广东省三株蚊媒黄病毒的鉴定和系统进化分析

为了解广东省蚊媒病毒的种类和分子特征,本研究对从蚊子中分离的三株黄病毒提取病毒核酸并进行二代测序,经序列拼接和比对,基因组序列中缺失的gap通过RT-PCR进行扩增填补。用MEGA软件登录GenBank下载黄病毒代表株序列并绘制进化树,同时用ClustalW2软件与新毒株进行氨基酸同源性比对和编码框序列的分析。结果显示利用二代测序技术获得了三株病毒基因组序列的96.35%～98.26%,并用RT-PCR成功补齐所有gap,序列比对显示其中一株与黄斑库蚊病毒核苷酸和氨基酸同源性98.51%,两株病毒与广平病毒的核苷酸同源性分别为97.30%和89.78%。三株病毒都属于黄病毒家族中的未分类黄病毒或昆虫特异性黄病毒,编码区序列中C蛋白的同源性最低,NS5的同源性最高。本研究发现的三株蚊媒黄病毒中,库蚊黄斑病毒在国内为首次鉴定;两株广平病毒核苷酸同源性存在较大差异,可能存在不同基因亚型。     

首次从我国蚊虫中分离到类似广平病毒

为掌握云南省西双版纳州勐海县蚊媒病毒分布状况,用诱蚊灯采集蚊虫标本,用细胞培养法分离病毒,对阳性分离物进行分子生物学鉴定。2012年7月,在勐海县打洛镇采集蚊虫32批共1 468只,其中三带喙库蚊(Culex tritaeniorhynchus)28批1 383只,致倦库蚊(Culex quinquefascitatus)2批66只,按蚊(Anopheles)2批19只。用金黄地鼠肾细胞(Golden hamster kidney cell,简称BHK-21)和白纹伊蚊细胞(Aedes albopictus cell,简称C6/36)进行病毒分离,结果有2批三带喙库蚊标本(BNDL1205和BNDL1227)能引起C6/36细胞产生聚合病变,而不能引起BHK-21细胞病变。用黄病毒属特异性引物对这2株阳性分离物进行RT-PCR,扩增得到800bp左右的条带并进行核苷酸序列测定。对核苷酸序列的系统进化分析表明,这2株病毒与分离自越南的广平病毒(Quang Binh virus,QBV)VN180株亲缘性最近,处在同一进化分支;与VN180株的核苷酸同源性分别为83.4%和82.9%,而与既往国内外分离的18株蚊虫黄病毒的核苷酸差异较大。结果表明BNDL1205和BNDL1227病毒为类似QBV,此为国内首次报道。     

三带喙库蚊中分离的西藏环状病毒全基因序列测定及分析

西藏环状病毒(Tibet orbivirus,TIBOV)是环状病毒属中的新成员,首次分离自中国西藏自治区的圆斑按蚊,本研究首次对从三带喙库蚊标本中分离到的西藏环状病毒(HN11121株)进行全基因组序列测定和分子遗传进化分析。结果显示除第2节段以外,三带喙库蚊分离的HN11121病毒株与从圆斑按蚊分离的西藏环状病毒原始分离株(XZ0906)第1~10节段核苷酸和其编码的氨基酸序列长度相同,核苷酸和氨基酸同源性分别为70.3%(S6)~98.7%(S5)和79.8%(S6)~99.8%(S10);而HN11121病毒株与XZ0906病毒株第2节段核苷酸长度分别为2 850bp和2 838bp,分别编码950和946个氨基酸,核苷酸和氨基酸同源性分别为55.8%和47.1%。病毒VP2蛋白氨基酸位点差异结果显示从三带喙库蚊分离的HN11121病毒与从圆斑按蚊分离的XZ0906相比存在390个氨基酸差异位点。基于HN11121病毒株VP1基因和VP3基因核苷酸序列的系统进化分析结果显示HN11121属于环状病毒属西藏环状病毒,但是从三带喙库蚊分离的HN11121病毒与在圆斑按蚊分离的西藏环状病毒原始分离株(XZ0906)处在不同的进化分支。以上结果提示,从三带喙库蚊分离的西藏环状病毒(HN11121)与从圆斑按蚊分离的西藏环状病毒在病毒基因组和病毒基因组分子遗传进化均存在较大差异,鉴于三带喙库蚊是我国乙脑病毒的主要传播媒介,同时该蚊种也是我国南方广泛存在的蚊虫种类,因此加强三带喙库蚊中西藏环状病毒监测对于了解西藏环状病毒的生物多态性具有重要意义。     

