Analysis of Occurrence and Mixed Infection of Sugarcane Bacilliform Virus Disease in Hainan Sugarcane-growing Area

[Objectives]The paper was to explore the occurrence and mixed infection of sugarcane bacilliform virus disease in Hainan sugarcane-growing area.[Methods]A total of 348 sugarcane leaf samples were collected from 7 sugarcane-growing areas in Hainan Province.Molecular detection of sugarcane bacilliform virus(SCBV)was carried out by PCR using specific primers.[Results]SCBV was detected in 244 out of 348 sugarcane samples,with an average detection rate of 70.11%.The highest detection rate was 76.66%in the Danzhou sugarcane-growing area,while the lowest was 57.14%in the Baisha sugarcane-growing area.The SCBV-positive samples were subjected to testing for SCYLV,SCSMV,SrMV,and SCMV,respectively.The results indicated that 106 out of 244 positive samples exhibited a single infection with SCBV,while 138 samples exhibited mixed infections with SCBV and other sugarcane viruses.The proportion of mixed infections among the SCBV-positive samples was as high as 56.56%.Among the various types of mixed infections,two-virus and three-virus mixed infections were the most prevalent.[Conclusions]SCBV has emerged as a significant threat to the secure production of sugarcane in the Hainan sugarcane-growing region.It presents an explosive infection in the Hainan sugarcane-growing region and frequently combines with other sugarcane viruses to infect sugarcane.The findings of this study will provide a theoretical foundation for the prevention and control of sugarcane bacilliform virus disease.

Development of a Liquid Chip Technique to Simultaneously Detect Taura Syndrome Virus( TSV) and Yellow Head Disease Virus( YHDV)

The aim is to develop a liquid chip technique to detect Taura syndrome virus( TSV) and yellow head disease virus( YHDV) on Penaeus orientalis simultaneously. The CP2 gene of TSV and N gene of YHDV in Gen Bank was analysed by using the software DNAStar 7. 0 to design the TSV-and YHDV-specific primers. The primers were labeled with biotin and subjected to amination modification. They were then coupled with fluorescence-coded microspheres and then used for hybridization with RT- PCR products of TSV and YHDV. The liquid chip detection technique for detection of TSV and YHDV was established by using BD FACSArray to detect fluorescence signal in the reaction system. This assay system had a high sensitivity to TSV and YHDV,with the detection of limit of 100 pg. Moreover,the assay was specific for the detection of TSV,YHDV and was not susceptible to cross with other viruses,including white spot syndrome virus( WSSV),spring viremia of carp virus( SVCV),infectious haematopoietic necrosis virus( IHNV). In conclusion,the liquid chip assay technique established in this study is highly sensitive and specific to TSV and YHDV detection. Moreover,it provides a novel,convenient and rapid approach for the detection of TSV and YHDV.

Advances in Research of Garlic Virus Diseases

Garlic virus infection is an important disease which affects garlic production,with the increasing years of planting,harm of virus is serious year by year,which seriously affect yield and quality of garlic.In order to know the garlic virus effectively,the paper reviewed the research situation of several important garlic virus in virus species,origin,distribution,host range,symptom,route of transmission,classification,genome and detection technique and the prevention technology of garlic viruses.At the same ...

Isolation of recombinant field strains of Marek's disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.

Analyzing the H19-and T65-epitopes in 38 kd phosphory-lated protein of Marek's disease viruses and comparing chicken immunological reactions to viruses point-mutated in the epitopes

DNA sequencing analysis in 38 kd phosphorylated protein (pp38) ORF of Marek's disease viruses (MDV) indicated that all tested 10 virulent strains with different pathotypes had 'A' at base #320 and glutamine at aa#107 while reacted with monoclonal antibody (Mab) H19 in indirect fluorescence antibody test (IFA). However, vaccine strain CVI988 had 'G' at base#320 and arginine at aa#107 instead, when it was negative in IFA with Mab H19. Some strains were also reactive with Mab T65 in IFA while there was 'G' at base #326 and glycine at aa#109, but the other strains, which had 'A' at base #326 and glutamic acid at aa#109, did not react with Mab T65. By comparison of CVI988 to its point mutants CVI/rpp38(AG) and CVI/rpp38(AA) with 1 or 2 base(s) changes at bases #320 and /or #326 of pp38 gene for their reactivity with Mab H19 and T65, it was confirmed that the glutamine at aa#107 and glycine at aa#109 were critical to epitopes H19 and T65 respectively. Immuno-reactions to MDV were compared in SPF chickens inoculated with cloned CVI988 and its mutant CVI/rpp38(AG). It was found that antibody responses to MDV in chickens inoculated with CVI/rpp38(AG) were delayed and significantly lower than that in chickens inoculated with the native CVI988. By differential comparison of antibody titers to different antigens, a third epitope specific to CVI988 and dependent on arginine at aa#107 was suggested to be responsible for the big difference in antibody responses induced by native CVI988 and its mutant.

