張奇亞

(1. 中國科學院水生生物研究所淡水生態(tài)與生物技術(shù)國家重點實驗室, 武漢 430072;2. 中國科學院種子設(shè)計創(chuàng)新研究院, 北京 100101)

為滿足全球日益增長的人口對優(yōu)質(zhì)蛋白質(zhì)的需求, 水產(chǎn)養(yǎng)殖業(yè)正快速發(fā)展[1,2], 且中國水產(chǎn)養(yǎng)殖的成功經(jīng)驗已提供給全球共享[3]。但仍面臨動物種類多、養(yǎng)殖密度大、在多變或劣質(zhì)水環(huán)境中易受流行病侵染的困擾[4]。尤其是病毒引起的水產(chǎn)動物疾病, 發(fā)病快、死亡率高、傳播廣、危害大, 尚無特效藥物與解決方案, 被認為是水產(chǎn)養(yǎng)殖業(yè)發(fā)展的限制因素之一[5—7]。而深入認識病毒病原本質(zhì)特征, 則成為有效檢測、預防控制水產(chǎn)動物病毒病的關(guān)鍵[6,8—11]。

具有雙鏈DNA基因組、其分子量接近或大于100 kb的病毒通常被稱為大DNA病毒(或核質(zhì)大DNA病毒, NCLDVs)[12,13]。包括感染動物的痘病毒科(Poxviridae)、虹彩病毒科(Iridoviridae)、魚蛙皰疹病毒科(Alloherpesviridae)、線頭病毒科(Nimaviridae)、非洲豬瘟病毒科(Asfarviridae)、桿狀病毒科(Baculoviridae)和囊泡病毒科(Ascoviridae)成員,感染真核藻的藻類DNA病毒科(Phycodnaviridae)成員, 感染原核藻或藍藻菌的肌尾病毒科(Myoviridae)成員, 感染原生生物的擬菌病毒科(Mimiviridae)、馬賽病毒科(Marseilleviridae)、潘多拉病毒科(Pandoraviridae)、闊口罐病毒科(Pithoviridae)成員等[14]。水生大DNA病毒變異率雖比RNA病毒要低, 但它們的宿主范圍及分布環(huán)境極廣, 存在于各種水體和沉積物中[15—17]。已開展針對大DNA病毒的遺傳進化及其宿主適應(yīng)性的研究[18—21], 其中有感染水產(chǎn)動物重要的病毒病原, 如: 虹彩病毒(Iridoviruses)、皰疹病毒(Herpesviruses)、線頭病毒(Nimaviruses)[22,23]和感染藍藻菌的肌尾噬藻體(Myovirus)[24]。近期查明水環(huán)境中大DNA病毒和巨病毒(Giant viruses)的部分基因來源于宿主[25], 且能增加病毒對宿主的適應(yīng)性[26]。相關(guān)研究不僅拓寬病毒知識的邊界, 也將促進水產(chǎn)動物病毒病遠程診斷、生物調(diào)控等智慧漁業(yè)水平的提升。

中國科學院水生生物研究所(簡稱中科院水生所)對魚病防控及魚池控藻的研究始于20世紀50年代, 由倪達書負責成立魚病工作站, 并在蘇、浙、粵等省展開魚病調(diào)查[27]。同時, 饒欽止[28]研究和報道了消滅魚池微囊藻湖靛(水華)的有效方法。20世紀70年代后期, 陳燕燊等[29]則對草魚出血病病毒病原開展研究, 以集體署名方式在《水生生物學集刊》上發(fā)表相關(guān)結(jié)果。1996年, 《水生生物學報》報道了我國分離鑒定的第一株水生脊椎動物大DNA病毒——沼澤綠牛蛙蛙病毒(Rana gryliovirus,RGV)的相關(guān)研究[30,31](圖 1), 并由此開始兩棲類以及魚類虹彩病毒的研究, 內(nèi)容涉及病原的分離鑒定和基因組測序, 如: 魚淋巴囊腫病毒中國株 (Lymphocystis disease virus-China, LCDV-C, AY380826)[32]、牛蛙蛙病毒RGV (JQ654586)[33]和大鯢蛙病毒 (Andrias davidianusranvirus, ADRV, KC865735)[34]; 確定病毒在宿主體內(nèi)或細胞中的分布與定位[35], 比較不同毒株主要結(jié)構(gòu)蛋白基因的異同[36]; 鑒定一批功能基因[37—41], 并闡明它們在病毒復制中的作用, 新建雙熒光標記可控基因表達重組病毒技術(shù)[42]; 揭示幾種大DNA病毒與水產(chǎn)動物宿主相互作用的分子機制[43]。病原體》(Ranaviruses: lethal pathogens of ectothermic vertebrates)的撰寫[46]、獲湖北省自然科學獎的“重要水產(chǎn)動物病毒病原的鑒定及致病機理研究” (2004Z-034-2-010-007)和“水產(chǎn)動物不同病毒基因組解析及病毒與宿主相互作的用分子機制”(2017Z-023-2-010-008)等工作, 記錄和見證了“水生病毒學”新枝萌發(fā)的過程。中科院水生所報道的蛙病毒RGV[31,33]、大鯢蛙病毒ADRV[34]及淋巴囊腫病毒LCDV-C[32], 中山大學報道的鱖魚傳染性脾腎壞死病毒ISKNV(AF371960)[47]和虎紋蛙病毒TFV(AF389451)、國家海洋局報道的大黃魚虹彩病毒LYCIV(AY779031)[48]、中國水產(chǎn)科學研究院黃海水產(chǎn)研究所報道的大菱鲆紅體病虹彩病毒TRBIV(GQ273492)[49]及新加坡石斑魚虹彩病毒SGIV(AY521625)[50]等作為虹彩病毒參考毒株, 被2020年更新發(fā)布的《國際病毒分類委員會報告》(The International Committee on Taxonomy of Viruses, ICTV Report)收錄[51,52], 為大DNA病毒種群共性特征提供借鑒[53]。