鹅源新城疫病毒ZJ1株全基因组的序列测定

依据GenBank上公布的新城疫病毒(NDV)的基因序列,自行设计了9对引物,运用反转录一聚合酶链反应(RT—PCR)获取覆盖NDV鹅源毒株ZJ1株全基因组的部分重叠片段。病毒RNA的5’及3’端的序列由cT)NA末端快速扩增技术(RACE)获取。整个病毒基因组序列由15192个碱基组成,比GenBank上公布的其它4个NDV毒株Beaudettec、B1、Lasota及Clone-30的全基因组序列长6个核苷酸。这6个核苷酸位于核衣壳蛋白(NP)基因内,相对于NDV毒株Lasota株全基因序列的1647nt～1648nt位。其它10个NDV毒株中的9个毒株也被验证具有此特征,且它们分属于3个不同的基因型。     

鸡传染性支气管炎病毒变异株（793／B）核蛋白基因的克隆与序列分析

根据GenBank已经发表的传染性支气管炎病毒(IBV)N全基因组序列设计引物,对IBV793/B分离毒株N基因进行克隆与序列分析。结果表明,IBV793/B的N基因由1229bp组成,与GenBank已发表的11株IBV的N基因相比较,IBV793/B的N基因共有88处点突变,在第991位发生了一个核苷酸的缺失。N基因的核苷酸同源性为86.9%～91.4%,氨基酸同源性为75.8%～77.5%。表明IBV93/B的N基因存在着较大的变异性。     

鸡传染性支气管炎病毒变异株(793/B)核蛋白基因的克隆与序列分析

根据GenBank已经发表的传染性支气管炎病毒(IBV)N全基因组序列设计引物,对IBV 793/B分离毒株N基因进行克隆与序列分析.结果表明,IBV 793/B的N基因由1229bp组成,与GenBank已发表的11株IBV的N基因相比较,IBV 793/B的N基因共有88处点突变,在第991位发生了一个核苷酸的缺失.N基因的核苷酸同源性为86.9%～91.4%,氨基酸同源性为75.8%～77.5%.表明IBV 93/B的N基因存在着较大的变异性.     

中国6株狂犬病病毒街毒株全基因组测序与分析

实验研究中对分离于中国的6株狂犬病病毒街毒株进行了全基因组测序,对基因组的5个结构基因(N、P、M、G和L)的核苷酸和推断的氨基酸序列以及非编码区序列进行了分析与比较,并与来自GenBank的40株毒株从全基因组水平进行了分子进化分析。所测6株中国狂犬病病毒街毒株的全基因组核苷酸序列长度介于11 907 nt(CQ92)和11 924 nt(SH06和gg4)之间,基因组结构相同,用全基因组和不同的结构基因构建的进化树拓扑结构相似,基因组3′和5′末端高度保守而且末端11个核苷酸互补配对,5个结构基因的保守性依次是NLMGP,核苷酸同源性的最小值依次分别是81.9%、81.7%、80.7%、78.3%和76.7%。     

我国新分离ECHO30病毒VP1序列分析

测定了引起2003年苏北地区无菌性脑膜炎暴发流行的病因病毒FDJS03分离株的VP1基因序列,并与国外同型流行毒株做比较,以了解本流行株的分子生物学特点及遗传变异规律。随机选取FDJS03分离毒株中的4株,用肠道病毒、VP1序列的特异性引物008／011进行RT-PCR,扩增产物经凝胶纯化后测序。将序列输入GenBank,用BLAgr程序进行核苷酸和氨基酸序列比对;选取32株不同地区不同年代的ECHO30分离毒株,在PHYLIP3．573C和TREE-PUZZLE5．0中构建进化树,比较它们完整VP1序列(876nt)的进化关系。核苷酸和氨基酸同源性比较结果证明：4株分离病毒均为ECHO30。进化树分析显示：本次FDJS03分离株与欧美20世纪70、80年代流行株亲缘关系最近,但自成一簇,与国外毒株仍然有地区差别。ECHO30的VP1基因进化有时间效应,但存在地区差异。本次流行的病原可能是单一基因型的ECHO30病毒。     

