鳗鲡疱疹病毒对欧洲鳗鲡的致病性研究

为了明确鳗鲡疱疹病毒(Anguillid herpesvirus, AngHV)的致病性,本实验采用一株从鳗鲡“脱黏败血综合征”病料中分离的AngHV(NA16108),研究了其对欧洲鳗鲡幼鳗的致病性。结果显示,注射AngHV的鳗鲡体表出现黏液脱落、鳍条出血、红头等症状;鳃部出现黏液增多、出血,肝脏褪色、肿大,脾脏和肾脏肿大等病变;进一步的组织病理学观察发现,鳗鲡的体表黏液及黏膜上皮细胞脱落,次级鳃瓣增生、呼吸细胞肿胀坏死,脾脏细胞坏死、黑色素细胞聚集,肾小管管壁上皮细胞坏死、管腔变窄等病理症状;这与鳗鲡“脱黏败血综合征”的发病特征一致。致死率分析显示,从第4天开始攻毒组鳗鲡开始出现死亡,第7天和第14天的累计死亡率分别达到26.7%和56.7%;荧光定量PCR检测显示,在攻毒鳗鲡肝脏、脾脏、肾脏、肠道、鳃和皮肤黏液中均可检测到AngHV;另外,用鳗鲡卵巢细胞系(eel ovary cell line, EO)从攻毒鳗鲡主要内脏器官中重新分离出AngHV,表明鳗鲡发生了AngHV的系统侵染。研究表明,AngHV是鳗鲡“脱黏败血综合征”的致病病原,这为深入开展该病的发病机制及防控研究奠定了基础。     

大鲵虹彩病毒理化及生物学特性研究

对大鲵虹彩病毒(Giant salamander iridovirus,GSIV)的理化特性及生物学特性进行了研究。结果表明:GSIV对热处理敏感,56℃和65℃处理30 min均可彻底灭活病毒;GSIV经酸(pH3)和碱(pH10)处理,病毒滴度(TCID50)与对照组相比较分别下降了8.58、9.04个对数级,差异极显著(P<0.01);GSIV经有机溶剂氯仿、乙醚以及胰蛋白酶处理,TCID50与对照组相比较分别下降了9.33、7.83、6.49个对数级,差异极显著(P<0.01)。冻融次数对GSIV滴度的影响不显著(P>0.05)。GSIV对细胞培养物的感染性试验结果表明,GSIV可在鲤上皮瘤细胞系(Epithelioma papilloma cyprini,EPC)、斑点叉尾鮰肾脏细胞系(Channel catfish kidney,CCK)、虹鳟鱼性腺细胞系(Rainbow trout gonadal,RTG-2)等细胞中增殖,但在EPC、CCK细胞中增殖速度快,TCID50高;GSIV在EPC细胞中的最适生长温度是25℃。GSIV在EPC细胞中增殖动态试验结果表明,GSIV感染细胞6 h后TCID50开始快速上升,进入对数增长期,72 h时TCID50达到最大值,以后趋于稳定。GSIV感染EPC细胞超薄切片透射电镜观察结果显示,在EPC细胞质中可见大量虹彩病毒样颗粒,呈晶格状排列,直径约140 nm。     

鲤疱疹病毒Ⅱ型的理化及生物学特性和超微形态发生

为了查明鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,Cy HV-2)的理化与生物学特性以及病毒在细胞内的超微形态发生过程,利用新建立的对Cy HV-2敏感的异育银鲫脑组织细胞系(Gi CB),对Cy HV-2的理化及生物学特性进行了详细研究,比较了不同来源鱼类细胞系对Cy HV-2感染的敏感性,并对体外培养细胞中Cy HV-2病毒粒子及其超微形态发生过程进行了电镜观察。结果显示,Cy HV-2对热、酸、碱、有机溶剂和冻融敏感;常用鱼类细胞系EPC、RTG-2、Koi-Fin、CIK、CCK、PF-Fin对Cy HV-2的感染不敏感,特异性巢式PCR检测盲传至第7代Cy HV-2细胞培养物,结果均为阴性;Cy HV-2在Gi CB细胞中的增殖动态研究结果表明:病毒感染细胞经过12 h的隐晦期,24 h开始进入对数生长期,96 h病毒滴度达到最高值(107.52±0.26 TCID50/m L),然后进入平台期;透射电子显微镜观察结果显示,Cy HV-2感染细胞可分为吸附与侵入、复制与装配、成熟与释放3个主要过程,病毒进入对数生长期后,被感染细胞内可见形态典型的疱疹病毒颗粒。     

鳗鲡疱疹病毒的分离与鉴定

为获知鳗鲡"脱粘败血病"与病毒的关系,实验用蔗糖密度梯度离心的方法从发病的欧洲鳗鲡内脏器官组织匀浆液中纯化了病毒粒子,负染后利用电镜观察;进一步用EO细胞对病毒进行了分离、培养,并对感染病毒的细胞进行超薄切片,电镜观察;然后,提取病毒DNA,利用鳗鲡疱疹病毒的PCR检测方法对其进行了鉴定。结果显示,接种匀浆上清液的EO细胞出现细胞融合的病变效应;分离病毒的病毒粒子具囊膜,大小约为200 nm;从感染病毒的细胞上清液DNA中扩增出特异性条带,序列测定与比对分析表明,该序列与鳗鲡疱疹病毒欧洲株(AngHV-1)的序列完全一致。研究表明,利用EO细胞分离了一株鳗鲡病毒,经形态观察和DNA分析,确认该病毒为鳗鲡疱疹病毒,命名为AngHV-FJ。该研究为深入开展鳗鲡疱疹病毒的致病机制及鳗鲡"脱粘败血病"的防控研究奠定了重要基础。     

虎纹蛙病毒体外培养及其理化特性

从发病濒死的虎纹蛙（Rana tigrina rugulosa)蝌蚪中分离到一种病毒，在25℃条件下，该病毒能在鲤鱼表皮瘤细胞系（EPC），胖头Shiu肌肉细胞系（FHM）和草鱼性腺细胞系（CO）三种鱼类细胞上产生空斑状的细胞病理变化（CPE）。该病毒对氯仿，热（56℃，20min)和酸（pH3)敏感，其体外培养的适合增殖温度范围为20-30℃，电镜下观察，病毒为对称的二十面体，切面正六边形，对角直径125nm左右。     

基于CPB细胞扩增体系的鳜弹状病毒增殖条件的优化

为获知鳜弹状病毒(Siniperca chuatsi rhabdovirus,SCRV)QY株在体外培养细胞中最适增殖条件,以鳜脑细胞系(Chinese perch brain cell line,CPB)为增殖体系,采用实时荧光定量PCR(quantitative real-time PCR,q RT-PCR)技术所测病毒拷贝数为判断指标,比较同步接毒和异步接毒2种接种方式以及病毒接种量、血清浓度等培养条件对病毒增殖的影响,确定SCRV-QY株在CPB细胞中的最适增殖条件。结果显示,单位体积病毒增殖量方面,异步接毒法优于同步接毒法,以感染复数(multiplicity of infection,MOI)为10将病毒接种长满单层的CPB细胞中,28°C吸附1.5 h,用含2%胎牛血清的L15培养液于28°C培养时,病毒增殖量最高,为5.59×10~(10)拷贝/mL;而从单位成本所获病毒量考虑,同步接毒法筛选出的4种增殖条件单位成本所获的病毒产量优于异步接毒法,其中当MOI=0.03、胎牛血清终浓度为6%时,同步接种对数生长中期的CPB细胞,28°C恒温培养70 h后收获病毒液,单位成本所获病毒量最高,为4.88×10~(13)个拷贝/元。综上所述,本研究以病毒增殖量和培养基成本作为考量,优化SCRV-QY株的增殖条件,可为SCRV疫苗低成本、规模化生产提供理论依据。     

草鱼呼肠孤病毒Ⅱ型在不同鱼类细胞中的增殖情况

草鱼呼肠孤病毒Ⅱ型(GCRVⅡ)是当前导致草鱼出血病流行和暴发的主要基因型,本研究拟通过分析GCRVⅡ代表株HZ08在不同鱼类细胞中的增殖情况来筛选GCRVⅡ的敏感细胞系。首先以不同浓度的HZ08株接种草鱼吻端成纤维细胞(PSF)和草鱼鳔细胞(GSB),以确定病毒接种的最佳浓度;然后以最佳接种浓度同时感染PSF、GSB、草鱼肝细胞(L8824)、草鱼卵巢细胞(CO)等10种鱼类细胞,接毒后每天观察细胞状态,用荧光定量PCR(q PCR)方法定量分析HZ08在各种细胞系中的增殖量,并在感染5 d后用间接免疫荧光定性分析病毒在各种细胞中的增殖情况。结果显示,HZ08感染细胞的最佳接种浓度为1.0×104拷贝/μL,该毒株接种的10种鱼类细胞均无明显的细胞病变效应(CPE);q PCR分析病毒感染5~8 d后的结果显示,HZ08在GSB、L8824、PSF、草鱼鳍条细胞(CF)、CO、草鱼脑细胞(CIB)、锦鲤吻端细胞(KS)7种细胞中均能增殖,其中在GSB、L8824和PSF细胞中的增殖量较大,最大分别为1.14×107、5.90×106和6.30×104拷贝/μL。而在鲤上皮细胞(EPC)、锦鲤脑细胞(KB)、鲫脑细胞(Cc B)中不增殖,免疫荧光定性检测结果与荧光定量检测结果相吻合,在GSB、L8824和PSF中的荧光信号较多、较强,其他细胞中则较弱或没有荧光信号。研究表明,GSB、L8824和PSF是GCRVⅡ较为敏感的细胞系,该研究结果对今后Ⅱ型GCRV的研究和防控产品开发具有重要意义。     

草鱼(Ctenopharyngodon idellus)性腺细胞系(GCO)特性和对鲤春病毒的敏感性

草鱼(Ctenopharyngodon idellus)性腺细胞系(GCO)是中国科学院水生生物研究所在20世纪70年代开展草鱼出血病研究时建立的一株细胞系,迄今已传至300多代,在中国鱼类病毒学研究领域发挥了重要作用.本研究采用形态学观察、细胞生长曲线测定、细胞周期测定、细胞核型分析、细胞凋亡检测、电镜观察等方法,对GCO的生长特性及鲤春病毒血症病毒(SvCv)在该细胞中的增殖特性等进行了研究.结果显示,GCO细胞的最适生长温度为25℃,在M199和MEM等细胞培养液中均能较好地生长,培养液中最适的胎牛血清浓度为10％.测定了GCO细胞系对SVCV病毒的敏感性,发现与鲤(Cyprinus carpio)上皮瘤细胞系(EPC)、肥头鲤(Pimephales promelas)细胞系(FHM)、大鳞大麻哈鱼(Oncorhynchus tshawytscha)胚胎细胞系(CHSE-214)等世界动物卫生组织(OIE)推荐和各检测实验室常用的鱼类细胞系相比,GCO细胞系对SVCV表现出非常高的敏感性.生长盐线、电镜观察和凋亡实验显示,SVCV能引起GCO细胞系出现明显而稳定的细胞病变,引起GCO细胞系出现凋亡,并在细胞质中大量增殖.结果表明,GCO细胞系适用于SVCV病毒的分离、检测以及病毒致病性的有关研究.GCO细胞的适宜生长温度为15-28℃,这一特点将使它可以广泛地用于多种水生动物病毒的分离.     

养殖场有效杀灭禽流感病毒的方法

禽流感病毒对热比较敏感，65℃加热30min或煮沸(100℃)2min以上可灭活。病毒在粪便中可存活1周，在水中可存活1个月，在pH＜4．1的条件下也具有存活能力。病毒对低温抵抗力较强，在有甘油保护的情况下可保持活力1年以上。病毒在直射阳光下40-48h即可灭活，如果用紫外线直接照射，可迅速破坏其传染性。禽流感病毒对乙醚、氯仿、丙酮等有机溶剂均敏感。     

两株草鱼呼肠孤病毒江西株的分离与鉴定

对从江西省南昌县莲塘镇的患病草鱼鱼种池塘采集到草鱼出血病疑似病样材料分离出的两株病毒株进行了鉴定。结果显示:用除菌过滤后的患病鱼肝脏、脾脏、肾脏组织浆滤液腹腔注射感染8～10 cm健康草鱼鱼种,5 d后试验鱼发病,可复制出自然发病症状,死亡率达50%以上,对照组未有死亡。用病样滤液接种草鱼肾细胞系(CIK),可产生细胞病变效应(CPE),病毒的TCID50分别为10-8.3/0.1 mL和10-8.0/0.1 mL。理化特性研究结果显示:氯仿、乙醚处理组病毒的感染力和对照组相比变化不显著。病毒基因组RT-PCR反应可扩增出目的片段,序列测定与分析结果表明,其与GenBank中GCRV(登录号为AF403392,AF239175)相应序列的同源性达99%以上。     

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency of CyHV‐3 by temperature shift in the cells

A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.     

Physiological investigations of a neurotoxin-producing phytoflagellate, Chattonella marina (Raphidophyceae)

The effects of temperature, salinity, light intensity and pH on the growth and morphology of Chattonella marina (Subrahmanyan) Hara & Chihara were examined. Optimal growth was observed at temperatures of 20-25°C, salinities of 20-30%o, light intensities of 60-140 μE m^?2 s^_1 and pH 7.5-8.5. Growth did not occur at temperatures below 15°C or above 30°C, and at salinities below 10%o. The morphology (shape) of the cells was strongly affected by temperature. At 20°C and 25°C, the population occurred mostly in a spindle-like form, whereas at 10°C, 90% of the cells became spherical within 10 days of inoculation and stationary phase cultures consisted entirely of spherical cells. Morphology was also markedly affected at 30°C. The number of spindle-like cells was highest at 20-30%o and was less at lower salinities. Light intensity and pH did not influence morphology markedly under the range of light intensities (20-180 μE m ^?2 s-^?1) and pH (6.5-8.5) tested.     

Litopenaeus vannamei (Boone) post-larval survival related to age, temperature, pH and ammonium concentration

Transport of post‐larvae shrimp used in aquaculture is an important element of successful cultivation because of the potential for stress during stocking procedures. To find optimum transport conditions, several bioassays were performed in the laboratory to evaluate survival of whiteleg shrimp Litopenaeus vannamei 5–30‐day‐old postlarvae under conditions similar to those encountered during transport from the hatchery to nursery and shrimp ponds. Postlarvae were exposed for 4 h to different temperatures and pH levels ammonia concentrations. Survival was significantly reduced after a 4 h exposure to pH 9 and was inversely related to temperature with or without 7 mg L^?1 of ammonia. The 15‐ and 20‐day‐old postlarvae had higher survival rates than other ages. The lowest survival occurred in alkali conditions (pH 9), with 7 mg L^?1ammonia at 30 and 32°C. To assure optimal survival of postlarvae during transfer from the hatchery to the nursery and shrimp ponds, we recommend temperatures below 28°C, pH no higher than 8, no ammonia and post‐larval age at least 15 days.     

Rapid visual detection of cyprinid herpesvirus 2 by recombinase polymerase amplification combined with a lateral flow dipstick

Herpesviral haematopoietic necrosis (HVHN), caused by cyprinid herpesvirus 2 (CyHV‐2), causes significant losses in crucian carp (Carassius carassius) aquaculture. Rapid and convenient DNA assay detection of CyHV‐2 is useful for field diagnosis. Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that can amplify DNA within 30 min at ~37°C by simulating in vivo DNA recombination. Herein, a rapid and convenient detection assay based on RPA with a lateral flow dipstick (LFD) was developed for detecting CyHV‐2. The highly conserved ORF72 of CyHV‐2 was targeted by specific and sensitive primers and probes. The optimized assay takes only 15 min at 38°C using a water bath, with analysis of products by 2% agarose gel electrophoresis within 30 min. A simple lateral flow strip based on the unique probe in reaction buffer was developed for visualization. The entire RPA‐LFD assay takes 50 min less than the routine PCR method, is 100 times more sensitive and displays no cross‐reaction with other aquatic viruses. The combined isothermal RPA and lateral flow assay (RPA‐LFD) provides a simple, rapid, reliable method that could improve field diagnosis of CyHV‐2 when resources are limited.     

Effect of thermal stress on Hsp70 gene expression and female reproductive performance of giant freshwater prawn,Macrobrachium rosenbergii

Using Hsp70 as a biomarker, thermal stress impinges on reproductive organs, ovary and hepatopancreas were being analyzed by determining the expression of Hsp70 mRNA inside the organs after the adult inter‐molt females were subjected to thermal treatment at 35, 30 and 28°C (Control). Results showed the expression of Hsp70 mRNA under thermal treatment of 35°C after 2 hr recovery in ovary were upregulated at 2, 4, 6, 12, 24 hr and 30 days compared to control whereas in hepatopancreas under similar treatment, the expression of Hsp70 mRNA were significantly higher than control at 6, 24 hr and 30 days. Frequency of reproductive molt at 35°C showed the ovary of females were failed to develop and only entered common molt along three consecutive molt cycles. For 30°C thermal treatment, the expression of Hsp70 mRNA was significantly higher than control after 2 hr recovery but returned to normal afterwards until 30 days’ thermal treatment. Maternal heat shock for 2 hr at 35°C were found to give significantly lower frequency of reproductive molt and longer duration of ovarian development and incubation period whereas maternal heat shock for 2 hr at 30°C gave lower frequency of reproductive molt, slower development of embryo and lower hatching success compared to untreated control. This study suggests that short and long‐term thermal stress at 30 and 35°C were found to affect the induction of Hsp70 mRNA in reproductive organs of Macrobrachium rosenbergii and also influence their reproductive performance.     

Acute Toxicity and Anesthetic Effects of Clove Oil in Penaeus semisulcatus under Various Water Quality Conditions

Acute toxicity and anesthetic effects of clove oil were studied in P. semisulcatus (1.8–2.1 g body weight). The EC₅₀ 1-h (the concentration effective for 50% of test animals), LC₅₀ 1-h (the concentration lethal to 50% of test animals after 1 h) and LC₅₀ 24-h (the concentration lethal to 50% of test animals after 24 h) were calculated at concentrations of 25, 130 and 30 mg/l, respectively, at 30°C, salinity 40 ppt, pH 8.6 and dissolved oxygen >6 mg/l. Generally, with increasing concentrations of clove oil, the times required for sedation and anesthesia decreased, while the recovery times increased. At concentrations 50, 100, 150 and 200 mg/l under temperature of 30°C and salinity of 40 ppt, the times required for sedation were 6 ± 0.2, 2.5 ± 0.3, 2 ± 0.08 and 0.5 ± 0.08 min, while times required for complete recovery were calculated to be 4.5 ± 0.3, 5.5 ± 0.17, 6.5 ± 0.25 and 11 ± 0.38 min, respectively. Also, the times required for deep anesthesia were 20 ± 1, 5 ± 0.5, 3 ± 0.4 and 2.2 ± 0.5 min in the above concentrations, while the times required for complete recovery were 10 ± 1, 11 ± 1.5, 14 ± 2.2 and 16 ± 3 min, respectively. Furthermore, considering the times to sedation, deep anesthesia and recovery at different temperatures of 20°C, 25°C, 30°C and 35°C and salinities of 25, 30, 35, 40 and 48 ppt; the combinations of salinity plus temperature and clove oil concentration plus salinity had the greatest and the least effects.