患病中国大鲵中分离到一株虹彩病毒及其特性的研究

从陕西某大鲵养殖场患病的大鲵体内分离到一株病毒。患病大鲵以体表溃疡,特别是肢体远端溃烂为主要临床特征。该病毒于10℃~30℃能在BF-2(Caudal trunk cells of blue-gillfry)、CO(Gorad cells of grass carp)、CHSE(Embryo cells of Chinook salmon)、FHM(cells of fathed minnow)等细胞中较好地增殖,最适生长温度为25℃~30℃。病毒对氯仿、热、pH3、pH10敏感,DNA抑制剂5-氟-2′-脱氧尿苷(5-fluro-2-′deoxyuridine,FUDR)能抑制病毒在细胞中的增殖,提示该病毒是有囊膜的DNA病毒。经电镜观察,在感染了病毒的细胞切片中可见到大量直径约130~150 nm有囊膜的六角形病毒颗粒成晶格排列在细胞质里,病毒呈典型的虹彩病毒形态。抽提病毒核酸后进行PCR扩增,用已知蛙病毒主要衣壳蛋白(MCP)基因的保守序列设计的引物能扩增出431bp的片段。扩增的片段测序后,和已知的几种蛙病毒属成员的主要衣壳蛋白基因中的相应片段进行比对,相似性在96%以上。血清学试验结果显示该病毒和IPNV(Infectious pancreatic necrosis virus,IPNV)、GCRV(Grass carp reovirus,GCRV)、SVCV(Spring viraemia of carp virus,SVCV)I、HNV(Infectious hematopoietic necrosis virus,IHNV)在血清学上没有相关性。以上结果提示该病毒可能是虹彩病毒科蛙病毒属的成员,暂时命名为大鲵虹彩病毒(Andrias davidianus iridovirus,ADIV)。该病毒与大鲵发病的关系有待进一步研究。     

鲤春血症病毒中国分离株糖蛋白基因和氨基酸序列的初步解析

2002～2004年间从国内养殖鲤科鱼类和观赏鱼类中分离出8株鲤春血症病毒(SVCV).根据SVCV参考株全序列,设计引物,用逆转录聚合酶链式反应扩增出8株SVCV糖蛋白编码基因片段,并对扩增产物进行了克隆和序列测定.用生物信息学方法对测得的序列进行分析,结果8个国内分离株的糖蛋白基因序列与参考株的基因序列相似性均在92%以上,8个国内分离株之间基因序列相似性均在97.7%以上;8个国内分离株之间糖蛋白推导出的氨基酸序列相似性均在94.5%以上,与参考株氨基酸序列相似性在92.9%-94.9%之间.系统发育树分析结果表明,SVCV国内分离株与USA株、980451株、980528株和970469株的进化方向一致,与其它SVCV毒株在进化方向上不同.8个毒株有19个共同的酶切位点,推导出的氨基酸序列中有10个亲水区、10个可能的抗原位点和10个跨膜蛋白区域,其峰值基本一致.对SVCV国内分离株的糖蛋白6个功能位点(天冬酰胺糖基化位点、精氨酸-甘氨酸-天冬氨酸序列、酪蛋白激酶Ⅱ磷酸化位点、蛋白激酶C磷酸化位点、酪氨酸磷酸化位点和肉豆蔻酰基化位点)进行了初步分析.     

鲤春血症病毒中国分离株糖蛋白基因和氨基酸序列的初步解析

2002～2004年间从国内养殖鲤科鱼类和观赏鱼类中分离出8株鲤春血症病毒(SVCV)。根据SVCV参考株 全序列,设计引物,用逆转录聚合酶链式反应扩增出8株SVCV糖蛋白编码基因片段,并对扩增产物进行了克隆和 序列测定。用生物信息学方法对测得的序列进行分析,结果8个国内分离株的糖蛋白基因序列与参考株的基因序 列相似性均在92％以上,8个国内分离株之间基因序列相似性均在97．7％以上;8个国内分离株之间糖蛋白推导 出的氨基酸序列相似性均在94．5％以上,与参考株氨基酸序列相似性在92．9％-94．9％之间。系统发育树分析结 果表明,SVCV国内分离株与USA株、980451株、980528株和970469株的进化方向一致,与其它SVCV毒株在进 化方向上不同。8个毒株有19个共同的酶切位点,推导出的氨基酸序列中有10个亲水区、10个可能的抗原位点 和10个跨膜蛋白区域,其峰值基本一致。对SVCV国内分离株的糖蛋白6个功能位点(天冬酰胺糖基化位点、精 氨酸-甘氨酸-天冬氨酸序列、酪蛋白激酶Ⅱ磷酸化位点、蛋白激酶C磷酸化位点、酪氨酸磷酸化位点和肉豆蔻酰 基化位点)进行了初步分析。     

从云豹分离的一株猫泛白细胞减少症病毒的特性

从病死的云豹体内分离到1株猫泛白细胞减少症病毒,进行了鉴定,研究了它的血凝特性和对猫与水貂的致病力,分析了它与猫泛白细胞减少症病毒参考毒株(FNF-8)抗原的相关性。 用中国兽药监察所提供的猫肾传代细胞(FK)进行病毒的分离鉴定。同步接种和异步接种测得病毒的TCID50均为4.8/ml。病毒经乙醚、酸(pH3.0)、热(56℃1小时)     

一株草鱼呼肠孤病毒弱毒株的分离、鉴定及免疫原性初步分析

从江西南昌患出血病草鱼体内分离出的草鱼病毒(暂命名为JX09-01)能使草鱼肾脏细胞(CIK)、草鱼肝细胞(L8824)、草鱼吻端成纤维细胞(PSF)产生明显的细胞病变效应(CPE)。感染CIK 细胞固定后经电镜观察,发现细胞质内有大量病毒聚集, 形态和排列方式与已报道的草鱼呼肠孤病毒(Grass carp reovirus, GCRV)相似。针对GCRV 873 株S6 基因设计的简并引物可以从病料组织和感染细胞中扩增出目的条带, 而针对GCRVHZ08 株S6 基因设计特异性引物未能扩增出目的条带。对JX09-01 株的S6 全基因进行序列分析表明, 其核苷酸序列同GCRV 873 株和HZ08 株的同源性分别是99.3%和30.4%, 推导出的氨基酸序列同源性分别是98.6%和30%, 说明草鱼病毒JX09-01 株为草鱼呼肠孤病毒。用JX09-01 株接种当年8-10 cm左右的草鱼, 没有明显的临床症状, 不能致草鱼死亡。用传代至15 代的CIK 细胞病毒液进行免疫保护试验, 结果显示其对强毒株的免疫保护率达到86.7%。实验结果初步显示, 新分离到的JX09-01 为草鱼呼肠孤病毒弱毒株, 可作为弱毒疫苗的候选毒株。       

鲤春病毒血症病毒G蛋白的研究进展

鲤春病毒血症病毒(Spring viremia of carp virus,SVCV)是引起鱼类传染性病毒病鲤春病毒血症(Spring viremia of carp,SVC)的病原,对鲤科鱼类养殖业造成巨大的经济损失。囊膜糖蛋白G可介导病毒内吞,还是最主要的抗原决定簇蛋白,已成为国内外研究的热点。综述G蛋白在表达技术、病毒检测、疫苗研制等方面的研究状况,旨在为G蛋白的深入研究及鲤春病毒血症的防治提供参考。     

鲤春病毒糖蛋白(G)基因的分离及同源性比较

通过RT-PCR和巢氏PCR方法从疑似鲤春病毒(SVCV)侵染的镜鲤肝组织中获得了鲤春病毒糖蛋白(G)基因。通过序列测定与分析,所获得的鲤春病毒糖蛋白(G)基因由606个核苷酸组成,编码一个由202个氨基酸组成的糖蛋白。通过NCBIblast与9个来自不同国家或地区的鲤春病毒糖蛋白(G)基因序列比对分析,发现获得的鲤鱼春季病毒糖蛋白G基因DNA序列与美国分离的AY527273株同源性最高为99.8%,与中国分离的AY842485株同源性次高为98.7%,与英国分离的两个序列SVI538065和SVI538066株的同源性为98.2%,而与其它国家的分离株如SVU18101、SVI318079、SVI538061、SVI538062、SVI538063的同源性最差,仅为89.4%~90.0%。氨基酸同源性分析结果与DNA同源性分析结果一致,所获得的鲤春病毒糖蛋白G基因氨基酸推导序列与其它9个分离株的氨基酸同源性在89.6%~99.5%之间。对SVCV不同分离株遗传变异和进化关系的分析为下一步开展SVCV快速诊断方法的研究和疫苗的研制奠定了基础。     

两株水生呼肠孤病毒部分特性的比较

水生呼肠孤病毒为感染水生动物的一类病原体,隶属于呼肠孤病毒科新建水生呼肠孤病毒属。草鱼呼肠孤病毒(Grass carp reovirus,GCRV)是引起中国南方淡水养殖草鱼暴发性出血病病原,鲅鱼呼肠孤病毒(Threadfin reovirus,TFV)是引起海水养殖鲅鱼病毒病病原。本研究将GCRV与新加坡TFV分离株进行了部分特性比较研究。结果表明,GCRV与TFV均能感染CIK细胞,但对其它鱼类细胞系的敏感性有所差异。此外,凝胶电泳与逆转录聚合酶链式扩增显示,GCRV与TFV核酸属不同的基因型。在多肽特性上,证实了GCRV的5条主要结构多肽具有与。FTV及水生呼肠孤病毒相似的特性。Westem blot检测显示,草鱼呼肠孤病毒与TFV结构蛋白拥有部分相同的抗原决定簇。     

两株水生呼肠孤病毒部分特性的比较

水生呼肠孤病毒为感染水生动物的一类病原体,隶属于呼肠孤病毒科新建水生呼肠孤病毒属.草鱼呼肠孤病毒(Grass carp reovirus,GCRV)是引起中国南方淡水养殖草鱼暴发性出血病病原,鮁鱼呼肠孤病毒(Threadfin reovirus,TFV)是引起海水养殖鮁鱼病毒病病原.本研究将GCRV与新加坡TFV分离株进行了部分特性比较研究.结果表明,GCRV与TFV均能感染CIK细胞,但对其它鱼类细胞系的敏感性有所差异.此外,凝胶电泳与逆转录聚合酶链式扩增显示,GCRV与TFV核酸属不同的基因型.在多肽特性上,证实了GCRV的5条主要结构多肽具有与FTV及水生呼肠孤病毒相似的特性.Western blot 检测显示,草鱼呼肠孤病毒与TFV结构蛋白拥有部分相同的抗原决定簇.     

猪Ⅱ型圆环病毒浙江株的分离及全基因组结构分析

用PCR方法对从浙江省猪场收集的、以淋巴结肿大和生长迟缓为特征的患猪腹股沟淋巴结进行PCV2检测.将检测为阳性的病料研磨、离心、过滤除菌后,接种PCV-free PK-15细胞进行病毒分离,分离获得3株病毒,命名为HZ0201、HZ0202、NB0301.PK-15细胞增殖所得病毒离心纯化后,在电镜下可观察到直径约为17～20nm,呈二十面体对称的病毒粒子.以组织和病毒的细胞DNA为模板进行PCV2的全基因扩增,测序结果显示,3株病毒分离物全基因序列均由1767bp组成,直接来自病料与细胞分离毒株的核苷酸同源性为100%.基因组内含有11个ORF.通过比较发现,分离毒株与PCV2参考株的同源性介于94.2%～99.7%之间;与PCV1参考株的同源性为77.2%～77.9%.     