猪戊型肝炎病毒swCH-GS189株ORF2基因的克隆及序列分析

为进行猪戊型肝炎病毒(HEV)ORF2基因特征研究,参照GenBank中已发表的戊型肝炎病毒(HEV)核酸序列,设计了一对扩增HEV ORF2基因的引物,利用RT-PCR等方法克隆出了一株猪戊型肝炎病毒甘肃分离株GS189的ORF2基因cDNA片段.序列测定结果表明,swCH-GS189株的ORF2基因长2 025 bp,编码674个氨基酸,与GenBank中公布的其它毒株间的核苷酸序列同源性为79.1%～91.8%,推导的氨基酸序列同源性为89.5%～98.8%.系统发育进化树结果表明,该分离株为基因IV型.     

节节麦-黑麦双二倍体α醇溶蛋白基因的克隆与序列分析

以人工合成节节麦-黑麦双二倍体基因组DNA为模板,用小麦种子醇溶蛋白保守引物进行PCR扩增,经克隆测序获得了843~897 bp共15个新的DNA序列(GenBank登录号为: JQ029719,JQ046392~JQ046405),分别编码280~298个氨基酸。序列比对结果表明,它们具有α-醇溶蛋白基因的典型结构特点,是α-醇溶蛋白基因家系成员,其中有两个序列为同义突变。利用14个新氨基酸序列与乳糜泻(celiac disease)病人毒性抗原相关序列的比对,发现有8个序列的Glia-α-2和Glia-α-9型抗原序列产生缺失和替换。与来自粗山羊草属和黑麦属的α-醇溶蛋白基因的编码氨基酸建立系统树,结果表明,14个DNA序列编码的氨基酸序列与粗山羊草属的相关序列聚在一起。     

节节麦-黑麦双二倍体α醇溶蛋白基因的克隆与序列分析

以人工合成节节麦-黑麦双二倍体基因组DNA为模板,用小麦种子醇溶蛋白保守引物进行PCR扩增,经克隆测序获得了843 ～ 897 bp共15个新的DNA序列(GenBank登录号为:JQ029719,JQ046392～JQ046405),分别编码280 ～298个氨基酸.序列比对结果表明,它们具有α-醇溶蛋白基因的典型结构特点,是α-醇溶蛋白基因家系成员,其中有两个序列为同义突变.利用14个新氨基酸序列与乳糜泻( celiac disease)病人毒性抗原相关序列的比对,发现有8个序列的Glia-α-2和Glia-α-9型抗原序列产生缺失和替换.与来自粗山羊草属和黑麦属的α-醇溶蛋白基因的编码氨基酸建立系统树,结果表明,14个DNA序列编码的氨基酸序列与粗山羊草属的相关序列聚在一起.     

Neoscardovia arbecensis gen. nov., sp. nov., isolated from porcine slurries

Three Gram-positive, anaerobic, pleomorphic strains (PG10(T), PG18 and PG22), were selected among five strains isolated from pig slurries while searching for host specific bifidobacteria to track the source of fecal pollution in water. Analysis of the 16S rRNA gene sequence showed a maximum identity of 94% to various species of the family Bifidobacteriaceae. However, phylogenetic analyses of 16S rRNA and HSP60 gene sequences revealed a closer relationship of these strains to members of the recently described Aeriscardovia, Parascardovia and Scardovia genera, than to other Bifidobacterium species. The names Neoscardovia gen. nov. and Neoscardovia arbecensis sp. nov. are proposed for a new genus and for the first species belonging to this genus, respectively, and for which PG10(T) (CECT 8111(T), DSM 25737(T)) was designated as the type strain. This new species should be placed in the Bifidobacteriaceae family within the class Actinobacteria, with Aeriscardovia aeriphila being the closest relative. The prevailing cellular fatty acids were C(16:0) and C(18:1)ω9c, and the major polar lipids consisted of a variety of glycolipids, diphosphatidyl glycerol, two unidentified phospholipids, and phosphatidyl glycerol. The peptidoglycan structure was A1γmeso-Dpm-direct. The GenBank accession numbers for the 16S rRNA gene and HSP60 gene sequences of strains PG10(T), PG18 and PG22 are JF519691, JF519693, JQ767128 and JQ767130, JQ767131, JQ767133, respectively.     

