猪流行性腹泻病毒AJ1102株的分离、鉴定及其分子进化特征
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摘要
猪流行性腹泻(porcine epidemicdiarrhea, PED)是由猪流行性腹泻病毒(PEDV)引起的一种以腹泻、呕吐和脱水为主要特征的猪肠道传染病。该病1971年首次在英国被发现,随后,其它欧洲国家和部分亚洲国家相继报道了该病的发生与流行。以前,美国从未出现PEDV,但从2013年5月以来,美国多个州发生PEDV的流行,引起了美国乃至全球养猪业的高度重视。
我国是上世纪80年代初首次发生PEDV。由于该病对新生仔猪的感染率和致死率均很高,给养猪业造成了巨大的经济损失。2010年冬季以来,流行性腹泻在我国10多个省市暴发并呈现新的流行特征：发病率和致死率高、流行广、损失大,而且以前接种过猪流行性腹泻疫苗的猪也未能幸免,提示该流行毒株毒力较强,而且可能与现有疫苗毒株存在较大的差异,导致现有疫苗难以提供免疫保护。有意义的是,美国2013年流行的PEDV在基因组水平上与我国流行的PEDV同源性高达99.5%,称为中国变异株。针对当前流行的猪流行性腹泻病毒,本研究开展了检测方法的建立、病毒的分离与鉴定,同时,对流行毒株的遗传进化特征进行了深入研究。主要研究内容如下：
1. PEDV RT-PCR检测方法的建立及其流行病学调查
针对PEDV高度保守的M基因设计了一对特异性引物,建立了PEDV的RT-PCR检测方法,该引物扩增片段大小为552bp。该方法特异性好、敏感性高,并且检测速度快,适于大样品的检测。利用建立的RT-PCR方法,对2010至2011年间从华中、华东、华南等地区9个省份采集的600多份临床样品进行检测,结果显示不同地区的临床样品其PEDV阳性率均超过50%,平均阳性率为58.32%,表明PEDV在我国流行十分广泛,感染率相当高。
2. PEDV AJ1102株的分离、鉴定尽管PEDV的感染率很高,但国内外的研究表明,猪流行性腹泻病毒的分离相当困难、分离的成功率极低。将采自湖北省某猪场发病仔猪的临床样品接种Vero细胞,盲传3代后有一份临床样品接种的细胞可观察到明显的细胞病变,主要表现为细胞形成空泡的合胞体的病变,最后细胞发生裂解,PEDV特异性RT-PCR检测也证实为阳性；进一步采用间接免疫荧光实验证实病变的细胞能与PEDV特异性单克隆抗体发生反应,空斑实验测定该分离病毒滴度达106.0PFU/ml(F5代)。对分离的病毒通过梯度离心纯化并采用电子显微镜进行鉴定,能观察到直径大小约为100nm的病毒粒子以及明显的囊膜和纤突,具有典型的PEDV病毒粒子形态特征,证实所分离的病毒为PEDV,命名为PEDVAJ1102株。
3. PEDV AJ1102株的全基因组序列测定与遗传进化分析参考以前所报道的PEDV全基因组序列,设计了16对引物,通过分段扩增的方法对PEDV AJ1102株进行了全基因组序列测定。结果表明：AJ1102株全基因组全长28044bp(GenBank accession number JX188454),基因组排列与PEDV经典毒株CV777一样,排列顺序均为5'-ORF1a-ORF1b-S-ORF3-E-M-N-3'.将AJ1102株以及目前国内外已测定全基因组序列的其它31株PEDV毒株进行比较,并利用MEGA4软件绘制系统发育树,发现32株PEDV可分为两个大群即Ⅰ群和Ⅱ群；其中Ⅰ群和Ⅱ群又被分为两个亚群即la、Ⅰb及Ha、Ⅱb。Ⅱ群主要由国内2010年以后报道的毒株以及2013年美国流行毒株组成。AJ1102株属于Ⅱa亚群,为新型的PEDV变异株,与处于Ⅰa亚群的经典毒株CV777遗传关系较远。
4.PEDV AJ1102株主要基因的遗传进化分析
本研究比较分析了AJ1102株5'UTR、3'UTR区域以及结构蛋白M、N、S、E和非结构蛋白ORF3的遗传进化特征。与经典毒株CV777相比,AJ1102株的5’UTR有5个碱基缺失、1个碱基插入和8个碱基突变；3’UTR无缺失或插入,但有10个碱基的突变；S基因的变异位点主要出现在S1区域(1-2217nt),包括3个插入区域(168nt,176-186nt,431-433nt)和1个缺失区域(493-498nt),而且AJ1102株S蛋白的核心抗原表位区(COE)有8个氨基酸突变,SS6(772LQDGQVKI779)抗原表位有1个氨基酸突变；M基因、N基因、ORF3和E基因均无插入或缺失,但存在一些碱基突变。对M、N、S、E、ORF3和E基因的进化分析显示,AJ1102株均与2010年以后分离的毒株处在同一分支,而与经典毒株CV777遗传关系较远,表明AJ1102株是当前的流行毒株,是研制针对当前PEDV流行毒株疫苗的候选毒株。
5. PEDV流行毒株的遗传标志
根据对全基因组以及主要结构蛋白的遗传进化分析,发现流行毒株S基因的S1区域具有较大的变异。为了探讨该区域是否具有目前我国PEDV流行毒株的特征性变异或遗传标记,针对S1区域的主要变异区设计了一对可扩增约450bp片段的特异性引物,对61份临床样品进行了RT-PCR扩增与序列分析,发现：目前在我国流行的PEDV毒株的S1基因具有特征性变异,提示这一特征性变异可以作为区分流行毒株和经典毒株的遗传标志,在实际操作中我们可以通过对该区域的检测,以确定特定猪场的PEDV是变异毒株还是经典毒株,为疫苗的选择提供理论依据。
6.姜黄素对PEDV增殖的抑制作用研究
