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1、石斑鱼神经坏死病毒传播途径阻断的初步研究林克冰 方琼珊 吴建绍 周宸 何丽斌 林琪 王涵生（福建省水产研究所，福建，厦门361012）摘要：本文报告了斜带石斑鱼(Epinephelus coioides)苗种培育中亲鱼、卵、饵料等几个关键环节的病毒携带检测以及几种药物对病毒的消除效果的试验。用RT-PCR法检测，斜带石斑鱼亲鱼的粪便、卵、稚鱼、仔鱼，饵料轮虫、桡足类中都有检出神经坏死病毒(nervous necrosis virus，NNV)。聚维酮碘50100 mg/L浸泡卵20min、浸泡轮虫、桡足类30min，盐酸吗啉胍35 mg/ L浸泡卵30min、浸泡轮虫、桡足类40min，聚维酮
2、碘50mg/L+盐酸吗啉胍3mg/L浸泡卵20min、浸泡轮虫、桡足类30min可有效灭活所携带的神经坏死病毒活性。关键词：斜带石斑鱼，神经坏死病毒，传播途径，阻断鱼类神经坏死病毒(nervous necrosis virus，NNV)1，是一种世界范围内流行的海水鱼类病毒病，主要发生在海水鱼类种苗生产阶段，是近几年来石斑鱼苗种培育中危害最大的病原，鱼苗死亡率高达90%以上，给石斑鱼养殖业的发展带来严重的危害34,7。目前普遍认为神经坏死病毒的传播有水平传播和垂直传播两个途径56，明确两个传播途径病毒的携带者以及具体的传播途径，是种苗培育生产中防治神经坏死病毒病的关键。本文报告了斜带石斑鱼苗种
3、培育中亲鱼、卵、饵料等几个关键环节的病毒携带检测以及几种药物对病毒的消除效果的试验，为深入研究神经坏死病毒传播途径奠定基础，同时也为石斑鱼苗种培育生产中神经坏死病毒病的防治提供理论依据。1材料和方法1.1.1实验鱼：斜带石斑鱼(Epinephelus coioides)亲鱼粪便分别于2007年8月16日取自福建省漳浦佛坛联合水产有限公司育苗室，2009年9月22日2010年10月18日取自诏安大华水产有限公司育苗场。斜带石斑鱼卵、孵化稚鱼分别于2007年8月16日和2010年8月21日取自福建省漳浦佛坛联合水产有限公司育苗室、2009年8月26日取自东山乌礁湾育苗室、2008年9月23日201
4、0年9月5日取自于澎湖渔业东山分公司育苗场。斜带石斑鱼稚鱼分别取自于诏安大华水产有限公司育苗场和诏安海康水产养殖有限公司育苗场。1.1.2饵料：轮虫分别取自诏安大华水产有限公司育苗场、诏安海康水产养殖有限公司育苗场、诏安梅岭海水鱼养殖土池，海区吊养牡蛎受精卵、海区捕捞桡足类、海区捕捞小杂鱼鱼糜。上述样品取后立即冷冻后转入-65冷冻保存。1.1.3 聚维酮碘：配成20mg/L、50 mg/L、100 mg/L、150 mg/L、200mg/L 盐酸吗啉胍：配成1 mg/L、3 mg/L、5 mg/L、7 mg/L、9 mg/L 聚维酮碘+盐酸吗啉胍：20mg/L、50 mg/L、100 mg/L
5、、150 mg/L、200mg/L分别加入3 mg/L的盐酸吗啉胍1.1.4 卵、轮虫、牡蛎受精卵、桡足类分别用上述海水溶液浸泡一定时间后取少量立即冷冻，然后转入-65冷冻保存1.1.5 试剂盒：国家海洋局第三海洋研究所研制的神经坏死病毒RT-PCR检测试剂盒。作者简介：林克冰,厦门东渡海山路7号9604，361012，Tel：,0592-6012063，E-mail：kebingl。1.1.6 聚维酮碘为北大泰普科技有限公司生产，盐酸吗啉胍为吉林省博大制药有限责任公司生产2结果表1 斜带石斑鱼亲鱼粪便、1d稚鱼、杂鱼糜、牡蛎受精卵神经坏死病毒RT-PCR检测结果Fig 1NNV RT-PCR
6、 detection of Epinephelus coioides dung, 1d old larvae, surimi, oyster oosperm 批次 RT-PCR批次 RT-PCR批次 RT-PCR粪便2007.08.16+稚鱼1（2007.08.06）+鱼糜（2007.08.20）粪便2009.09.22稚鱼2（2009.08.26）+鱼糜（2008.09.25）粪便2010.10.18稚鱼3（2008.09.23）ML1（2007.08.07）稚鱼4（2010.08.21）+ML2（2007.09.13）稚鱼5（2010.09.05）ML3（2008.08.20）+ 表示强阳
7、性，+ 表示弱阳性， 表示阴性，ML 表示牡蛎受精卵表2 聚维酮碘浸泡斜带石斑鱼卵20min前、后RT-PCR检测结果Fig 2 NNV RT-PCR detection of Epinephelus coioides eggs before and after exposure to Povidone Iodine 20min聚维酮碘mg/L2050100150200处理前 处理后处理前 处理后处理前 处理后待添加的隐藏文字内容2处理前 处理后处理前 处理后1（2007.08.06）+ + + + + 2 （2008.09.23） 3（2009.08.26）+ + + + + 4（2010.
8、08.21）+ + + + + 5（2010.09.05） 表3 盐酸吗啉胍处理斜带石斑鱼卵30min前、后RT-PCR检测结果Fig 3 NNV RT-PCR detection of Epinephelus coioideseggs before and after exposure to Moroxydine Hydrochloride 30min盐酸吗啉胍mg/L13579处理前 处理后处理前 处理后处理前 处理后处理前 处理后处理前 处理后1（2007.08.06）+ + + + + 2 （2008.09.23） 3（2009.08.26）+ + + + + 4（2010.08.21
