Genetic diversity and recombination analysis based on capsid protein gene of <Emphasis Type="Italic">Chilli veinal mottle virus</Emphasis> isolates from Pakistan

Chilli veinal mottle virus (ChiVMV), is a Potyvirus that causes severe yield losses in capsicum worldwide including Pakistan. In the current study, genetic diversity and molecular evolution of ChiVMV were explored based on the CP gene sequences. In multiple sequence alignments of the CP gene of 29 ChiVMV isolates, Pakistani isolates shared 82–92% and 78–96% nucleotide and amino acid identities, respectively with other ChiVMV isolates. In nucleotide and amino acid based phylogenetic analysis of the CP gene, the Pakistani isolates clustered with Indian (JN692501 and JN624776) and Chinese (KC711055, KC711055, JX088636 and HQ218936) isolates in a separate clade. In all Pakistani isolates, conserved motifs (DAG, WCIEN, QMKAAL, and AFDF) were located at 6–8, 141–145, 222–225, and 242-248th amino acid positions, respectively. Eleven recombination events were detected in the isolates investigated. One Pakistani isolate KX236451 was suggested to be a recombinant between the Pakistani isolate (KT876050) and the Indian isolate (JN692501). Most of the codons were found under negative selection except for codons at 28, 34, and 38th positions that were found under positive selection by REL method. An infrequent gene flow was observed between the ChiVMV isolates from Pakistan and other countries of the world. To our knowledge, this is the first report on genetic diversity of Pakistani isolates of ChiVMV based on recombination and phylogenetic analysis. Findings of this study may be helpful in developing sustainable management strategies against ChiVMV not only in Pakistan but also in other countries, ultimately resulting in enhanced and good quality production of chilli crop.     

ChiVMV在滇重楼上的首次发现及部分基因序列分析

滇重楼(Paris yunnanensis)是主要分布在我国西南地区尤其是云南的珍贵药材.2016年,从云南省德宏州芒市的滇重楼种植基地上观察到叶片斑驳、皱缩、坏死和花叶的滇重楼罹病植株.利用马铃薯Y病毒属(Potyvirus)、烟草花叶病毒属(Tobamovirus)、黄症病毒属(Luteovirus)和菜豆金色花叶...     

甜菜花叶病毒新疆分离物基因组3′末端序列分析

甜菜花叶病毒(Beet mosaic virus,BtMV)属马铃薯Y病毒科、马铃薯Y病毒属,可经多种蚜虫以非持久性方式传播,病毒粒子为弯曲线状,核酸为单分子正义ssRNA。目前只有美国华盛顿分离物的全序列以及斯洛伐克和英国少数几个分离物3′端的部分序列被报道[1,2]。美国分离物全长9591nt,3′端具有PolyA尾,编码一个由3086个氨基酸组成的多聚蛋白,与其它Potyvirus病毒一样可切割成10个蛋白,从N到C端依次为P1、HC-Pro、P3、6K1、CI、6K2、NIa-Vpg、NIa-Pro、NIb和CP[2]。对于我国发生的BtMV,1981年Liu等[3]报道了发生于北京地区菠菜上的Bt…     

云南烟草产区辣椒脉斑驳病毒的调查及其遗传多样性分析

明确辣椒脉斑驳病毒(chilli veinal mottle virus, ChiVMV)在云南6个州(市)烟草生产区的分布、遗传多样性和群体遗传结构,对病害的防控预警具有重要意义。本研究以2022年采集自云南6个州(市)不同烟草产区的96份疑似感染ChiVMV的烟草样品为试验材料,利用电子显微镜负染色观察和分子生物学方法对病毒进行检测和鉴定,获得21个ChiVMV云南烟草分离物的cp基因序列。利用SDT、MEGA、RDP、DnaSP及Arlequin等生物学软件对21个云南烟草分离物及NCBI上下载的38个来自不同国家、地区及寄主的ChiVMV分离物cp基因序列的系统进化、遗传多样性和群体遗传结构特征进行分析。结果显示,从云南6个州(市)烟草生产区采集的96份烟草样品中,ChiVMV检出率为51.04%;序列比对发现,本研究获得的cp基因与NCBI中38个ChiVMV分离物cp基因的核苷酸一致性在84.1%以上;系统发育分析发现,59个ChiVMV分离物按照地理位置的远近被划分为4个分支,聚类结果具有明显的地理分布特征,而与寄主植物无关;遗传多样性和遗传分化分析表明,4个地理种群的ChiVMV cp基因遗传多样性水平均较高,中国种群与印度、泰国和其他国家种群间发生了很大的遗传分化且差异显著(P<0.05);遗传力分析显示,基因交流、遗传漂变和基因的负选择压力是ChiVMV分离物适应性进化的重要方式。     

