2. 新乡市动物疫病预防控制中心, 新乡 453003
2. Animal Disease Prevention and Control Center of Xinxiang, Xinxiang 453003, China
根据冠状病毒基因组特征和遗传关系, 可将其分为α-冠状病毒(Alphacoronavirus，CoV-α)、β-冠状病毒(Betacoronavirus，CoV-β)、γ-冠状病毒(Gammacoronavirus，CoV-γ)和δ-冠状病毒(Deltacoronavirus，CoV-δ)四个属，其中CoV-α和CoV-β只感染哺乳动物，而CoV-γ和CoV-δ主要感染鸟类，某些哺乳动物也可感染[1]。目前, 已知6种冠状病毒可感染猪，其中, 有4种属于CoV-α，包括猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)、猪传染性胃肠炎病毒(transmissible gastroenteritis virus，TGEV)、猪呼吸道冠状病毒(porcine respiratory coronavirus，PRCV)和猪急性腹泻综合征冠状病毒(swine acute diarrhea syndrome-coronavirus，SADS-CoV)，而猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus，PHEV)和猪德尔塔冠状病毒(porcine deltacoronavirus，PDCoV)分别属于CoV-β和CoV-δ[2]。近年发生的PDCoV和SADS-CoV引发的猪新发冠状病毒感染在我国局部地区及国外一些猪场流行，并对人类健康造成潜在威胁[3-6]。作者从PDCoV和SADS-CoV的病原学、病毒起源与进化、致病性以及检测诊断技术等方面进行综述，为后续研究提供参考借鉴。
1 PDCoV和SADS-CoV的病原学PDCoV属于套式病毒目(Nidovirales)、冠状病毒科(Coronaviridae)、δ-冠状病毒属(Deltacoronavirus)成员。病毒粒子呈球形或椭圆形，直径120~180 nm，具有囊膜结构，囊膜上分布有花瓣状纤突。PDCoV的核酸类型为不分节段的单股正链RNA，基因组全长约25 400 nt，编码4种结构蛋白：纤突蛋白(spike，S)、膜蛋白(membrane，M)、小包膜蛋白(envelope，E)和衣壳蛋白(nucleocapsid，N)。基因组构成为：5′非翻译区(untranslated region, UTR)-两个重叠的开放阅读框(open reading frame, ORF)ORF1a和ORF1b(ORF1a/1b)-S-E-M-非结构蛋白6(nonstructural protein 6, NS6)-N-NS7-UTR-3′[7-8]。其中，ORF1a/1b编码的两个多聚蛋白pp1a和pp1b被蛋白酶切割成14~16种非结构蛋白，参与病毒基因组转录和复制[2]。S蛋白负责病毒与细胞受体结合，介导病毒入侵和感染。同时，由于S蛋白能够诱导机体产生中和性抗体，可作为研制PDCoV疫苗的重要靶标[2, 9-10]。N蛋白主要定位于细胞核，通过自身非共价交联形成寡聚体，与病毒的组装、复制以及感染后的细胞应激反应密切相关[11]。M蛋白和E蛋白是构成PDCoV外膜的重要成分，同时M蛋白也可用于PDCoV的检测诊断[12]。位于M基因和N基因之间的NS6基因与病毒的复制和毒力相关[13]，并具有拮抗干扰素的功能[14]。NS7的编码区位于N基因内部，其编码蛋白定位于宿主细胞的线粒体，可能与病毒复制及拮抗宿主天然免疫有关[15]。位于NS7 3′末端的NS7a由一段单独的亚基因组mRNA编码，其具体的生物学功能仍有待研究[16]。
