Genome architecture changes and major gene variations of Andrias davidianus ranavirus (ADRV)

Ranaviruses are emerging pathogens that have led to global impact and public concern. As a rarely endangered species and the largest amphibian in the world, the Chinese giant salamander, Andrias davidianus, has recently undergone outbreaks of epidemic diseases with high mortality. In this study, we isolated and identified a novel ranavirus from the Chinese giant salamanders that exhibited systemic hemorrhage and swelling syndrome with high death rate in China during May 2011 to August 2012. The isolate, designated Andrias davidianus ranavirus (ADRV), not only could induce cytopathic effects in different fish cell lines and yield high viral titers, but also caused severely hemorrhagic lesions and resulted in 100% mortality in experimental infections of salamanders. The complete genome of ADRV was sequenced and compared with other sequenced amphibian ranaviruses. Gene content and phylogenetic analyses revealed that ADRV should belong to an amphibian subgroup in genus Ranavirus, and is more closely related to frog ranaviruses than to other salamander ranaviruses. Homologous gene comparisons show that ADRV contains 99%, 97%, 94%, 93% and 85% homologues in RGV, FV3, CMTV, TFV and ATV genomes respectively. In addition, several variable major genes, such as duplicate US22 family-like genes, viral eukaryotic translation initiation factor 2 alpha gene and novel 75L gene with both motifs of nuclear localization signal (NLS) and nuclear export signal (NES), were predicted to contribute to pathogen virulence and host susceptibility. These findings confirm the etiologic role of ADRV in epidemic diseases of Chinese giant salamanders, and broaden our understanding of evolutionary emergence of ranaviruses.     

Molecular Cloning,Characterization, and Expression Analysis of Cathepsin A in the Chinese Giant Salamander Andrias davidianus

Abstract

Cathepsin A (CTSA) is serine carboxypeptidase, an important protease in the lysosome. In this study, the full complementary DNA (cDNA) sequence of CTSA in Chinese giant salamanders Andrias davidianus was cloned, and its sequence features were analyzed. Tissue expression patterns of CTSA in healthy and Aeromonas hydrophila-infected salamanders were also investigated. The full cDNA sequence of salamander CTSA was 1,620 base pairs in length, encoding 472 amino acids. Salamander CTSA shared high sequence identities with other vertebrates’ CTSAs, ranging from 62.7% to 68.9%. In healthy salamanders, CTSA was highly expressed in spleen, followed by brain, intestine, and stomach. After A. hydrophila infection, salamander CTSA was significantly upregulated in lung, heart, muscle, and kidney; was downregulated in liver, spleen, and intestine; and exhibited no significant changes in stomach and skin, indicating that salamander CTSA might play defense roles in multiple tissues during bacterial infection. These results provide a solid basis for further study of the immune function of amphibian CTSA.

Received September 18, 2016; accepted June 18, 2017 Published online October 9, 2017     

养殖大鲵蛙病毒自然感染的病理形态学观察

对经PCR确诊为蛙病毒感染的养殖大鲵自然病例进行病理形态学变化观察。剖检发现病鲵腹部膨大,皮肤上出现白点、出血斑,溃疡,头部与四肢肿胀;肝肿大,呈灰白色或斑点状出血;脾、肾肿大,淤血、斑点状出血;肠壁变薄,肠腔内充满大量含血的或淡黄色的液体。镜检主要见全身组织器官广泛性水肿、出血、变性、坏死和炎症细胞浸润,特别是肾、肝、胃肠道、脾和皮肤肌肉的损伤较为严重,并在病变组织细胞见圆形或椭圆形嗜酸性胞浆包涵体。肾表现为渗出性肾小球肾炎及肾小管上皮的空泡变性与坏死;肝细胞广泛性空泡变性与灶性坏死;脾淋巴细胞坏死,数量减少;胃肠道为卡他性一出血性炎;皮肤水肿,表皮细胞空泡变性,坏死脱落,形成渍疡。电镜显示肝、脾、肾的细胞发生明显的病变,线粒体肿胀,嵴断裂,溶解,粗面内质网扩张,核糖体颗粒脱落,细胞核染色质浓缩,边集,并在一些病变细胞浆内见晶格状排列或散在的虹彩病毒样颗粒。     

