番木瓜环斑病毒畸叶株系的CP基因克隆和序列分析

采用RT－PCR方法合成了本研究室保存的番木瓜畸叶病毒（PMaLV）的外壳蛋白（CP）基因,将其CP基因克隆进Promega公司的pGEM-T and pGEM-T Easy Vector System（简称T－载体）,并进行了序列分析。结果表明,PMaLV CP基因核苷酸序列全长为861nt,推导其编码287个氨基酸。与番木瓜环斑病毒（PRSV）美国夏威HA株系和澳大利亚W株系的CP基因相比,在第66nt处开始连续缺失3个核苷酸。与PRSV的华南Ys、Sm和G株系以及夏威夷的HA和澳大利亚的W株系相比,其CP基因序列同源率分别为96％、98％、95％、89％和89％。其的氨基酸序列同源率分别为98％、97％、97％、96％和95％。此结果表明,PMaLV属于PRSV的一个株系,不是一种新病毒。因此,我们称其为番木瓜环斑病毒畸叶株系（ML株系）。     

Variation in the Coat Protein Gene of Papaya ringspot Virus Isolates from Multiple Locations of China

The potyvirus Papaya ringspot virus （PRSV） is an important pathogen of papaya that causes severe losses in economic crops for papaya production globally. The coat protein （CP） genes of five PRSV isolates originating from different locations in China were cloned and sequenced. The CP-coding region varied in size from 864-873 nucleotides, encoding proteins of 288-291 amino acids. The five Chinese isolates of PRSV have been characterized as papaya-infecting （PRSV-P）. The CP sequences of the Chinese isolates were compared with those of previously published PRSV isolates originating from different countries at amino acid levels. A number of KE repeat boxes in the N terminus of the PRSV-CP were found in all Chinese isolates. The phylogenetic branching pattern revealed that there was certain extended grouping between geographic locations, and the Asian type probably represents the oldest population of PRSV. The information of CP genes will be useful in designing and developing durable virus resistant-PRSV transgenic papaya in China. Meanwhile broad-spectrum-virus resistant, strongly resistant-PRSV and good safe papaya lines are required.     

番木瓜环斑病毒海南分离株全基因组序列分析

番木瓜环斑病毒(Papaya ringspot virus,PRSV)是马铃薯Y病毒属(genus Potyvirus)的成员之一.病毒粒体呈线状,长700～900nm,直径为12.5nm.为单分体正链RNA病毒,由多种蚜虫以非持久方式传播.依寄主范围可划分为P型和W型两种.其中P型株系(PRSV-P)是制约生产的重要病原,除了给番木瓜生产带来严重危害,也会危害葫芦科作物.W型株系(PRSV-W)是危害葫芦科作物的主要病原,虽然和PRSV-P型株系血清学反应密切相关,但不侵染番木瓜.     

香蕉束顶病毒基因克隆和序列分析

对香蕉束顶病毒（ＢＢＴＶ）中国分离株ＤＮＡ组份Ｉ（ＤＮＡ－１）、外壳蛋白（ＣＰ）和运转蛋白（ＭＰ）基因进行了克隆和序列分析。ＢＢＴＶＤＮＡ－１含有１１０３个核苷酸,与南太平洋和亚洲分离株分别有８７％－８８％ ９６．９－９８％的核苷酸序列同源性。由ＤＮＡ－１编码的复制酶含有１８６个在酸残基。与南太平洋和亚洲分离株分别有８４．４％－９５．８％和９７．６％、９８．０％的氨基酸序列同源性。外壳蛋白基因由５     

番木瓜环斑病毒复制酶(亚基)基因的克隆、序列分析及其植物表达载体的构建

近二年报道,在TMV^⑴、PVX^⑵、PEBV^⑶和CMV^⑷等病毒中,利用病毒编码的复制酶(亚基)基因或相关cDNA片段转化烟草,工程植株获得绝对或极高的病毒抗性。大量研究认为：马铃薯Y病毒组的核内含体大分子量蛋白(Nib)是依赖于RNA的RNA复制酶(或核心亚基),Nlb与上述病毒的复制酶有广泛的同源保守区^⑸。因此,Nib基因的克隆,不但在植物抗病基因工程方面,而且在马铃薯Y病毒组基因组的复制研究方面．均有重要的意义。本文以马铃薯Y病毒组的重要成员番木瓜环斑病毒的华南强株系(PRSV—sM)为材料,克隆了PRSV的Nib基因,并完成其全序列测定和植物表达载体的构建,为探索马铃薯Y病毒组复制酶可能介导的抗病性打下了基础。     