The enhancement effect of pp38 gene product on the activity of its upstream bi-directional promoter in Marek's disease virus

There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene; 1.8-kb mRNA transcript gene family in the genome of Marek's disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) reporter plamids, pP(pp38)-EGFP; pP(1.8-kb)-EGFP, were constructed under this bi-directional promoter in two directions. The two plasmids were transfected into uninfected chicken embryo fibroblast (CEF), MDV clone rMd5 infected CEF (rMd5-CEF); pp38-deleted derivative rMd5Δpp38 infected CEF (rMd5Δpp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF,; no EGFP could be detected in uninfected CEF or rMd5Δpp38-CEF, implying that pp38 was a factor influencing the activity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to co-transfect CEF or rMd5Δpp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5Δpp38-CEF but still not in uninfected CEF, implying that pp38 needs other protein(s) to work together for the complete trans-acting activity. Another MDV gene, 24 kd phosphorylated protein pp24 gene was cloned into pcDNA3.1 as a pp24-expressing recombinant plasmid pcDNA-pp24. When uninfected CEF was co-transfected with pcDNA-pp38, pcDNA-pp24; EGFP expressing plasmids pP(pp38)-EGFP or pP(1.8-kb)-EGFP, the EGFP could be detected. These results indicated that pp38; pp24 could enhance the activity of the promoter when they worked together. DNA mobility shift assay showed that pp38 would bind to the bi-directional promoter with the co-existing of pp24, although neither of them alone influenced mobility of the promoter DNA. All the above suggested that MDV pp38 could transactivate the bi-directional promoter when combined with pp24. The results also indicated that the activity of the promoter in the direction of 1.8-kb mRNA was significantly stronger than that of pp38 direction.

基于双特异纳米抗体FMDV血凝检测方法的建立

本研究利用基因工程方法将抗口蹄疫病毒(FMDV)纳米抗体Nb205和抗鸡醛化红细胞(cRBC)纳米抗体NbRBC48的基因片段连接,构建双特异纳米抗体Nb205-48,其相对分子质量为3.5×10^(4),能同时与FMDV和cRBC反应。利用Nb205-48成功建立了可用于FMDV抗原检测的血凝方法,该方法检测灵敏度为1μg/ml,与其他病毒无交叉反应,且批内和批间重复性好。分别采用血凝法和夹心ELISA方法对3批次FMDV抗原进行了检测,二者检测结果基本吻合。本研究建立了基于双特异纳米抗体Nb205-48的血凝检测方法,该方法具有较好的灵敏度、特异性和重复性,且简便、快捷、成本低,其检测结果与传统的FMDV定量检测方法的检测结果相关性好,为检测口蹄疫疫苗生产过程中FMDV抗原的含量提供了新方法。

水貂阿留申病毒的微滴式数字PCR方法的建立及初步应用

为建立水貂阿留申病毒(AMDV)微滴式数字PCR (ddPCR)检测方法,本研究根据AMDV VP2基因的保守区设计特异性引物和探针,通过PCR扩增AMDV VP2基因并克隆至p MD19-T载体,构建重组质粒p MD19-T-AMDV,并经PCR和测序鉴定后利用该质粒标准品作为模板,通过各反应条件的优化,初步建立了检测AMDV的ddPCR方法。分别以AMDV、水貂肠炎细小病毒、犬瘟热病毒、犬细小病毒、水貂流感病毒基因组为模板,按照本研究建立的ddPCR方法检测,评估其特异性;将1.43×10^(7)拷贝/μL~1.43×10^(0)拷贝/μL的重组质粒标准品作为模板,采用优化后的ddPCR和文献中的荧光定量PCR (qPCR)检测,评估该方法的敏感性;分别以不同浓度的重组质粒标准品作为模板,采用优化后的ddPCR分别进行批内和批间重复性试验,以变异系数(CV)评估该方法的重复性。优化结果显示,该方法的最佳引物终浓度为400 nmol/L (1.2μL)、探针终浓度为200 nmol/L (0.6μL)、退火温度为60℃。特异性试验结果显示,该方法仅可检测到AMDV和质粒标准品,而其他相关病毒均为阴性结果。敏感性试验结果显示,该方法对重组质粒标准品的检测限为1.43拷贝/μL,qPCR对质粒标准品的检测限为1.43×10^(2)拷贝/μL,前者比后者的敏感性高100倍。重复性试验结果,批内和批间重复性试验的变异系数均小于3%。对采集的70份病貂肝脏、脾、肾脏、环境拭子、肛拭子样品进行ddPCR和qPCR检测,结果显示,ddPCR对AMDV的检出率为24.28%(17/70);qPCR的检出率为15.7%(11/70),二者的阳性符合率为64.7%,阴性符合率为89.83%,总符合率为91.42%。上述结果表明,本研究建立的ddPCR方法特异性强、敏感性高和重复性好,可以检测各种临床样品及含量极低样品中的AMDV,为AMDV早期感染的检测、流行病学调查及AMD的防控提供有力技术手段。