中草药姜黄素不仅具有抗炎、抗肿瘤、抗氧化、抗诱变的作用,而且还具有抗病毒的作用,其对乙型肝炎病毒(HBV),丙型肝炎病毒(HCV)、人类免疫缺陷病毒等多种病毒均具有明显的抑制效果。然而姜黄素对PEDV是否具有抗病毒作用还未见报道。本研究利用10uM、20μM及30uM的姜黄素处理细胞后,再接种PEDVAJ1102毒株,并于感染后6h、12h和18h收集样品,通过空斑实验测定病毒滴度,发现姜黄素能够显著抑制PEDV的增殖,且呈时间和剂量依赖性。
Porcine epidemic diarrhea (PED) is a kind of contagious enteric disease caused by porcine epidemic diarrhea viruse (PEDV) characterized by acute watery diarrhea, vomiting and dehydration, which was first reported in England in1971. The occurrence and prevelance were consecutively reported in other Europe countries and part of Asia countries. America never has PEDV previously, there were prevalence of PEDV in several states of United States since May,2013which was paid the high attention in porcine industry of America and the whole world.
In1980s, this disease first occurred in our country. It has caused severe economic loss to swine industry due to the high morbidity and mortality to newborn piglets. Since winter in2010, epidemic disease erupted in10provinces of China with new characteristics:pandemic, high morbidity and mortality with severe loss. The pigs vaccinated previously were also infected which incidicated that the epidemic strain with strong virulence had big difference with current vaccine strain, as a result, the current vaccine strain was invalid to provide protection. It is meaningful that the genome sequence of PEDV strain isolated in Ameirica in2013shared99.5%sequence identity with the epidemic PEDV strain isolated in our country which is called Chinese variant strain. For this current porcine epidemic diarrhae virus, we established RT-PCR detection, isolation and identification method, meanwhile, we had a intensive study to genetic evolution characteristics of epidemic strain. The major studies include:
1. The establishment of RT-PCR detective method diagnosis and epidemiological survey for PEDV.
To the highly conservative M gene, a pair of specific primer were degined to establish the RT-PCR detective method, the amplified segment was552bp. This method is specific, sensitive and rapid which is propriate for the detection of big samples. The600clinic specimen from9provinces in central, east and south of China were detected, the result indicated the average positive rate of PEDV was58.32%, which showed the high infection rate of pandemic PEDV in China.