Spontaneous nephroblastoma in a Japanese giant salamander (Andrias japonicus)

An adult male Japanese giant salamander (Andrias japonicus) died accidentally, and necropsy showed a white mass (23 × 15 mm) in the left kidney and hepatorrhexis with hemoperitoneum. Histologically, the renal mass was mainly composed of immature nephroblastic tumor cells. In the tumor tissue, a trabecular pattern lined by oval to polygonal tumor cells with a rich interstitium, solid growth and a few tubular structures was observed. Nephroblastic tumor cells were strongly positive for vimentin and weakly positive, and epithelium-like tumor cells were strongly positive for cytokeratin. However, antibody for Wilms' tumor protein 1 did not react with the salamander's cells. On electron microscopy, a desmosome junction was observed between tumor cells. This is the first report of nephroblastoma in a Japanese giant salamander.     

大鲵α1-血红蛋白基因的生物信息学分析及组织表达研究

 从大鲵皮肤cDNA文库中分离获得大鲵α1 血红蛋白基因全长cDNA序列,生物信息学分析表明,大鲵α 血红蛋白基因全长cDNA序列为594 bp,开放阅读框共432 bp,编码144个氨基酸,编码的大鲵α1 血红蛋白推测的理论分子量为16048.3 u,等电点为6.44。氨基酸组成中酸性氨基酸11.9%,中性氨基酸69.9%,碱性氨基酸18.2%。结构分析表明,该蛋白没有信号肽,但在100~122个氨基酸处以及98~122个氨基酸处分别有由里向外、由外向里的跨膜螺旋区,二级结构以α 螺旋为主。氨基酸序进行同源性列比对表明与人、猕猴的亲缘关系最近,和牛蛙的亲缘关系最低只有35%。荧光定量PCR结果表明,α1 血红蛋白基因在肌肉中表达量最高,在胃中最低。     

Identification and determination of antigenic proteins of Korean ranavirus-1 (KRV-1) using MALDI-TOF/TOF MS analysis

Ranaviruses are serious pathogens of fish, amphibians, and reptiles, and pose a major threat to global biodiversity. A ranavirus isolated from tissues of diseased tadpoles and frogs in Gangwon province, Korea, in 2006 and 2007, was designated Korean ranavirus-1 (KRV-1) and was infectious in a variety of fish cell lines with highest titers (10¹⁰ TCID₅₀/ml) in Epithelioma papulosum cyprini cells (EPCs) and baby hamster kidney-21 (BHK-21) cells. Bullfrog (Rana catesbeiana) tadpoles challenged by immersion in 10⁵ TCID₅₀/ml of KRV-1 showed 60% mortality within 10 days. SDS-PAGE of frog virus 3 (FV3) and KRV-1 proteins yielded several bands 35-49 kDa in size, which were identified as major capsid proteins (MCPs) by MALDI-TOF MS. Immunoblotting of FV3 proteins showed antigenic bands 34 kDa and 93 kDa in size which were identified by MALDI-TOF/TOF MS as MCP and neurofilament triplet H1-like protein (NF-H1), respectively. In KRV-1, antigenic bands at 32 kDa, 69 kDa, and 72 kDa were identified as MCP, Hypothetical protein, and NF-H1, respectively. The genes encoding these KRV-1 proteins were sequenced. KRV-1 appeared to be closely related to the soft-shelled turtle iridovirus (STIV), based on alignments of amino acid sequences from various ranaviruses. Variability in ranavirus antigenic proteins was apparent in an earlier study. It is expected that use of the methods employed here, together with the results of the present work, will contribute to an understanding of the pathogenesis of ranaviruses, and will further the development of DNA- or protein-based bait vaccines for conservation of natural habitats.     

