小反刍兽疫研究概况

小反刍兽疫是严重危害山羊和绵羊等家畜的A类动物传染病,目前该病已经给我国养羊业造成了巨大的威胁。近年来,国内外学者对该病开展了许多研究工作,本文从病原学、流行病学、临床症状、发病机理、病理变化、诊断、防控措施等几个方面对该病研究概况进行了综述,为该病的有效防控提供有益的参考。     

小反刍兽疫病毒研究进展

小反刍兽疫(PPR)是由小反刍兽疫病毒(PPRV)引起的一种主要感染小反刍动物的急性、烈性、接触性A类传染病,患病率、死亡率高.本文就世界PPR流行状况、PPRV基因组及病毒结构蛋白、PPRV检测方法、最新的药物及疫苗、存在的问题等方面做了简要综述.     

小反刍兽疫病毒研究进展

小反刍兽疫（PPR）是由小反刍兽疫病毒（PPRV）引起的一种主要感染小反刍动物的急性、烈性、接触性A类传染病,患病率、死亡率高。本文就世界PPR流行状况、PPRV基因组及病毒结构蛋白、PPRV检测方法、最新的药物及疫苗、存在的问题等方面做了简要综述。     

中国小反刍兽疫疫情分析

为分析中国PPRV毒株分子演变特点和疫情传播路线,从NCBI下载中国和世界其他国家全部PPRV毒株N和F基因全序列,应用分子生物学软件,并结合疫情发生的地理位置进行系统分析。结果显示,中国西藏阿里地区PPRV毒株之间核苷酸同源性为100%,其与西藏那曲地区毒株核苷酸同源性为99.9%;中国毒株与其他国家毒株F基因核苷酸序列分析显示,同源性为89.7%～98.8%,同源性最低的为科特迪瓦1989年毒株,同源性最高的为孟加拉国2010年毒株;N 基因核苷酸序列分析显示,同源性为69.5%～98.5%,同源性最低的为朝鲜2002年毒株,同源性最高的为印度1995年毒株;F和N 基因系统进化关系分析均显示,中国西藏3株PPRV均属于基因Ⅳ系;截止2014年3月30日,中国由西至东9省份发生该疫情。更加全面的分析结果,有待于更多PPRV流行毒株N或F基因全序列在GenBank的公布。     

小反刍兽疫病毒结构与功能的研究进展

小反刍兽疫病毒属于副黏病毒科麻疹病毒属,主要感染山羊、绵羊和野生小反刍兽,临床症状以发热、肺炎、腹泻及呼吸道和消化道黏膜炎症为主要特征。迄今为止对于小反刍兽疫无特效药物进行治疗,该病可对家畜养殖业造成一定的经济损失,因此对小反刍兽疫病毒病原学、结构和功能的研究已成为迫切需求。主要综述了小反刍兽疫病毒的六种结构蛋白N、P、M、F、HN、L和两种非结构蛋白C、V的基因组结构及功能,探讨了新型疫苗的研发方向,以期为小反刍兽疫病毒的深入研究、小反刍兽疫的临床防控提供参考。     

小反刍兽疫病毒基因组全长cDNA的构建及其序列的测定

根据小反刍兽疫Nigeria75/1株全基因组序列设计并合成PPRV特异引物,进而应用RT-PCR技术分5段扩增了PPRV全基因组cDNA。将扩增的各个cDNA重叠片段JF1、JF2、JF3、JF4和JF5分别克隆到载体上,建立了PPRV的初级cDNA克隆。在扩增5′末端时,引入AscI酶切位点和T7启动子序列;在基因组3′末段引入PacI酶切位点,后者供cDNA模板的线性化之用。将扩增片段再依次连接,最后亚克隆到质粒pok12中,获得了PPRV全长基因组cDNA克隆pok12-PPRV。通过序列同源性和进化树分析,结果表明,Nigeria75/1与MV和RPV的遗传关系最近。Nigeria75/1株全长cDNA的序列测定及构建,为拯救PPRV和在分子水平进一步深入研究PPRV打下坚实的基础。     