圖1 蛙病毒RGV引起的蛙致死性出血綜合癥及其負染電鏡圖(黃曉紅 圖)Fig. 1 Rana grylio virus (RGV) caused frog disease with lethal hemorrhagic syndrome and negative staining electron micrograph of the ranavirus particles. Bar=200 nm

盡管人類病毒性疫病會引起公共衛(wèi)生挑戰(zhàn), 甚至引發(fā)全球政治經(jīng)濟格局變化[54,55], 但病毒的感染與毒性有特定宿主范圍[56,57]?，F(xiàn)有研究表明, 水生病毒僅感染低等脊椎動物與其他水生生物, 尚無魚類病毒會感染人類的直接證據(jù)。水產(chǎn)品是人類安全和高質(zhì)量蛋白質(zhì)的重要來源。世界動物衛(wèi)生組織 (OIE)指出: 水產(chǎn)養(yǎng)殖的好處是無窮的[58]。水產(chǎn)健康養(yǎng)殖能更好地促進水產(chǎn)動物、水環(huán)境與人類的整體健康[59]。水生病毒學當前的重點任務(wù)就是要在闡釋病毒本質(zhì)及其與宿主和水環(huán)境相互作用的基礎(chǔ)上, 降低水生動物發(fā)生病毒病的風險, 并借助噬藻體等藍藻菌病毒優(yōu)化水生態(tài)系統(tǒng), 促進生態(tài)健康的水產(chǎn)養(yǎng)殖業(yè)可持續(xù)增長[9]。

1 研究動態(tài)

歷經(jīng)半個世紀, “水生病毒學”學科已發(fā)展成水生生物學的特色學科之一?！端《緦W》與中英文雙語專著《水生病毒和及水生病毒病圖鑒》[44,45]、由國際知名蛙病毒專家、美國Chinchar和Gray教授為主編的英文專著《蛙病毒:變溫脊椎動物的致命淡水大DNA病毒在形態(tài)、基因結(jié)構(gòu)、進化及生態(tài)作用等方面都具有多樣性[21,60,61]。本節(jié)主要就兩棲類蛙病毒、鯽皰疹病毒、克氏原螯蝦(小龍蝦)線頭病毒及藍藻菌噬藻體這幾種淡水大DNA病毒的研究動態(tài)做一簡介。

1.1 沼澤綠牛蛙蛙病毒(Rana grylio virus, RGV)和大鯢蛙病毒(Andrias davidianus ranavirus, ADRV)

蛙病毒是能感染世界各地養(yǎng)殖和野生水生動物、有囊膜、基因組大小105—150 kb、直徑為100—200 nm的球形大DNA病毒[62], 已測全基因組序列的蛙病毒毒株超過22株[52]。在同一物種中分離到不同蛙毒株的事件也時有發(fā)生, 如從牛蛙中分離鑒定了RGV-9506、RGV-9807、RGV-9808等毒株[36]; 而從發(fā)病大鯢中分離鑒定CGSIV-HN1104(KF512820)[63]、ADRV’(KF033124)[64]及未分類毒株(KC243313)等。蛙病毒還能跨種感染不同水生動物[65,66]。因此, 專家呼吁要重視并避免這類病毒病傳播[67]。

大鯢細胞系的建立與蛙病毒研究已建立的魚類細胞系現(xiàn)超過300個[68—70], 與之相比, 兩棲動物細胞系則要少得多, 并限于無尾(如蛙類)動物細胞系[71—73]。直至2015年, 由中科院水生所建立大鯢胸腺細胞系(Chinese giant salamander thymus cell line, GSTC)、大鯢脾細胞系(Chinese giant salamander spleen cell line, GSSC)及大鯢腎細胞系(Chinese giant salamander kidney cell line, GSKC)之后, 方見有尾動物(如大鯢)細胞系用于科研的報道[74]。其中, GSTC也是源于兩棲動物胸腺組織的第一株細胞系。分別測試大鯢胸腺細胞系GSTC、爪蟾腎細胞系A(chǔ)6及鯉上皮瘤細胞系EPC這三種來源不同物種的細胞系對大鯢蛙病毒ADRV的敏感性。引起GSTC細胞病變所需時間最短、病變程度最嚴重、病毒滴度最高,TCID50達108/mL, 顯示GSTC細胞對ADRV很敏感。再用帶綠色熒光蛋白標記的重組牛蛙蛙病毒(rRGV)感染, 不僅很快形成空斑, 且與rRGV產(chǎn)生的綠色熒光信號相吻合[75]?？梢? GSTC不僅可用于測試蛙病毒感染, 也可用做蛙病毒基因擴增、表達及與宿主相互作用研究的工具[76]。