2. Isolation and identification of AJ1102strain
According to the research at home and abroad, the isolation of PEDV is extremely difficult although the infection rate is high. The clinic specimen of diseased piglets from Hubei pig farm was innoculated in Vero cell, after3blind passages, the apparent CPE could be observed, the specific RT-PCR decteted it positive, the further indirect immunofluorescence test veified that the isolated strain reacted with specific monoclonal antibody. Plaque assay idendified the titer of isolated strain was106.0PFU/ml. Electron microscopy after gradient centrifugation showed virion in100nm diameter with obvious envelope and spike which were the typical morphology of PEDV. All the above conformed the isolated strain PEDV named PEDV AJ1102.
3. Determination and phylogenetic analysis of the full-length genomic sequences of PEDV AJ1102
Refer to complete genome sequence of PEDV reported previously,16pairs of primers were designed to determinate complete genome sequence of PEDV AJ1102strain by RT-PCR segmented amplifying method. The result showed, the complete genome sequence of PEDV AJ1102strain was28044bp (GenBank accession number JX188454), arranged in5'-ORFla-ORFlb-S-ORF3-E-M-N-3'with the same sequnce as the PEDV typical strain CV777. Compared AJ1102strain with other31strains at home and aborad whose complete sequneces determined and pictured the phylogenetic tree by MEGA4, which showed the32strains of PEDV divided into two classes:group Ⅰ and group Ⅱ which had two subtypes respectively:Ⅰa、Ⅰb and Ⅱa、Ⅱb. Group Ⅰ was composed by the strains reported after2010inland and America epidemic strains in2013. AJ1102belongs to subtype Ⅱa, the neotype variant strain which is remote to classical strain CV777in Ⅰa.
4. the phylogenetic analysis for major genes of AJ1102
The study compared the difference in5'UTR,3'UTR, M, N, S, E and ORF3between AJ1102and classical strain CV777. AJ1102had5bases deletion,1base insertion and8bases mutation in5'UTR; there was no insertion and deletion in3'UTR except10bases mutation; the major mutation area was in S1area (1-2217nt) including3insertion areas(168nt,176-186nt,431-433nt) and1deletion area(493-498nt), and there were8amino acids mutation in COE epitope of S protein,1amino acid mutation in SS6(772LQDGQVKI779) epitope; there was no insertion and deletion in M, N, ORF3and E genes except some bases mutation. The phylogenetic analysis of M, N, S, E, ORF3and E genes showed that AJ1102was in the same branch with the other isolated strains after2010, and remote to classical strain CV777, thus proved AJ1102was the current epidemic strain and the exactly the cadidate strain for vaccine preparation of the current PEDV.
5. The genetic marker for current strain of PEDV
According to the genetic evolution analysis of complete gene and major structure proteins, we found there was comparatively big variant in SI area of S gene. In order to explore if this area had the characteristic or genetic marker of current PEDV strain, we designed a pair of specific primer to amplify the450bp segments of major variant area of S1area to do RT-PCR amplifying and sequence analysis which indicated that there was representative variation could be used as the genetic marker to distinguish between epidemic strain and classical strain to provide theoretical basis for vaccine selection.
6. Analysis for the anti-PEDV effects of curcumin
Curcumin is a kind of Chinese herb has not only anti-inflammation, anti-tumor, anti-oxidation and antimutagenesis effect but also anti-virus effect which can remarkably inhibit HBV, HCV, HIV and other various viruses. However the anti-virus effect of curcumin hasn't been reported. This study utilized curcumin of lOuM,20uM and30uM to take effects on cells and then AJ1102strain was inoculated, the cells were collected after6h,12h and18h, the plague assay was used to identify the virus titer. It was found that the curcumin remarkably inhibited the proliferationof PEDV, with time and dose dependent.