小反刍兽疫病毒H蛋白的原核表达及免疫原性测定

小反刍兽疫是由小反刍兽疫病毒（peste des petits ruminants virus,PPRV）引起的急性、接触性传染病,对我国的畜牧业发展造成了严重的影响,目前主要通过疫苗进行防控。为检测PPRV主要抗原蛋白血凝素（hemagglutinin,H）蛋白的免疫原性,从GenBank数据库中查找了近年来公布的H蛋白氨基酸序列,选择1条符合我国流行趋势的序列,对其基因序列进行优化合成后克隆至pET28a载体上。筛选出表达量高的Rosetta（DE3）菌株进行表达,表达产物经SDS?PAGE、Western Blot及质谱分析鉴定。经镍柱亲和层析纯化出单一H蛋白,取20 μg与佐剂混合后免疫小鼠,收集血清进行抗体效价检测,结果显示,血清中H蛋白抗体滴度在二免2周时达到1∶6 400,表明H蛋白具有较好的免疫原性。进一步对二免2周时血清进行中和抗体检测显示,小鼠血清对PPRV疫苗株具有中和效应,中和效价不超过1∶40。研究结果对H蛋白用于PPRV疫苗的研发提供了理论依据。     

小反刍兽疫病毒H和F蛋白在细胞融合中的作用

【目的】研究小反刍兽疫病毒囊膜糖蛋白(血凝素蛋白和融合蛋白)在病毒囊膜和宿主细胞膜融合过程中所发挥的作用。【方法】制备构建成功的小反刍兽疫病毒囊膜糖蛋白和病毒受体SLAM、Nectin4的真核表达质粒pCMV-HA-H、pCAGGS-Flag-F、pCMV-Myc-SLAM和pCMV-Myc-Nectin 4,将其组合转染至CHO-K1细胞,通过显微观察和间接免疫荧光技术分析小反刍兽疫病毒H和F蛋白在病毒融合过程中的功能。【结果】除空白对照组和重组质粒单独转染组细胞中没有发现合胞体外,其余组细胞中均出现了合胞体,而且F和H蛋白共转染组合胞体的数目明显较多;并在共表达H、F蛋白的细胞中观察到了蛋白分布极化的帽子现象。【结论】PPRV F蛋白是病毒囊膜和细胞膜融合的必需蛋白,但需要与PPRV H共同作用才能使病毒成功入侵靶细胞。     

小反刍兽疫病毒N蛋白原核表达条件的优化及反应活性

目的 探讨小反刍兽疫病毒（Peste des Petits Ruminants Virus,PPRV）N蛋白原核表达、条件优化及反应活性。方法 参照GenBank中PPRV(Nigeria/75/1)N基因序列,人工合成该基因,构建重组表达质粒pET-30a(+)-N,转化至BL21(DE3)感受态细胞,IPTG诱导表达,进行SDS-PAGE电泳、Western-blot分析。在不同温度、时间、IPTG浓度条件下诱导表达N蛋白,确定最佳表达条件。结果 PPRV N蛋白（64.4 kD）成功表达,能被羊PPRV免疫血清所识别。N蛋白主要以包涵体存在,其最佳诱导条件：诱导温度28℃、IPTG终浓度2.0 mmol/L、诱导时间16 h。结论 原核表达的PPRV N蛋白具有良好的特异性和反应活性,为单克隆抗体的制备及快速诊断方法的建立提供了技术资料。     

小反刍兽疫病毒诱导巨噬细胞产生炎症反应

[目的]探究小反刍兽疫病毒(Peste des petits ruminants virus, PPRV)感染引起的炎症反应并解析其致病机制。[方法]分离山羊肺泡巨噬细胞(PAM),利用qRT-PCR、ELISA及组织病理切片HE染色等方法分别检测PPRV感染后引起的炎性细胞因子表达、一氧化氮(NO)含量和炎性细胞侵润等现象。进一步利用转录组学测序分析PPRV感染PAM引起的差异基因、信号通路以及差异基因的表达情况。[结果]与正常山羊相比,感染PPRV的山羊其肝脏、肾脏及肺脏的部分细胞坏死并且呈现炎性细胞侵润的现象;与对照组相比,感染后的PAM细胞显著增加了NO含量(P<0.001);与未感染组相比,PPRV感染的PAM其炎性细胞因子IL-1β、TNF-α、IL-10等mRNA表达水平显著升高(P<0.05);此外,PPRV感染诱导的差异基因主要集中在炎症通路及NF-κB信号通路等。[结论]PPRV通过诱导大量炎性细胞侵润、增加炎性细胞因子表达,激活炎症信号通路从而产生炎症反应。     

Epigallocatechin gallate (EGCG) combined with zinc sulfate inhibits Peste des petits ruminants virus entry and replication

