 viruses circulating in the operation during this 5 year period were evolving, as shown by antigenic variations between viruses manifested by reactivity with polyclonal antisera and monoclonal antibodies, by haemagglutination with erythrocytes from different animals, by amino acid differences in haemagglutinin and neuraminidase proteins, and by variation in their ability to replicate in the respiratory and intestinal tract and to be transmitted by aerosol. Phylogenetic analysis revealed that the internal genes from some H5N1 viruses of duck origin clustered together with those from H9N2 virus and that the RNP genes of these H5N1 viruses isolated after 2001 are more closely related to the genes of the Ck/SH/F/98-like H9N2 viruses, indicating more recent reassortment events between these two subtypes of viruses. Continuous surveillance of influenza virus in poultry and waterfowl is critical for monitoring the genesis and emergence of potentially pandemic strains in this region.

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Avian influenza viruses of subtype H9N2 are widely prevalent in poultry in many Asian countries, and the segmented nature of the viral genome results in multiple distinct genotypes via reassortment. In this study, genetic evolution of H9N2 viruses circulating in eastern China during 2007-2013 was analyzed. The results showed that the diversity of the gene constellations generated six distinct genotypes, in which a novel genotype (S) bearing the backbone of A/chicken/Shanghai/F/98-like viruses by acquiring A/quail/Hong Kong/G1/97-like polymerase basic subunit 2 and matrix genes has gradually established its ecological niche and been consistently prevalent in chicken flocks in eastern China since its first detection in 2007. Furthermore, genotype S possessed the peculiarity to donate most of its gene segments to other emerging influenza A viruses in China, including the novel reassortant highly pathogenic avian influenza H5N2, the 2013 novel H7N7, H7N9 and the latest reassortant H10N8 viruses, with potential threat to poultry industry and human health.

[5] GUAN Y, SHORTRIDGE K F, KRAUSS S, CHIN P S, DYRTING K C, ELLIS T M, WEBSTER R G, PEIRIS M . H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in southeastern China
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The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1) (abbreviated HK/156/97) genome complex may be present in H9N2 viruses in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Serological studies indicated that H9N2 influenza viruses had infected a high proportion of chickens and other land-based birds (pigeon, pheasant, quail, guinea fowl, and chukka) from southeastern China. Two lineages of H9N2 influenza viruses present in the live-poultry markets were represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97)-like and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like viruses. Up to 16% of cages of quail in the poultry markets contained Qa/HK/G1/97-like viruses, while about 5% of cages of other land-based birds were infected with Dk/HK/Y280/97-like viruses. No reassortant between the two H9N2 virus lineages was detected despite their cocirculation in the poultry markets. Reassortant viruses represented by A/Chicken/Hong Kong/G9/97 (H9N2) were the major H9N2 influenza viruses circulating in the Hong Kong markets in 1997 but have not been detected since the chicken slaughter in 1997. The Qa/HK/G1/97-like viruses were frequently isolated from quail, while Dk/HK/Y280/97-like viruses were predominately associated with chickens. The Qa/HK/G1/97-like viruses were evolving relatively rapidly, especially in their PB2, HA, NP, and NA genes, suggesting that they are in the process of adapting to a new host. Experimental studies showed that both H9N2 lineages were primarily spread by the aerosol route and that neither quail nor chickens showed evidence of disease. The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to HK/156/97 (H5N1) virus emphasizes the need for surveillance of mammals including humans.

[6] SHEN Y Y, KE C W, LI Q, YUAN R Y, XIANG D, JIA W X, YU Y D, LIU L, HUANG C, QI W B, SIKKEMA R, WU J, KOOPMANS M, LIAO M . Novel Reassortant Avian Influenza A(H5N6) viruses in humans, Guangdong, China, 2015
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[7] ZHANG Z, LI R, JIANG L, XIONG C, CHEN Y, ZHAO G, JIANG Q . The complexity of human infected AIV H5N6 isolated from China
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Novel avian influenza viruses (AIVs) of H7N9, H10N8, and H5N6 are currently circulating in China's poultry flocks, occasionally infecting human and other mammals. Human infected AIV H5N6 in China during 2014-2015 is believed to be a triple reassortant originated from H6N6 and two clades of H5 viruses. The current report suggests that its reassortment history is more complicated.

[8] RAHIMIRAD S, ALIZADEH A, ALIZADEH E, HOSSEINI S M . The avian influenza H9N2 at avian-human interface: A possible risk for the future pandemics
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The avian influenza subtype H9N2 is considered a low pathogenic virus which is endemic in domestic poultry of a majority of Asian countries. Many reports of seropositivity in occupationally poultry-exposed workers and a number of confirmed human infections with an H9N2 subtype of avian influenza have been documented up to now. Recently, the human infections with both H7N9 and H10N8 viruses highlighted that H9N2 has a great potential for taking a part in the emergence of new human-infecting viruses. This review aimed at discussing the great potential of H9N2 virus which is circulating at avian-human interface, for cross-species transmission, contribution in the production of new reassortants and emergence of new pandemic subtypes. An intensified surveillance is needed for controlling the future risks which would be created by H9N2 circulation at avian-human interfaces.

[9] GU M, XU L, WANG X, LIU X . Current situation of H9N2 subtype avian influenza in China
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In China, H9N2 subtype avian influenza outbreak is firstly reported in Guangdong province in 1992. Subsequently, the disease spreads into vast majority regions nationwide and has currently become endemic there. Over vicennial genetic evolution, the viral pathogenicity and transmissibility have showed an increasing trend as year goes by, posing serious threat to poultry industry. In addition, H9N2 has demonstrated significance to public health as it could not only directly infect mankind, but also donate partial or even whole cassette of internal genes to generate novel human-lethal reassortants like H5N1, H7N9, H10N8 and H5N6 viruses. In this review, we mainly focused on the epidemiological dynamics, biological characteristics, molecular phylogeny and vaccine strategy of H9N2 subtype avian influenza virus in China to present an overview of the situation of H9N2 in China.

[10] ROCHE B, LEBARBENCHON C, GAUTHIER-CLERC M, CHANG C M, THOMAS F, RENAUD F, VAN DER WERF S, GUEGAN J F . Water-borne transmission drives avian influenza dynamics in wild birds: the case of the 2005-2006 epidemics in the Camargue area
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Transmission and persistence of avian influenza viruses (AIV) among wildlife remains an unresolved issue because it depends both on the ecology of the host (e.g. population density, migration) and on the environment (e.g. AIV persistence in water). We have developed a mathematical model that accounts for both AIV epidemics and bird community dynamics. The model is parameterized using bird counts and AIV prevalence data. Results suggest that the transmission patterns driving the dynamics of infection at our study site (Camargue, South of France) involved both a density-dependent and a water-borne transmission processes. Water-borne transmission is, however, the main determinant of the disease dynamics and observed prevalence level. This pattern of transmission highlights the importance of the persistence of viral particles in water in AIV dynamics in wild birds.

