Isolation, characterisation and identification of a potyvirus from Dioscorea alata L. (water yam) in Nigeria

A yam potyvirus was isolated from Dioscorea alata samples collected in Nigeria. The virus was not transmissible mechanically but was transmitted by Aphis craccivora to four cowpea cultivars (Ife Brown, IT84S-2114, IT82E-10 and TVu2657), and from which it could be mechanically transmitted between the cowpea cultivars. In infectivity- tests using cowpea extracts, the virus had a dilution end point of 10^-4, a thermal inactivation point of 60–65°C and longevity in vitro of 2 days at room temperature. The virus coat protein had an estimated molecular weight of 32 100 daltons. The virus was identified as an isolate of Dioscorea alata virus (DAV; syn. yam virus 1) due to its biological characteristics and its serological reaction with antiserum raised against DAV. The virus is not related to yam mosaic virus, but distantly related to blackeye cowpea mosaic virus and cowpea aphid-borne mosaic virus.     

Inheritance of resistance to Yam mosaic virus, genus Potyvirus, in white yam (Dioscorea rotundata)

Yam mosaic virus (YMV) causes the most-widespread and economically important viral disease affecting white yam (Dioscorea rotundata) in West Africa. The genetic basis of resistance in white yam to a Nigerian isolate of YMV was investigated in three tetraploid D. rotundata genotypes: TDr 93–1, TDr 93–2 and TDr 89/01444. F₁ progeny were produced using TDr 87/00571 and TDr 87/00211 as the susceptible parents. Segregation ratios indicated that a single dominant gene in a simplex condition governs the resistance in TDr 89/01444, while the resistance in TDr 93–2 is associated with the presence of a major recessive gene in duplex configuration. Segregation of progeny of the cross TDr 93–1×TDr 87/00211 fitted a genetic ratio of 2.48:1 resistant:susceptible, which can be expected when two simplex heterozygotes are crossed, indicating the possible modifying effect of the susceptible parent. A triple antibody immunosorbent assay (TAS-ELISA) was used for virus detection in inoculated plants. Slight mosaic symptoms appeared on most resistant individuals, while asymptomatic resistant genotypes with high ELISA (A₄₀₅) values were observed in all crosses. Such a heterogeneous response suggests the influence of additional modifier genes that segregate in the progeny. The finding that resistance can be inherited as a dominant or recessive character has important implications for YMV resistance breeding. Received: 15 August 2000 / Accepted: 12 April 2001     

Occurrence of latent virus infection in visually-rated cowpea (Vigna unguiculata L. Walp) seedlings

Abstract

The presence of latent infections was studied in five cowpeas varieties. Seeds of the varieties were planted and the seedlings inoculated with antigens from Cucumber mosaic virus (CMV) genus Cucumovirus, Bean common mosaic virus (BCMV) genus Potyvirus (Blackeye cowpea mosaic virus strain), Southern bean mosaic virus (SBMV) genus Sobemovirus and Cowpea mottle virus (CPMoV) genus Carmovirus seven days after planting. Seedlings expressing symptoms were rouged at two weeks after inoculation, while asymptomatic ones were subjected to serological indexing to detect the presence/absence of latent infection. Protein A-sandwich enzyme-linked immunosorbent assay (PAS ELISA) was employed for the serological detection of CMV, SBMV and CPMoV, while antigen-coated plate (ACP) ELISA was used to detect BCMV in the asymptomatic plants. Cowpea seedlings without virus symptoms but with positive serological reactions were considered as being latently infected. All of the inoculated TVu 1272 and SuVita-2 plants showed symptoms consistent with CMV and CPMoV infections, respectively. The rate of CMV latent infection was high in TVu 1179 (14.5%), low in SuVita-2 (1.3%) but not recorded in TVu 1272.     