重組蛙病毒構(gòu)建及其應(yīng)用推導蛙病毒基因組可編碼95—162個基因, 其中僅三分之一可利用序列同源性推定功能, 而對蛙病毒與宿主相互作用基因則知之甚少[77]。病毒重組技術(shù)是研究基因功能的重要技術(shù)之一, 還可用于篩選免疫活性分子、基因表達調(diào)控及疫苗研發(fā)[78]、模擬病毒入侵宿主過程和提供病毒與宿主之間相互作用的研究模型[79]等?；趯ν懿《綬GV尿嘧啶脫氧核糖核苷三磷酸酶基因RGV-dUTP(RGV-67R)[80]、淋巴囊腫病毒-中國株胸苷酸合成酶基因LCDV-CTS(LCDV-C11L)的鑒定[81], 以蛙病毒RGV的胸苷激酶基因TK(RGV-92R)和囊膜蛋白基因(RGV-53R)作為外源基因靶點,構(gòu)建熒光報告基因或缺失特定基因位點重組病毒[82],獲得既保留親本毒株生物學特性, 又攜帶外源基因的幾種重組蛙病毒[42,83,84]; 還構(gòu)建可調(diào)節(jié)特定基因表達、含乳糖操縱子和雙熒光標記的條件致死型重組蛙病毒[85]。能否成功構(gòu)建重組病毒, 選擇適合的外源基因插入位點很重要[86]。

蛙病毒功能蛋白的鑒定有囊膜病毒可借助囊膜受體識別分子與細胞受體結(jié)合, 介導病毒內(nèi)吞進入宿主細胞[87]。為查明蛙病毒囊膜蛋白在入侵時有何作用, 選擇與蛙病毒屬成員有高度同源性的膜蛋白RGV-43R進行分析。結(jié)果顯示, 該蛋白跨膜結(jié)構(gòu)域決定其在胞質(zhì)中的定位; 而缺失基因43R的重組蛙病毒?43R-RGV與野生型RGV相比,前者DNA復制及超微形態(tài)不受影響, 但使細胞病變程度及病毒滴度卻顯著降低, 表明該蛋白是蛙病毒入侵的關(guān)鍵蛋白[88]。

病毒核心蛋白(Core proteins)指不同種屬毒株之間高度同源、涉及結(jié)構(gòu)及與復制的病毒蛋白。虹彩病毒科成員有26個核心蛋白[89], 其中, 蛙病毒蛋白RGV-63R被推導為DNA聚合酶, 具有3′－5′外切酶結(jié)構(gòu)域和DNA聚合酶B家族催化結(jié)構(gòu)域。研究揭示該蛋白不僅與病毒加工廠共定位, 還能與作為增殖細胞核抗原(Proliferating cell nuclear antigen,PCNA)的RGV-91R蛋白相互作用, 當這兩個蛋白單獨或共同過表達時, 均能促進蛙病毒RGV在不同來源細胞系中復制[90]。對大鯢蛙病毒ADRV-96L蛋白進行分析, 顯示這是一個具有ATPase活性、促進宿主細胞增殖和生長、有助于產(chǎn)生更多子代病毒[91]的重要蛋白。測試兩種蛙病毒的同源核心蛋白RGV-27R和ADRV-85L, 結(jié)果顯示它們不僅能在兩棲動物細胞中高效表達, 且都具有相同的抗原特性[92]。

蛙病毒與宿主的相互作用靶向破壞病毒囊膜就能抑制經(jīng)囊膜蛋白吸附細胞表面受體而入侵的病毒[93]。盡管糖蛋白或糖脂可作為哺乳動物病毒受體, 但不同的表面分子結(jié)構(gòu)會導致病毒對物種或組織的取向不同[94]。研究揭示蛙病毒RGV和ADRV能以不同細胞表面的硫酸乙酰肝素作為受體, 經(jīng)此跨種入侵[95]。蛙病毒成熟和囊膜形成也可發(fā)生在不同細胞的囊泡中[31,96,97]。這些研究為蛙病毒跨種感染提供了新注釋。已知病毒能用miRNA操縱宿主細胞和病毒基因的表達[98], 借助miRNAs干擾與病毒重組等進行分析, 結(jié)果顯示敲除病毒膜蛋白基因RGV-2L和RGV-53R能顯著抑制病毒裝配[42,99]。這表明蛙病毒囊膜蛋白不僅是其吸附入侵的要素, 且能顯著影響病毒裝配與成熟。分別對大鯢正常血清和感染蛙病毒血清及正常黏液和感染蛙病毒黏液的蛋白圖譜進行測試比較, 顯示不同程度發(fā)生變化[100], 預示蛙病毒感染可引起宿主機體的生理生化反應(yīng)。蛙病毒RGV和ADRV的基因有99%同源性, 將其分別感染養(yǎng)殖大鯢,構(gòu)建15個轉(zhuǎn)錄組文庫, 并進行測序, 結(jié)果從8.2億個有效讀數(shù)中獲得12.8萬個注釋基因; 在蛙病毒感染自然宿主或跨種感染過程中具有不同的基因表達模式, 所引起宿主應(yīng)答也各有不同。在跨種感染時,蛙病毒進入宿主后, 迅速表達自身基因、快速復制,但宿主應(yīng)答較弱, 以此提升其適應(yīng)性進化及在種間傳播的時效性[101]。

以蛙病毒囊膜蛋白基因ADRV-2L和ADRV-58L分別構(gòu)建重組質(zhì)粒pcDNA-2L和pcDNA-58L, 并就其對大鯢的免疫保護作用進行評價。對經(jīng)pcDNA-2L免疫過的大鯢進行蛙病毒ADRV攻毒,其I型干擾素(IFN-1)、抗病毒蛋白(Mx)、主要組織相容性復合物(MHC-IA)和免疫球蛋白M (IgM)表達水平都能明顯上調(diào), 存活率為 66.7%, 顯著高于用pcDNA-58L免疫的大鯢存活率(3.3%)[78], 經(jīng)比較可篩出候選疫苗。蛙病毒會采取拮抗或免疫逃逸策略[102,103], 或有效變異, 增強對新物種宿主的適應(yīng)性, 以突破物種屏障感染新物種[104—106]。

1.2 鯽皰疹病毒(Crucian carp herpesvirus, CaHV)

皰疹病毒目(Herpesvirales)成員是有囊膜、基因組大小為125—290kb的大DNA病毒,在二十面體核衣殼外有層蛋白質(zhì)基質(zhì)被膜, 再被囊膜包裹[107]。魚蛙皰疹病毒科(Alloherpesviridae)以魚類和兩棲類為宿主[108],分為蛙皰疹病毒屬(Batrachovirus)、鯉皰疹病毒屬(Cyprinivirus)、鮰皰疹病毒屬(Ictalurivirus)和鮭皰疹病毒屬(Salmonivirus)[6,109,110]。2016年從急性鰓出血癥的鯽中分離鯽皰疹病毒CaHV[111](圖 2), 基因組為275 kb的線性雙鏈DNA(KU199244)[112], 推測可編碼150個基因, 是基因組架構(gòu)及引發(fā)病癥與已知鯉皰疹病毒都不同的新毒株[23]。

CaHV的G蛋白偶聯(lián)受體G蛋白偶聯(lián)受體(G Protein-Coupled Receptors, GPCR)是一類有七個跨膜結(jié)構(gòu)域的膜蛋白受體, 可作為信號通路或參與信號轉(zhuǎn)導, 并可利用激活細胞內(nèi)信號通路為病毒的復制提供保障[113]。鯽皰疹病毒G蛋白偶聯(lián)受體CaHV-25L(或稱CaHV-GPCR), 其C端含賴氨酸殘基、蛋白激酶C磷酸化位點及豆蔻?；稽c。經(jīng)截短、缺失或替換等方式, 構(gòu)建了CaHV-GPCR的C端系列突變子, 并在魚類細胞中表達。結(jié)果證實其C端不同氨基酸對蛋白亞細胞定位與分布狀態(tài)有不同影響[114]。

鯽皰疹病毒膜蛋白及其靶向分子有研究表明皰疹病毒膜蛋白呈動態(tài)分布且有不同作用[115]。CaHV-138L是有兩個跨膜結(jié)構(gòu)域的鯽皰疹病毒膜蛋白, 分析顯示全長CaHV-138L呈點狀分布于質(zhì)膜或核膜周圍, 且與線粒體共定位。當截短其單一或雙跨膜結(jié)構(gòu)域時, 就會改變其亞細胞定位, 使之在胞質(zhì)和胞核中呈斑塊狀分布。經(jīng)酵母雙雜交和免疫共沉淀篩查到能與該病毒蛋白相互作用的宿主線粒體蛋白FoF1-ATP酶, 并證實CaHV-138L能靶向線粒體蛋白FoF1-ATP酶[116]。這預示該蛋白可通過介導線粒體ATP合成, 為病毒復制提供能量。另外, 對CaHV與CyHV-2高度同源、且含RNase E/G家族典型結(jié)構(gòu)域的蛋白CaHV-31R進行分析, 結(jié)果表明它能與內(nèi)質(zhì)網(wǎng)和高爾基體等有單層膜結(jié)構(gòu)的細胞器共定位, 可能涉及病毒胞內(nèi)運輸與釋放[117]。

圖2 鯽皰疹病毒CaHV引起的高致死系統(tǒng)性出血癥及感染鯽頭腎超薄切片的電鏡圖 (方進 圖)Fig. 2 Crucian carp herpesvirus (CaHV) caused disease with highlylethal systemic hemorrhagic symptoms and electron electronmicrograph of the infected Carassius auratus head kidney ultrathin section. Bar=200 nm

鯽皰疹病毒病防控對鯽皰疹病毒CaHV攻毒和感染的不同品系異育銀鯽轉(zhuǎn)錄組進行分析, 測試其對病毒的抗性。結(jié)果, 三個雌核發(fā)育異育銀鯽品系對鯽皰疹病毒分別顯示出高(H)、中(F)和低(A+)抗性。又從不同品系中鑒定顯著差異表達的基因、免疫相關(guān)途徑及干擾素系統(tǒng)基因等[118]。在H、F和A+品系中, 依次有26條、7條和15條途徑與感染或免疫相關(guān)基因。鑒定出與病毒載量呈正相關(guān)或負相關(guān)的表達模塊[119]。這不僅顯示H品系的免疫力更強, 且為分子標記輔助選擇育種及鯽抗病毒分子育種實踐提供了新思路。還從被CaHV感染的異育銀鯽中, 鑒定出28個含不同免疫球蛋白結(jié)構(gòu)域的蛋白會上調(diào)表達; 而且鯽蛋白DICPs能通過激活脂質(zhì)A驅(qū)動熒光素酶, 與肉瘤病毒Src基因同源結(jié)構(gòu)域1蛋白酪氨酸磷酸酶(src-homology 1 protein tyrosine phosphatase, SHP-1)及SHP-2相互作用, 從而抑制干擾素及干擾素刺激基因(Interferon-stimulated genes, ISGs)表達[120]。所鑒定的干擾素系統(tǒng)基因有RIG-Is、LGP2s、IRF1-B、IRF3s、IRF7s、IRF9-B、Mxs及干擾素刺激因子Viperins等。進一步研究顯示, CaHV侵染會啟動干擾素調(diào)節(jié)因子RIG-I、遺傳學和生理學實驗室蛋白2 (Laboratory of genetics and physiology 2, LGP2)表達, 并激活線粒體抗病毒信號通路, 誘導表達干擾素調(diào)節(jié)因子[121]。從中等抗性的F品系中, 鑒定兩個大小不同的3′UTRs干擾素基因, 證實3′UTR參與干擾素基因的轉(zhuǎn)錄和翻譯, 是調(diào)節(jié)抗病毒免疫的潛在因素[122]。

通過替代藥物來控制水產(chǎn)病害越來越受到關(guān)注, 如有益微生物(益生菌probiotics)就被認為是抗生素的有效且生態(tài)友好替代品[123,124]。經(jīng)對未喂餌益生菌而直接用CaHV攻毒的鯽, 與已喂餌益生菌后再攻毒的鯽, 分別測試其成活率和免疫相關(guān)基因。結(jié)果表明: 喂餌益生菌使鯽抗病毒的應(yīng)答水平及群體存活率顯著提高[125], 該研究為魚類抗病毒病添加了候選方案。

1.3 克氏螯蝦線頭病毒(Procambarus clarkia nimavirus, PCV)

克氏螯蝦(Procambarus clarkia)也稱小龍蝦。中國已成為世界養(yǎng)殖小龍蝦的最大生產(chǎn)國[126], 其需求和產(chǎn)量仍在增長, 但小龍蝦病毒病種類及其危害也隨之增加[127—131]。線頭病毒是有雙層囊膜、基因組大小 280—309 kb、一端帶尾、囊膜大小約430 nm×120 nm 的大DNA病毒。曾有小龍蝦受線頭病毒科(Nimaviridae)成員白班綜合癥病毒(White spot syndrome virus, WSSV)感染, 并出現(xiàn)白斑癥狀的報道[132,133]?；?qū)⑿↓埼r作為WSSV的實驗動物[134], 感染后也能觀察到白斑癥狀。但從自然感染小龍蝦中分離鑒定線頭病毒的文獻仍很少見。下面簡介相關(guān)研究。

PCV的核酸檢測與超微形態(tài)某養(yǎng)殖場蝦群突然大量死亡卻無體表病癥, 采集幸存小龍蝦樣本, 并對這些無典型白斑癥的蝦解剖觀察。腸道無食物, 但因出血(或充血)而呈淡藍色, 肝胰腺呈淡黃或白色, 部分蝦鰓發(fā)黑。以幸存小龍蝦核酸作為模板, 設(shè)計小龍蝦線頭病毒PCV特有基因PCV-87R及五種對蝦病毒(WSSV、IHHNV、TSV、YHV及MrNV)保守基因的引物[135—137], 進行PCR或RTPCR檢測。結(jié)果檢出PCV-87R與wssv-vp28為陽性,其他均呈陰性。這預示PCV是一株新線頭病毒, 與已知白斑病毒成員之間存在關(guān)鍵基因的遺傳與變異。

對自然感染無病癥小龍蝦組織制備的超薄切進行電鏡觀察, 可見病毒顆粒存在于不同組織和細胞中。如在鰓和腸細胞中, 有大量病毒分布在胞質(zhì)和核質(zhì)中, 或規(guī)則排列在核膜周圍, 并伴隨廣泛的組織病變。完整PCV顆粒大小約300 nm×110 nm、兩端鈍圓呈短桿狀。負染電鏡圖顯示: PCV核衣殼呈有節(jié)桿狀[109](圖 3)。

圖3 小龍蝦線頭病毒PCV無癥狀感染的小龍蝦及病毒核衣殼負染電鏡圖 (李濤 圖)Fig. 3 Procambarus clarkia nimavirus (PCV) infected red swamp crayfish were asymptomatic and the negative staining electron micrograph of the viral nucleocapsid. Bar=200 nm

PCV基因組架構(gòu)及其多變區(qū)PCV基因組DNA大小為287 kb (MH663976), 推定可編碼180個基因。有觀點認為可將對蝦白斑綜合征病毒基因大片段缺失作為時空進化的標記[138]。將PCV基因組與已知對蝦白斑綜合征病毒基因組進行比較, 顯示PCV在易重組、有進化意義的核酸片段相應(yīng)位置[139]也有缺失。此外, PCV另有一個差異顯著的核酸片段及普遍存在的核酸插入、缺失、替換及基因突變。

1.4 淡水藍藻菌噬藻體及水生大DNA病毒新成員

病毒歷史遠比人類史悠遠, 而且數(shù)量極大, 地球被稱為 “病毒星球” (A Planet of Viruses)[140]。地球上的病毒已超過宇宙中繁星的數(shù)量, 若將預測地球上的1031個病毒首尾相連, 其長度將達1億光年[141]。肌尾噬藻體(Cyanophage)是感染原核藍藻菌的病毒, 屬有尾目(Caudovirales)中肌尾噬藻體科(Myoviridae)的成員, 病毒顆粒由頭部和尾部兩部分組成, 呈蜊蚪形, 其DNA基因組大小約120 kb[142]。噬藻體可感染藍藻甚至操控水華藍藻的種群密度, 并將宿主機體和細胞轉(zhuǎn)化為有機物, 從而驅(qū)動地球物理化學循環(huán); 在介導微生物之間的基因水平轉(zhuǎn)移、維持水生微生物群落多樣性等方面也發(fā)揮重要作用[143—147]。水體中有許多感染真核藻的大DNA病毒, 如藻DNA病毒(Phycodnavirus), 屬于藻類DNA病毒科(Phycodnaviridae), 通常為二十面體, 其DNA基因組的分子量為160—380 kb。還有感染原生動物變形蟲的巨病毒(Giant virus), 其基因組大小甚至可達500 kb, 被認為是規(guī)模迅速擴張的水生大DNA病毒家族新成員[148]。相關(guān)新認知拓展了水生大DNA病毒的范疇, 挑戰(zhàn)了對病毒的傳統(tǒng)認識[149], 日漸模糊了病毒與細胞間的界限[150]。

噬藻體感染藍藻菌不僅改變宿主種群密度, 也促進噬藻體的適應(yīng)性及與宿主的共同進化[151]。噬藻體修飾宿主細胞膜能增強光保護及病毒編碼光合蛋白的表達, 產(chǎn)生新的代謝通路或網(wǎng)絡(luò)[61]。水體中病毒對宿主的致死率很高, 同時, 包括藍藻菌在內(nèi)的水生細菌對病毒的抵抗力也在增強[152]。噬藻體感染通常取決宿主的防御效果[153], 并由此可獲得高突變率及重組率[154]。由于從基因組數(shù)據(jù)所獲微生物的功能信息及其可信度都有限。因此, 純培養(yǎng)仍是微生物利用的前提與基礎(chǔ)[155]。運用無菌技術(shù), 經(jīng)優(yōu)化條件、反復篩選、純化鑒定、培養(yǎng)和保藏等過程, 才可獲得很少量噬藻體純培養(yǎng)物[156]。自然界中不可培養(yǎng)微生物(Viable but non-culturable, VBNC)仍占絕大多數(shù)[157], 導致對微生物活體總數(shù)的低估[158], 也突顯基礎(chǔ)微生物研究的難點及蘊藏開發(fā)微生物資源的潛能[159]。

銅綠微囊藻肌尾噬藻體-滇池株(Microcystis aeruginosa myovirus in Lake Dianchi, MaMV-DC)的分離及超微形態(tài)銅綠微囊藻肌尾噬藻體-滇池株MaMV-DC是利用微囊藻、魚腥藻、聚球藻等21株藍藻菌, 對昆明滇池采集的水樣進行篩查, 并經(jīng)噬藻體單斑分離、擴大培養(yǎng)及純化所獲得的噬藻體。其宿主范圍窄, 僅感染銅綠微囊藻株Microcystic aeruginosaFACHB-524, 噬藻斑為圓形透亮空斑; 噬藻體呈蝌蚪形, 二十面體頭部直徑約70 nm,收縮尾長約160 nm (圖 4)。MaMV-DC的裂解量約為80個感染活性單位[160]。此外, 還有些噬藻體有形態(tài)獨特的噬斑, 如噬藻體A-4L可形成同心圓噬斑, 而同心圓形成與光照節(jié)律有關(guān)[161]。

圖4 銅綠微囊藻肌尾噬藻體-滇池株MaMV-DC的噬斑、負染電鏡圖及基因組圖譜 (歐銅 圖)Fig. 4 The plaque, negative staining electron micrograph and genome map of Microcystis aeruginosa myovirus in Lake Dianchi(MaMV-DC). Bar=100 nm