9、）+ + + + + 5（2010.09.05） 表4 聚维酮碘、盐酸吗啉胍处理斜带石斑鱼卵20min前、后RT-PCR检测结果Fig 4 NNV RT-PCR detection of Epinephelus coioides eggs before and after exposure to Povidone Iodine Moroxydine Hydrochloride 20min聚维酮碘mg/L盐酸吗啉胍mg/L20+350+3100+3150+3200+3处理前 处理后处理前 处理后处理前 处理后处理前 处理后处理前 处理后1（2007.08.06）+ + + + + 2 （2008
10、.09.23） 3（2009.08.26）+ + + + + 4（2010.08.21）+ + + + + 5（2010.09.05） 表5 聚维酮碘浸泡轮虫、桡足类30min前、后RT-PCR检测结果Fig 5 NNV RT-PCR detection of rotiferscopepods before and after exposure to Povidone Iodine 30min聚维酮碘mg/L2050100150200处理前 处理后处理前 处理后处理前 处理后处理前 处理后处理前 处理后轮1（2007.08.06）+ + + + + 轮2（2007.08.10） 轮3（2008
11、.08.13）+ + + + + 轮4（2009.09.02） 轮5（2010.09.15） 桡1（2007.08.30）+ + + + + 桡2（2007.09.20） 桡3（2008.09.06）+ + + + + 桡4（2008.09.15） 桡5（2009.09.02） 桡6（2007.09.19） 轮：表示虫，桡：表示桡足类表6 盐酸吗啉胍浸泡轮虫、桡足类40min前、后RT-PCR检测结果Fig 6 NNV RT-PCR detection of rotiferscopepods before and after exposure to Moroxydine Hydrochlori
12、de 40min盐酸吗啉胍mg/L13579处理前 处理后处理前 处理后处理前 处理后处理前 处理后处理前 处理后轮1（2007.08.06）+ + + + + 轮2（2007.08.10） 轮3（2008.08.13）+ + + + + 轮4（2009.09.02） 轮5（2010.09.15） 桡1（2007.08.30）+ + + + + 桡2（2007.09.20） 桡3（2008.09.06）+ + + + + 桡4（2008.09.15） 桡5（2009.09.02） 桡6（2007.09.19） 轮：表示虫，桡：表示桡足类表7 聚维酮碘、盐酸吗啉胍浸泡轮虫、桡足类30min前、后
13、RT-PCR检测结果Fig 7 NNV RT-PCR detection of rotiferscopepods before and after exposure to Moroxydine HydrochloridePovidone Iodine 30min聚维酮碘mg/L盐酸吗啉胍mg/L20+350+3100+3150+3200+3处理前 处理后处理前 处理后处理前 处理后处理前 处理后处理前 处理后轮1（2007.08.06）+ + + + + 轮2（2007.08.10） 轮3（2008.08.13）+ + + + + 轮4（2009.09.02） 轮5（2010.09.15） 桡
14、1（2007.08.30）+ + + + + 桡2（2007.09.20） 桡3（2008.09.06）+ + + + + 桡4（2008.09.15） 桡5（2009.09.02） 桡6（2007.09.19） 轮：表示虫，桡：表示桡足类上述实验结果表明聚维酮碘50100 mg/L浸泡卵20min、浸泡轮虫、桡足类30min，盐酸吗啉胍35 mg/ L浸泡卵30min、浸泡轮虫、桡足类40min，聚维酮碘50mg/L+盐酸吗啉胍3mg/L浸泡卵20min、浸泡轮虫、桡足类30min可有效灭活所携带的神经坏死病毒活性。3讨论石斑鱼神经坏死病毒的垂直传播两个可能的方式一种是病毒粒子存在于卵细胞之
15、内，从而传给稚鱼，如果是这种传播方式，理、化处理都难于灭活细胞内的病毒粒子而不影响到鱼受精卵的活性。另一种可能方式是带毒亲鱼粪便、分泌物、脱落细胞里含有病毒粒子，附着在受精卵表面，这就可以通过处理受精卵灭活表面的病毒粒子，而基本不影响或很小影响鱼受精卵的活性。本实验中，在斜带石斑鱼亲鱼粪便里有检出病毒阳性，而且3批不同来源的受精卵以及对应的未处理孵化稚鱼都有检出病毒，可以表明存在着病毒粒子附着在受精卵表面的这种垂直传播方式。因此，受精卵的抗病毒预处理对阻断石斑鱼苗种期神经坏死病毒病的传播十分重要。石斑鱼神经坏死病毒的水平传播基本有饵料、水环境、养殖环境、养殖器具移动、苗种在养殖场内的移动与场间
16、的流动等途径。石斑鱼从开口稚鱼到仔鱼饵料品种繁多，这个环节是病毒传播最直接而又比较难于控制，养殖生产中常常受到忽视而未采取灭毒措施。本实验分别在养殖鮸鱼土池培育的轮虫和别育苗场培育的轮虫都检出病毒阳性，海区和土池捕捞的桡足类也各有1批检出病毒，说明轮虫和桡足类会携带病毒粒子传播给稚鱼，尤其是土池培育的轮虫和桡足类存在较大风险，发病场培育的轮虫，携带病毒的可能性也较大，活体饵料投喂前的去毒处理是阻断病毒水平传播的重要手段之一。聚维酮碘和盐酸吗啉胍是较常见的抗病毒药物，使用较为安全方便，浸泡石斑鱼受精卵、轮虫、桡足类可以较有效的灭活所携带的神经坏死病毒。PCR诊断技术灵敏简便，广泛应用于鱼类病毒病
17、的检测，RT-PCR方法是OIE推荐的神经坏死病毒标准诊断程序的诊断方法之一,具有比免疫组织化学方法、荧光抗体技术和细胞培养等诊断方法更快速、灵敏、简便、准确的特点。但由于取样量很少，反应步骤多，取样的部位以及实验室的条件和操作过程都有可能影响实验结果的准确性，本实验检测的2批鱼糜，3批牡蛎受精卵都未检出神经坏死病毒，这不能排除二者有携带病毒的可能性。参考文献：1 Yoshikoshi K, Inoue K. Viral nervous necrosis in hatchery reared larvae and juveniles of Japanese parrotfish, Oplegn
18、athus fasciatus (Temminck & Schlegel) J. J Fish Dis,1990,13(1): 6977. 2 Mori K, Nakai T, Muroga K, etal. Properties of a new virus be longing to Nodaviridea found in larval striped jack (Pseudocaranxdentex) with necrosis disease J. Virology,1992,187:368-371.3 Munday B L,Langdon J S, Hyatt A, etal. M
19、ass mortalityassociated with a viral- induced vacuolating encephalopathy and retinopathy of larval and juvenile barramundi, Lates calcarifer BlochJ.Aquac,1992,103(3-4):197211.4 Munday B L, Kwang J, Moody N. Betanodavirus in fections of teleost fish: a reviewJ.J Fish Dis,2002,25(3):127142.5 Iwamoto T
20、, Mise k，Mori k, etal. Estabilsment of an infectious RNA transcription system for Striped jack nervous necrosis virus, the type species of the betanodavirysesJ Journal of General virology,2001,82:26532662.6 Grove S, Johansen R, Dannevig B H, etal. Experimental infection of Atlantic halibut Hippoglos
21、sus hippoglossus with nodavirus: tissues distribution and immuneresponse. Diseases of Aquatic Organisms,2003,53:211221.7 陈信忠，龚艳清，苏永全，等。闽南地区紫石斑(Epinephelus lanceolatus)幼鱼爆发性传染病的病原学研究J。厦门大学学报(自然科学版)，2004,43(4):5571562.Preliminary study on breaking transmission of NNV in Epinephelus coioidesLin Kebing
22、Fang Qionshan Wu JianshaoFujian Fisheries Reserch Institute Haishan Rd Dongdu Xiamen China, 361012Abstract: To study the effect of breaking transmission of nervous necrosis virus NNV，some medication of This report detected NNV from the key parts of hatching Epinephelus coioides such as parent fish,
23、eggs, feeds by reverse transcription poly merase chain reaction(RT-PCR).The detection of the soil, eggs, larvae, yang fishes, rotifers and copepods by RT-PCR indicate that the NNV challenged were all have positively infected samples. NNV took with eggs, rotifers and copepods was completely inactivit
24、y within 20min and 30min after exposure to a solution of 50100 mg/L Povidone Iodine. NNV took with eggs, rotifers and copepods was completely inactivity within 30min and 40min after exposure to a solution of 35 mg/ L Moroxydine Hydrochloride. NNV took with eggs, rotifers and copepods was completely
25、inactivity within 20min and 30min after exposure to a solution of 50mg/L Povidone Iodine and 3mg/L Moroxydine Hydrochloride.Key words: Epinephelus coioides, NNV, transmission, breakingEditors note: Judson Jones is a meteorologist, journalist and photographer. He has freelanced with CNN for four year
26、s, covering severe weather from tornadoes to typhoons. Follow him on Twitter: jnjonesjr (CNN) - I will always wonder what it was like to huddle around a shortwave radio and through the crackling static from space hear the faint beeps of the worlds first satellite - Sputnik. I also missed watching Ne
27、il Armstrong step foot on the moon and the first space shuttle take off for the stars. Those events were way before my time.As a kid, I was fascinated with what goes on in the sky, and when NASA pulled the plug on the shuttle program I was heartbroken. Yet the privatized space race has renewed my ch
28、ildhood dreams to reach for the stars.As a meteorologist, Ive still seen many important weather and space events, but right now, if you were sitting next to me, youd hear my foot tapping rapidly under my desk. Im anxious for the next one: a space capsule hanging from a crane in the New Mexico desert
29、.Its like the set for a George Lucas movie floating to the edge of space.You and I will have the chance to watch a man take a leap into an unimaginable free fall from the edge of space - live.The (lack of) air up there Watch man jump from 96,000 feet Tuesday, I sat at work glued to the live stream o
30、f the Red Bull Stratos Mission. I watched the balloons positioned at different altitudes in the sky to test the winds, knowing that if they would just line up in a vertical straight line we would be go for launch.I feel this mission was created for me because I am also a journalist and a photographe
31、r, but above all I live for taking a leap of faith - the feeling of pushing the envelope into uncharted territory.The guy who is going to do this, Felix Baumgartner, must have that same feeling, at a level I will never reach. However, it did not stop me from feeling his pain when a gust of swirling
32、wind kicked up and twisted the partially filled balloon that would take him to the upper end of our atmosphere. As soon as the 40-acre balloon, with skin no thicker than a dry cleaning bag, scraped the ground I knew it was over.How claustrophobia almost grounded supersonic skydiverWith each twist, y
33、ou could see the wrinkles of disappointment on the face of the current record holder and capcom (capsule communications), Col. Joe Kittinger. He hung his head low in mission control as he told Baumgartner the disappointing news: Mission aborted.The supersonic descent could happen as early as Sunday.