Identification and potential use of RAPD markers linked to Yam mosaic virus resistance in white yam (Dioscorea rotundatd)

Resistance to Yam mosaic virus (YMV) in tetraploid white yam (Dioscorea rotundatd) is inherited differentially as a dominant and recessive character. Elite D. rotundata breeding lines with durable resistance to YMV can be developed by pyramiding major dominant and recessive genes using marker‐assisted selection (MAS). The tetraploid breeding line, TDr 89/01444, is a source of dominant genetic resistance to yam mosaic disease. Bulked segregant analysis was used to search for random amplified polymorphic DNA (RAPD) markers linked to YMV resistance in F₁ progeny derived from a cross between TDr 89/01444 and the susceptible female parent, TDr 87/00571. The F₁ progeny segregated 1:1 (resistantsusceptible) when inoculated with a Nigerian isolate of YMV, confirming that resistance to YMV in TDr 89/01444 was dominantly inherited. A single locus that contributes to YMV resistance in TDr 89/01444 was identified and tentatively named Ymv‐1. Two RAPD markers closely linked in coupling phase with Ymv‐1 were identified, both of which were mapped on the same linkage group: OPW18₈₅₀ (3.0 centiMorgans [cM]) and OPX15₈₅₀ (2.0 cM). Both markers successfully identified Ymv‐1 in resistant genotypes among 12 D. rotundata varieties and in resistant F1 individuals from the cross TDr 93–1 × TDr 877 00211, indicating their potential for use in marker‐assisted selection. OPW18₈₅₀ and OPX15₈₅₀ are the first DNA markers for YMV resistance and represent a starting point in the use of molecular markers to assist breeding for resistance to YMV.     

侵染菊花的黄瓜花叶病毒的初步鉴定和血清学检测

侵染菊花的黄瓜花叶病毒的初步鉴定和血清学检测张海保,朱西儒,张云开（中国科学院华南植物研究所广州５１０６５０）关键词菊花,黄瓜花叶病毒,间接ＥＬＩＳＡ菊花（Ｃｈｒａｙｓａｎｔｈｅｍｕｍｍｏｒｉｆｏｌｉｕｍ）病毒病是危害菊花的一类主要病害。国外文献报道...     

几种葫芦科蔬菜病毒病的鉴定及药剂防治研究

分别在甘肃酒泉、陕西户县及临潼3地区蔬菜大田采集黄瓜、西葫芦、南瓜3种蔬菜上表现病毒病症状的毒原标样135个,采用鉴别寄主的生物学反应和双抗夹心ELISA法鉴定,结果表明引起上述地区黄瓜、西葫芦、南瓜3种蔬菜病毒病的毒原主要是黄瓜花叶病毒。3．95％“病毒必克”可湿性粉剂防治3种蔬菜病毒病的结果表明,300倍、500倍的病毒必克预防效果可达到75％～78％,且治疗效果明显,药效持久。因此,认为病毒必克是目前理想的植物病毒抑制剂。     

Preponderance and distribution of plant-parasitic nematodes in Nassarawa State,North Central Nigeria: Consequences for healthy tuber production and export

One of the most cosmopolitan pests responsible for considerable negative economic impacts on yam is the plant-parasitic nematodes. They are also capable of facilitating fungal, bacterial and viral infections of yams. In order to produce healthy yam seeds to meet local demand and tubers for export, there is a need to identify nematode-free areas in Nassarawa, one of the hubs of yam production in Nigeria. To this end, 30 major yam growing communities of the state were surveyed to identify nematode-free areas. Structured questionnaires were administered to three farmers in each of the 10 local government areas selected. Results showed that 97% of the study areas investigated was seriously infested with the three key genera of plant-parasitic nematodes namely Scutellonema, Pratylenchus and Meloidogyne. Incidence and severity of tuber deterioration in transit threatened acceptability of the tubers when it eventually reached England. Healthy yams would attract more income than damaged ones.     