[11] BARBER M R, ALDRIDGE J R, JR., WEBSTER R G, MAGOR K E , Association of RIG-I with innate immunity of ducks to influenza
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Ducks and wild waterfowl perpetuate all strains of influenza viruses in nature. In their natural host, influenza viruses typically cause asymptomatic infection and little pathology. Ducks are often resistant to influenza viruses capable of killing chickens. Here, we show that the influenza virus sensor, RIG-I, is present in ducks and plays a role in clearing an influenza infection. We show evidence suggesting that RIG-I may be absent in chickens, providing a plausible explanation for their increased susceptibility to influenza viruses compared with ducks. RIG-I detects RNA ligands derived from uncapped viral transcripts and initiates the IFN response. In this study, we show that the chicken embryonic fibroblast cell line, DF-1, cannot respond to a RIG-I ligand. However, transfection of duck RIG-I into DF-1 cells rescues the detection of ligand and induces IFN-beta promoter activity. Additionally, DF-1 cells expressing duck RIG-I have an augmented IFN response resulting in decreased influenza replication after challenge with either low or highly pathogenic avian influenza virus. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression is induced 200 fold, early in an innate immune response in ducks challenged with the H5N1 virus A/Vietnam/1203/04. Finding this natural disease resistance gene in ducks opens the possibility of increasing influenza resistance through creation of a transgenic chicken.

[12] MARKWELL D D, SHORTRIDGE K F . Possible waterborne transmission and maintenance of influenza viruses in domestic ducks
Applied and Environmental Microbiology, 1982,43(1):110-115.
URLPMID:7055370 [本文引用: 1]
Two duck farms in Hong Kong were examined monthly for 1 year for the occurrence and persistence of influenza viruses within the duck communities. The predominant virus in one community was H3N2, a virus antigenically related to the pandemic Hong Kong strain. This virus was isolated monthly throughout the year from feces or pond water or both, indicating a cycle of waterborne transmission. Viruses of the same antigenic combination were isolated 1 and 2 years after the last sampling occasion, implying persistence in the community. Infection was asymptomatic. Maintenance of virus appeared to be dependent upon the continual introduction of ducklings susceptible to infection onto virus-contaminated water; the feces of ducks 70 to 80 days old were generally free of detectable virus despite the exposure of the ducks to virus in pond water. In the second community, in which ducklings were not introduced after the initial sampling, the prevailing viruses, H7N1 and H7N2, also present asymptomatically, ceased to be detected once the ducks were 70 to 80 days old. The normal practice of raising ducks of different ages on the same farm, wherein the water supplies are shared, as typified by the first community, appears to be instrumental in maintaining a large reservoir of influenza viruses in the duck population of southern China.

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[本文引用: 2]

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[15] WANG J, HOPER D, BEER M, OSTERRIEDER N . Complete genome sequence of virulent duck enteritis virus (DEV) strain 2085 and comparison with genome sequences of virulent and attenuated DEV strains
Virus Research, 2011,160(1-2):316-325. doi: 10.1016/ j.virusres.2011.07.004.
DOI:10.1016/j.virusres.2011.07.004URLPMID:21802458 [本文引用: 1]
We here report the complete genome sequence of the duck enteritis virus (DEV) wild-type strain 2085, an avian herpesvirus (GenBank ID: JF999965). The nucleotide sequence was derived from the 2085 genome cloned as an infectious bacterial artificial chromosome (BAC) clone. The DEV 2085 genome is 160,649-bp in length and encodes 78 predicted open reading frames (ORFs), a number identical to that identified for the attenuated DEV VAC strain (GenBank ID: EU082088.2). Comparison of the genome sequences DEV 2085 and VAC with partial sequences of the virulent CHv strain and the attenuated strain Clone-03 was carried out to identify nucleotide or amino acid polymorphisms that potentially contribute to DEV virulence. No amino acid changes were identified in 24 of the 78 ORFs, a result indicating high conservation in DEV independently of strain origin or virulence. In addition, 39 ORFs contain non-synonymous nucleotide substitutions, while 15 ORFs had nucleotide insertions or deletions, frame-shift mutations and/or non-synonymous nucleotide substitutions with an effect on ORF initiation or termination. In 7 of the 15 ORFs with high and 27 of the 39 ORFs with low variability, polymorphisms were exclusively found in DEV 2085, a finding that likely is a result of a different origin of 2085 (Europe) or VAC, Clone-03 and CHv (Eastern Asia). Five ORFs (UL2, UL12, US10, UL47 and UL41) with polymorphisms were identical between the virulent DEV 2085 and CHv but different from VAC or Clone-03. They, individually or in combination, may therefore represent DEV virulence factors. Our comparative analysis of four DEV sequences provides a comprehensive overview of DEV genome structure and identifies ORFs that are changed during serial virus passage.

[16] YANG C, LI Q, LI J, ZHANG G, LI H, XIA Y, YANG H, YU K . Comparative genomic sequence analysis between a standard challenge strain and a vaccine strain of duck enteritis virus in China
Virus Genes, 2014,48(2):296-303. doi: 10.1007/s11262-013-1009-9.
DOI:10.1007/s11262-013-1009-9URLPMID:24287923 [本文引用: 1]
Here, we present the complete genomic sequence of the Chinese standard challenge strain (CSC) of duck enteritis virus (DEV), which was isolated in China in 1962. The DEV CSC genome is 162,131 bp long and contains 78 predicted open reading frames (ORFs). Comparison of the genomic sequences of DEV CSC and DEV live vaccine strain K at passage 63 (DEV K p63) revealed that the DEV CSC genome is 4,040 bp longer than the DEV K p63 genome, mainly because of 3,513-bp and 528-bp insertions at the 5' and 3' ends of the unique long segment, respectively. At the nucleotide level, 63 of the 76 ORFs in the DEV CSC genome were 100 % identical to the ORFs in the DEV K p63 genome. Two ORFs (UL56 and US10) had frameshift mutations in the C-terminal regions, while LORF5 was unique to the DEV K p63 genome. It is difficult to assign attenuated virulence to changes in specific genes. However, the complete DEV CSC genome will further advance our understanding of the genes involved in virulence and evolution. The DEV CSC genome sequence has been deposited in GenBank under accession number JQ673560.

[17] YANG C, LI J, LI Q, LI L, SUN M, LI H, XIA Y, YANG H, YU K . Biological properties of a duck enteritis virus attenuated via serial passaging in chick embryo fibroblasts
Archives of Virology, 2015, 160(1):267-274. doi: 10.1007/s00705-014-2275-0.
DOI:10.1007/s00705-014-2275-0URLPMID:25392272 [本文引用: 2]
To gain a better understanding of the genetic changes required for attenuation of duck enteritis virus (DEV), the Chinese standard challenge strain of DEV (DEV CSC) was serially passaged 80 times in chick embryo fibroblasts. We plaque-purified the virus after the 25th passage (DEV p25) and the 80th passage (DEV p80) and investigated its in vitro and in vivo properties. Average plaque sizes for DEV p25 and p80 were significantly smaller than those for their parental DEV CSC. The results from an in vivo experiment revealed that DEV p25 and p80 were avirulent in ducks and protected them from virulent DEV challenge. The complete genome sequence of DEV p80 was determined and compared with that of the parent virus. An 1801-bp deletion was identified in the genome of DEV p80, which affected the genes encoding gI and gE. Moreover, there were 11 base substitutions, which led to seven amino acid changes in open reading frames LORF9, UL51, UL9, UL7, UL4, ICP4 and US3. Further DNA sequence analysis showed that the 1801-bp deletion was also present in DEV p25. Our findings suggest that DEV gE and/or gI are nonessential for virus growth and might, as with other herpesviruses, play an important role in cell-to-cell spread and virulence. Our experiments provide more genetic information about DEV attenuation and further advance our understanding of the molecular basis of DEV pathogenesis.