我国是上世纪80年代初首次发生PEDV。由于该病对新生仔猪的感染率和致死率均很高,给养猪业造成了巨大的经济损失。2010年冬季以来,流行性腹泻在我国10多个省市暴发并呈现新的流行特征：发病率和致死率高、流行广、损失大,而且以前接种过猪流行性腹泻疫苗的猪也未能幸免,提示该流行毒株毒力较强,而且可能与现有疫苗毒株存在较大的差异,导致现有疫苗难以提供免疫保护。有意义的是,美国2013年流行的PEDV在基因组水平上与我国流行的PEDV同源性高达99.5%,称为中国变异株。针对当前流行的猪流行性腹泻病毒,本研究开展了检测方法的建立、病毒的分离与鉴定,同时,对流行毒株的遗传进化特征进行了深入研究。主要研究内容如下：
1. PEDV RT-PCR检测方法的建立及其流行病学调查
针对PEDV高度保守的M基因设计了一对特异性引物,建立了PEDV的RT-PCR检测方法,该引物扩增片段大小为552bp。该方法特异性好、敏感性高,并且检测速度快,适于大样品的检测。利用建立的RT-PCR方法,对2010至2011年间从华中、华东、华南等地区9个省份采集的600多份临床样品进行检测,结果显示不同地区的临床样品其PEDV阳性率均超过50%,平均阳性率为58.32%,表明PEDV在我国流行十分广泛,感染率相当高。
2. PEDV AJ1102株的分离、鉴定尽管PEDV的感染率很高,但国内外的研究表明,猪流行性腹泻病毒的分离相当困难、分离的成功率极低。将采自湖北省某猪场发病仔猪的临床样品接种Vero细胞,盲传3代后有一份临床样品接种的细胞可观察到明显的细胞病变,主要表现为细胞形成空泡的合胞体的病变,最后细胞发生裂解,PEDV特异性RT-PCR检测也证实为阳性；进一步采用间接免疫荧光实验证实病变的细胞能与PEDV特异性单克隆抗体发生反应,空斑实验测定该分离病毒滴度达106.0PFU/ml(F5代)。对分离的病毒通过梯度离心纯化并采用电子显微镜进行鉴定,能观察到直径大小约为100nm的病毒粒子以及明显的囊膜和纤突,具有典型的PEDV病毒粒子形态特征,证实所分离的病毒为PEDV,命名为PEDVAJ1102株。
3. PEDV AJ1102株的全基因组序列测定与遗传进化分析参考以前所报道的PEDV全基因组序列,设计了16对引物,通过分段扩增的方法对PEDV AJ1102株进行了全基因组序列测定。结果表明：AJ1102株全基因组全长28044bp(GenBank accession number JX188454),基因组排列与PEDV经典毒株CV777一样,排列顺序均为5'-ORF1a-ORF1b-S-ORF3-E-M-N-3'.将AJ1102株以及目前国内外已测定全基因组序列的其它31株PEDV毒株进行比较,并利用MEGA4软件绘制系统发育树,发现32株PEDV可分为两个大群即Ⅰ群和Ⅱ群；其中Ⅰ群和Ⅱ群又被分为两个亚群即la、Ⅰb及Ha、Ⅱb。Ⅱ群主要由国内2010年以后报道的毒株以及2013年美国流行毒株组成。AJ1102株属于Ⅱa亚群,为新型的PEDV变异株,与处于Ⅰa亚群的经典毒株CV777遗传关系较远。
4.PEDV AJ1102株主要基因的遗传进化分析
本研究比较分析了AJ1102株5'UTR、3'UTR区域以及结构蛋白M、N、S、E和非结构蛋白ORF3的遗传进化特征。与经典毒株CV777相比,AJ1102株的5’UTR有5个碱基缺失、1个碱基插入和8个碱基突变；3’UTR无缺失或插入,但有10个碱基的突变；S基因的变异位点主要出现在S1区域(1-2217nt),包括3个插入区域(168nt,176-186nt,431-433nt)和1个缺失区域(493-498nt),而且AJ1102株S蛋白的核心抗原表位区(COE)有8个氨基酸突变,SS6(772LQDGQVKI779)抗原表位有1个氨基酸突变；M基因、N基因、ORF3和E基因均无插入或缺失,但存在一些碱基突变。对M、N、S、E、ORF3和E基因的进化分析显示,AJ1102株均与2010年以后分离的毒株处在同一分支,而与经典毒株CV777遗传关系较远,表明AJ1102株是当前的流行毒株,是研制针对当前PEDV流行毒株疫苗的候选毒株。
5. PEDV流行毒株的遗传标志
根据对全基因组以及主要结构蛋白的遗传进化分析,发现流行毒株S基因的S1区域具有较大的变异。为了探讨该区域是否具有目前我国PEDV流行毒株的特征性变异或遗传标记,针对S1区域的主要变异区设计了一对可扩增约450bp片段的特异性引物,对61份临床样品进行了RT-PCR扩增与序列分析,发现：目前在我国流行的PEDV毒株的S1基因具有特征性变异,提示这一特征性变异可以作为区分流行毒株和经典毒株的遗传标志,在实际操作中我们可以通过对该区域的检测,以确定特定猪场的PEDV是变异毒株还是经典毒株,为疫苗的选择提供理论依据。
6.姜黄素对PEDV增殖的抑制作用研究
中草药姜黄素不仅具有抗炎、抗肿瘤、抗氧化、抗诱变的作用,而且还具有抗病毒的作用,其对乙型肝炎病毒(HBV),丙型肝炎病毒(HCV)、人类免疫缺陷病毒等多种病毒均具有明显的抑制效果。然而姜黄素对PEDV是否具有抗病毒作用还未见报道。本研究利用10uM、20μM及30uM的姜黄素处理细胞后,再接种PEDVAJ1102毒株,并于感染后6h、12h和18h收集样品,通过空斑实验测定病毒滴度,发现姜黄素能够显著抑制PEDV的增殖,且呈时间和剂量依赖性。