Isolation and characterization of culturable halophilic microorganisms of salt ponds in Lianyungang,China

The Lianyungang salt ponds are an extreme saline environment, and their microbial communities have not been characterized. A typical extreme halophilic archaeon strain designated as HBCC-2 (GenBank accession number: EF687739) was isolated from the salt ponds of Lianyungang in Jiangsu Province, P. R. China, using conventional microbial culture methods. The other halotolerant bacterial strain designated as HBCC-3 (GenBank accession number: EU377478) was isolated from the same sampling sites. The morphological and physiological characteristics of HBCC-2 and HBCC-3 were observed and examined. G+C content of HBCC-2 and HBCC-3 were determined using high-performance liquid chromatography. The cellular phospholipid fatty acids were analyzed using gas chromatography-mass spectrometry. The 16S rRNA gene sequences of the strains HBCC-2 and HBCC-3 were amplified by PCR using archaeal primers and bacterial primers, respectively. Homology of 16S rRNA gene sequences of the strains HBCC-2 and HBCC-3 were compared with the other similar sequences obtained from GenBank using the BLAST program. Phylogenetic analysis was performed using the software MEGA 4.0 after multiple alignments of sequence data using software CLUSTALW 1.8. The evolutional distances (by Kimura’s model) were calculated and the clusters were performed with the neighbor-joining method. The results showed that the 16S rRNA gene sequences of the strains HBCC-2 and HBCC-3 are related to the genera Halorubrum and Alkalibacillus, respectively. Two phylogenetic trees were constructed by phylogenetic analysis based on the 16S rRNA gene sequence. Based on the above results, the strains HBCC-2 and HBCC-3 were finally identified. The discovery of the two species provides an opportunity to further study these halophilic microorganisms in the Lianyungang salt ponds.     

First report of Fusarium euwallaceae on avocado trees in Palestine

Abstract

A new die back symptoms in many avocado orchards had been reported in Palestine. The disease is associated with the Ambrosia beetle Euwallacea fornicatus. Stem samples from infected avocado trees with obvious symptoms were collected from different regions in Palestine. Stem cuttings and dissected adult and galleries of the insect were placed on potato dextrose agar media and incubated for 5–7?days at 25?°C. PCR amplification using EF1/2 specific primers was performed to identify the isolated fungus. The resulting PCR products were sequenced. BLASTn search showed 99% similarity with Fusarium euwallaceae (Accession Nos. JX891785.1, JQ723763.1, JQ723762.1 and JQ723761.1). The isolated fungus was identified as F. euwallaceae (Genbank accession no. MK054177).     

Characteristics and metabolic pathway of Alcaligenes sp. TB for simultaneous heterotrophic nitrification-aerobic denitrification

Applied Microbiology and Biotechnology - A novel heterotrophic nitrification-aerobic denitrification bacterium, Alcaligenes sp. TB (GenBank accession no. JQ044686), was isolated from a rotating...     

A Pair of Novel Primers for Universal Detection of the NS1 Gene from Various Bluetongue Virus Serotypes

Twenty five serotypes of Bluetongue virus （BTV） have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue （BT）. We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 （VP7） and the non-structural protein （NS1） gene from different serotypes of BTV by DNAstar showed that the 5＇end of the NS1 gene is the most conserved region. The primer pairs （P1 and P2） were designed based on the highly conserved region of NS 1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus （EHDV） serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes     

A pair of novel primers for universal detection of the NS1 gene from various bluetongue virus serotypes

Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 (VP7) and the non-structural protein (NS1) gene from different serotypes of BTV by DNAstar showed that the 5' end of the NS1 gene is the most conserved region. The primer pairs (P1 and P2) were designed based on the highly conserved region of NS1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus (EHDV) serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes.