中华鳖病毒（TSV）的致病性及其电镜观察

本文应用流行病学调查和电镜超薄切片技术,对福建省４个中华鳖养殖场的病毒性传染病进行观察,发现在病鳖肝细胞中的中华鳖病毒（ＴＳＶ）是导致幼鳖大批死亡的主要致病原,还讨论了该病毒的超微结构以及对宿主细胞的损害情况。     

动物病毒与宿主细胞的细胞凋亡

细胞凋亡(apoptosis)，又称programmed cell death(PCD)，是细胞在内外物理、化学、生物因素作用下启动系列自身调节基因而发生的一种自主死亡过程。细胞凋亡中发生了系列不同于细胞坏死(necrosis)的形态学和生化事件，其在许多生理和病理过程中具有重要生物学意义。在病毒与宿主细胞     

中华鳖的一种致病性病毒及其电镜观察

应用流行病学调查方法和电镜超薄切片技术,对福建省４个中华鳖养殖场发生的病毒性传染病进行了观察。在肝细胞中发现的球形病毒是导致幼鳖大批死亡的主要致病原。还讨论了该病毒的超微结构以及对宿主细胞的损害作用。     

外泌体携带miRNA进入邻近细胞能够促进新城疫病毒(NDV)感染

外泌体(Exosome)是一类由细胞主动分泌释放到胞外的小囊泡,直径大小约为30 nm^150 nm。它凭借自身能在细胞间传递蛋白、核糖核酸、脂类等小分子物质而在细胞间通讯中发挥着关键作用。近年来,关于外泌体与病毒感染的研究报道主要集中在人类病毒,尤其是外泌体在人类免疫缺陷病毒(Human immunodeficiency virus,HIV)和丙型肝炎病毒(The Hepatitis C virus,HCV)感染中生物功能的研究。然而,禽类病毒外泌体的相关报道较为鲜见。     

犬瘟热病毒在Vero细胞上培养条件的优化研究

犬瘟热病毒(CDV)在Vero细胞上增殖条件研究结果表明，血清含量5％，2倍的细胞密度，24h接毒，48～96h有利于CDV的增殖。     

重组禽流感病毒灭活疫苗(细胞源)保存期试验的研究

为保证重组禽流感病毒灭活疫苗(细胞源)在有效期内使用是安全、高效、稳定、均一的,将公司生产的3批疫苗置2～8 ℃保存,分别于保存0、4、8、12、15个月时进行性状检验、效力检验及甲醛残留量测定。结果表明,保存15个月内疫苗性状稳定,免疫SPF鸡后血清抗体效价无明显变化,甲醛残留量含量稳定。实际应用中,疫苗按规定条件储存,在有效期内使用是有质量保证的。     

不同细胞进行牛血清中牛病毒检测的比较试验

选出最适合牛血清中外源病毒增殖的细胞,用于牛血清中病毒检测,可以提高血清中病毒的检出率,以确保用于疫苗生产的牛血清是安全可靠的。将牛腹泻病毒、牛腺病毒、牛细小病毒、牛副流感病毒、呼肠孤病毒和狂犬病毒接种到培养好的MDBK细胞、VERO细胞、BT细胞中,逐日观察细胞的病变情况,待观察到10%细胞出现病变或者培养7天后,进行免疫荧光染色,选择出适合用于此六种病毒增殖的细胞系。结果显示BT细胞对所有病毒敏感,vero细胞对BAV、BVDV病毒不敏感,MDBK对REO、BVDV、RV三种病毒不敏感。综合比较后,选择BT细胞为BPIV、BPV、BAV、BVDV的增殖及实验用细胞,vero细胞为REO、RV的增殖及实验用细胞,为了提高血清中病毒的检出率,可以选用多种细胞进行同时实验。     

Susceptibility of Four New Salmonid Cell Lines to Infectious Hematopoietic Necrosis Virus

Abstract

Four salmonid cell lines, CoE 45, CoE 115, CoE 345, and RBTE 45, were established from embryonic tissues of coho salmon Oncorhynchus kisutch and rainbow trout O. mykiss. In vitro challenges of the new lines were conducted with four isolates of infectious hematopoietic necrosis virus (IHNV). Two of the IHNV isolates used for the challenges were derived from infected tissues of rainbow trout, one was derived from chinook salmon O. tshawytscha, and the other isolate was derived from coho salmon. To standardize the virus challenges of the new cell lines, several established piscine cell lines (EPC, CHSE 214, CSE-119, RTH-149, RTG, and RTS) were challenged in the same way as the new lines. Each of the lines was challenged with virus at a single low multiplicity of infection (0.01 plaque-forming unit per cell). Virus yields were quantitated by plaque assay on epithelioma papulosum cyprini (EPC) cells on day 3. Results of the challenge experiments revealed different levels of production of virus for each isolate on the various cell lines. Overall, the new cell line derived from rainbow trout, RBTE 45, was quite susceptible to all viruses tested. The three cell lines newly derived from coho salmon embryo were not as resistant to the replication of IHNV as was the established coho salmon cell line, CSE-119. An established cell line, EPC, derived from an epithelial tumor of common carp Cyprinus carpio, remained the most susceptible to all four IHNV isolates tested.     