[18] YANG C, LI J, LI Q, LI H, XIA Y, GUO X, YU K, YANG H . Complete genome sequence of an attenuated duck enteritis virus obtained by in vitro serial passage
Genome Announc, 2013, 1(5): 10.1128/genomeA.00685-3.
DOI:10.1128/genomeA.00919-13URLPMID:24179116 [本文引用: 1]
Pseudomonas alcaligenes, a Gram-negative aerobic bacterium, is a rare opportunistic human pathogen. Here, we report the whole-genome sequence of P. alcaligenes strain MRY13-0052, which was isolated from a bloodstream infection in a medical institution in Japan and is resistant to antimicrobial agents, including broad-spectrum cephalosporins and monobactams.

[19] LI Y, HUANG B, MA X, WU J, LI F, AI W, SONG M, YANG H . Molecular characterization of the genome of duck enteritis virus
Virology, 2009, 391(2):151-161. doi: 10.1016/j.virol.2009.06.018.
DOI:10.1016/j.virol.2009.06.018URLPMID:19595405 [本文引用: 1]
The genomic sequence of a strain of duck enteritis virus (DEV) was determined and analyzed in this study. The size of its genome is 158,091 bp in length and the genome is predicted to encode 78 putative proteins and resembles the members of the Alphaherpesvirinae in genomic organization and gene composition. The genome of the virus is composed of a unique long (UL) region, a unique short (US) region, a unique short internal repeat (IRS) region and a unique short terminal repeat (TRS) region. Its genomic arrangement pattern (UL-IRS-US-TRS) corresponds to D-type herpesvirus and is consistent with the members of Varicellovirus and Iltovirus genera. Sequence analysis reveals that the genome of the virus contains 67 genes having homologs in most members of the Alphaherpesvirinae. Out of these genes, one gene has a homolog in cercopithecine herpesvirus 8 which is a virus of Betaherpesvirinae, and 5 genes have homologs in avian herpesviruses. Furthermore, the genome possesses three unique genes without homologs in any other herpesviruses. Like most members of the Alphaherpesvirinae, the genes in the UL region of its genome are well conserved, whereas the gene arrangement of IRS-US is similar to that of Marek's disease virus and equine herpesviruses 1. Therefore, our data based on the genomic analysis suggest that DEV represents an osculant taxonomic entity within the Alphaherpesvirinae.

[20] WU Y, CHENG A, WANG M, YANG Q, ZHU D, JIA R, CHEN S, ZHOU Y, WANG X, CHEN X . Complete genomic sequence of Chinese virulent duck enteritis virus
Journal of Virology, 2012, 86(10):5965. doi: 10.1128/ JVI.00529-12.
DOI:10.1128/JVI.00529-12URL [本文引用: 1]
The Chinese virulent (CHv) strain of duck enteritis virus (DEV) has a genome of approximately 162,175 nucleotides with a GC content of 44.89%. Here we report the complete genomic sequence and annotation of DEV CHv, which offer an effective platform for providing authentic research experiences to novice scientists. In addition, knowledge of this virus will extend our general knowledge of DEV and will be useful for further studies of the mechanisms of virus replication and pathogenesis.

[21] WANG J, OSTERRIEDER N . Generation of an infectious clone of duck enteritis virus (DEV) and of a vectored DEV expressing hemagglutinin of H5N1 avian influenza virus
Virus Research, 2011,159(1):23-31. doi: 10.1016 /j.virusres.2011.04.013.
DOI:10.1186/s12985-015-0354-9URLPMID:26263920 [本文引用: 2]
Highly pathogenic avian influenza virus (AIV) subtype H5N1 remains a threat to poultry. Duck enteritis virus (DEV)-vectored vaccines expressing AIV H5N1 hemagglutinin (HA) may be viable AIV and DEV vaccine candidates.

[22] LIU J, CHEN P, JIANG Y, WU L, ZENG X, TIAN G, GE J, KAWAOKA Y, BU Z, CHEN H . A duck enteritis virus-vectored bivalent live vaccine provides fast and complete protection against H5N1 avian influenza virus infection in ducks
Journal of Virology, 2011,85(21):10989-10998. doi: 10.1128/JVI. 05420-11.
DOI:10.1128/JVI.05420-11URLPMID:21865383 [本文引用: 2]
Ducks play an important role in the maintenance of highly pathogenic H5N1 avian influenza viruses (AIVs) in nature, and the successful control of AIVs in ducks has important implications for the eradication of the disease in poultry and its prevention in humans. The inactivated influenza vaccine is expensive, labor-intensive, and usually needs 2 to 3 weeks to induce protective immunity in ducks. Live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine is used routinely to control lethal DEV infections in many duck-producing areas. Here, we first established a system to generate the DEV vaccine strain by using the transfection of overlapping fosmid DNAs. Using this system, we constructed two recombinant viruses, rDEV-ul41HA and rDEV-us78HA, in which the hemagglutinin (HA) gene of the H5N1 virus A/duck/Anhui/1/06 was inserted and stably maintained within the ul41 gene or between the us7 and us8 genes of the DEV genome. Duck studies indicated that rDEV-us78HA had protective efficacy similar to that of the live DEV vaccine against lethal DEV challenge; importantly, a single dose of 10(6) PFU of rDEV-us78HA induced complete protection against a lethal H5N1 virus challenge in as little as 3 days postvaccination. The protective efficacy against both lethal DEV and H5N1 challenge provided by rDEV-ul41HA inoculation in ducks was slightly weaker than that provided by rDEV-us78HA. These results demonstrate, for the first time, that recombinant DEV is suitable for use as a bivalent live attenuated vaccine, providing rapid protection against both DEV and H5N1 virus infection in ducks.