MaMV-DC基因組的結(jié)構(gòu)測序分析顯示MaMV-DC基因組(KF356199)為末端循環(huán)冗余、雙鏈線性DNA大小169 kb, 推測可編碼170個基因, 其中含一個轉(zhuǎn)運RNA (tRNA)基因。MaMV-DC與日本報道的銅綠微囊藻噬藻體Ma-LMM01(AB231700)基因組序列相似性為86%, 有150個同源基因[162]; 而與宿主銅綠微囊藻有29個同源基因。當用主要衣殼蛋白構(gòu)建進化樹時, MaMV-DC與Ma-LMM01聚在一簇, 但兩者所攜帶的宿主或其他物種核酸片段卻差異顯著[163]。另外, 短尾噬藻體A-4L, 基因組DNA大小約為42 kb, 推測可編碼38個基因[164], 不屬大DNA病毒類群, 但能感染模式生物魚腥藻Anabaenasp. PCC 7120, 可作為載體, 用于研究噬藻體基因功能。

噬藻體功能基因噬藻體的生態(tài)功能是要憑借與宿主相互作用而體現(xiàn)[165]。對肌尾噬藻蛋白A-1(L)-ORF36功能進行了鑒定, 顯示這是一個能與細胞表面脂多糖(LPS) O抗原結(jié)合的蛋白。還證實噬藻體正是利用該蛋白與細胞表面脂多糖特異性吸附而入侵藍藻菌的[166]。從絲狀藍藻噬藻體(Planktothrix agardhiivirus isolated from Lake Donghu,PaV-LD, NC_016564) 中鑒定藻膽體降解蛋白基因(NblA)、穿孔素基因和內(nèi)肽酶基因等[167,168]。銅綠微囊藻噬藻體基因MaMV-DC-5L也是編碼藻膽體降解蛋白基因, 能在模式集胞藻Synechocystissp.PCC 6803中表達, 顯著消減宿主藻藍蛋白吸收峰,促進噬藻體釋放[163]。已鑒定的噬藻體基因還有不同光合作用蛋白基因[169]和能量與代謝相關(guān)基因[170]。

噬藻體與宿主的相互作用微生物基因組所含規(guī)律間隔短回文重復序列及相關(guān)系統(tǒng)(Clustered regularly interspaced short palindromic repeats-associated endonuclease, CRISPR-Cas), 因為它能與crRNA(CRISPR-derived RNA)堿基配對、識別并使入侵的噬菌體及其他病原體被Cas蛋白切割降解, 而被證明是細菌等原核生物的防御系統(tǒng)[171,172]。鑒定了功能多樣化、且有不同切割效率的V型CRISPRCas系統(tǒng)及由RNA引導的效應(yīng)蛋白Cas12[173]。還鑒定了絲狀藍藻菌長尾噬菌體vB_AphaS-CL131編碼的V-U2 CRISPR-Cas系統(tǒng)[174], 顯示在藍藻菌中普遍存在V-U2的效應(yīng)蛋白[175]。而噬菌體也進化出不同抗-CRISPR的蛋白(Acr), 能抑制或逃逸宿主CRISPR-Cas的防御作用[176]。將銅綠微囊藻肌尾噬藻體-滇池株MaMV-DC置于低溫冰箱(-80℃)中保存超過3年, 不僅保留對Microcystis aeruginosaFACHB-524的感染性, 也能感染微囊藻Microcystis flosaquaeTF09、Microcystis aeruginosaTA09和Microcystis wesenbergiiDW09, 但敏感性不同。再對不同微囊藻株的防御系統(tǒng)CRISPR-Cas進行分析比較,顯示其分子結(jié)構(gòu)與含量都有變化。這證明CRISPR-Cas會影響微囊藻對噬藻體的敏感性, 或能決定噬藻體的宿主范圍[177]。CRISPR-Cas已作為基因編輯工具廣泛應(yīng)用[178], 并取得相應(yīng)成果, 將噬菌體酶靶向嵌入細菌特定基因位點, 則能破壞細菌生物膜;而利用噬菌體將抗生素敏感基因?qū)肽退幘? 則可使耐藥菌致敏[179], 由此能獲得抗病毒新對策[180]。

2 機遇與挑戰(zhàn)

我國漁業(yè)已進入設(shè)施化、智能化與生態(tài)化的歷史變革時期[181], 對水產(chǎn)健康生態(tài)養(yǎng)殖有更高要求, 也給水生病毒學學科發(fā)展帶來難得契機。水生大DNA病毒的性質(zhì)特征、遺傳進化及與宿主及水生態(tài)關(guān)聯(lián)的研究基礎(chǔ)相對薄弱, 對水產(chǎn)動物病毒病預測預警、智能健康水產(chǎn)養(yǎng)殖體系構(gòu)建、大DNA病毒群落多樣性及其水生態(tài)閾值評估的認知也參差不齊, 正面臨前所未有的挑戰(zhàn)[182]。