Porcine epidemic diarrhea (PED) is a kind of contagious enteric disease caused by porcine epidemic diarrhea viruse (PEDV) characterized by acute watery diarrhea, vomiting and dehydration, which was first reported in England in1971. The occurrence and prevelance were consecutively reported in other Europe countries and part of Asia countries. America never has PEDV previously, there were prevalence of PEDV in several states of United States since May,2013which was paid the high attention in porcine industry of America and the whole world.
In1980s, this disease first occurred in our country. It has caused severe economic loss to swine industry due to the high morbidity and mortality to newborn piglets. Since winter in2010, epidemic disease erupted in10provinces of China with new characteristics:pandemic, high morbidity and mortality with severe loss. The pigs vaccinated previously were also infected which incidicated that the epidemic strain with strong virulence had big difference with current vaccine strain, as a result, the current vaccine strain was invalid to provide protection. It is meaningful that the genome sequence of PEDV strain isolated in Ameirica in2013shared99.5%sequence identity with the epidemic PEDV strain isolated in our country which is called Chinese variant strain. For this current porcine epidemic diarrhae virus, we established RT-PCR detection, isolation and identification method, meanwhile, we had a intensive study to genetic evolution characteristics of epidemic strain. The major studies include:
1. The establishment of RT-PCR detective method diagnosis and epidemiological survey for PEDV.
To the highly conservative M gene, a pair of specific primer were degined to establish the RT-PCR detective method, the amplified segment was552bp. This method is specific, sensitive and rapid which is propriate for the detection of big samples. The600clinic specimen from9provinces in central, east and south of China were detected, the result indicated the average positive rate of PEDV was58.32%, which showed the high infection rate of pandemic PEDV in China.