与PDCoV不同，SADS-CoV属于冠状病毒科中的CoV-α成员。尽管SADS-CoV在形态上与其他冠状病毒相似，但其基因组结构与PEDV、TGEV、PDCoV等冠状病毒明显不同。SADS-CoV的基因组全长约27 100 nt，基因组结构：5′-UTR-ORF1a/1b (ORF1ab)-S-NS3a-E-M-N-NS7a-NS7b-UTR-3′，核酸类型为单股正链RNA。由于SADS-CoV发现较晚，其编码蛋白(尤其是非结构蛋白)的生物学功能尚需进一步研究[2-3, 17]。
2 PDCoV和SADS-CoV的起源与进化2012年，Woo等[18]首次报道在猪的粪便样品中检测到PDCoV，并对其中两个毒株HKU15-44和HKU15-155的全基因组进行了测序和分析，发现PDCoV与来自麻雀的冠状病毒(SpCoV HKU17)亲缘关系最近，推测PDCoV可能来源于鸟类的跨种传播。后续的报道进一步支持了该假说[19-20]。另外，亚洲豹猫和中华雪貂可能是PDCoV传播的重要媒介[7]，发生在S编码区的基因重组是导致鸟类CoV-δ跨种传播的重要因素[19]。
Dong等[21]在一份采自2004年安徽某猪场的腹泻样品中检测到PDCoV(CHN-AH-2004)，暗示PDCoV可能早就在我国猪群中存在。CHN-AH-2004与HKU15-44及湖北毒株CHN-HB-2014亲缘关系较近，三者可能起源于同一毒株。来自中国境内的大部分PDCoV毒株(HKU15-155、HN-2014、HB-2014、JS-2014、SXD1、CHJXNI2、CHN-GD-2016)基因组序列相似性较高，亲缘关系较近，遗传相对稳定，尚未出现大的变异[22-25]。
2014年2月，PDCoV在美国暴发，给美国的养猪业造成了重大损失。美国毒株Ohio CVM1和来自中国香港的两个毒株(HKU15-44和HKU15-155)的基因组序列分别具有99%和99.1%的相似性，遗传关系较近[8]。韩国毒株KOR/KNU14-04/2014与美国PDCoV毒株的全基因组序列相似性在99.6%~99.9%，并在进化树上处于同一分支，表明它们可能起源于同一祖先[26]。泰国PDCoV毒株(S5011和S5015L)显示出独特的基因组序列特征，其5′UTR分别存在3个碱基(TCT)和1个碱基(A)的缺失，同时在ORF1a/b编码区分别存在6个碱基(AGTTTG)和9个碱基(GAGCCAGTC)的缺失，3′UTR区域存在4个碱基(CTCT)的插入，这些序列变化可能是导致泰国PDCoV毒力较强的原因[27]。
SADS-CoV是最近才被发现的猪新型冠状病毒。2017年，研究人员在我国广东省发生腹泻的猪群中检测到SADS-CoV，并对其进行了全基因组测序和遗传进化分析。发现SADS-CoV的基因组序列与猪的其他冠状病毒相似度较低，而与蝙蝠冠状病毒HKU2-CoV存在较高的相似性(≈95%)，并且遗传距离较近，推测二者可能起源于共同祖先[3, 28]，或者HKU2-CoV通过基因重组进化为SADS-CoV样病毒，从而获得对猪的感染能力[29]。总之，蝙蝠在SADS-CoV传播中发挥重要作用。与HKU2-CoV相比，SADS-CoV毒株CH/GD-01/2017/P2和CH/FJWT/2018的基因组多个区域存在碱基插入或缺失:在位于4 554~4 555 nt和20 504~20 505 nt位置分别存在3个碱基(TTG)和6个碱基(GGCCTC)的插入，在22 463~22 465 nt和24 773~24 775 nt位置分别存在3个碱基(GGC和GTA)的缺失，特别是发生在S蛋白75个氨基酸的替换和2个氨基酸的插入，可能与SADS-CoV的感染谱转换相关[17, 30]。另有研究发现，SADS-CoV与人冠状病毒229E/NL63进化距离较近，而且SADS-CoV与NL63具有相似的受体结合域，二者均可利用人的血管紧张素转换酶2(angiotensin-converting enzyme 2，ACE2)作为入侵受体，提示应重视对于SADS-CoV跨种传播到人类的潜在威胁(图 1)[17, 29, 31]。
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虚线代表潜在的跨种传播 The broken line denote potential interspecies transmission 图 1 PDCoV和SADS-CoV的起源及传播途径 Fig. 1 Origin and transmission routes of PDCoV and SADS-CoV |
各种年龄阶段的猪对PDCoV均易感，典型症状包括呕吐、水样腹泻、脱水等。新生仔猪感染后引起的死亡率较高，日龄较大的猪感染后通常能够恢复[32-33]。PDCoV除了能感染猪以外，也能感染牛和鸡[34-36]。与PEDV相似，PDCoV主要感染猪的小肠，空肠和回肠是其主要靶器官[37]。感染后引起小肠上皮细胞急性坏死，肠绒毛皱缩、脱落；肠壁变薄，肠道功能紊乱，机体对水分的吸收减少，从而导致腹泻[33, 38]。在PDCoV感染的早期，只能在小肠绒毛上皮细胞及固有层的淋巴细胞中检测到PDCoV抗原，当感染猪出现病毒血症时，在胃、肺、心、扁桃体、脾、肝及肾等器官也可检测到低拷贝的PDCoV RNA[39]。
关于PDCoV感染的细胞受体目前仍存在争议。有研究认为氨基肽酶(aminopeptidase N，APN)是PDCoV感染的关键受体[6, 40]。卢曼曼等[41]认为猪的APN并非PDCoV入侵的功能性受体，PDCoV的感染能力不受APN表达的影响。而Zhu等[42]认为，尽管APN不是PDCoV的功能性受体，但APN有助于增强PDCoV的感染能力。除APN外，PDCoV感染可能还需要第二受体(共受体)的参与[43]。尽管如此，由于多种哺乳动物、禽类以及人源的APN相对保守，且在体外均能与PDCoV的S1蛋白结合，提示对于PDCoV跨种传播的风险及其对人类健康的潜在威胁，应引起高度重视[6]。此外，PDCoV可利用N蛋白和非结构蛋白nsp5抑制干扰素产生，从而拮抗机体天然免疫，促进自身感染和复制[44-45]。
与PDCoV类似，SADS-CoV也可感染各个年龄阶段的猪，发病程度与猪的日龄有关，5日龄以内的仔猪感染后出现急性水样腹泻、呕吐、脱水等症状，病猪常在出现症状后2~6 d内死亡，死亡率高达90%左右。而母猪感染SADS-CoV后通常只表现出轻微的一过性腹泻，2 d后即可恢复[3]。SADS-CoV感染后，病变主要发生在小肠，空肠和回肠最为明显，导致肠壁变薄，肠腔内充盈大量黄色水样粪便。小肠绒毛变短，其中的毛细血管和中央乳糜管破坏严重，这可能是肠道功能丧失的主要原因[17]。将SADS-CoV通过Vero细胞传代后发现。高代次病毒(第83代)的毒力明显低于低代次(第7代)，表明SADS-CoV可以通过细胞传代降低毒力，该研究为疫苗的研制奠定了基础[46]。
另外，SADS-CoV能通过阻碍IRF3的激活干扰RIG-I信号通路，从而抑制IFN-β的产生[47]。SADS-CoV感染后，由FasL介导的外源性凋亡通路(caspase-8)和线粒体介导的内源性凋亡通路(caspase-9)共同作用导致细胞凋亡。环孢菌素A(CsA)能够通过阻止细胞凋亡和干扰病毒蛋白合成发挥抗病毒作用[48]。以上研究为寻找抗SADS-CoV的药物靶点提供了重要参考。