中国大鲵子二代规模化人工孵化技术的研究

系统研究了影响大鲵子二代孵化率的环境因子(包括水质、水温、孵化密度)、硬件设施和软件管理技术。从理论上阐明孵化水质的离子比例平衡直接影响孵化率和苗种成活率,试验结果表明,广州华宝珍稀水产养殖有限公司养殖场调节水和水库水适合孵化,孵化率分别为53.3%和46.7%;过高、过低孵化水温都可导致大鲵胚胎发育停止,适宜孵化水温为16~22℃,最适水温为(20±1)℃;孵化密度以每筛(25 cm×35 cm×6 cm)放150颗受精卵为宜(孵化率为48%)。在本试验设定的条件下,大鲵孵化率逐年提高,由1998年的平均3%,提高到2003年的平均18%。     

桑叶提取物和1-脱氧野尻霉素对大鲵生长性能、肝脏功能及免疫能力的影响

本试验旨在研究桑叶提取物(MLE)和1-脱氧野尻霉素(DNJ)对大鲵生长性能、肝脏功能及免疫能力的影响.选择初始均重为(47.81±0.23)g的大鲵72尾,随机分为3组,每组3个重复,每个重复8尾.对照组饲喂基础饲料,试验组在基础饲料中分别添加7.5 g/kg MLE(MLE组)和0.04 g/kg DNJ(DNJ组...     

中国大鲵子二代制种技术的研究

为了推动中国大鲵人工生态繁殖和迁地保护的成功 ,系统介绍了中国大鲵子二代制种的技术路线 ,首先收集野生中国大鲵经人工驯养成为亲本种鲵 ,种鲵进行人工催产获得卵子、精子 ,经人工授精产生受精卵 ,再经人工孵化获得子一代幼苗 ,子一代经人工培育 2～ 3年产生子一代后备种鲵 ,再通过生理生态人工强化培育获得 1998年、1999年子一代亲本 ,然后经人工繁殖产生子二代大鲵 ,子二代大鲵可以放归自然保护区或供科研、观赏、商品鲵等利用。从 1998～ 2 0 0 3年共生产子一代 1 5万余尾 ,2 0 0 2年生产子二代 12 6 0尾 ,2 0 0 3年 4 80 6尾 (2年合计子二代 6 6 0 6尾 ) ,种群数量从最初的 1996年的 10 0尾增加到 2 0 0 3年的 2 31万尾 ,增长2 31倍 ,实践证明 ,此技术路线正确、可行。     

Trichinella spiralis—A potential anti-tumor agent

Murine forestomach carcinoma (cell line MFC), ascitic hepatoma (cell line H22) and sarcoma (cell line S180) solid tumor models were used to test the anti-tumor effect of Trichinella spiralis in vivo. Mice previously infected by oral administration of 400 viable T. spiralis larvae per mouse for 7 days were grafted with various solid tumor cell lines. Other groups of tumor-bearing mice were given caudal vein injection of crude extracts of adult and newborn larvae at 17.5, 35.0 or 70.0 mg kg^?1. These treatments to inhibit tumor growth were dose-dependent (p < 0.05). The anti-proliferative activity of crude T. spiralis extract was examined in vitro at 0.035, 0.070 or 0.140 mg ml^?1 using MFC, H22, S180, human chronic myeloid leukemia cell line (K562) and hepatoma cell line (H7402), tumor cell proliferation in vitro was measured by methyl thiazolium stain and was inhibited in dose-dependent manner (p < 0.05). At the same doses, crude T. spiralis extracts induced apoptosis of K562 and H7402 as detected by DNA fragmentation. Cell cycle analysis indicated that crude T. spiralis extracts, at 0.140 mg ml^?1, arrested the cell cycle of K562 and H7402 in G1 or S phase. It is concluded that T. spiralis contains anti-tumor active agent.     

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.     

