牛病毒性腹泻病毒的分离与鉴定

[目的]为了解牛病毒性腹泻/黏膜病在新疆的流行现状,对疑似该病病料进行地方毒株的分离,并对其进行鉴定.[方法]将采自新疆不同地区疑似牛病毒性腹泻的病料接种MDBK细胞,并盲传2～4代;对分离株进行毒力测定及中和试验,应用RT-PCR方法扩增分离株的5'UTR基因片段并克隆测序分析.[结果]分离到的8 株病毒可发生细胞病变,在MDBK细胞上的TCID50为103～106.毒株血清中和实验结果,RT-PCR反应结果,克隆后重组质粒经BamH I、Hind III双酶切鉴定、重组质粒PCR鉴定结果均得到预计扩增片段.重组质粒测序结果与GenBank中已发表的牛病毒性腹泻病毒NADL株序列同源性为82.0;～94.8;.[结论]证实所分离到的病毒为BVDV,命名为B-S-1、B-S-2、B-S-3、B-S-4、B-S-5、B-S-6、B-G-1和B-G-2.     

2016——2018年我国部分猪群塞尼卡病毒回顾性监测

塞尼卡病毒(Senecavirus A,SVA)是最近新出现的可引起猪水泡样病变的单股小RNA病毒。为了解SVA的流行状况,采集对2016—2018年从我国辽宁等省份猪群的164份病料和2018年从我国福建等7省份35个屠宰场的458份组织混合样品和95份血清样品,用荧光实时定量RT-PCR方法进行SVA检测和分析。结果显示:在2016年的48份样品中,从3个省份检测出7份阳性样品,阳性率为14.6%。2017年的32份病料中,从5个省份检测出7份阳性样品,占比为21.9%;2018年的84份病料中,从3个省份检测出19份阳性样品,占比为22.6%。屠宰场组织样品中,从6个省份检出55份SVA阳性样品,阳性率为12.0%;血清样品中,从1个省份检出7份阳性样品,阳性率为7.4%。结果表明,我国猪群SVA感染年份至少可追溯到2016年;SVA在我国流行面相对较广,且猪群中存在隐性带毒,因此须重视猪群的SVA监视和监测。     

大鼠RVLG选择性剪接变异体的鉴定和分析

[目的]确认大鼠RVLG(rat vasa-Iike gene)的第7、8两个内含子的部分片段在卵巢中可能存在选择性剪接现象,进一步检测大鼠RVLG的第7、8两个内含子在卵巢和睾丸中的选择性剪接形式.[方法]从雌雄各5只Wistar大鼠的卵巢和睾丸组织中提取总RNA,反转录成cDNA后作为模板,用PCR扩增RVLG起始密码后约620-690 bp的编码区cDNA.分别选卵巢和睾丸各7个独立克隆测序,将测序结果与大鼠RVL6基因组DNA比对,分析是否有选择性剪接.[结果]大鼠卵巢和睾丸组织中各7个RVLG cDNA 编码区5'端620-690 bp的片段测序结果均存在45 bp和15 bp两处选择性剪接.在7个卵巢RVLG cDNA独立克隆中,有5个仅存45 bp而缺少15 bp,两个仅存15 bp而缺少45 bp;7个睾丸RVLG cDNA独立克隆中,4个仅存在45 bp而缺少15 bp,两个既含有15bp又含有45 bp,一个既没有45 bp也没有15 bp.经与大鼠RVLG基因组DNA序列比对后确认45 bp位于其第7内含子5'端的两个GT之间,15 bp位于第8内含子3'端的两个AG之间,剪掉或保留这两段均符合典型的GT-AG内含子剪接原则,属选择性剪接.[结论]大鼠RVLG的第7、8两个内含子在卵巢和睾丸中均存在多种选择性剪接形式,体内的标准剪接方式应该是第7外显子3'端保留45 bp,第9外显子不保留来自第8内含子的15 bp.     

鹅副粘病毒上海株的分子鉴定

对新近分离的鹅副粘病毒SF02株采用RT－PCR方法，扩增部分F基因进行测序，其裂解位点的序列为112R－Q－K－R－L^117，与新城疫病毒强毒株的特征相符，通过对其F基因与我国大陆新城疫强毒F48E9株，疫苗毒Lasota株比较，发现该毒株的F基因已发生了较大的变异，而与1999年在我国台湾省流行的新城疫病毒基因Ⅶ型有很高的同源性。     

进境番茄种子中番茄花叶病毒的检测与鉴定

利用双抗夹心 酶联免疫吸附试验(DAS-ELISA)和反转录 聚合酶链式反应（RT-PCR）方法对来自韩国的番茄种子进行种传病毒检测。结果表明,在该批番茄种子中检测到番茄花叶病毒（ToMV）和烟草花叶病毒（TMV）。PCR产物测序结果表明,ToMV特异引物（ToA/ToB）与TMV特异引物（TA/TB）扩增的片段均为ToMV的外壳蛋白（CP）基因及3′非编码区（3′ UTR）,该片段与其他ToMV分离物的核苷酸序列相似性为98.2%~99.9%。本研究利用重新设计合成TMV和ToMV特异引物对该批种子进行RT PCR检测,仅ToMV特异性引物（ToMf1/ToMr1）扩增到670 bp的预期片段,TMV特异引物（TMf1/TMr1）则未出现特异性扩增,表明重新设计的引物可准确区分ToMV和TMV。以上结果证实该批番茄种子仅携带ToMV。     

Sequence comparison and transmission of <Emphasis Type="Italic">Blackcurrant reversion virus</Emphasis> isolates in black,red and white currants with black currant reversion disease and full blossom disease symptoms

We collected samples from black, red and white currants showing symptoms of blackcurrant reversion disease (BRD) and full blossom disease (FBD), cultivated in the Czech Republic. Blackcurrant reversion virus (BRV) was detected in all symptomatic plants. After amplification, a substantial part of the 3′ non-translated region (3′-NTR) of RNA2 of 15 new isolates of BRV was sequenced and compared with sequences available in the literature and GenBank. We did not find significant sequence diversity among isolates associated with either FBD or BRD. BRV was graft-transmitted from FBD infected red currant to black currant where symptoms of BRD were observed. Further sequence analysis of BRV isolates resulted in a phylogenetic tree with four branches, each consisting of six to nine isolates. No correlation with geographic origin was visible on the tree as isolates from various countries occurred in all four branches. We also found no correlation between the host and the topology of the tree: most of black currant isolates occurred in branches 3 and 4, but also occurred in branches 1 and 2. Only one white currant and one red currant isolate occurred in branches 3 and 4, respectively. The sequence identity of the Czech isolates in this region ranged from 91.9 to 99.8%. The 17 plant species growing within and in the close vicinity of the BRD-infested plantation were tested negative for BRV by RT-PCR as natural hosts of BRV. BRV was successfully transmitted by mechanical inoculation from black currant to Nicotiana occidentalis and N. tabacum cv. Xanthi, the latter being a new host for BRV. The infection was confirmed by PCR and sequencing.     

Identification of divergent variants of Grapevine virus A

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.     

苹果果实中细胞质型苹果酸酶基因（NADP-ME）的克隆与表达分析

【研究目的】从苹果果实中克隆细胞质型NADP-苹果酸酶(NADP-ME)基因,并且从基因转录水平上研究该基因与苹果果实酸度的关系。【方法】利用RT-PCR和RACE技术克隆NADP-ME基因,半定量RT-PCR分析该基因在高酸和低酸个体不同发育时期果实中的转录情况,并进行原核表达分析。【结果】成功克隆了细胞质型NADP-ME基因的cDNA全长序列,命名为Mal-ME(GenBank注册号:DQ280492);该基因表达一个约66kD的蛋白;半定量RT-PCR分析表明:不同发育时期的果实中Mal-ME的转录与苹果酸含量呈负相关,即随着Mal-ME转录水平的增强果实含酸量下降。【结论】成功从苹果果实中克隆了细胞质型Mal-ME基因,Mal-ME在果实发育过程中对苹果酸的积累起负调控作用。