4 PDCoV和SADS-CoV的检测诊断技术由于猪的肠道冠状病毒引起的临床症状和病理变化非常相似，通过肉眼难以临床鉴别诊断，需借助实验室检测诊断技术确诊。目前，PDCoV的实验室检测诊断方法主要包括核酸检测和抗体检测。在核酸检测方面，由于PDCoV的M基因和N基因相对保守，常被作为PDCoV的基因检测靶标。基于M基因和N基因保守区的常规RT-PCR或巢式PCR方法已被用于临床检测及流行病学研究[24, 49-50]。针对临床上PDCoV容易与其他肠道病毒混合感染的情况，研究人员建立了一系列多重RT-PCR方法，用于检测多种病毒混合感染[51-54]。另外，基于环介导等温扩增技术(loop-mediated isothermal amplification，LAMP)、荧光定量及纳米技术的PCR方法也被用于PDCoV的诊断[55-59]。Gao等[60]以N蛋白为靶标，采用重组酶聚合酶扩增(recombinase polymerase amplification，RPA)与侧向层析技术(lateral flow dipstick，LFD)相结合，建立了用于检测PDCoV的LFD-RPA方法，该方法灵敏度可达1×102 copies·μL-1，在10~37 ℃环境下均可操作，10 min内即可得到检测结果，具有广阔的应用前景。在抗体检测方面，以重组M蛋白和N蛋白为包被抗原的ELISA方法取得了很好的临床检测效果[12, 61]。基于重组S蛋白的ELISA方法不仅可以用于临床诊断，也可用于抗体水平评价[62-63]。
与PDCoV类似，许多实验室建立了用于检测SADS-CoV的RT-PCR或荧光定量PCR方法。Zhou等[4]基于SADS-CoV的N基因设计引物，建立了常规RT-PCR方法，采用该方法对来自广东省45个猪场的236份腹泻样本进行了检测，发现SADS-CoV阳性率(43.5%)仅低于PEDV(78.2%)，明显高于猪轮状病毒(21.8%)和PDCoV(8.8%)的阳性率。同时发现SADS-CoV和PEDV混合感染的比例最高(17.65%)，需引起猪场对PDCoV的关注。另外，基于TaqMan和SYBR的实时荧光定量PCR技术以及RT-LAMP方法也为PDCoV的诊断提供了重要技术支撑[64-66]。
5 研究展望 5.1 病毒溯源及传播机制研究尽管基于PDCoV和SADS-CoV的基因组序列相似性分析推测其分别与鸟类和蝙蝠来源的冠状病毒进化距离较近，但仍然不能据此确证其源头。需加强对病毒溯源、变异及演化机制方面的研究，明确PDCoV和SADS-CoV的传播规律，弄清病毒变异与致病性之间的关系。
5.2 病毒感染与免疫机制研究由PDCoV和SADS-CoV引起的临床表现与其他肠道冠状病毒(PEDV、TGEV等)较为相似，但以上病毒的基因组结构存在差异，特别是编码的非结构蛋白生物学功能并不十分明确，不同病毒之间是否存在交叉保护尚不清楚，病毒感染后的免疫应答机制仍需研究阐明。
5.3 加强病毒监测及流行病学研究当前流行的SARS-CoV-2让公众更加认识到动物在人类疾病传播中的作用。特别需要指出的是，冠状病毒具有很强的重组能力，这是导致其跨种传播的重要因素[67]。鸟类和蝙蝠是冠状病毒的天然宿主，而猪是人类与其接触的重要媒介，同时，猪又是许多病毒传播的混合器，能为病毒重组创造良好条件。因此，加强对猪源PDCoV和SADS-CoV的监测及流行病学研究具有十分重要的公共卫生意义。
此外，疫苗接种是控制传染病最有效手段之一。应该汲取PEDV和SARS的流行暴发初期无有效疫苗可用导致的惨痛历史教训，加强PDCoV和SADS-CoV的疫苗研究，提前做好疫苗技术储备。在目前仍缺乏疫苗的情况下，猪场除做好日常管理外，加强生物安全是有效防控PDCoV和SADS-CoV的关键。
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