The detection of hydrogen peroxide involved in plant virus infection by fluorescence spectroscopy

The production of reactive oxygen species (ROS) forms part of the defense reaction of plants against invading pathogens. ROS have multifaceted signaling functions in mediating the establishment of multiple responses. To verify whether hydrogen peroxide (H₂O₂) contributes to plant virus infection and the development of induced symptoms, we used fluorescence to monitor the generation of H₂O₂ and confocal laser scanning microscopy (CLSM) to investigate the subcellular distribution of H₂O₂ in leaves. In this study, the M strain of Cucumber mosaic virus (M‐CMV) induced heavy chlorotic symptoms in Nicotiana tabacum cv. white burley during systemic infection. Compared with mock‐inoculated leaves, H₂O₂ accumulation in inoculated leaves increased after inoculation, then decreased after 4 days. For systemically infected leaves that showed chlorotic symptoms, H₂O₂ accumulation was always higher than in healthy leaves. Subcellular H₂O₂ localization observed using CLSM showed that H₂O₂ in inoculated leaves was generated mainly in the chloroplasts and cell wall, whereas in systemically infected leaves H₂O₂ was generated mainly in the cytosol. The levels of coat protein in inoculated and systemically infected leaves might be associated with changes in the level of H₂O₂ and symptom development. Further research is needed to elucidate the generation mechanism and the relationship between coat protein and oxidative stress during infection and symptom development. Copyright © 2016 John Wiley & Sons, Ltd.     

The incidence and distribution of viruses infecting lettuce, cultivated Brassica and associated natural vegetation in Spain

A virus survey was conducted during the spring and autumn of 2001 and 2002 to determine the presence, prevalence and distribution in Spain of the viruses that are most commonly found infecting lettuce and Brassica worldwide. Crop plants showing virus symptoms from the principal lettuce and Brassica-growing regions of Spain, and some samples of the annual and perennial flora nearby, were tested by enzyme-linked immunosorbent assays using specific commercial antibodies against the following viruses: Alfalfa mosaic virus (AMV), Broad bean wilt virus 1 (BBWV-1), Beet western yellows virus (BWYV), Cauliflower mosaic virus (CaMV), Cucumber mosaic virus (CMV), Lettuce mosaic virus (LMV), Pea seed-borne mosaic virus (PSbMV), Turnip mosaic virus (TuMV) and Tomato spotted wilt virus (TSWV). Samples were also tested with a Potyvirus genus antibody. Virus incidence was much lower in spring than in autumn, especially in 2001. In spring 2002, CMV and LMV were the most prevalent viruses in lettuce, while CaMV was the most important virus present in Brassica crops grown in Navarra, followed by CMV and BWYV. In the autumn, the spectrum of viruses was different; potyviruses were widespread in lettuce grown in Madrid, but TSWV and BWYV were predominant in the Murcia region. The prevalent Potyvirus detected in lettuce fields was LMV, but none of the samples collected were positive for PSbMV or TuMV. In Brassica crops, TSWV was the most abundant in autumn-sown crops, especially in the Navarra region. All of the viruses present in lettuce and Brassica were also frequently detected in their associated natural vegetation at the same time, suggesting that they probably play an important role as virus reservoirs. Sonchus spp. were particularly common and were frequently infected with CMV, LMV and BWYV. Another common species, Chenopodium album, was often infected with TSWV and BWYV. Multiple infections were common, especially in non-crop plants, and the most common combination was BWYV and TSWV. The role of weeds in the epidemiology of viruses that infect lettuce and Brassica crops in Spain is discussed.     

核酸酶保护试验在黄瓜花叶病毒株系鉴定中的初步应用

采用黄瓜花叶病毒（ＣＭＶ）亚组Ⅰ株系Ｆｎｙ－ＣＭＶＲＮＡ＿２的１２０９～１６２６核苷酸片段和亚组Ⅱ株系Ｌｓ－ＣＭＶＲＮＡ＿２的２００２～２４３３核苷酸片段的ｃＤＮＡ克隆,体外转录,同时掺入￣（３２）Ｐ获得负链ＲＮＡ探针,与纯化的番茄和甜椒上的ＣＭＶ中国分离物的ＲＮＡ杂交,结果表明：ＣＭＶ番茄和甜椒中国分离物与Ｆｎｙ－ＣＭＶ的核苷酸有高度同源性,隶属于Ｆｎｙ－ＣＭＶ为代表的亚组Ⅰ株系。并利用Ｋ－ＣＭＶ株系（亚组Ⅰ,源于中国）的ＲＮＡ＿２全长ｃＤＮＡ克隆的两个ＥｃｏＲＩ位点间的核苷酸序列（１６５７～２１２５ｎｔ）作探针,与上述两种ＣＭＶ中国分离物的ＲＮＡ杂交,进一步比较分析了这两个分离物和Ｋ－ＣＭＶ株系的关系。讨论了核酸酶保护法在ＣＭＶ株系鉴定中的作用。     