34、The weather plays an important role in this mission. Starting at the ground, conditions have to be very calm - winds less than 2 mph, with no precipitation or humidity and limited cloud cover. The balloon, with capsule attached, will move through the lower level of the atmosphere (the troposphere) w
35、here our day-to-day weather lives. It will climb higher than the tip of Mount Everest (5.5 miles/8.85 kilometers), drifting even higher than the cruising altitude of commercial airliners (5.6 miles/9.17 kilometers) and into the stratosphere. As he crosses the boundary layer (called the tropopause),
36、he can expect a lot of turbulence.The balloon will slowly drift to the edge of space at 120,000 feet (22.7 miles/36.53 kilometers). Here, Fearless Felix will unclip. He will roll back the door.Then, I would assume, he will slowly step out onto something resembling an Olympic diving platform.Below, t
37、he Earth becomes the concrete bottom of a swimming pool that he wants to land on, but not too hard. Still, hell be traveling fast, so despite the distance, it will not be like diving into the deep end of a pool. It will be like he is diving into the shallow end.Skydiver preps for the big jumpWhen he
38、 jumps, he is expected to reach the speed of sound - 690 mph (1,110 kph) - in less than 40 seconds. Like hitting the top of the water, he will begin to slow as he approaches the more dense air closer to Earth. But this will not be enough to stop him completely.If he goes too fast or spins out of con
39、trol, he has a stabilization parachute that can be deployed to slow him down. His team hopes its not needed. Instead, he plans to deploy his 270-square-foot (25-square-meter) main chute at an altitude of around 5,000 feet (1,524 meters).In order to deploy this chute successfully, he will have to slo
40、w to 172 mph (277 kph). He will have a reserve parachute that will open automatically if he loses consciousness at mach speeds.Even if everything goes as planned, it wont. Baumgartner still will free fall at a speed that would cause you and me to pass out, and no parachute is guaranteed to work high
41、er than 25,000 feet (7,620 meters).It might not be the moon, but Kittinger free fell from 102,800 feet in 1960 - at the dawn of an infamous space race that captured the hearts of many. Baumgartner will attempt to break that record, a feat that boggles the mind. This is one of those monumental moments I will always remember, because there is no way Id miss this.