水貂阿留申病毒微滴式数字PCR检测方法的建立与应用

为建立一种准确且灵敏的水貂阿留申病毒(AMDV)微滴式数字PCR检测方法(dd PCR),本研究根据AMDV-G基因保守区,设计特异性引物及探针,并对反应条件进行优化,初步建立检测AMDV的dd PCR方法。同时对该方法的特异性、敏感性和重复性进行分析。结果显示,dd PCR的退火温度为55℃,最佳引物和探针终浓度均为10μmol/μL。绘制的标准曲线结果显示,核酸标准物质稀释倍数的对数与对应检出拷贝数的对数之间呈良好的线性关系(R^(2)=0.9945>0.99)。建立的dd PCR方法的特异性强,除能检测到AMDV以外,对相关病原如水貂犬瘟热病毒、犬细小病毒、貂肠炎病毒、犬冠状病毒、犬腺病毒I型、狂犬病毒的检测结果均为阴性。对AMDV核酸标准物质的检测限为6.2拷贝/μL,敏感性高,比荧光定量PCR(q PCR)敏感性高10倍。对不同稀释度AMDV核酸标准物质的批内和批间重复性试验的变异系数均<5%,重复性好。采用建立的dd PCR方法和荧光定量PCR方法对貂场采集的40份水貂脏器组织样品进行检测,两种检测结果基本一致,但dd PCR敏感性更高。上述结果表明,本研究建立了一种特异性强、灵敏度高、重复性好的AMDV定量检测方法,为水貂阿留申病毒的有效防控提供技术支持。

裂皮病病毒在柑橘不同部位的分布研究

柑橘裂皮病类病毒(Citrus exocortis viroid,CEVd)主要为害以枳和枳橙作砧木的柑橘品种,引起砧木树皮开裂、植株矮化,导致病树枯枝、落花落果,严重时可致全株死亡,严重阻碍了柑橘的优质生产.前期研究发现,柑橘样品的采集部位对CEVd检测具有显著影响.为进一步探究不同部位样品中CEVd滴度情况及对检测的影响,从感染柑橘裂皮病的白金柚和脐橙植株上分别取嫩叶、成熟叶、老叶及不同年龄枝条的树皮提取总RNA,进行一步法反转录多聚酶链式反应(RT-PCR)检测,分析CEVd在柑橘体内的分布情况.结果表明成熟叶、老叶和树皮都可以检测到CEVd特异性亮条带,但是嫩叶中CEVd滴度低,容易造成检测结果的假阴性.

不同症状类型苜蓿病毒病AMV病原检测及其寄主范围测定

为明确采自田间不同症状类型苜蓿病毒病病样中苜蓿花叶病毒(alfalfa mosaic virus,AMV)的带毒情况、症状表现与叶绿素含量的相关性及AMV的寄主范围,本试验通过田间调查采样、症状归类、丙酮乙醇混合液法和双抗体夹心酶联免疫吸附测定(DAS-ELISA)法对不同病样的叶绿素和AMV含量进行测定和检测,并对提纯至不同症状类型病样中AMV对9科32种植物的致病性进行测定。结果表明田间苜蓿病毒病病样有轻花叶、重花叶、叶片边缘褪绿黄化型和叶片畸形皱缩花叶矮化型4种症状类型,均带有AMV,且叶绿素含量、AMV含量与症状表现之间具有正相关性,症状表现最严重的叶片畸形皱缩花叶矮化型病样的叶绿素a、b、总叶绿素含量均比对照低58.00%以上,类胡萝卜素含量比对照高134.06%,AMV含量最高,为252.96 pg·mL^(-1);寄主范围测定表明AMV可侵染7科27种植物,对西葫芦致病性最强,症状表现为局部枯斑,30 d时AMV浓度为316.19 pg·mL^(-1)。