Despite the fact that the Peste des petits ruminants virus (PPRV) leads to high morbidity and mortality (up to 100%), antiviral drugs against PPRV are not available. The aim of this study was to estimate the dose of epigallocatechin gallate (EGCG) co-administered with zinc (II) ions as an antiviral agent against PPRV. Treatment of PPRV-infectedVero cells with EGCG and zinc sulfate (zinc II) was administered, and antiviral activities against PPRV in infected Vero cells was evaluated by determination of virus yields, expressed as logTCID₅₀/mL. Cytotoxicity was determined using the tetrazolium-based MTS test. Zinc sulfate at 1.1 mg/mL and EGCG at 25 μM showed low potentiated and potentiated antiviral activities against PPRV, respectively. These agents caused significant inhibition of PPRV in Vero cells (p < 0.05) with a reduction in logTCID₅₀/mL by up to 3-fold. The combination of EGCG (25 μM) and zinc sulfate (1.1 mg/mL) was observed to have strong antiviral activity (p < 0.01) against PPRV with a reduction in logTCID₅₀/mL of the virus up to 4-times without causing any host cell cytotoxicity. This study is the first one to prove that the zinc II has the capability of stimulating EGCG to inhibit in vitro PPRV entry. Moreover, this combination appears capable of reducing infection resistance by hindering viral adaptation.     

小反刍兽疫病毒Ｍ基因的截短表达及多抗血清的制备

目的 截短表达小反刍兽疫病毒M蛋白基因并将其用于多抗血清的制备。方法 之前有研究未能表达完整的M蛋白,而若在抗原性较弱的区域将其一分为二,以截短的形式进行表达,却能达到较理想的水平。因此根据GenBank上公布的PPRV M基因的序列,设计1对特异性引物,扩增出480 bp的目的基因,将其克隆至原核表达载体pET-32a(+)中,得到重组表达质粒pET-32a-PPRV-M1,后转化至Rosetta感受态细胞中,IPTG诱导表达后,通过SDS-PAGE和Western blot试验对重组蛋白进行鉴定,将纯化后的重组蛋白免疫6周龄BALB/c雌鼠,制备多克隆抗体血清。结果 经SDS-PAGE及Western blot鉴定,证明截短的M基因的蛋白主要以包涵体形式高效表达并具有良好的反应原性。结论 成功克隆表达了截短的小反刍兽疫病毒M基因的蛋白并制备了多抗血清,为建立血清学相关的检测方法及临床治疗奠定了基础。     

从消灭牛瘟分析全球根除小反刍兽疫的可行性

根除,又称为消灭,是指某传染病的传播永远停止,不再发生。2011年,联合国粮农组织和国际兽疫局联合发布了全球根除牛瘟的消息。这两大国际组织的官方信息预示着,牛瘟病毒,正如天花病毒,将来只能在授权的实验室中保存了。牛瘟根除之后,相关学者不禁把目光投向了下一个目标-小反刍兽疫,因为从病原学和病理学等诸多方面考虑,二者最为相似。本文首先分析了前者根除的主客观因素,然后从正反两方面评述了后者全球性根除的可行性。     

Cytokines expression profile and kinetics of Peste des petits ruminants virus antigen and antibody in infected and vaccinated goats

The present study deals with the co-ordination of cytokine (IL-4 and IFN-γ) expression and kinetics of peste des petits ruminants (PPR) virus antigen and antibody in PPRV infected and vaccinated goats. The infected animals exhibited mixed cytokine (both T_H1 and T_H2) responses in the initial phase of the disease. The infected and dead goats had increased IFN-γ response before their death; while IL-4 remained at the base level. The cytokine expression in recovered animals was almost similar to that of vaccinated ones, where a unique biphasic response of IL-4 expression was observed with an up-regulation of IFN-γ on 7^th days post vaccination (dpv). Analysis of PPR virus antigen and antibody kinetics in different components of blood from infected and vaccinated animals revealed that the PPR virus antigen load was highest in plasma followed by serum and blood of the infected animals, whereas vaccinated animals showed only marginal positivity on 9^th dpv. The antibody titer was high in serum followed by plasma and blood in both vaccinated and infected animals. Therefore, it is inferred that the presence of antigen and antibody were significant with the expression of cytokine, and that a decreased response of IL-4 was noticed during intermediate phase of the disease i.e., 7 to 12^th days post infection (dpi). This indicates the ability to mount a functional T_H2 response after 14^th dpi could be a critical determinant in deciding the survival of the PPR infected animal.     