A proteomic analysis of the effect of growth hormone on mammary alveolar cell-T (MAC-T) cells in the presence of lactogenic hormones

The bovine mammary alveolar cell-T (MAC-T) cell line is able to uniformly differentiate and secrete casein proteins in response to dexamethasone, insulin, and prolactin and is extensively used to study bovine mammary epithelial cell (MEC) function. Somatotropin, or growth hormone (GH), has been shown to increase milk protein synthesis both in vivo and in mammary cell models and to induce cytoskeletal rearrangement in a 3T3 fibroblast cell line and a Chinese hamster ovary cell line. To identify the nature of the effects of GH in MECs cultured with lactogenic hormones, changes in global protein expression were assessed in the MAC-T cell line with the use of two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry. Forty proteins were differentially expressed in response to GH (P < 0.05) and were related to metabolism, the cytoskeleton, protein folding, RNA and DNA processing, and oxidant stress. These widespread changes in protein expression are indicative of a global role of GH in overall cellular differentiation that may underlie the direct modulation of milk component synthesis in MEC models that have been described to date.     

Bighorn sheep fetal lung cell line for detection of respiratory viruses

Pneumonia is an important disease of bighorn sheep (BHS) that is primarily responsible for the drastic decline in numbers of these animals in North America. Members of the genus Mannheimia and Pasteurella have frequently been isolated from the pneumonic lungs of BHS. Antibodies to several respiratory viruses, including bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), and bovine herpesvirus 1 (BoHV-1), have been detected in herds of BHS. The availability of BHS fetal lung cell lines is likely to enhance the chances of isolation of these viruses. Here we report the development of such a cell line. This line is permissive for BPIV-3, BRSV, BVDV, and BoHV-1 infection, as revealed by an enzyme immunoassay of virus-infected cells with antibodies specific for each of these viruses. This cell line should be valuable for detecting these 4, and possibly other, respiratory viruses in BHS.     

不同饵料对大鲵稚体生长性能、体组成和消化酶活性的影响

为确定大鲵稚体的最佳饵料,本试验研究了不同饵料对大鲵稚体生长性能、体组成和消化酶活性的影响。试验选取平均体重为(1.08±0.01)g的大鲵稚体300尾,随机分成5组,每组3个重复,每个重复20尾。5组大鲵稚体投喂5种不同的饵料,分别为鱼块、黄粉虫、水蚯蚓、卤虫和米虾,养殖时间为60 d。结果显示:不同饵料对大鲵稚体的存活率以及全鱼水分、粗灰分含量均无显著影响(P0.05);米虾组的末均重、增重率和特定生长率均为各组最高,显著高于水蚯蚓组和卤虫组(P0.05);黄粉虫组和水蚯蚓组的肥满度显著高于卤虫组(P0.05)。干物质效率表现为鱼块组和水蚯蚓组显著高于其他各组(P0.05),米虾组显著高于卤虫组和黄粉虫组(P0.05),卤虫组显著高于黄粉虫组(P0.05);各组间大鲵稚体蛋白质效率差异显著(P0.05),表现为水蚯蚓组米虾组鱼块组卤虫组黄粉虫组;鱼块组脂肪效率显著高于其他组(P0.05),卤虫组和米虾组显著高于水蚯蚓组和黄粉虫组(P0.05),水蚯蚓组显著高于黄粉虫组(P0.05)。米虾组全鱼粗蛋白质含量为各组最高,显著高于黄粉虫组和卤虫组(P0.05);而全鱼粗脂肪含量最高的是黄粉虫组,显著高于其他各组(P0.05)。各组大鲵稚体全鱼17种氨基酸含量、氨基酸总量、必需氨基酸总量和鲜味氨基酸总量差异不显著(P0.05),但水蚯蚓组、卤虫组和米虾组的必需氨基酸与非必需氨基酸的比值显著高于鱼块组和黄粉虫组(P0.05)。鱼块组和米虾组大鲵稚体胃肠道蛋白酶活性显著高于黄粉虫组和卤虫组(P0.05);黄粉虫组胃肠道脂肪酶活性显著高于其他各组(P0.05);胃肠道淀粉酶活性仅卤虫组显著低于其他各组(P0.05)。综上所述,米虾是大鲵稚体的最佳饵料,鱼块次之,水蚯蚓和黄粉虫最后选用。     