我国北方部分苹果主产区病毒病的发生与检测

苹果病毒病在我国广泛发生,已成为限制我国苹果优质高产的关键因素。20世纪80年代曾对其做过详细的调查和研究,但近年来由于各地农业产业结构调整等因素,苹果病毒病的发生特点有了不同程度的变化。为了解目前我国苹果病毒病的发生情况,在我国北方苹果主产区山东、陕西、山西、辽宁、北京和黑龙江6个省市的部分地区采集苹果样品共计267份,经RT-PCR检测和扩增产物的克隆与测序分析表明在上述地区采集的样品中,苹果褪绿叶斑病毒（ACLSV）、苹果茎沟病毒（ASGV）、苹果茎痘病毒（ASPV）、苹果锈果类病毒（ASSVd）的发生率分别为66.7%~100.0%、38.1%~94.1%、4.8%~85.7%和4.8%~48.6%;苹果凹果类病毒（ADFVd）仅在山东的两个果园零星发生;6个省市样品中病毒复合侵染率分别为67.1%、92.1%、75.0%、88.2%、94.1%和76.2%。

我国部分地区樱桃病毒病害初步调查和病原检测

对山东泰安、辽宁大连和北京的樱桃病毒病发生情况进行调查,发现8个果园/栽培区均有病毒病发生,主要症状为叶片皱缩、畸形、卷叶、花叶、植株矮缩等。采集20份样品,利用12种病毒的引物进行RT-PCR检测。结果表明,在样品中扩增出与樱桃病毒A(Cherry virus A,CVA)、李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV),李矮缩病毒(Prune dwarf virus,PDV)、李树皮坏死与茎痘伴随病毒(Plum bark necrosis stem pitting-associated virus,PBNSPaV)、樱桃绿环斑驳病毒(Cherry green ring mottle virus,CGRMV)、樱桃小果病毒-1(Little cherry virus-1,LChV-1)预期大小一致的目的片段;序列分析表明,与GenBank中注册所测的病毒核苷酸序列均具有较高的一致性。其中,大连、泰安和北京样品均检测到CVA;大连和北京样品中检测到PNRSV和PDV;北京样品中检测到PBNSPaV;大连苗木样品枝条中检测到CGRMV和LChV-1。这是在我国樱桃上首次检测到LChV-1。

云南甘蔗花叶病病原检测及一个分离物的分子鉴定

调查表明甘蔗花叶病在云南发生普遍。电镜检测采自云南6个蔗区主栽品种上的28个甘蔗花叶病病样(分离物),其中25个病样的病叶汁液中观察到弯曲线状的病毒粒体,病叶组织中有风轮状和卷筒状内含体;对这25个分离物进行间接ELISA检测,16个与马铃薯Y病毒属抗血清呈阳性反应,其余呈阴性反应。根据蔗区及其主栽品种的不同,挑选7个分离物进行鉴别寄主测定,结果显示不同分离物鉴别寄主范围和致病性存在明显差异,分离物HH-1有范围最广的鉴别寄主和较强的致病性。克隆并测定HH-1基因组3′末端序列,序列分析发现HH-1的外壳蛋白(CP)基因共864个核苷酸,编码287个氨基酸,与高粱花叶病毒(Sorghum mosaic virus,SrMV)余杭分离物CP氨基酸序列的同源性最高,为97.7%;因此推定HH-1属于SrMV的一个新分离物。

应用多重聚合酶链反应同时检测鉴别鸡新城疫病毒、鸡传染性支气管炎病毒、鸡传染性喉气管炎病毒和鸡毒支原体的研究

本文建立了一种同时检测NDV、IBV、ILTV和MG 4种病原体的多重PCR技术。根据NDV、IBV、ILTV和MG的基因文库 ,设计了 4对分别与NDV、IBV、ILTV和MG某段基因序列互补的引物 ,用这 4对引物对同一样品中的NDV、IBV、ILTV、MGRVA或DNA模板进行多重PCR扩增 ,结果均同时得到了 4条特异性大小与实验设计相符的 310bp(NDV)、172 0bp(IBV)、6 47bp(ILTV)、和 732bp(MG)多重PCR扩增带 ,而对其他 6种禽病病原的PCR扩增结果均为阴性 ;敏感性测定结果表明 ,该多重PCR技术能检出 10pg的IBV、1pg的NDVRNA模板和 10pg的ILTV、1pg的MGDNA模板。