Microarray multiplex assay for the simultaneous detection and discrimination of hepatitis B, hepatitis C, and human immunodeficiency type-1 viruses in human blood samples

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.     

Rapid detection of viruses, transgenes, and mRNAs in small plant leaf samples

A simple method for the detection of DNA and RNA from small tissue samples (<-1 mg) is described. Stabs of fresh leaves from wheat, clover, tobacco, broad bean, grape, tomato, lettuce, or asparagus were taken using glass microcapillaries, combined with RNase inhibitor and subjected to RT-PCR either directly or after a DNase treatment. This method was used to successfully detect the presence of (1) virus in infected plants, (2) transgenes such asneomycin phosphotransferase II in transformed plants and (3) various plant genes including RubiscoL and 1,3-β-D-glucanase. With the described DNase treatment, the technique was shown to effectively detect RNA and, therefore, can be used to localize and study patterns of gene expression, virus distribution, and/or transgene expression. The benefits of the described tissue stab technique are that it is quick, it requires little manipulation, and is applicable to a variety of plants.     

Prevalence of three shrimp viruses in Zhejiang Province in 2008

White spot syndrome virus (WSSV), Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province, China, in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations, 8 farms were positive for WSSV, 8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV, while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples: Bingjiang (93.3%), liuao (66.7%), Jianshan (46.7%) and Xianxiang (46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.     

Monoclonal Antibody-Based Serological Detection of Rice Stripe Mosaic Virus Infection in Rice Plants or Leafhoppers

Rice stripe mosaic virus(RSMV) is a rhabdovirus recently found in southern part of China and can cause severe reduction in rice production. To establish serological methods for RSMV epidemiological studies and to establish a control strategy for this virus, we first purified RSMV virions from infected rice plants and then used them as an immunogen to produce four RSMV-specific monoclonal antibodies(MAbs)(i.e.,1D4, 4A8, 8E4 and 11F11). With these MAbs, we have developed a highly specific and sensitive antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA), a Dot-ELISA and a Tissue print-ELISA for rapid detections of RSMV infection in rice plants or in leafhoppers. Our results showed that RSMV can be readily detected in RSMV-infected rice plant tissue crude extracts diluted at 1:20,971,520(w/v, g/m L)through ACP-ELISA or diluted at 1:327,680(w/v, g/m L) through Dot-ELISA. Both ACP-ELISA and Dot-ELISA can also be used to detect RSMV infection in individual RSMV viruliferous leafhopper(Recilia dorsalis) homogenate diluted at 1:307,200 and 1:163,840(individual leafhopper/l L), respectively. Detection of RSMV infection in field-collected rice samples or in RSMV viruliferous leafhoppers indicated that the three serological methods can produce same results with that produced by RT-PCR(19 of the 33 rice samples and 5 of the 16 leafhoppers were RSMV-positive). We consider that the four MAbs produced in this study are very specific and sensitive, and the three new serological methods are very useful for detections of RSMV infection in rice plants or in leafhoppers and the establishment of the disease control strategies.     

六种猪呼吸道RNA病毒MALDI-TOF MS同步检测体系的构建

【目的】建立猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)、猪瘟病毒(classical swine fever virus,CSFV)、口蹄疫病毒(foot-and-mouth disease virus,FMDV)和猪流感病毒(swine influenza virus,SIV)的基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)多目标检测方法,同时对SIV进行通用型、H1型及H3型分型检测。【方法】本研究根据6种病原体基因的保守序列,设计了6对加标引物及对应的延伸探针并进行单反应试验。通过体系优化引物浓度和反应条件,以及方法特异性、重复性及灵敏度分析,使用MALDI-TOF MS检测方法及荧光定量PCR方法分别对临床样本和猪源产制品进行检测,并对结果进行对比验证。【结果】质谱结果显示,6种产物峰仅在靶标病毒对应的产物位置出现峰...     

基于基质辅助激光解吸/电离飞行时间质谱的猪呼吸道病毒多目标鉴定方法的建立和应用北大核心

【目的】建立能高效同步鉴定猪伪狂犬病毒(porcine pseudorabies virus,PRV)、猪圆环病毒2型(porcine circovirus 2,PCV-2)和3型(porcine circovirus 3,PCV-3)、非洲猪瘟病毒(African swine fever virus,ASFV)以及猪博卡病毒1型(porcine bocavirus group 1,PBoV-G1)、2型(porcine bocavirus group 2,PBoV-G2)和3型(porcine bocavirus group 3,PBoV-G3)等呼吸道病毒的核酸基质辅助激光解吸/电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)高通量多目标检测技术。【方法】根据7种病原体基因的保守序列,分别设计不同病原的引物及对应的单碱基延伸探针,通过引物浓度和反应条件优化,方法特异性、敏感性和稳定性分析,以及临床样本和猪源产制品的检测验证,建立常见猪呼吸道DNA病毒的MALDI-TOF MS多目标检测体系。【结果】质谱分析显示,多目标检测体系的7种靶标产物峰只在特定病毒阳性样品检测时产生,与其他病原体检测无交叉反应,表明该方法对7种靶标病毒检测特异性良好。重复性试验结果分析显示,体系中每种病毒在高、中、低浓度时批内阳性符合率均≥98.0%,批间均≥98.3%,表明该方法具有较高的稳定性。体系中7种病原体每种病毒最低检测限在8.65–26.27拷贝/μL之间,与荧光PCR(real-time fluorescence quantitative PCR,RT-qPCR)检测方法相当。采用MALDI-TOF MS多重检测方法对100份组织、饲料和猪肉样品进行检测应用,检出2种及以上混合感染样品39份,其中5份样本同步检出5种病原体阳性;对8份ASFV-p72假病毒人工污染样品进行验证,均可检出ASFV阳性。将以上样本检测应用结果与荧光PCR方法进行比对验证,2种方法对于不同病原体检测结果的符合率高达94.4%–100%。【结论】本研究建立的基于MALDI-TOF MS的猪呼吸道常见DNA病毒多重检测方法为猪群相关疫病快速监测和鉴别诊断,以及便利化进出口动物检疫等提供了一种新的敏感、特异的高通量多目标检测技术。     

Propagation of plant viruses in hairy root cultures: a potential method for in vitro production of epitope vaccines and foreign proteins

Hairy roots were used as an in vitro culture system for the propagation of wild-type and transgenic plant viruses. Tobacco mosaic virus (TMV) was added to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by viral infection. Maximum concentrations of TMV in Nicotiana benthamiana hairy roots were 1-2 orders of magnitude greater than in suspended N. benthamiana cells and reached levels of 1-2 mg g(-1) dry weight or 20-28% total soluble protein. Virus accumulated in the roots initially with a constant doubling time of about 1.0 day; subsequent reductions in viral growth rate were correlated with a significant decline in infectivity relative to the inoculum virus. The morphological integrity of the viral particles was maintained during propagation in hairy roots. The contribution to the overall viral titer of passive association of virus with the biomass, for example, by surface adsorption, was negligible compared with active viral replication. N. benthamiana hairy roots were also infected with a TMV-based viral vector developed to express green fluorescent protein (GFP). This vector was about 260-fold less infectious than wild-type TMV and accumulated much more slowly in the roots. Maximum levels of TMV-GFP in the biomass were about 65-fold lower than for TMV. This work demonstrates that hairy root cultures are a feasible means for in vitro propagation of wild-type and transgenic plant viruses under conditions that allow a high degree of environmental containment and control.