Molecular Analysis of Spring Viraemia of Carp Virus in China: A Fatal Aquatic Viral Disease that Might Spread in East Asian

Spring viraemia of carp (SVC) is a fatal viral disease for cyprinid fish, which is caused by spring viraemia of carp virus (SVCV). To date, no SVC outbreak has been reported in China. Between 1998 and 2002, outbreaks of SVC were reported in ornamental and wild fish in Europe and America, imported from multiple sources including China. Based on phylogenetic analysis, the viral strain isolated from America was shown to be originated from Asia. These outbreaks not only resulted in huge economic losses, but also raise an interesting question as to whether SVCV really exists in China and if so, is it responsible for SVC outbreaks? From 2002 to 2006, we screened 6700 samples from ornamental fish farms using the cell culture method of the Office International des Epizooties (OIE), and further verified the presence of SVCV by ELISA and real-time quantitative RT-PCR. Two infected samples were found and the complete genome of SVCV was sequenced from one of the isolates, termed SVCV-C1. Several unique hallmarks of SVCV-C1 were identified, including six amino acid (KSLANA) insertion in the viral RNA-dependent RNA polymerase (L) protein and ten nucleotide insertion in the region between glycoprotein (G) and L genes in European SVCV strains. Phylogenetic tree analysis of the full-length G protein of selected SVCV isolates from the United Kingdom and United States revealed that G proteins could be classified into Ia and Id sub genogroups. The Ia sub genogroup can be further divided into newly defined sub genogroups Ia-A and Ia-B. The isolates derived from the United States and China including the SVCV-C1 belongs to in the Ia-A sub genogroup. The SVCV-C1 G protein shares more than 99% homology with the G proteins of the SVCV strains from England and the United States, making it difficult to compare their pathogenicity. Comparison of the predicted three-dimensional structure based on the published G protein sequences from five SVCV strains revealed that the main differences were in the loops of the pleckstrin homology domains. Since SVCV is highly pathogenic, we speculate that SVC may therefore pose a serious threat to farmed cyprinid fish in China.     

Phylogenetic analysis of spring virema of carp virus reveals distinct subgroups with common origins for recent isolates in North America and the UK

Genetic relationships between 35 spring viremia of carp virus (SVCV) genogroup Ia isolates were determined based on the nucleotide sequences of the phosphoprotein (P) gene and glycoprotein (G) genes. Phylogenetic analysis based on P gene sequences revealed 2 distinct subgroups within SVCV genogroup Ia, designated SVCV Iai and Iaii, and suggests at least 2 independent introductions of the virus into the USA in 2002. Combined P- and G-sequence data support the emergence of SVCV in Illinois, USA, and in Lake Ontario, Canada, from the initial outbreak in Wisconsin, USA, and demonstrate a close genetic link to viruses isolated during routine import checks on fish brought into the UK from Asia. The data also showed a genetic link between SVCV isolations made in Missouri and Washington, USA, in 2004 and the earlier isolation made in North Carolina, USA, in 2002. However, based on the close relationship to a 2004 UK isolate, the data suggest than the Washington isolate represents a third introduction into the US from a common source, rather than a reemergence from the 2002 isolate. There was strong phylogenetic support for an Asian origin for 9 of 16 UK viruses isolated either from imported fish, or shown to have been in direct contact with fish imported from Asia. In one case, there was 100% nucleotide identity in the G-gene with a virus isolated in China.     

Activation of ergot alkaloid biosynthesis in prototrophic isolates by Claviceps purpurea protoplast fusion.

Intraspecific protoplast fusions were carried out with active ergocornine-ergokryptine and inactive ergocristine Claviceps purpurea strains and vice versa. The isolated prototrophic strains from both types of crosings produced all three alkaloid types, showing that biosynthesis of distinct alkaloid was activated in an inactive partner strain. The prototrophic isolates were stable on minimal medium but they segregated by subculturing on complete medium. In comparison with the original partner strains, differences in morphological and cytological characteristics were also established.     