樱桃病毒病脱毒检测技术及抗病毒基因工程研究进展

综述了国内外樱桃病毒病的研究进展,包括李属坏死环斑病毒(PNRSV)、李矮缩病毒(PDV)、苹果褪绿叶病毒(ACLSV)、樱桃绿环斑驳病毒(CGRMV)、樱桃小果病毒(LCh V)、樱桃病毒A(CVA)、樱桃卷叶病毒(CLRV)7种主要樱桃病毒病的分类地位、地理分布、传播途径、分子生物学特性、检测方法等,阐述了微茎尖组织培养法、热处理法、化学药剂法3种樱桃常用脱毒技术,总结了抗病毒基因工程在樱桃抗病育种中的作用。

4种重要虫媒病的核酸液相芯片高通量检测方法的建立

为建立可检测鹿流行性出血热病毒（EHDV）、阿卡斑病毒（AKV）、蓝舌病病毒（BTV）和水泡性口炎病毒（VSV）的液相芯片快速检测技术,用DNAStar软件对GenBank中BTV的VP7基因、EHDV的VP7基因、AKV的N基因和VSV的NP基因序列进行序列分析,设计针对这些基因的特异性探针并标记生物素,分别与不同编号的荧光编码微球偶联后再与这些病毒相应基因的PCR产物杂交反应,用液相芯片检测仪（Liquichip 200）检测荧光信号建立了以上4种虫媒病的快速液相芯片检测方法。检测结果显示,该方法具有较好的特异性,偶联特异性探针的微球只与相应的病毒基因的PCR产物反应,而不与其他虫媒病病毒反应;检测灵敏度达到50~100个TCID50。本研究建立了可以同时检测鹿流行性出血热病毒、阿卡斑病毒、蓝舌病病毒和水泡性口炎病毒的快速高通量液相芯片技术,为其他类似病毒的快速高通量检测提供了借鉴和经验。

赤羽病病毒RT-LAMP检测方法的建立

为建立一种赤羽病病毒(AKAV)的快速检测方法,本研究根据环介导等温扩增技术(LAMP)原理,设计了针对AKAV S基因的特异性引物,并对反应条件和反应体系进行优化,建立了AKAV的RT-LAMP方法。特异性和灵敏度试验结果显示,所建立的RT-LAMP检测方法具有良好的特异性,与蓝舌病、牛病毒性腹泻及传染性牛鼻气管炎等病毒无交叉反应,敏感度可达11.5拷贝/μL,采用SYBR Green I染色,肉眼即可判定检测结果。该方法能够简便、快速、灵敏、特异地检测AKAV。

多重PCR同时检测口蹄疫病毒、猪水疱病病毒和水疱性口炎病毒

根据基因库中的口蹄疫病毒(FM-DV),猪水疱病病毒(SVDV)和水疱性口炎病毒(VSV)各基因序列,设计了与FMDV,SVDV和VSV互补的3对特异性引物,对样品中的cDNA模板进行了多重PCR扩增及反应条件的优化,结果同时得到与设计相符合的3条特异性条带,分别为189 bp,125 bp和300 bp。用这3对引物对病毒样品cDNA模板进行多次扩增,均能稳定得到与设计相符合的3条特异性条带。本试验能特异、敏感、快速地鉴定FMDV,SVDV和VSV。

二重荧光RT-LAMP方法鉴别检测口蹄疫病毒和水泡性口炎病毒

口蹄疫和水泡性口炎是牛常见高度急性病毒传染病,在全球范围内广泛存在。本研究旨在建立一种可同时鉴别口蹄疫病毒和水泡性口炎病毒的二重荧光RT-LAMP检测方法。根据口蹄疫病毒(FMDV)3D基因和水泡性口炎病毒(VSV)N基因的保守序列,设计了2套特异性引物,在每条内引物的5′端标记荧光基团,通过扩增产物颜色判断检测结果。优化反应条件,建立了可同时检测口蹄疫病毒和水泡性口炎病毒的二重荧光RT-LAMP方法。结果显示,该方法灵敏性高,每个反应最低能够检测到100个拷贝混合模板;特异性好,能在同一个反应管里检测到两种病毒,对其他牛病原体无扩增;干扰性小,扩增效率不受模板浓度影响。本研究建立的口蹄疫病毒和水泡性口炎病毒二重荧光RT-LAMP方法具有简便、快速、特异、敏感等优点,可用于FMDV和VSV的临床检测和流行病学调查。