Characterization of Three Continuous Cell Lines from Marine Fish

Abstract

Three continuous cell lines were established: JSKG from gonads of Japanese striped knife jaw Oplegnathus fasciatus, KRE from embryos of a hybrid of kelp Epinephelus moara and red spotted grouper E. akaara, and PAS from the skin of greater amberjack (also called purplish amberjack) Seriola dumerili; these cell lines were passed 60, 89, 120 times, respectively. Although initially cultured in Leibovitz's L-15 medium, two of the cell lines, JSKG and PAS, exhibited optimal growth response in Eagle's minimum essential medium buffered with a combination of tris and sodium bicarbonate. These cell lines were initiated at a higher NaCl concentration of 0.206 M but gradually adapted to the low NaCl concentration of 0.116 M after several subcultures. Optimum growth temperature was 25°C for JSKG and PAS cells, and 30°C for KRE cells. The modal chromosome number is 83 for the JSKG cell line, 92 for the KRE cell line, and 96 for the PAS cell line. Results for efficiency of plating indicate that all three cell lines are composed of transformed cells. Cell lines JSKG and PAS are susceptible to nine fish viruses, including channel catfish virus (CCV) and chum salmon virus (CSV). The KRE cell line is susceptible to CCV and fish rhabdoviruses of the vesiculovirus group. None of the cells showed cytopathic effect for Oncorhynchus masou virus (OMV) or Herpesvirus salmonis. Yields of infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), hirame rhabdovirus (HRV), and CSV were relatively low in these cell lines.     

Nucleotide Sequences Homologous to Mammalian Proto-oncogenes and Their Expression in Fish Cell Lines

Abstract

It has been shown that mammalian proto-oncogenes may be centrally involved in cell transformation in vitro and the formation of tumors in vivo. As part of a study on the evolutionary aspects of tumor formation, we have screened cell lines derived from brown bullhead Ictalurus nebulosus (BB), rainbow trout Oncorhynchus mykiss (RTG-2), common carp Cyprinus carpio (EPC), and chinook salmon Oncorhynchus tshawytscha (CHSE-214) for the presence or expression of sequences homologous to the mammalian proto-oncogenes. Southern blotting of DNA isolated from the fish cell lines showed multiple fragments having homology to v-src, v-raf, v-ras ^h, and v-erb-b DNA probes. In addition, monoclonal antibodies against the viral or cellular oncogenes of mammalian cells were able to recognize fish proteins that may be related to the myc, abl, fos, ras ^k, and p53 gene products. This study shows that proto-oncogenes are well conserved evolutionarily in vertebrates and may be factors involved in the acquisition of the transformed phenotype in fish.     

Establishment and Characterization of Seven Continuous Cell Lines from Freshwater Fish

Abstract

Seven continuous cell lines were established from salmonid and nonsalmonid fishes. Salmonid cell lines derived from rainbow trout Oncorhynchus mykiss and chum salmon O. keta were designated RTE and RTE-2 (rainbow trout embryo), RTT (rainbow trout tail), and SEH (“sake” or chum salmon embryo head). Nonsalmonid cell lines derived from pond smelt Hypomesus olidus, chevron snakehead Channa striata, and goldfish Carassius auratus were designated WF-1 (“wakasagi” fin), SHH (snakehead heart), and EPG (epithelioma papulosum of goldfish), respectively. Optimum growth for most of the cell lines was observed in Eagle's minimum essential medium buffered with sodium bicarbonate (26 mM) or a combination of sodium bicarbonate (8.9 mM) and tris (16 mM). Likewise, most of the cell lines showed optimum growth at the lowest NaCl concentration tested (0.116 M). Optimum growth temperatures ranged from 15 to 20°C for the salmonid cell lines and from 15 to 30°C for nonsalmonid cell lines. Except for RTT, the cell lines were heteroploid. Eleven fish viruses were used to test the susceptibility of these cell lines. Cell lines derived from salmonids developed cytopathic effects (CPE) when infected with 10 of the 11 fish viruses tested, except for RTT, which produced CPE with only 8 of the fish viruses. Six fish rhabdoviruses used in this study elicited a pronounced CPE when inoculated into nonsalmonid cell lines EPG, WF-1, and SHH. Among the new cell lines, RTE-2 showed the best potential for the isolation of fish viruses.     