Morphological and Molecular Differences in Two Strains of <Emphasis Type="Italic">Ustilago esculenta</Emphasis>

Ustilago esculenta is a fungal endophyte of Zizania latifolia that plays an important agricultural role in this vegetable crop. The purpose of this study was to characterize sporidial (T) and mycelial (M-T) strains of U. esculenta isolated from sporulating and non-sporulating galls on plants growing in Zhejiang province, China. Morphological comparisons of the T strain and M-T strain were made by optical and scanning electron microscope observation. Genetic differences were examined by sequencing the ITS region of the fungus and examining differential protein expression by two-dimensional gel electrophoresis and MALDI-TOF-MS/MS. The sporidial (T) and mycelial (M-T) strains differed in morphological characteristics of their in vitro single colony formations and in cell shape. Alignment of ITS sequences of the T strain and M-T strain revealed a single mutation between the T strain and M-T strain, but the sequences were the same within strains. A total of 146 proteins were only expressed in the M-T strain, and 242 proteins were only expressed in the T strain isolated from infected plants. A total of 222 proteins were up-regulated or down-regulated in the T strain when compared with the M-T strain. Of these, 18 proteins were identified and eight were associated with processes involving energy metabolism and the cytoskeleton. Two morphology-related proteins, MAP kinase kinase and actin, were differentially expressed. The differences noted in the T strain and M-T strain may lead to a better understanding of the life cycle and morphogenesis in U. esculenta.     

Genotypic and Phenotypic Characterization of Chikungunya Virus of Different Genotypes from Malaysia

Background

Mosquito-borne Chikungunya virus (CHIKV) has recently re-emerged globally. The epidemic East/Central/South African (ECSA) strains have spread for the first time to Asia, which previously only had endemic Asian strains. In Malaysia, the ECSA strain caused an extensive nationwide outbreak in 2008, while the Asian strains only caused limited outbreaks prior to this. To gain insight into these observed epidemiological differences, we compared genotypic and phenotypic characteristics of CHIKV of Asian and ECSA genotypes isolated in Malaysia.

Methods and Findings

CHIKV of Asian and ECSA genotypes were isolated from patients during outbreaks in Bagan Panchor in 2006, and Johor in 2008. Sequencing of the CHIKV strains revealed 96.8% amino acid similarity, including an unusual 7 residue deletion in the nsP3 protein of the Asian strain. CHIKV replication in cells and Aedes mosquitoes was measured by virus titration. There were no differences in mammalian cell lines. The ECSA strain reached significantly higher titres in Ae. albopictus cells (C6/36). Both CHIKV strains infected Ae. albopictus mosquitoes at a higher rate than Ae. aegypti, but when compared to each other, the ECSA strain had much higher midgut infection and replication, and salivary gland dissemination, while the Asian strain infected Ae. aegypti at higher rates.

Conclusions

The greater ability of the ECSA strain to replicate in Ae. albopictus may explain why it spread far more quickly and extensively in humans in Malaysia than the Asian strain ever did, particularly in rural areas where Ae. albopictus predominates. Intergenotypic genetic differences were found at E1, E2, and nsP3 sites previously reported to be determinants of host adaptability in alphaviruses. Transmission of CHIKV in humans is influenced by virus strain and vector species, which has implications for regions with more than one circulating CHIKV genotype and Aedes species.     

Sequence comparison of the capsid region of hepatitis E viruses isolated from Myanmar and China.

Hepatitis E viruses (HEVs) were isolated during epidemics, one from Myanmar (formerly called Burma) and one from China and were partially sequenced. Another HEV Myanmar strain from sporadic hepatitis was previously sequenced by us. A cDNA sequence comparison was performed among them in the 3'-terminal region, approximately 750-base long. This region contained at least two immunological epitopes and was considered to correspond to the structural protein. The nucleotide sequence identity was 97.2% between the two Myanmar strains and 93.3 and 92.5% between the two Myanmar and the China strain. The deduced amino acid sequence identity ranged from 98.4 to 100.0% among the three strains. Thus this segment was well conserved on the amino acid level among the different strains isolated from these two Asian countries, although the China strain diverged more from the Myanmar strains on the nucleotide sequence level. This data may provide important information for the development of a vaccine and for identification of the virological link between different geographical locations.     

Twenty Years of DENV-2 Activity in Brazil: Molecular Characterization and Phylogeny of Strains Isolated from 1990 to 2010

In Brazil, dengue has been a major public health problem since its introduction in the 1980s. Phylogenetic studies constitute a valuable tool to monitor the introduction and spread of viruses as well as to predict the potential epidemiological consequences of such events. Aiming to perform the molecular characterization and phylogenetic analysis of DENV-2 during twenty years of viral activity in the country, viral strains isolated from patients presenting different disease manifestations (n = 34), representing six states of the country, from 1990 to 2010, were sequenced. Partial genome sequencing (genes C/prM/M/E) was performed in 25 DENV-2 strains and full-length genome sequencing (coding region) was performed in 9 strains. The percentage of similarity among the DENV-2 strains in this study and reference strains available in Genbank identified two groups epidemiologically distinct: one represented by strains isolated from 1990 to 2003 and one from strains isolated from 2007 to 2010. No consistent differences were observed on the E gene from strains isolated from cases with different clinical manifestations analyzed, suggesting that if the disease severity has a genetic origin, it is not only due to the differences observed on the E gene. The results obtained by the DENV-2 full-length genome sequencing did not point out consistent differences related to a more severe disease either. The analysis based on the partial and/or complete genome sequencing has characterized the Brazilian DENV-2 strains as belonging to the Southeast Asian genotype, however a distinction of two Lineages within this genotype has been identified. It was established that strains circulating prior DENV-2 emergence (1990–2003) belong to Southeast Asian genotype, Lineage I and strains isolated after DENV-2 emergence in 2007 belong to Southeast Asian genotype, Lineage II. Furthermore, all DENV-2 strains analyzed presented an asparagine (N) in E₃₉₀, previously identified as a probable genetic marker of virulence observed in DHF strains from Asian origin. The percentage of identity of the latter with the Dominican Republic strain isolated in 2001 combined to the percentage of divergence with the strains first introduced in the country in the 1990s suggests that those viruses did not evolve locally but were due to a new viral Lineage introduction in the country from the Caribbean.     

新疆泥火山产酶嗜盐放线菌的筛选及多样性

[目的]了解新疆乌苏泥火山嗜盐放线菌及其产酶功能多样性.[方法]分别采用含有5%与10%NaCl的5种分离培养基,稀释平板涂布法对泥火山土壤样品进行分离;利用五种筛选培养基定性检测酶活性;在形态特征、耐盐性实验及16S rDNA基因测序的基础上进行系统发育学分析.[结果]获得嗜盐放线菌43株,极端嗜盐放线菌3株.4株嗜盐放线菌产脂肪酶,30株产半乳糖苷酶,27株产淀粉酶,6株产酯酶,4株产纤维素酶,1株同时产4种酶.系统发育学分析结果表明其中24株为拟诺卡氏菌属(Nocardiopsis),1株为链霉菌属(Streptomyces).产两种酶的菌株10006与Nocardiopsis exhalans(AY03600)相似性为96.64%(小于97%),可能是潜在的新种.[结论]本研究表明新疆乌苏泥火山中存在大量的产半乳糖苷酶及淀粉酶的嗜盐放线菌,所分离到的拟诺卡氏菌属产酶多样性比较高,并且潜藏着新的微生物资源.     

空肠弯曲菌生物学特性上的一些歧异

对195例不同年龄腹泻患者的粪便、690只健康和腹泻畜禽的直肠和泄殖腔拭子及108份腹泻死亡畜禽的脏器材料进行了空肠弯曲菌培养,共分离鉴定458株弯曲菌(其中空肠弯曲菌445株、结肠弯曲菌13株),就其一些生物学特性进行了观察,采用Lior生物学分型法进行了354株弯曲菌(空肠弯曲菌352株、结肠弯曲菌2株)的分型。虽然表明绝大多数生物学性状符合已有文献描述,但也发现在形态、培养和生理生化特性及抗菌药抗性上存有一些歧异,其中最为主要的是483%(215/445)的空肠弯曲菌和231%(3/13)的结肠弯曲菌有萘啶酮酸抗性,11%(5/445)的空肠弯曲菌和76%(1/13)的结肠弯曲菌有噻孢霉素抗性,抗菌药抗性与菌株来源有关(P<0005)。352株空肠弯曲菌生物学分型结果表明在这些动物体内生物型Ⅰ(409%)和Ⅱ(582%)占优势,同一动物体内可有该菌的2个生物型分布。