Occurrence and distribution of viruses infecting tomato and pepper in Alibori in northern Benin

In surveys conducted in 2011 and 2012 to identify the viruses causing diseases on pepper and tomato in the department of Alibori in northern Benin, 451 samples of pepper and tomato were analyzed by ELISA using 11 specific antibodies. The highest virus incidence among the surveyed districts was recorded on pepper in Malanville (56.18%), followed by Karimama (39.32%). The most frequently found viruses were Pepper veinal mottle virus (PVMV), Cucumber mosaic virus (CMV), and Potato virus Y-necrotic (PVY-n), accounting respectively for 22.39%, 21.73% and 15.96% of the collected samples. Tomato yellow leaf curl virus (TYLCV) was detected in only 2.43% of the samples, whereas Alfalfa mosaic virus (AMV), Chilli veinal mottle virus (ChiVMV), Tomato mosaic virus (ToMV) and Tomato spotted wilt virus (TSWV) were not detected in any of the samples tested. Double and triple infections involving different virus combinations were found, respectively, in 14.86% and 4% of the samples. Five plant species (Euphorbia hirta Linnaeus, Moringa oleifera Lam, Leucas martinicencis (Jacquin) R. Brown, Combretum micranthum G. Don, Abelmoschus esculentus L. Moench.) out of 30 samples belonging to 13 botanical families, collected within or nearby tomato and pepper fields, were found infected with PVMV, PVY-n, and CMV. Control measures to reduce the impact of viruses on pepper and tomato production are discussed. This is the first report of viruses infecting pepper and tomato in Benin.     

Molecular and biological characterization of Cowpea mild mottle virus isolates infecting soybean in Brazil and evidence of recombination

The biological and molecular characterization of six isolates of a new Cowpea mild mottle virus strain (CPMMV; Carlavirus, Betaflexiviridae) are reported. Soybean plants with mosaic and stem necrosis were collected in Bahia, Goiás, Mato Grosso and Minas Gerais states, Brazil. Complete genomes of the CPMMV isolates are 8180–8198 nucleotides (nt) long, excluding the 3′‐polyadenylated tail, and have 67–68% nt sequence identity with a Ghana isolate of CPMMV, the only CPMMV isolate for which the genome has previously been sequenced. The replicase has only 60–61% nt sequence identity with the Ghana CPMMV isolate, and the coat protein (CP) is highly conserved (79% nt sequence identity and 95–96% amino acid sequence identity). The high CP identity and the phylogenetic analyses supported the classification of the Brazilian isolates as CPMMV. Biological and molecular differences with the Ghana CPMMV isolate were found and indicated that the six isolates represent a distinct CPMMV strain denominated as CPMMV‐BR. Furthermore, it is shown that recombination occurred mainly in the polymerase gene, and may occur less frequently in other regions of the CPMMV genome.     

甜菜花叶病毒新疆分离物基因组3'末端序列分析

甜菜花叶病毒（Beet mosaic virus，BtMV）属马铃薯Y病毒科、马铃薯Y病毒属，可经多种蚜虫以非持久性方式传播，病毒粒子为弯曲线状，核酸为单分子正义ssRNA。目前只有美国华盛顿分离物的全序列以及斯洛伐克和英国少数几个分离物3’端的部分序列被报道。美国分离物全长9591 nt，3’端具有PolyA尾，编码一个由3086个氨基酸组成的多聚蛋白，与其它Potyvirus病毒一样可切割成10个蛋白，从N到C端依次为P1、HC—Pro、P3、6K1、CI、6K2、NIa—Vpg、NIa～Pro、NIb和CP。对于我国发生的BtMV，1981年Liu等报道了发生于北京地区菠菜上的BtMV，之后研究人员相继报道了黑龙江、内蒙古和新疆等甜菜主产区甜菜花叶病的发生及危害情况，并陆续开展了对BtMV的生物学特性、外壳蛋白分子量测定和氨基酸组分分析、细胞病理学等研究，目前对于我国发生的BtMV的分子结构特征还未见报道。本文报道了甜菜花叶病毒新疆分离物（BtMV—XJ）3’端的核酸序列，并与国外已报道序列进行了比较分析，为从分子水平上明确我国BtMV的分子结构特点、深入研究其编码蛋白的功能打下了基础。     