[23] LIU X, WEI S, LIU Y, FU P, GAO M, MU X, LIU H, XING M, MA B, WANG J . Recombinant duck enteritis virus expressing the HA gene from goose H5 subtype avian influenza virus
Vaccine, 2013,31(50):5953-5959. doi: 10.1016/j.vaccine.2013.10.035.
DOI:10.1016/j.vaccine.2013.10.035URLPMID:24144474 [本文引用: 2]
The duck enteritis virus (DEV) may be a promising candidate viral vector for an aquatic poultry vaccination that can protect against multiple pathogens because it has a very large genome and a narrow host range. Recently, we described two DEV recombinants that contained deletions of the viral US2 or gIgE genes. The hemagglutinin (HA) gene of an H5N1-type highly pathogenic avian influenza virus (HPAIV) of goose origin was inserted into the deletion sites to construct two rDEVs expressing the AIV HA antigen. The resulting rDEV-ΔgIgE-HA or rDEV-ΔUS2-HA recombinant DEV viruses were used to infect duck embryo fibroblasts. Reverse transcription PCR, immunofluorescence and western blot analysis results indicated that rDEV-ΔgIgE-HA and rDEV-ΔUS2-HA were successfully expressed in duck embryo fibroblasts (DEFs). To investigate whether the HA gene could be stably maintained in the recombinant viruses, the viruses were passaged in DEFs 18 times. The HA gene in both recombinants could be detected by PCR amplification. The immunized four-week-old ducks induced specific antibodies against DEV and AIV HA and were protected against challenge infections with DEV AV1221 viruses.

[24] ZOU Z, HU Y, LIU Z, ZHONG W, CAO H, CHEN H, JIN M . Efficient strategy for constructing duck enteritis virus-based live attenuated vaccine against homologous and heterologous H5N1 avian influenza virus and duck enteritis virus infection
Veterinary Research, 2015,46(1):42. doi: 10.1186/s13567-015-0174-3.
DOI:10.1186/s13567-015-0174-3URLPMID:25889564 [本文引用: 3]
Duck is susceptible to many pathogens, such as duck hepatitis virus, duck enteritis virus (DEV), duck tembusu virus, H5N1 highly pathogenic avian influenza virus (HPAIV) in particular. With the significant role of duck in the evolution of H5N1 HPAIV, control and eradication of H5N1 HPAIV in duck through vaccine immunization is considered an effective method in minimizing the threat of a pandemic outbreak. Consequently, a practical strategy to construct a vaccine against these pathogens should be determined. In this study, the DEV was examined as a candidate vaccine vector to deliver the hemagglutinin (HA) gene of H5N1, and its potential as a polyvalent vaccine was evaluated. A modified mini-F vector was inserted into the gB and UL26 gene junction of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The HA gene of A/duck/Hubei/xn/2007 (H5N1) was inserted into the C-KCE genome via the mating-assisted genetically integrated cloning (MAGIC) to generate the recombinant vector pBAC-C-KCE-HA. A bivalent vaccine C-KCE-HA was developed by eliminating the BAC backbone. Ducks immunized with C-KCE-HA induced both the cross-reactive antibodies and T cell response against H5. Moreover, C-KCE-HA-immunized ducks provided rapid and long-lasting protection against homologous and heterologous HPAIV H5N1 and DEV clinical signs, death, and primary viral replication. In conclusion, our BAC-C-KCE is a promising platform for developing a polyvalent live attenuated vaccine.

[25] SUN Y, YANG C, LI J, LI L, CAO M, LI Q, LI H . Construction of a recombinant duck enteritis cirus vaccine expressing hemagglutinin of H9N2 avian influenza virus and evaluation of its efficacy in ducks
Archives of Virology, 2017,162(1):9. doi: DOIhttps://doi.org/10.1007/ s00705-016-3077-3.
DOI:10.1007/s00705-016-3077-3URLPMID:27709401 [本文引用: 3]
H9 subtype avian influenza viruses (AIVs) remain a significant burden in the poultry industry and are considered to be one of the most likely causes of any new influenza pandemic in humans. As ducks play an important role in the maintenance of H9 viruses in nature, successful control of the spread of H9 AIVs in ducks will have significant beneficial effects on public health. Duck enteritis virus (DEV) may be a promising candidate viral vector for aquatic poultry vaccination. In this study, we constructed a recombinant DEV, rDEV-?UL2-HA, inserting the hemagglutinin (HA) gene from duck-origin H9N2 AIV into the UL2 gene by homologous recombination. One-step growth analyses showed that the HA gene insertion had no effect on viral replication and suggested that the UL2 gene was nonessential for virus growth in vitro. In vivo tests further showed that the insertion of the HA gene in place of the UL2 gene did not affect the immunogenicity of the virus. Moreover, a single dose of 10³ TCID₅₀ of rDEV-?UL2-HA induced solid protection against lethal DEV challenge and completely prevented H9N2 AIV viral shedding. To our knowledge, this is the first report of a DEV-vectored vaccine providing robust protection against both DEV and H9N2 AIV virus infections in ducks.

[26] GE J, DENG G, WEN Z, TIAN G, WANG Y, SHI J, WANG X, LI Y, HU S, JIANG Y, YANG C, YU K, BU Z, CHEN H . Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses
Journal of Virology, 2007,81(1):150-158. doi: 10.1128/jvi.01514-06.
DOI:10.1128/JVI.01514-06URLPMID:17050610 [本文引用: 2]
H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.

[27] QIAO C, YU K, JIANG Y, LI C, TIAN G, WANG X, CHEN H . Development of a recombinant fowlpox virus vector-based vaccine of H5N1 subtype avian influenza
Developments in Biologicals, 2006,124:127-132.
URLPMID:16447503 [本文引用: 1]
The genetic stability of the recombinant fowlpox virus (named rFPV-HA-NA) was confirmed by serial passage on chicken embryo fibroblast (CEF) cells. The immune efficacy, safety, the minimum immunising dose, the time of immunity induced and the immune duration of the vector-based vaccine was evaluated in specific-pathogen-free (SPF) chickens. The recombinant virus vaccine containing 100 plaque form units (PFU) could induce complete protection against challenge with H5N1 highly pathogenic avian influenza virus (HPAIV). The immune efficacy, protecting chickens from clinical signs and death after challenge, was obtained one week after the immunisation with this vaccine. Protective immunity could last for 40 weeks post-immunisation. So the recombinant fowlpox vaccine is a safe and highly effective gene engineering vaccine candidate, and will be used to prevent H5 subtype avian influenza in the future.

[28] 陈柳, 余斌, 倪征, 华炯钢, 叶伟成, 云涛, 张存 . 表达小鹅瘟病毒VP2蛋白重组鸭瘟病毒的构建及其生物学特性
中国农业科学, 2016,49(14):2813-2821.
DOI:10.3864/j.issn.0578-1752.2016.14.015URL [本文引用: 1]
【Objective】Duck enteritis virus (DEV) and goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting ducklings, Muscovy ducklings and goslings. According to the most recent virus taxonomy reported in 2012 by the International Committee on Taxonomy of Viruses (ICTV), DEV (also referred to Anatid herpesvirus 1) is classified into the genus Mardivirus, the subfamily Alphaherpesvirinae of Herpesviridae. Many herpesviruses, such as Pseudorabies virus (PRV), Marek's disease virus (MDV), Herpesvirus of turkey（HVT）have been widely made as live viral vector for the expression of foreign antigens, and there were some reports about DEV as live viral vector in recent years. To control DEV and GPV infection, a recombinant vectored DEV expressing GPV VP2 was constructed in this study based on the bacterial artificial chromosome (BAC) clone pDEV-EF1 which carries DEV full-length genome (Chen L, et al. , 2015), and then the biological characteristics of the obtained recombinant virus rDEV-VP2 were analyzed to explore the possibility of rDEV-VP2 as duplex live carrier vaccine. 【Method】 The recombinant BAC clone pDEV-VP2 carrying GPV VP2 gene was generated by two-step Red/ET recombination in E. coli. pDEV-VP2 was constructed by inserting codon optimized-GPV VP2 expression cassette between DEV US7 and US8 genes on pDEV-EF1. The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre without BAC sequence were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. And the growth curve in vitro, plaque size and expression of GPV VP2 in CEFs were analyzed. The antibody level of GPV VP2 in sera of rDEV-VP2-incoculated ducklings was detected by an indirect-ELISA method based on the GPV VP2 protein. 【Result】 The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Growth curves show that the growth kinetics of rDEV-VP2 was basically consistent with those of parental virus in vitro. And the plaque size of rDEV-VP2 was slightly increased compared to the parental virus rDEV-BAC. Immunofluorescence assay and Western blot analysis showed that GPV VP2 protein is expressed in recombinant virus-infected CEFs. And the rDEV-VP2 infection could induce 7-day-old Muscovy ducklings to produce antibody specific for GPV VP2. 【Conclusion】 In this study, the antigen gene VP2 of GPV was inserted into the genome of DEV US7 and US8, and an recombinant infectious BAC clone of DEV was successfully constructed. Then the corresponding recombinant virus rDEV-VP2 was rescued, and its cellular growth characteristics were basically consistent with those of parental virus, and rDEV-VP2 could induce Muscovy ducklings to produce VP2-specific antibody. These studies have laid a foundation for developing bivalent vaccine controlling DEV and GPV infection.
CHEN L, YU B, NI Z, HUA J G, YE W C, YUN T, ZHANG C . Construction and characterization of a recombinant duck enteritis virus expressing VP2 gene of goose parvovirus
Scientia Agricultura Sinica, 2016,49(14):2813-2821.(in Chinese)
DOI:10.3864/j.issn.0578-1752.2016.14.015URL [本文引用: 1]
【Objective】Duck enteritis virus (DEV) and goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting ducklings, Muscovy ducklings and goslings. According to the most recent virus taxonomy reported in 2012 by the International Committee on Taxonomy of Viruses (ICTV), DEV (also referred to Anatid herpesvirus 1) is classified into the genus Mardivirus, the subfamily Alphaherpesvirinae of Herpesviridae. Many herpesviruses, such as Pseudorabies virus (PRV), Marek's disease virus (MDV), Herpesvirus of turkey（HVT）have been widely made as live viral vector for the expression of foreign antigens, and there were some reports about DEV as live viral vector in recent years. To control DEV and GPV infection, a recombinant vectored DEV expressing GPV VP2 was constructed in this study based on the bacterial artificial chromosome (BAC) clone pDEV-EF1 which carries DEV full-length genome (Chen L, et al. , 2015), and then the biological characteristics of the obtained recombinant virus rDEV-VP2 were analyzed to explore the possibility of rDEV-VP2 as duplex live carrier vaccine. 【Method】 The recombinant BAC clone pDEV-VP2 carrying GPV VP2 gene was generated by two-step Red/ET recombination in E. coli. pDEV-VP2 was constructed by inserting codon optimized-GPV VP2 expression cassette between DEV US7 and US8 genes on pDEV-EF1. The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre without BAC sequence were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. And the growth curve in vitro, plaque size and expression of GPV VP2 in CEFs were analyzed. The antibody level of GPV VP2 in sera of rDEV-VP2-incoculated ducklings was detected by an indirect-ELISA method based on the GPV VP2 protein. 【Result】 The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Growth curves show that the growth kinetics of rDEV-VP2 was basically consistent with those of parental virus in vitro. And the plaque size of rDEV-VP2 was slightly increased compared to the parental virus rDEV-BAC. Immunofluorescence assay and Western blot analysis showed that GPV VP2 protein is expressed in recombinant virus-infected CEFs. And the rDEV-VP2 infection could induce 7-day-old Muscovy ducklings to produce antibody specific for GPV VP2. 【Conclusion】 In this study, the antigen gene VP2 of GPV was inserted into the genome of DEV US7 and US8, and an recombinant infectious BAC clone of DEV was successfully constructed. Then the corresponding recombinant virus rDEV-VP2 was rescued, and its cellular growth characteristics were basically consistent with those of parental virus, and rDEV-VP2 could induce Muscovy ducklings to produce VP2-specific antibody. These studies have laid a foundation for developing bivalent vaccine controlling DEV and GPV infection.

[29] ZOU Z, LIU Z, JIN M . Efficient strategy to generate a vectored duck enteritis virus delivering envelope of duck tembusu virus
Viruses, 2014, 6(6):2428-2443. doi: 10.3390/v6062428.
DOI:10.3390/v6062428URLPMID:24956180 [本文引用: 2]
Duck Tembusu virus (DTMUV) is a recently emerging pathogenic flavivirus that has resulted in a huge economic loss in the duck industry. However, no vaccine is currently available to control this pathogen. Consequently, a practical strategy to construct a vaccine against this pathogen should be determined. In this study, duck enteritis virus (DEV) was examined as a candidate vaccine vector to deliver the envelope (E) of DTMUV. A modified mini-F vector was inserted into the SORF3 and US2 gene junctions of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The envelope (E) gene of DTMUV was inserted into the C-KCE genome through the mating-assisted genetically integrated cloning (MAGIC) strategy, resulting in the recombinant vector, pBAC-C-KCE-E. A bivalent vaccine C-KCE-E was generated by eliminating the BAC backbone. Immunofluorescence and western blot analysis results indicated that the E proteins were vigorously expressed in C-KCE-E-infected chicken embryo fibroblasts (CEFs). Duck experiments demonstrated that the insertion of the E gene did not alter the protective efficacy of C-KCE. Moreover, C-KCE-E-immunized ducks induced neutralization antibodies against DTMUV. These results demonstrated, for the first time, that recombinant C-KCE-E can serve as a potential bivalent vaccine against DEV and DTMUV.

[30] CHEN P, LIU J, JIANG Y, ZHAO Y, LI Q, WU L, HE X, CHEN H . The vaccine efficacy of recombinant duck enteritis virus expressing secreted E with or without PrM proteins of duck tembusu virus
Vaccine, 2014, 32(41):5271-5277. doi: 10.1016/j.vaccine. 2014.07.082.
DOI:10.1016/j.vaccine.2014.07.082URL [本文引用: 2]
A newly emerged tembusu virus that causes egg-drop has been affecting ducks in China since 2010. Currently, no vaccine is available for this disease. A live attenuated duck enteritis virus (DEV; a herpesvirus) vaccine has been used routinely to control lethal DEV in ducks since the 1960s. Here, we constructed two recombinant DEVs by transfecting overlapping fosmid DNAs. One virus, rDEV-TE, expresses the truncated form of the envelope glycoprotein (TE) of duck tembusu virus (DTMUV), and the other virus, rDEV-PrM/TE, expresses both the TE and pre-membrane proteins (PrM). Animal study demonstrated that both recombinant viruses induced measurable anti-DTMUV neutralizing antibodies in ducks. After two doses of recombinant virus, rDEV-PrM/TE completely protected ducks from DTMUV challenge, whereas rDEV-TE only conferred partial protection. These results demonstrate that recombinant DEV expressing the TE and pre-membrane proteins is protective and can serve as a potential candidate vaccine to prevent DTMUV infection in ducks. (C) 2014 Elsevier Ltd.

[31] LI H, WANG Y, HAN Z, WANG Y, LIANG S, JIANG L, HU Y, KONG X, LIU S . Recombinant duck enteritis viruses expressing major structural proteins of the infectious bronchitis virus provide protection against infectious bronchitis in chickens
Antiviral Research, 2016, 130:19-26. doi: 10.1016/j.antiviral.2016.03. 003.
DOI:10.1016/j.antiviral.2016.03.003URLPMID:26946113 [本文引用: 2]
To design an alternative vaccine for control of infectious bronchitis in chickens, three recombinant duck enteritis viruses (rDEVs) expressing the N, S, or S1 protein of infectious bronchitis virus (IBV) were constructed using conventional homologous recombination methods, and were designated as rDEV-N, rDEV-S, and rDEV-S1, respectively. Chickens were divided into five vaccinated groups, which were each immunized with one of the rDEVs, covalent vaccination with rDEV-N &amp;amp; rDEV-S, or covalent vaccination with rDEV-N &amp;amp; rDEV-S1, and a control group. An antibody response against IBV was detectable and the ratio of CD4(+)/CD8(+) T-lymphocytes decreased at 7 days post-vaccination in each vaccinated group, suggesting that humoral and cellular responses were elicited in each group as early as 7 days post-immunization. After challenge with a homologous virulent IBV strain at 21 days post-immunization, vaccinated groups showed significant differences in the percentage of birds with clinical signs, as compared to the control group (p?&amp;lt;?0.01), as the two covalent-vaccination groups and the rDEV-S group provided better protection than the rDEV-N- or rDEV-S1-vaccinated group. There was less viral shedding in the rDEV-N &amp;amp; rDEV-S- (2/10) and rDEV-N &amp;amp; rDEV-S1- (2/10) vaccinated groups than the other three vaccinated groups. Based on the clinical signs, viral shedding, and mortality rates, rDEV-N &amp;amp; rDEV-S1 covalent vaccination conferred better protection than use of any of the single rDEVs.

[32] 孙莹, 李俊平, 黄小洁, 李岭, 曹明慧, 李启红, 李慧姣, 杨承槐 . 表达绿色荧光蛋白重组鸭肠炎病毒构建
中国农业科学, 2016,49(14):2805-2812.
DOI:10.3864/j.issn.0578-1752.2016.14.014URL [本文引用: 1]
【Objective】Compared with duck enteritis virus(DEV) virulent strain, the vaccine strain has a 528 bp deletions at the UL2, resulting to a 176 aa deletion after amino acids 65. To study the effect of UL2 gene on virus biological properties and explore the feasibility of the DEV as a carrier to express foreign gene, a recombinant DEV expressing the green fluorescent protein (GFP) were constructed;【Method】In this study, the UL2 gene of DEV was chosen as a target site and homologous arm for recombination. Two fragments of UL2 gene were amplified by polymerase chain reaction (PCR) with DNA of DEV cell-adapted strain as template, and were cloned into the pMD-18T vector. The expression cassette including GFP gene and gpt gene controlled by CMV promoter was cloned into UL2 gene as a transfer vector pT-UL2-GFP-gpt. Confluent CEF monolayers were transfected with DEV and Lipofectamine 2000 was used as the transfer vector. When the cytopathic effect (CPE) was observed, the total supernatant and cells were harvested. The infected virus was diluted and then plated on the fresh CEF, and overlaid with M199-FBS containing 1% agarose. When green fluorescent plaques were observed, plaque-purification was carried out to obtain a green fluorescent plaque population termed rDEV-&#x025B3;UL2-GFP-gpt, PCR and sequencing assay were used to identify the recombinant virus. CEF cells cultured in 25cm2 flasks were inoculated with recombinant virus at an MOI of 0.01. The cells and supernatants were harvested respectively every 12 hours, the titer of virus were measured and the one-step growth analyses was performed; To evaluate the genetic stability of GFP gene in the recombinant virus, the virus was passaged in primary CEF 20 times. Four-week-old specific- pathogen-free (SPF) ducks were inoculated intramuscularly with the recombinant virus, and the ducks were challenged with lethal DEV (CVCC AV1221) by intramuscular injection at 14 days post vaccination, then the ducks were observed for symptom of disease and death.【Result】The recombinant expression vector pT-UL2-GFP-gpt was correctly constructed, identified by double-enzyme digestion. After 8 hours of transfection, spindle cells with green fluorescent were appeared. After 8 rounds of plaque-purification, the purified rDEV-&#x025B3;UL2-GFP-gpt were obtained. The results of the PCR and sequencing indicated that the GFP expression cassette has already successfully insert into the DEV genome, which replaced 196-723 nucleotide of UL2. The recombinant virus possessed growth kinetics were similar to that of the parental virus, the cell titer peaked at 36 hours with the peak titer 106.2TCID50/0.1mL, and the supernatant titer peaked at 72 hours with the peak titer 105.5TCID50/0.1mL. The virus were passaged in CEF cells 20 times, the GFP gene was stably maintained in 1st to 5th passages, however, from the 6th passage, there was little CPE without green fluorescent, and in 15th to 20th passages, most CPE had no green fluorescent, GFP mutated during subculture. All immunized animals were protected against subsequent challenge with lethal DEV, the insertion of the GFP gene did not alter the protective efficacy of parental virus. 【Conclusion】In this research, the recombinant DEV expressing the green fluorescent protein were successfully constructed, and firstly has confirmed that the deletion of UL2 gene has no effect on virus replication in cells and the immunogenicity in ducks. This study laid a foundation for the research of the function of the DEV UL2 gene and the DEV vector vaccine.
SUN Y, LI J P, HUANG X J, LI L, CAO M H, LI Q H, LI H J, YANG C H . Construction and characterization of recombinant duck enteritis virus expressing the green fluorescent protein
Scientia Agricultura Sinica, 2016, 49(14):2805-2812.(in Chinese)
DOI:10.3864/j.issn.0578-1752.2016.14.014URL [本文引用: 1]
【Objective】Compared with duck enteritis virus(DEV) virulent strain, the vaccine strain has a 528 bp deletions at the UL2, resulting to a 176 aa deletion after amino acids 65. To study the effect of UL2 gene on virus biological properties and explore the feasibility of the DEV as a carrier to express foreign gene, a recombinant DEV expressing the green fluorescent protein (GFP) were constructed;【Method】In this study, the UL2 gene of DEV was chosen as a target site and homologous arm for recombination. Two fragments of UL2 gene were amplified by polymerase chain reaction (PCR) with DNA of DEV cell-adapted strain as template, and were cloned into the pMD-18T vector. The expression cassette including GFP gene and gpt gene controlled by CMV promoter was cloned into UL2 gene as a transfer vector pT-UL2-GFP-gpt. Confluent CEF monolayers were transfected with DEV and Lipofectamine 2000 was used as the transfer vector. When the cytopathic effect (CPE) was observed, the total supernatant and cells were harvested. The infected virus was diluted and then plated on the fresh CEF, and overlaid with M199-FBS containing 1% agarose. When green fluorescent plaques were observed, plaque-purification was carried out to obtain a green fluorescent plaque population termed rDEV-&#x025B3;UL2-GFP-gpt, PCR and sequencing assay were used to identify the recombinant virus. CEF cells cultured in 25cm2 flasks were inoculated with recombinant virus at an MOI of 0.01. The cells and supernatants were harvested respectively every 12 hours, the titer of virus were measured and the one-step growth analyses was performed; To evaluate the genetic stability of GFP gene in the recombinant virus, the virus was passaged in primary CEF 20 times. Four-week-old specific- pathogen-free (SPF) ducks were inoculated intramuscularly with the recombinant virus, and the ducks were challenged with lethal DEV (CVCC AV1221) by intramuscular injection at 14 days post vaccination, then the ducks were observed for symptom of disease and death.【Result】The recombinant expression vector pT-UL2-GFP-gpt was correctly constructed, identified by double-enzyme digestion. After 8 hours of transfection, spindle cells with green fluorescent were appeared. After 8 rounds of plaque-purification, the purified rDEV-&#x025B3;UL2-GFP-gpt were obtained. The results of the PCR and sequencing indicated that the GFP expression cassette has already successfully insert into the DEV genome, which replaced 196-723 nucleotide of UL2. The recombinant virus possessed growth kinetics were similar to that of the parental virus, the cell titer peaked at 36 hours with the peak titer 106.2TCID50/0.1mL, and the supernatant titer peaked at 72 hours with the peak titer 105.5TCID50/0.1mL. The virus were passaged in CEF cells 20 times, the GFP gene was stably maintained in 1st to 5th passages, however, from the 6th passage, there was little CPE without green fluorescent, and in 15th to 20th passages, most CPE had no green fluorescent, GFP mutated during subculture. All immunized animals were protected against subsequent challenge with lethal DEV, the insertion of the GFP gene did not alter the protective efficacy of parental virus. 【Conclusion】In this research, the recombinant DEV expressing the green fluorescent protein were successfully constructed, and firstly has confirmed that the deletion of UL2 gene has no effect on virus replication in cells and the immunogenicity in ducks. This study laid a foundation for the research of the function of the DEV UL2 gene and the DEV vector vaccine.

[33] BUBLOT M, PRITCHARD N, LE GROS F X, GOUTEBROZE S . Use of a vectored vaccine against infectious bursal disease of chickens in the face of high-titred maternally derived antibody
Journal of Comparative Pathology, 2007, 137(Suppl 1):S81-84. doi: 10.1016/j. jcpa.2007.04.017.
DOI:10.1016/j.jcpa.2007.04.017URLPMID:17560594 [本文引用: 1]
Interference by maternally derived antibody (MDA) is a major problem for the vaccination of young chickens against infectious bursal disease (IBD). The choice of the timing of vaccination and of the type (degree of attenuation) of modified-live vaccine (MLV) to use is often difficult. An IBD vectored vaccine (vHVT13), in which turkey herpesvirus (HVT) is used as the vector, was recently developed. This vaccine is administered once at the hatchery, either in ovo or by the subcutaneous route, to 1-day-old chicks at a time when MDA is maximal. In terms of safety, the vHVT13 vaccine had negligible impact on the bursa of Fabricius when compared with classical IBD MLV. Vaccination and challenge studies demonstrated that this vaccine is able to protect chickens against various IBD virus (IBDV) challenge strains including very virulent, classical, and USA variant IBDV, despite the presence of high-titred IBD MDA at the time of vaccination. These data show that the vector vaccine combines a safety and efficacy profile that cannot be achieved with classical IBD vaccines.

[34] MATSUMURA T, KONDO T, SUGITA S, DAMIANI A M, O'CALLAGHAN D J, IMAGAWA H , An equine herpesvirus type 1 recombinant with a deletion in the gE and gI genes is avirulent in young horses
Virology, 1998,242(1):68-79. doi: 10.1006/viro.1997. 8984.
DOI:10.1006/viro.1997.8984URLPMID:9501037 [本文引用: 1]
The cell culture-adapted KyA strain of equine herpesvirus type 1 (EHV-1) has been found to be attenuated in young horses (Matsumura et al., 1996, Vet. Microbiol. 48, 353-365). The KyA strain lacks at least six genes in its genome, including those encoding glycoproteins gE and gI. To elucidate whether EHV-1 glycoproteins gE and gI play a role in viral virulence, we have constructed an EHV-1 recombinant that has the genes encoding both gE and gI deleted from its genome and its revertant. Growth properties of the deletion mutant virus in vitro were compared with those of the parent and the revertant viruses. Plaque size of the mutant virus in fetal horse kidney (FHK) cells was significantly smaller than those of the parent and the revertant viruses. In one-step growth experiments, however, the yields of infectious virus from FHK cells infected with the deletion mutant, the parent, or the revertant virus were approximately the same. The results suggested that gE and/or gI of EHV-1 promoted cell-to-cell spread of the virus, but that these glycoproteins were not involved in the process of virus maturation and release or in virus attachment and penetration. Subsequently, the virulence of mutant and revertant viruses was examined in young horses. No clinical signs were observed in six horses, including three colostrum-deprived foals inoculated intranasally with the deletion mutant virus, whereas three colostrum-deprived foals inoculated intranasally with the revertant virus manifested clinical signs typical for EHV-1 respiratory infection (i.e., pyrexia, nasal discharge, and swelling of submandibular lymph nodes). The results obtained from in vivo studies revealed that the EHV-1 mutant defective in both gE and gI genes was avirulent in young horses, suggesting that gE and/or gI of the EHV-1 have an important role in EHV-1 virulence. However, the EHV-1 mutant defective in both gE and gI genes induced only a partial protectivity in inoculated foals from manifestation of respiratory symptoms after challenge infection.

[35] DAMIANI A M, MATSUMURA T, YOKOYAMA N, MIKAMI T, TAKAHASHI E . A deletion in the gI and gE genes of equine herpesvirus type 4 reduces viral virulence in the natural host and affects virus transmission during cell-to-cell spread
Virus Research, 2000,67(2):189-202. doi: 10.1016/ S0168-1702(00)00146-5.
DOI:10.1016/s0168-1702(00)00146-5URLPMID:10867198 [本文引用: 1]
In order to identify the role of the equine herpesvirus type 4 (EHV-4) glycoprotein I (gI) and E (gE) genes in determining viral virulence and their affect on the infection cycle, we constructed an EHV-4 recombinant strain containing a deletion in both gI and gE genes and its revertant. The recombinant was assayed in vitro in order to compare its growth kinetics with the parent and revertant viruses. Our results indicated that a deletion in the genes encoding gI and gE affected cell-to-cell spread of the virus in vitro. In order to assess the pathogenicity and vaccine efficacy of the recombinant in a natural host, colostrum-deprived foals were inoculated intranasally with the recombinant. Clinical signs obtained in foals upon the inoculation with the recombinant were milder than that for the revertant. This suggests that intact gI and/or gE genes are important factors in the expression of virulence in EHV-4 as in seen in the case of other herpesviruses. In addition, full protection against challenge infection was observed in foals, which had undergone a previous inoculation of the recombinant.