西藏株小反刍兽疫病毒H蛋白的原核表达及其多克隆抗体的制备

【背景】小反刍兽疫是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起的一种急性、烈性、接触性传染病,严重威胁我国养羊业的发展。【目的】原核表达PPRVH蛋白,并制备其多克隆抗体。【方法】根据GenBank中PPRV西藏株h基因序列,对其进行密码子大肠杆菌偏爱性优化,采用两步PCR法全化学合成全长h基因。将测序验证正确的h基因克隆至原核表达载体pET-28a、pET-30a、pET-32a,转化E. coli BL21(DE3)并利用IPTG诱导H蛋白表达。以经SDS-PAGE割胶纯化的重组H蛋白免疫新西兰大白兔制备抗PPRV H蛋白多克隆抗体。【结果】重组E. coli [pET-28a(-30a,-32a)-H]表达的重组H蛋白相对分子质量分别约为70、68和86 kD;诱导7 h时PRRV H蛋白表达量最高,而且主要以包涵体形式表达;重组E.coli(pET-30a-H)表达的H蛋白经SDS-PAGE割胶纯化后免疫新西兰大白兔制备的多抗血清能与表达的重组H蛋白发生特异性反应;ELISA法检测抗体效价在1:6400-1:25600之间。【结论】原核表达了PPRVH蛋白,并制备了高效价的抗H蛋白多克隆抗体,为进一步研究PPRV H蛋白的功能及H蛋白的线性B细胞表位作图奠定了基础。     

青海岩羊源小反刍兽疫病毒N、F基因的遗传进化分析

2021年1月19日,青海省海西州都兰县巴隆乡伊克高里村发生岩羊不明原因的死亡,表现为离群独处、卧地不起、体质虚弱、觅食困难、肛门周围黑色附着物等现象。通过临床症状、病理学解剖及实时荧光RT-PCR诊断,为小反刍兽疫病毒(PPRV)感染。采用RT-PCR技术从病死岩羊病料组织中扩增出了PPRV N、F基因的部分片段。采用MegAlig、NT1和MEGA6.0软件对岩羊PPRV株N、F基因序列进行了比对和分析,绘制系统进化树,结果显示：此次岩羊感染的PPRV株N、F基因片段与新疆株(China/Xinjiang/2015/16)序列片段的同源性分别为99.43%和99.73%。遗传进化分析,该病原属于基因Ⅳ系。在N、F基因核酸序列水平上,与国内新疆地区分离毒株亲缘关系最近,同在一个小的分支;与国外毒株相比,N基因与塞内加尔和尼日利亚分离毒株亲缘关系较远,F基因与国外分离毒株亲缘关系较远。综上所述,青海岩羊源PPRV株属于基因Ⅳ系,与当前我国流行的野毒株属于同一个谱系。     

Study on Passive Immunity:time of Vaccination in Kids Born to Goats Vaccinated Against Peste des petits ruminants

In this study,the decay of maternal peste des petits ruminants virus(PPRV) antibodies in kids born to goats vaccinated with Asian lineage IV PPR vaccine and the efficacy of passive immunity against PPRV was assessed to determine the appropriate period for vaccination in kids.Serum samples collected from kids born to vaccinated,unvaccinated and infected goats at different time intervals were tested by PPR competitive ELISA and serum neutralization test(SNT).Maternal antibodies in kids were detectable up to 6 months with a decline trend from the third month onwards and receded below the protective level by the fourth month.The kid with an SN titre of 1:8 at the time of immunization showed significant PPRV specific antibody response(percentage inhibition of 76;SN titers >1:16),when tested on 21 day post-vaccination and was completely protected from infection upon virulent PPRV challenge.Similarly,the kid with 1:8 SN titers was completely protected from PPR infection on active challenge.Therefore,PPR vaccination is recommended in kids,aged 4 months and born to immunized or exposed goats.This could be a suitable period to avoid window of susceptibility in kids to PPRV and the effort to eliminate PPR infection from susceptible populations.     

Study on passive immunity: Time of vaccination in kids born to goats vaccinated against Peste des petits ruminants

In this study, the decay of maternal peste des petits ruminants virus (PPRV) antibodies in kids born to goats vaccinated with Asian lineage IV PPR vaccine and the efficacy of passive immunity against PPRV was assessed to determine the appropriate period for vaccination in kids. Serum samples collected from kids born to vaccinated, unvaccinated and infected goats at different time intervals were tested by PPR competitive ELISA and serum neutralization test (SNT). Maternal antibodies in kids were detectable up to 6 months with a decline trend from the third month onwards and receded below the protective level by the fourth month. The kid with an SN titre of 1:8 at the time of immunization showed significant PPRV specific antibody response (percentage inhibition of 76; SN titers >1:16), when tested on 21 day post-vaccination and was completely protected from infection upon virulent PPRV challenge. Similarly, the kid with 1:8 SN titers was completely protected from PPR infection on active challenge. Therefore, PPR vaccination is recommended in kids, aged 4 months and born to immunized or exposed goats. This could be a suitable period to avoid window of susceptibility in kids to PPRV and the effort to eliminate PPR infection from susceptible populations.