辣椒轻斑驳病毒湖南分离物的全基因序列测定及结构分析

采自湖南地区的辣椒轻斑驳病毒Pepper mild mottle virus(PMMoV)样品经单斑分离后,根据已经报道的PMMoV序列基因保守区设计6对简并引物,采用片段重叠法和RACE方法扩增、克隆获得一个全长为6 356bp的湖南分离物(PMMoV-HN1,登录号:KP345899)全基因组序列,编码4个蛋白,分别为126kD蛋白(70~3 423nt)、183kD蛋白(70~4 908nt)、28kD蛋白(4 909~5 682nt)和17.5kD蛋白(5 685~6 158nt),5′-非编码区(5′-UTR)和3′-非编码区(3′-UTR)分别含有69和198个碱基,其中5′-UTR存在一个序列为m7G5′pppG的甲基化核苷酸帽子结构。一致性分析发现PMMoV-HN1与PMMoV其他分离物的核酸一致性为94%~99%,编码的氨基酸一致性为94%~99%。全基因组序列系统进化分析表明PMMoV-HN1分离物与中国首次报道的PMMoV-CN分离物亲缘关系最近。本研究是国内报道的第二例PMMoV全基因组序列。     

小西葫芦黄花叶病毒北京分离物的鉴定及其基因组3''端特性分析

在北京东郊自然感病的南瓜Cucurbita moschata上获得一病毒分离物（BJ-1），经生物学、血清学和分子生物学鉴定，确定为小西葫芦黄花叶病毒（Zucchini yellow mosaic virus，ZYMV）。为分析其基因组3’端特性，以发病叶片中提取的总RNA为模板，对基因组3’端进行RT-PCR扩增，产物克隆到pMD18-T栽体上进行序列分析，共测定了该病毒分离物包括全部CP基因在内的1269bp。该分离物CP基因由837个核苷酸组成，编码279个氨基酸。对包括该分离物在内的30个序列的760bp（含NIb基因3’端56bp和CP基因中的704bp）片段、NIb蛋白与CP蛋白的切割位点、蚜传必需基序的变异、寄主来源及地域来源进行了分析。结果表明，ZYMV不同分离物的基因分型与上述五个因素无明显关系。     

Molecular characterization of the Cassava brown streak virus coat protein

A partial sequence of 1114 nucleotides of a virus from cassava brown streak diseased (CBSD) material was obtained. Alignment of the predicted amino acid sequence with those of other members of the Potyviridae showed closest identity with the coat protein of Sweet potato mild mottle virus (genus Ipomovirus ). The predicted amino acid sequence has one open reading frame with a 3' untranslated region of 144 nucleotides and a poly(A) tail. The expressed protein was shown to cross-react with an antiserum raised previously to a virus isolated from CBSD material. Evidence presented suggests that CBSD is caused by Cassava brown streak virus , a tentative member of the genus Ipomovirus , as this virus is consistently found associated with CBSD.     

Survey of viruses infecting open‐field pepper crops in Côte d'Ivoire and diversity of Pepper veinal mottle virus and Cucumber mosaic virus

The prevalence of viruses in pepper crops grown in open fields in the different agro‐ecological zones (AEZs) of Côte d'Ivoire was surveyed. Pepper veinal mottle virus (PVMV; genus Potyvirus) and Cucumber mosaic virus (CMV; genus Cucumovirus) were the most frequent viruses among those surveyed, while tobamoviruses (genus Tobamovirus) were detected at low frequency. PVMV showed a high heterogeneity across AEZs, which may be related to climatic, ecological or agronomical conditions, whereas CMV was more homogeneously distributed. The molecular diversity of CMV and PVMV were analysed from partial genome sequences. Despite the low number of CMV isolates characterized, two molecular groups were revealed, one corresponding to subgroup IA and the other to reassortants between subgroups IA and IB. RNAs 1 and 3 of the reassortants clustered with the IB subgroup of CMV isolates, whereas their RNA 2 clustered with the IA subgroup. Importantly, RNA 1 of CMV isolates of the IB subgroup has been shown to be responsible for adaptation to pepper resistance. The diversity of PVMV in the VPg‐ and coat protein‐coding regions revealed multiple clades. The central part of the VPg showed a high level of amino acid diversity and evidence of positive selection, which may be a signature of adaptation to plant recessive resistance. As a consequence, for efficient deployment of resistant pepper cultivars, it would be desirable to examine the occurrence of virulent isolates in the CMV or PVMV populations in Côte d'Ivoire and to follow their evolution as the resistance becomes more widely deployed.     

A potyvirus isolated from solanaceous hosts

A potyvirus was isolated from Datura stramonium, Lycopersicon esculentum (tomato) and Solanum nigrum in the Yemen. It was transmitted mechanically and by Myzus persicae in a non-persistent manner. Its flexuous rod-shaped particles had a mean length of 719 nm and some of its pinwheel inclusion bodies in infected Nicotiana clevelandii leaves were unusual in that they were dichotomously branched. The virus infected various solanaceous species, but the symptoms it induced were distinct from those of pepper veinal mottle (PVMV) and potato Y viruses. Its particles were purified from N. glutinosa and their coat protein had an atypically high molecular mass a potyvirus of 41·5 kDa. They showed a distant serological relationship to those of PVMV and potato virus V in ISEM decoration tests, but did not react with antisera to particles of any other potyvirus tested. The virus has been tentatively named tomato mild mottle virus.     

Diversity of Plum pox virus isolates in Bosnia and Herzegovina

Sixteen Plum pox virus (PPV) isolates from several stone fruit cultivars, host species, orchards and geographical areas of Bosnia and Herzegovina were selected for typing, using serotype-specific monoclonal antibodies (MAbs) and PCR–RFLP, targeting the 3' terminal region of the coat protein (CP) and P3-6K₁ with restriction enzymes Rsa I and Dde I. Four PPV isolates were identified as PPV-M by serology and PCR; eight isolates were identified as PPV-D based on PCR–RFLP on both genomic regions, but were not recognized by the D-specific MAb4DG5. Four isolates from plum were identified as natural D/M recombinants (PPV-Rec), based on conflicting results of CP and P3-6K₁ typing. To investigate the genetic diversity of Bosnian PPV isolates in more detail, five isolates (three PPV-Rec, one PPV-M and one PPV-D) were partially sequenced in the region spanning the 3' terminal part of the NIb gene and the 5'-terminal part of the CP gene, corresponding to nucleotides 8056–8884. Nucleotide sequence alignment of recombinant isolates showed that they were closely related at the molecular level to previously characterized recombinants from other European countries, and shared the same recombination break point in the 3' terminal part of the NIb gene. This is the first report of naturally infected Prunus trees with PPV-M, PPV-D and PPV-Rec in Bosnia and Herzegovina. The high variability of the Bosnian PPV isolates fits with the presence of this virus in the country over a long period.     

<Emphasis Type="Italic">Amaranthus leaf mottle virus</Emphasis>: 3′-end RNA sequence proves classification as distinct virus and reveals affinities within the genus <Emphasis Type="Italic">Potyvirus</Emphasis>

Amaranthus leaf mottle virus (AmLMV) was classified as a member of the genus Potyvirus on the basis of its particle morphology, serology, and biological properties (Casetta et al., 1986). Based on these properties, an Amaranthus viridis-infecting virus isolated in Spain, causing mottle and leaf blistering as well as reduced growth has been identified as AmLMV. The 3′ terminal genomic region of this and a reference isolate from Italy has been sequenced and reveals a 95% nucleotide identity between the two isolates. The sequenced part comprises the coat protein with 281 amino acids and 315 nucleotides of the 3′ untranslated region (UTR) preceding a polyadenylated tail. Pairwise comparisons and phylogenetic analysis of the nucleotide and deduced amino acid sequences of the CP and 3′ UTR of the cloned cDNAs with those of other potyviruses shows that AmLMV is a distinct potyvirus closely related to Potato virus Y.     

Phenotype and spectrum of action of the Pvr4 resistance in pepper against potyviruses, and selection for virulent variants

The dominant Pvr4 gene identified in Capsicum annuum cv. Criollo de Morelos 334 (CM334) is frequently used in pepper cultivars because it possesses one of the largest spectra of action among plant virus resistance genes. This gene was previously shown to confer efficient resistance to all known Potato virus Y isolates, to Pepper mottle virus , to Pepper yellow mosaic virus and to Ecuadorian rocoto virus. This study showed that the W4 line, derived from CM334 and carrying Pvr4 , was also resistant to Peru tomato mosaic virus and Pepper severe mosaic virus , but not to Pepper veinal mottle virus , Chilli veinal mottle virus or Tobacco etch virus . It was noticed that the phenotype of the resistance was atypical since, in the W4 line, hypersensitive reaction or extreme resistance could be observed, depending on virus isolates and inoculated organs. Despite the large deployment of Pvr4 in hybrid cultivars, the numerous tests performed in controlled conditions and the use of W4 serial back-inoculations with potyvirus isolates controlled by this line, no virulent variant isolates were obtained. However, it was shown that the use of graft inoculation experiments allow PVY virulent variants to be selected.     

一种侵染滇重楼的Potexvirus病毒分离物分子鉴定及其3'末端序列分析

 从云南武定的滇重楼上得到一个病毒分离物Paris-YN,病毒粒体为弯曲线状。利用RT-PCR扩增获得一条1074bp的片段,序列比较分析发现其与马铃薯X病毒属(Potexvirus)病毒3'末端的结构最为相似,且与属内的白三叶草花叶病毒等20个不同分离物3'末端有36.7%~58.9%的同源性;该病毒cp基因长639个核苷酸,编码212个氨基酸(22.8kDa),与20个Potexvirus病毒分离物的CP氨基酸序列比较发现,Paris-YN与白三叶草花叶病毒的CP氨基酸同源性最高(60.1%)。证据表明,该分离物可能为Potexvirus的新成员,暂命名为重楼X病毒(Paris polyphylla virus X)。     

我国北方稻区水稻条纹病毒分子变异和水稻品种抗病性分析

为了明确我国北方稻区水稻条纹病毒(Rice stripe virus,RSV)群体的分子变异和水稻品种的抗性情况,测定了2004年和2005年从我国6省9个地区采集的34个RSV分离物的外壳蛋白(coat protein,CP)基因的序列,并对其进行了分析,同时用强致病性江苏分离物(RSV-JS)对河南、安徽、山东、河北等省的25个推广品种进行了抗性研究。结果表明,供试RSV分离物间的核苷酸序列一致性和氨基酸序列的一致性分别在93.9%～100%和96.3%～100.0%之间。根据CP基因核苷酸序列一致性,所有分离物可以分为2组。云南4个分离物为一组,其余为一组。在第2组中各分离物CP基因的核苷酸序列和氨基酸序列的一致性与地域无必然联系。且在2年之内,RSVCP基因变异不大。抗病性鉴定表明同一分离物在不同水稻品种上表现不同症状。表现高抗(HR)的品种占供试品种的24%;60%以上的品种表现为感病,且不同水稻品种上表现不同症状。因此,我国北方稻区RSV的CP基因非常保守,但同一分离物在不同水稻品种上可能表现不同症状,不同水稻品种对RSV抗性有显著差异。这些结果为我国北方稻区水稻条纹叶枯病防治和抗病毒基因工程提供了理论依据。     

云南怒江番茄种植新区番茄病毒检测及病毒种传特性

病毒病对番茄生产造成严重危害, 近年来在番茄种植新区发生严重, 疑似为种子带毒传播。本研究通过对云南省怒江州番茄种植新区的番茄病毒病样品采用RNA-seq高通量测序, RT-PCR验证的方法检测病毒种类;对番茄病果种子进行超薄切片制样透射电子显微镜观察, 将病果种子播种后对种苗进行RT-PCR带毒检测。结果表明, RNA-seq高通量测序及RT-PCR检测到的病毒有番茄环纹斑点病毒(tomato zonate spot orthotospovirus, TZSV)、番茄黄斑驳相关病毒(tomato yellow mottle-associated virus, TYMaV)、辣椒脉斑驳病毒(chili veinal mottle virus, ChiVMV)、南方番茄病毒(southern tomato virus, STV)。透射电镜观察到种胚细胞及胚乳细胞中分布典型的正番茄斑萎病毒属Orthotospovirus病毒粒体。病果种子播种28 d后的种苗具有病毒病症状, 通过RT-PCR检出TZSV、ChiVMV、STV, 检出率分别为60%、100%、80%。上述研究结果为TZSV通过种子传播提供了有利的证据, 并为源头防控番茄病毒病提供了依据。