猪瘟病毒广西流行毒株E0基因的克隆与分析

应用RT-PCR方法对猪瘟病毒E0基因进行扩增、克隆及测序,用DNA Star分析软件对6株毒株与猪瘟兔化弱毒苗(HCLV)、石门(Shimen)株、Paderborn株、GXWZ02株的相应片段进行比较分析,构建了CSFV遗传进化树。序列分析表明,广西流行株与HCLV株、Shimen株、Paderborn株、GXWZ02株之间核苷酸的同源性分别为80.8%~81.5%、82.8%~83.3%、93.1%~94.2%、93.7%~94.2%,推导的氨基酸同源性分别为88.3%~89.7%、89.7%~91.1%、96.2%~97.7%、97.7%~99.1%;6株广西CSFV流行毒株与国内外已发表的14株病毒相应序列进行比较,构建遗传进化树,结果表明所比较的20株毒株分为2个基因群,广西流行毒株均属于基因群Ⅱ。本研究成功地对广西流行猪瘟病毒株的E0基因进行了测序分析,表明近年来广西流行毒株未发生较大的变异,但病毒株有远离疫苗株发展的趋势。     

中华鳖病毒和克氏原螯虾白斑综合征病毒危害机理和集成防治

由于集约化养殖的快速发展,病害频繁发生,特别是近年来暴发的按常规方法难以控制的突发性中华鳖病毒(trionyx sinesis virus,TSV)和克氏原螯虾白斑综合征病毒(white spot syndrome virus,WSSV)感染,使水产业蒙受较大的经济损失.主要对中华鳖病毒和克氏原螯虾白斑综合征病毒的危害机理和集成防治进行了阐述,以期为有效防治上述2种病毒的感染提供参考.     

鸭肝炎病毒A66弱毒株在鸡胚成纤维细胞上的培养

将鸡胚致弱的鸭肝炎病毒 ( DH V) A6 6 株接种于鸡胚成纤维细胞 ( CEF)进行细胞培养 ,通过对细胞培养物进行病毒滴度测定、鸡胚中和试验和电镜检查 ,证明了 DHV A6 6 毒株能够在 CEF上增殖。试验还表明 ,犊牛血清对 DH V增殖具有明显的抑制作用。此外 ,还根据细胞培养物病毒滴度测定结果绘制出了病毒在 CEF上的生长曲线。     

Invasion and Replication of Photobacterium damselae subsp. piscicida in Fish Cell Lines

Abstract

The objective of this study was to evaluate the ability of Photobacterium damselae subsp. piscicida to invade and replicate within different fish cell lines. Channel catfish ovary (CCO), fathead minnow (FHM), and epithelioma papillosum cyprini (EPC) cell lines were all susceptible to invasion and supported replication of P. damselae in an in vitro invasion assay in which extracellular growth was controlled with gentamicin. The number of bacteria recovered from EPC and CCO cells increased significantly after 6, 12, and 18 h, indicating that P. damselae replicated within those cells. There was also a significant increase in EPC cells at 3 h. Although the number of bacteria recovered from FHM cells increased slightly after 3 and 6 h, it declined at 12 and 18 h. The decline, however, was due to the early release of bacteria from FHM cells associated with a greater susceptibility to initial infection, which resulted in early cell lysis and subsequent exposure to gentamicin in the media. Using light and electron microscopy, we observed the invasion of bacteria as early as 30 min after infection. Intracellular bacteria were initially contained in small, close-fitting vacuoles that developed into large, clear, spacious vacuoles over time. There was no evidence of bacteria free in the cytoplasm. The intracellular location of P. damselae was confirmed by means of transmission electron microscopy with ruthenium red staining to discriminate between the extra- and intracellular spaces. This is the first report that provides evidence of intracellular replication of P. damselae in cell lines.     

H4亚型禽流感病毒套式RT-PCR检测方法的建立

旨在建立一种快速、准确检测H4亚型禽流感病毒(AIV)的方法,针对H4亚型AIV HA基因保守序列,设计并筛选出2对特异性引物,通过优化反应条件,建立了H4亚型AIV套式RT-PCR检测方法。特异性试验结果显示,该法可特异性检测出H4亚型AIV,对其他常见禽病病原体无交叉;敏感性试验结果显示,该法对H4亚型AIV检测下限为360fg/μL;用该法对106份临床样品进行检测,临床样品检测结果与病毒分离结果一致。本研究建立的套式RT-PCR方法为H4亚型AIV的早期诊断及有效防控提供了快速、特异和敏感的检测方法。     

H3N8亚型马流感病毒RT-LAMP检测方法的建立

为了建立检测H3N8亚型马流感病毒的RT-LAMP方法,根据H3N8亚型马流感病毒HA基因序列,设计了2对LAMP引物,经过优化反应条件,建立了RT-LAMP检测H3N8亚型马流感病毒方法。结果表明,RT-LAMP方法能够在63℃恒温条件下、75min内实现目的基因片段的大量特异性扩增,通过荧光显色就可直接用肉眼判断结果。对H7N7亚型马流感病毒、马动脉炎病毒、马鼻肺炎病毒的核酸无交叉反应,具有较好的特异性;方法的灵敏度比常规RT-PCR的高10倍;该方法操作简便快速、省时省力,而且灵敏度高、特异性强,对H3N8亚型马流感病毒的检测具有实际的应用价值。