[36] JOHNSON D C, WEBB M, WISNER T W, BRUNETTI C . Herpes simplex virus gE/gI sorts nascent virions to epithelial cell junctions, promoting virus spread
Journal of Virology, 2001,75(2):821-833. doi: 10.1128/ jvi.75.2.821-833.2001.
DOI:10.1128/JVI.75.2.821-833.2001URLPMID:11134295 [本文引用: 1]
Alphaherpesviruses spread rapidly through dermal tissues and within synaptically connected neuronal circuitry. Spread of virus particles in epithelial tissues involves movement across cell junctions. Herpes simplex virus (HSV), varicella-zoster virus (VZV), and pseudorabies virus (PRV) all utilize a complex of two glycoproteins, gE and gI, to move from cell to cell. HSV gE/gI appears to function primarily, if not exclusively, in polarized cells such as epithelial cells and neurons and not in nonpolarized cells or cells that form less extensive cell junctions. Here, we show that HSV particles are specifically sorted to cell junctions and few virions reach the apical surfaces of polarized epithelial cells. gE/gI participates in this sorting. Mutant HSV virions lacking gE or just the cytoplasmic domain of gE were rarely found at cell junctions; instead, they were found on apical surfaces and in cell culture fluids and accumulated in the cytoplasm. A component of the AP-1 clathrin adapter complexes, mu1B, that is involved in sorting of proteins to basolateral surfaces was involved in targeting of PRV particles to lateral surfaces. These results are related to recent observations that (i) HSV gE/gI localizes specifically to the trans-Golgi network (TGN) during early phases of infection but moves out to cell junctions at intermediate to late times (T. McMillan and D. C. Johnson, J. Virol., in press) and (ii) PRV gE/gI participates in envelopment of nucleocapsids into cytoplasmic membrane vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000). Therefore, interactions between the cytoplasmic domains of gE/gI and the AP-1 cellular sorting machinery cause glycoprotein accumulation and envelopment into specific TGN compartments that are sorted to lateral cell surfaces. Delivery of virus particles to cell junctions would be expected to enhance virus spread and enable viruses to avoid host immune defenses.

[37] JACOBS L, RZIHA H J, KIMMAN T G, GIELKENS A L, VAN OIRSCHOT J T . Deleting valine-125 and cysteine-126 in glycoprotein gI of pseudorabies virus strain NIA-3 decreases plaque size and reduces virulence in mice
Archives of Virology, 1993,131(3/4):251-264.
DOI:10.1007/bf01378630URLPMID:8394068 [本文引用: 1]
We investigated the function of antigenic domains on gI in virulence and immunogenicity. Three PRV gI mutants were constructed by deleting nucleotides coding for the following amino acids: valine-125 and cysteine-126, located in a discontinuous antigenic domain (M 303); glycine-59 and aspartic acid-60 located in a continuous antigenic domain (M304); and arginine-67 and alanine-68, located in a discontinuous antigenic domain (M305). Mismatch primers in the polymerase chain reaction were used to introduce the deletions. Anti-gI monoclonal antibodies were used in an immunoperoxidase monolayer assay to distinguish PRV gI mutants from wild-type PRV. The gI mutant viruses were tested for their growth in vitro and for their virulence in mice. The growth properties of PRV gI mutant virus M303 were comparable to the growth properties of a PRV gI-negative mutant (M301): both mutants produced small plaques in various cells, and when grown on swine kidney cells and chicken embryo fibroblasts, their growth was disadvantaged compared to wild-type PRV. However, in embryonal Balb/c mouse cells expressing gI, gI mutant viruses and wild-type PRV produced plaques of the same size, confirming that the mutations in gI are responsible for the small plaque phenotype. The growth properties of PRV gI mutant viruses M 304 and M 305 were comparable to the growth properties of wild-type PRV. When the mean time to death was used as the criterion, the gI mutant viruses M 301 and M 303 were significantly less virulent in mice than wild-type PRV. Four other, independently obtained, PRV mutants all carrying the valine-125 and cysteine-126 deletion (M 308, M 309, M 310 and M 311 respectively) exhibit the same phenotype. Our results show that deleting valine-125 and cysteine-126 in gI decreases plaque size and reduces virulence in mice to the same degree as deleting the gI protein.

[38] TRAPP S, OSTERRIEDER N, KEIL G M, BEER M . Mutagenesis of a bovine herpesvirus type 1 genome cloned as an infectious bacterial artificial chromosome: analysis of glycoprotein E and G double deletion mutants
Journal of General Virology, 2003, 84(Pt 2):301-306.
DOI:10.1099/vir.0.18682-0URLPMID:12560561 [本文引用: 1]
The genome of bovine herpesvirus type 1 Sch?nb?ken was cloned as a bacterial artificial chromosome (BAC) by inserting mini F plasmid sequences into the glycoprotein (g) E gene. The resulting BAC clone, pBHV-1DeltagE, was transfected into bovine kidney cells and viable gE-negative BHV-1 (BHV-1DeltagE) was recovered. By RecE/T mutagenesis in Escherichia coli, the gG open reading frame was deleted from pBHV-1DeltagE. From the mutated BAC, double negative BHV-1DeltagE-gG was reconstituted and its growth properties were compared to those of rescuant viruses in which the gE gene was restored (BHV-1rev, BHV-1DeltagG). The mutant viruses did not exhibit markedly lowered virus titres. Plaque sizes of BHV-1DeltagE, BHV-1DeltagE-gG and BHV-1DeltagG, however, were reduced by 19 to 55 % compared to parental strain Sch?nb?ken or BHV-1rev. Our results suggested that gE and gG function independently from each other in cell-to-cell spread, because an additive effect on plaque formation was observed in the gE/gG double deletion mutant.

[39] SUSSMAN M D, MAES R K, KRUGER J M, SPATZ S J, VENTA P J . A feline herpesvirus-1 recombinant with a deletion in the genes for glycoproteins gI and gE is effective as a vaccine for feline rhinotracheitis
Virology, 1995,214(1):12-20. doi: 10.1006/viro.1995.9959.
DOI:10.1006/viro.1995.9959URLPMID:8525607 [本文引用: 1]
Using a site-directed mutagenesis technique we constructed a new feline herpesvirus-1 recombinant strain containing a deletion in two genes encoding glycoproteins gI and gE. These proteins may have a role in virulence, the establishment of latency, and viral recurrence as shown in other herpesviruses of the varicella and simplex types. This recombinant was characterized and used to immunize juvenile cats against virulent virus challenge. Significant protection resulted from vaccination of cats by the subcutaneous route.