Replication of bovine respiratory syncytial virus in bovine and ovine peripheral blood lymphocytes and monocytes and monocytic cell lines

The present study compared the replication of bovine respiratory syncytial virus (BRSV) in bovine and ovine peripheral blood mononuclear cells, ovine and bovine monocytic cell lines and ovine alveolar macrophages. Low titres of virus were detected in ovine and bovine lymphocytes and monocytes 24–96 h post-exposure to the virus but there was no apparent replication of the virus in ovine alveolar macrophages during the culture period. The virus replicated to higher but statistically insignificant titres in ovine and bovine peripheral blood monocytes than in lymphocytes, with lymphocytes yielding peak titres significantly earlier. The secondary cell lines obtained from ovine liver and bone marrow also supported the replication of BRSV to high titres. The titres of BRSV in ovine and bovine lymphocytes and monocytes were significantly lower than in secondary cell lines. The addition of human recombinant tumour necrosis factor alpha after exposure to the virus or pre-incubation of ovine or bovine monocytic cells with either human recombinant interleukin 2 or phorbol myristate acetate before exposure to BRSV, did not significantly affect virus titre. Pre-incubation of cells with indomethacin or actinomycin significantly lowered virus titre (p<0.05).     

Characterization of Murine Pituitary-Derived Cell Lines Tpit/F1, Tpit/E and TtT/GF

The pituitary is an important endocrine tissue of the vertebrate that produces and secretes many hormones. Accumulating data suggest that several types of cells compose the pituitary, and there is growing interest in elucidating the origin of these cell types and their roles in pituitary organogenesis. Therein, the histogenous cell line is an extremely valuable experimental tool for investigating the function of derived tissue. In this study, we compared gene expression profiles by microarray analysis and real-time PCR for murine pituitary tumor-derived non-hormone-producing cell lines TtT/GF, Tpit/F1 and Tpit/E. Several genes are characteristically expressed in each cell line: Abcg2, Nestin, Prrx1, Prrx2, CD34, Eng, Cspg4 (Ng2), S100β and nNos in TtT/GF; Cxcl12, Raldh1, Msx1 and Twist1 in Tpit/F1; and Cxadr, Sox9, Cdh1, EpCAM and Krt8 in Tpit/E. Ultimately, we came to the following conclusions: TtT/GF cells show the most differentiated state, and may have some properties of the pituitary vascular endothelial cell and/or pericyte. Tpit/F1 cells show the epithelial and mesenchymal phenotypes with stemness still in a transiting state. Tpit/E cells have a phenotype of epithelial cells and are the most immature cells in the progression of differentiation or in the initial endothelial-mesenchymal transition (EMT). Thus, these three cell lines must be useful model cell lines for investigating pituitary stem/progenitor cells as well as organogenesis.     

人血小板生成素基因的克隆及CHO稳定细胞系的建立

 以人肝cDNA为模板克隆了人血小板生成素（Human Thrombopoietin,hTPO）基因,利用基因重组技术构建了带有新霉素抗性基因（neo）筛选标记的pcDNA3.1(+)-hTPO真核表达载体,将重组质粒瞬时转染293T细胞,用鼠抗人TPO 单抗Western blot 检测 TPO蛋白的瞬时表达;再将重组质粒转染中国仓鼠卵巢细胞(Chinese Hamster Ovary,CHO),应用400 μg/mL的G418筛选克隆,经PCR及Western blot验证,获得了3株hTPO蛋白表达水平不同的CHO细胞系,为获得大量蛋白并进行活性功能试验及临床应用奠定基础。