2. Isolation and identification of AJ1102strain
According to the research at home and abroad, the isolation of PEDV is extremely difficult although the infection rate is high. The clinic specimen of diseased piglets from Hubei pig farm was innoculated in Vero cell, after3blind passages, the apparent CPE could be observed, the specific RT-PCR decteted it positive, the further indirect immunofluorescence test veified that the isolated strain reacted with specific monoclonal antibody. Plaque assay idendified the titer of isolated strain was106.0PFU/ml. Electron microscopy after gradient centrifugation showed virion in100nm diameter with obvious envelope and spike which were the typical morphology of PEDV. All the above conformed the isolated strain PEDV named PEDV AJ1102.
3. Determination and phylogenetic analysis of the full-length genomic sequences of PEDV AJ1102
Refer to complete genome sequence of PEDV reported previously,16pairs of primers were designed to determinate complete genome sequence of PEDV AJ1102strain by RT-PCR segmented amplifying method. The result showed, the complete genome sequence of PEDV AJ1102strain was28044bp (GenBank accession number JX188454), arranged in5'-ORFla-ORFlb-S-ORF3-E-M-N-3'with the same sequnce as the PEDV typical strain CV777. Compared AJ1102strain with other31strains at home and aborad whose complete sequneces determined and pictured the phylogenetic tree by MEGA4, which showed the32strains of PEDV divided into two classes:group Ⅰ and group Ⅱ which had two subtypes respectively:Ⅰa、Ⅰb and Ⅱa、Ⅱb. Group Ⅰ was composed by the strains reported after2010inland and America epidemic strains in2013. AJ1102belongs to subtype Ⅱa, the neotype variant strain which is remote to classical strain CV777in Ⅰa.
4. the phylogenetic analysis for major genes of AJ1102
The study compared the difference in5'UTR,3'UTR, M, N, S, E and ORF3between AJ1102and classical strain CV777. AJ1102had5bases deletion,1base insertion and8bases mutation in5'UTR; there was no insertion and deletion in3'UTR except10bases mutation; the major mutation area was in S1area (1-2217nt) including3insertion areas(168nt,176-186nt,431-433nt) and1deletion area(493-498nt), and there were8amino acids mutation in COE epitope of S protein,1amino acid mutation in SS6(772LQDGQVKI779) epitope; there was no insertion and deletion in M, N, ORF3and E genes except some bases mutation. The phylogenetic analysis of M, N, S, E, ORF3and E genes showed that AJ1102was in the same branch with the other isolated strains after2010, and remote to classical strain CV777, thus proved AJ1102was the current epidemic strain and the exactly the cadidate strain for vaccine preparation of the current PEDV.
5. The genetic marker for current strain of PEDV
According to the genetic evolution analysis of complete gene and major structure proteins, we found there was comparatively big variant in SI area of S gene. In order to explore if this area had the characteristic or genetic marker of current PEDV strain, we designed a pair of specific primer to amplify the450bp segments of major variant area of S1area to do RT-PCR amplifying and sequence analysis which indicated that there was representative variation could be used as the genetic marker to distinguish between epidemic strain and classical strain to provide theoretical basis for vaccine selection.
6. Analysis for the anti-PEDV effects of curcumin
Curcumin is a kind of Chinese herb has not only anti-inflammation, anti-tumor, anti-oxidation and antimutagenesis effect but also anti-virus effect which can remarkably inhibit HBV, HCV, HIV and other various viruses. However the anti-virus effect of curcumin hasn't been reported. This study utilized curcumin of lOuM,20uM and30uM to take effects on cells and then AJ1102strain was inoculated, the cells were collected after6h,12h and18h, the plague assay was used to identify the virus titer. It was found that the curcumin remarkably inhibited the proliferationof PEDV, with time and dose dependent.
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