2.1 先進技術(shù)是提升水生大DNA病毒研究水平的載體

構(gòu)建和運用大數(shù)據(jù)網(wǎng)絡(luò), 宏觀與微觀結(jié)合開展研究是水生大DNA病毒學新的生長點。多國學者合作, 已從全球不同水體采集樣品重建大DNA病毒基因組信息, 圍繞其地理分布、基因多樣性、代謝特征等開展研究。闡明水體大DNA病毒全球分布的模式, 使其系統(tǒng)發(fā)育多樣性及功能多樣性數(shù)據(jù)各提升11倍和10倍; 發(fā)現(xiàn)病毒基因組編碼與光合作用及底物運輸過程相關(guān)蛋白; 揭示大DNA病毒普遍具備使宿主重編程的功能[148]。單病毒顆粒示蹤、基因組解析、轉(zhuǎn)錄組分析也運用于水生大DNA病毒研究中[101,183]; 剖析鑒定與疾病相關(guān)的DNA序列[184],可極大提高對疾病發(fā)生機制及病毒與水環(huán)境相關(guān)性的認識, 并推動水產(chǎn)動物病毒病由隨機檢測向系統(tǒng)防控戰(zhàn)略布局轉(zhuǎn)變。也由此可見, 掌握和運用關(guān)鍵核心技術(shù)將為拓展水生病毒學學科注入鮮活動力, 而新技術(shù)突破與經(jīng)典技術(shù)融合集成則會加速科研成果向?qū)嶋H應(yīng)用轉(zhuǎn)化。

2.2 整合、集成水生病毒學知識體系

淡水水生大DNA病毒研究是水生病毒學學科的重要內(nèi)容及原有基礎(chǔ)。隨著對水產(chǎn)品需求的不斷增長及水產(chǎn)養(yǎng)殖規(guī)模持續(xù)擴大, 水產(chǎn)動物疾病也如影隨形。因此, 應(yīng)使水生病毒學知識從零星分散向交匯集成轉(zhuǎn)化, 尤其要強化水生病毒分類、溯源與遺傳進化研究。

分類完全開放、不斷更新發(fā)布的《病毒分類報告》(http://ictv.global/report/), 是由國際病毒分類與命名的權(quán)威機構(gòu)——國際病毒分類委員會(The International Committee on Taxonomy of Viruses, ICTV), 按病毒性質(zhì)、親緣關(guān)系進行歸類編排,為學者與公眾隨時了解新病毒的共性與特征而提供的綱要。隨著對更多水生大DNA病毒新成員及其與宿主相互作用、協(xié)同進化軌跡的認知[185], 將會使大DNA病毒的知識更加豐富和立體。

溯源尋覓病毒傳播源頭有益摸清病毒初始發(fā)生過程與傳播途徑, 預測病毒病流行趨勢與潛在風險, 為疫病防控提供科學依據(jù)。但病毒只能在活細胞中復制, 沒留下化石, 其起源很難追蹤, 須從宿主或其他物種演化史中找答案。部分大DNA病毒有共同的祖先和起源[186], 或源自不同水生生物群落[187,188]; 有些大DNA病毒除了編碼復制和結(jié)構(gòu)形成所必需的蛋白質(zhì)核心基因外, 還會伴隨基因丟失或募集其他物種基因而形成新病毒[189]。但許多病毒的起源尚不清楚, 尤其是一些新發(fā)病毒病原。因此, 應(yīng)強化對水生大DNA病毒的溯源研究。

進化轉(zhuǎn)換、插入、缺失、顛換、重組、重配及自然選擇等基因突變方式是病毒進化與多樣性的源泉。病毒復制頻率高, 發(fā)生變異也較其他生物要快。病毒除了具備遺傳可變性, 還能通過跨宿主感染, 高效擴大病毒的多樣性。有觀點認為,病毒在地球生命的進化中扮演重要的角色, 可導致重大進化飛躍[190], 甚至還存在與宿主互惠共生病毒(Mutualistic viruses)。

2.3 魚水共護, 促生態(tài)健康漁業(yè)發(fā)展

水生大DNA病毒病的流行方式與病毒、水生生物及水環(huán)境存在耦合關(guān)系[191]。病毒導致水產(chǎn)動物病毒病流行, 或是噬藻體不作為任由水華暴發(fā)都會影響水生態(tài)健康及人與自然和諧共處[192]。以魚護水、以水養(yǎng)魚, 只有魚水共養(yǎng)護的健康生態(tài)漁業(yè)才可持續(xù)發(fā)展和有更旺盛的生命力。因此, 可利用分子生物學檢測方法和模型構(gòu)建預警網(wǎng)絡(luò)等途徑, 對機體及水生動物健康狀況進行評估[193]。但試圖通過投放藥物, 來防控水產(chǎn)動物疾病的可操作性仍然很低[194,195]?？傊? 面向綠色水產(chǎn)的全球重大需求, 要實現(xiàn)產(chǎn)品優(yōu)質(zhì)、環(huán)境優(yōu)美、生態(tài)優(yōu)穩(wěn)、養(yǎng)魚護水的漁業(yè)健康、可持續(xù)發(fā)展愿景(https://baijiahao.baidu.com/s?id=1654687744824397971&wfr=spider&for=pc), 就要拓展水生病毒學新方法、新技術(shù)與新知識的儲備; 探索水生大DNA病毒的感染、復制、發(fā)病機制、細胞/宿主取向, 及病毒、宿主與水環(huán)境相互作用等基礎(chǔ)理論問題; 深入研究水生動物病毒病因、流行方式、預警閾值及防控管理等實踐中的新問題, 尋求噬藻體調(diào)控水環(huán)境核心技術(shù)的突破。研究者不斷探尋防御病毒新策略,為水生動物提供更先進的衛(wèi)生服務(wù), 以保障水產(chǎn)動物健康, 創(chuàng)建魚水共護新機制。

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