Book Review

To study the variability and to identify the species of Begomovirus associated with yellow mosaic disease of blackgram in Andhra Pradesh, India, infected blackgram samples were collected from six districts belonging to three regions of Andhra Pradesh. The total DNA was isolated by modified CTAB method and amplified with coat protein gene-specific primers (RHA-F and AC abut) resulting in 900?bp gene product. The PCR products were cloned, sequenced and deposited in GenBank. The sequence analysis of six clones showed that the size of amplified CP gene of YMV was 920?bp. Based on nucleotide sequence identity of six isolates representing three regions of Andhra Pradesh, the isolates from Rayalaseema and Telangana region are the same variant of YMV (>99.5% identity) and isolate from coastal Andhra is another variant of YMV (>95.4%) when compared with other region isolates. Comparison of CP gene sequence of YMV-TPT isolate with 27 other isolates in database revealed more than 93.2 and 86.2% identity with MYMIV isolates and less than 80 and 64% identity with MYMY isolates that originate from Indian sub-continent and South-East Asia at nucleotide and amino acid level, respectively. Phylogenetic tree based on CP gene sequences of six isolates with other isolates from GenBank formed unique cluster with MYMIV. Hence the YMV infecting blackgram in Andhra Pradesh is caused by MYMIV rather than MYMY as reported in Tamil Nadu which is adjoining state in southern India.     

Identification and Characterization of a Badnavirus Infecting Betel Vine and Indian Long Pepper

Badnavirus infecting betel vine (Piper betle L) and Indian long pepper (P. longum L) were detected with primers designed from the open reading frame (ORF III) region of the virus using Polymerase Chain Reaction (PCR). The amplicons obtained from these infected hosts were cloned and sequenced. The sequenced region of ORF III contained 597 nucleotides in both the isolates. Sequence analysis with recognized badnaviruses revealed that Badnavirus infecting P. betle and P. longum had highest sequence identity (>89.1% at nucleotide level and >93.4% at amino acid level) with an Indian isolate of Piper yellow mottle virus (PYMoV) infecting black pepper. Based on the sequence identity and phylogenetic relationship studies, it was concluded that Badnavirus infecting P. betle and P. longum in India is a strain of PYMoV. To our knowledge, this is the first report on the identification and characterization of PYMoV infecting P. betle in India and PYMoV infecting P. longum in India and elsewhere.     

A single amino acid of niapro of papaya ringspot virus determines host specificity for infection of papaya

Most strains of Papaya ringspot virus (PRSV) belong to type W, causing severe loss on cucurbits worldwide, or type P, devastating papaya in tropical areas. While the host range of PRSV W is limited to plants of the families Chenopodiaceae and Cucuribitaceae, PRSV P, in addition, infects plants of the family Caricaceae (papaya family). To investigate one or more viral genetic determinants for papaya infection, recombinant viruses were constructed between PRSV P-YK and PRSV W-CI. Host reactions to recombinant viruses indicated that the viral genomic region covering the C-terminal region (142 residues) of NIaVPg, full NIaPro, and N-terminal region (18 residues) of NIb, is critical for papaya infection. Sequence analysis of this region revealed residue variations at position 176 of NIaVPg and positions 27 and 205 of NIaPro between type P and W viruses. Host reactions to the constructed mutants indicated that the amino acid Lys27 of NIaPro determines the host-specificity of PRSV for papaya infection. Predicted three-dimensional structures of NIaPros of parental viruses suggested that Lys27 does not affect the protease activity of NIaPro. Recovery of the infected plants from certain papaya-infecting mutants implied involvement of other viral factors for enhancing virulence and adaptation of PRSV on papaya.     

Papaya ringspot virus-P: characteristics, pathogenicity, sequence variability and control

TAXONOMY: Papaya ringspot virus (PRSV) is an aphid-transmitted plant virus belonging to the genus Potyvirus, family Potyviridae, with a positive sense RNA genome. PRSV isolates belong to either one of two major strains, P or W. The P strains infect both papaya and cucurbits whereas the W strains infect only cucurbits. GEOGRAPHICAL DISTRIBUTION: PRSV-P is found in all major papaya-growing areas. PHYSICAL PROPERTIES: Virions are filamentous, non-enveloped and flexuous measuring 760-800 x 12 nm. Virus particles contain 94.5% protein and 5.5% nucleic acid. The protein component consists of the virus coat protein (CP), which has a molecular weight of about 36 kDa as estimated by Western blot analysis. Density of the sedimenting component in purified PRSV preparations is 1.32 g/cm(3) in CsCl. GENOME: The PRSV genome consists of a unipartite linear single-stranded positive sense RNA of 10 326 nucleotides with a 5' terminus, genome-linked protein, VPg. TRANSMISSION: The virus is naturally transmitted via aphids in a non-persistent manner. Both the CP and helper component (HC-Pro) are required for vector transmission. This virus can also be transmitted mechanically, and is typically not seed-transmitted. HOSTS: PRSV has a limited number of hosts belonging to the families Caricaceae, Chenopodiaceae and Cucurbitaceae. Propagation hosts are: Carica papaya, Cucurbita pepo and Cucumis metuliferus cv. accession 2459. Local lesion assay hosts are: Chenopodium quinoa and Chenopodium amaranticolor. CONTROL: Two transgenic papaya varieties, Rainbow and SunUp, with engineered resistance to PRSV have been commercially grown in Hawaii since 1998. Besides transgenic resistance, tolerant varieties, cross-protection and other cultural practices such as isolation and rogueing of infected plants are used to manage the disease. VIRUS CODE: 00.057.0.01.045. VIRUS ACCESSION NUMBER: 57010045. USEFUL LINK: http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/57010045.htm.     

Natural Infection by Tospoviruses of Cucurbitaceous and Fabaceous Vegetable Crops in India

Natural infection of tospoviruses on three cucurbitaceous (Cucumis sativus, cucumber; Luffa acutangula, ridge gourd; Citrullus lanatus, watermelon) and three fabaceous (Vigna unguiculata, cowpea; Phaseolus vulgaris, French bean; Dolichos lablab, sem) vegetable crops in India was identified on the basis of nucleocapsid protein (NP) gene characteristics. The complete NP gene of the cowpea isolate from Kerala and the sem isolate from Tamil Nadu was 831 nucleotides long, encoding a protein of 276 amino acids. For other Tospovirus isolates from cucumber, French bean, ridge gourd and watermelon, the partial NP gene (291 nt) was sequenced. Comparative NP gene sequence analyses revealed that fabaceous isolates shared maximum identity both at the nucleotide (92–97%) and amino acid (93–97%) levels with the corresponding region of Groundnut bud necrosis virus (GBNV), whereas cucurbitaceous isolates shared maximum identity both at nucleotide (93–99%) and amino acid (95–98%) levels with the corresponding region of Watermelon bud necrosis virus (WBNV), results suggesting that the Tospovirus isolates infecting fabaceous hosts should be regarded as strain of GBNV, whereas those infecting cucurbitaceous hosts as a strain of WBNV. Nucleocapsid protein gene was conserved both in GBNV and WBNV isolates originating from different hosts and locations.     

Konjac mosaic virus Naturally Infecting Three Aroid Plant Species in Andhra Pradesh,India

The genomes of three potyvirus isolates from, respectively, naturally infected Colocasia esculenta, Caladium spp. and Dieffenbachia spp. in Andhra Pradesh, India, were amplified by RT‐PCR using degenerate potyvirus primers. Sequence analysis of RT‐PCR amplicons (1599 nucleotides) showed maximum identity of 97% with the KoMV‐Zan isolate of Konjac mosaic virus (KoMV) from Taiwan (A/C AF332872). The three isolates had a maximum identity of 99.4%. The length of coat protein (CP) gene of three isolates was 846 nucleotides encoding 282 amino acids with a deduced size of 32.25 kDa. The CP gene of the isolates had, respectively, 78.1–95.7% and 88.2–96.4% identity at nucleotide and amino acid levels with KoMV isolates. The CP gene of the three isolates had 93.1–100% (nucleotide) and 98.2–100% (amino acid) identity. The 3′‐UTR of the three isolates showed maximum identity of 91.1–100% identity between and with other KoMV isolates. In the CP amino acid–based phylogenetic analyses, the isolates branched as a distinct cluster along with known KoMV isolates. The three potyvirus isolates associated with mosaic, chlorotic feathery mottling, chlorotic spots, leaf deformation and chlorotic ring spots on three aroids were identified as isolates of KoMV for the first time from Andhra Pradesh, India.     

Role of Genetic Recombination in the Molecular Architecture of Papaya ringspot virus

Papaya ringspot virus (PRSV) has a single-stranded RNA genome and causes severe economic losses both in cucurbits and papaya worldwide. The extent to which the genome of PRSV is shaped by recombination provides an understanding of the molecular evolution of PRSV and helps in studying features such as host specificity, geographic distribution, and its emergence as new epidemics. The PRSV-P-Indian isolate was completely sequenced and compared with 14 other isolates reported from the rest of the world for their phylogenetic survey of recombination events. Cistron-by-cistron sequence comparison and phylogenetic analysis based on full-genome polyprotein showed two distinct groupings of Asian and American isolates, although PRSV-P and W-India clustered along with the American isolates. Recombination sites were found throughout the genomes, except in the small 6K1 protein gene. A significant proportion of recombination hotspots was found in the P1 gene, followed by P3, cylindrical inclusion (CI), and helper component proteinase (HcPro). Correlations between the presence of recombination sites, geographic distribution, and phylogenetic relationship provide an opportunity to establish the molecular evolution and geographic route of PRSV.     

Genetic variability of tobacco streak virus in South India based on the analysis of coat protein gene

Tobacco streak virus (TSV), a member of the genus Ilarvirus, family Bromoviridae is an important viral pathogen in peanut and other crops in South India. Fifteen TSV isolates naturally infecting groundnut, sunflower, onion, black gram, green gram, jute, tagetes, calotropis, pumpkin, watermelon and kenaf plants were collected from fields in different regions of Andhra Pradesh, Tamil Nadu and Karnataka. Virus was identified as TSV by direct antigen coating enzyme linked immunosorbent assay using TSV antiserum. The CP gene from each isolate was amplified using TSV coat protein specific primers. About 700 bp product was amplified, cloned, sequenced and determined its length as 717 nucleotides and codes for 239 amino acids. The sequence analysis revealed that the CP gene shared 91–100% and 91–99% sequence identity with TSV at nucleotide and amino acid level, respectively. The phylogenetic relationship based on the nucleotide sequence of these isolates from different geographical regions was also analysed in this study.     

Complete nucleotide sequence of the 6 kb element and conserved cytochrome b gene sequences among Indian isolates of Plasmodium falciparum.

The malaria parasite contains a nuclear genome with 14 chromosomes and two extrachromosomal DNA molecules of 6 kb and 35 kb in size. The smallest genome, known as the 6 kb element or mitochondrial DNA, has been sequenced from several Plasmodium falciparum isolates because this is a potential drug target. Here we describe the complete nucleotide sequence of this element from an Indian isolate of P. falciparum. It is 5967 bp in size and shows 99.6% homology with the 6 kb element of other isolates. The element contains three open reading frames for mitochondrial proteins-cytochrome oxidase subunit I (CoI), subunit III (CoIII) and cytochrome b (Cyb) which were found to be expressed during blood stages of the parasite. We have also sequenced the entire cyb gene from several Indian isolates of P. falciparum. The rate of mutation in this gene was very low since 12 of 14 isolates showed the identical sequence. Only one isolate showed a maximum change in five amino acids whereas the other isolate showed only one amino acid change. However, none of the Indian isolates showed any change in those amino acids of cyb which are associated with resistance to various drugs as these drugs are not yet commonly used in India.