Incidence and Distribution of Grapevine Leafroll-associated viruses in Tunisian Vineyards

Surveys were carried out in Tunisian table grape vineyards for assessing the occurrence and distribution of grapevine leafroll-associated viruses (GLRaVs). Leafroll symptoms were commonly observed in most of the surveyed vineyards. Samples were randomly collected from 712 individual vines for laboratory testing. Enzyme-linked immunosorbent assay (ELISA) tests showed that 81.5% of the vines were infected by one (35.7%) or more (45.8%) viruses. GLRaV-3 was the most widespread virus (76.3%), followed by GLRaV-5 (38.5 %), GLRaV-6 (13.2%), GLRaV-1 (9.1%), GLRaV-2 (6.3%), and GLRaV-7 (0.9%). GLRaV-3 and GLRaV–5, two mealybug-transmissible Ampeloviruses, were present in mixture in 35.9% of samples. The highest infection rate was found in Cape Bon region (81.7 %), where cv. Italia had an infection rate of 79.5%. Superior seedless, the main cultivar in Sidi Bouzid, had 75% infection. GLRaV-6 and -7 were detected for the first time in Tunisia.     

High-Sensitivity Detection of Fruit Tree Viruses Using Bacterial Magnetic Particles

Prunus necrotic ring spot virus (PNRSV) and grapevine fanleaf virus (GFLV) were detected by fluoroimmunoassay using bacterial magnetic particles (BMPs),and a double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA).For the fluoroimmunoassay,fluorescein isothiocyanate labeled anti-PNRSV antibody or anti-GFLV antibody was conjugated onto BMPs of Magnetospirillum gryphiswaldense MSR-I.With this method,a very low minimum antigen concentration (1 x 106 dilution of the original sample concentration) could be detected.Using DAS-ELISA,the minimum antigen detection concentration was the original sample concentration.Thus,comparing these two methods,a BMP-based method could increase the sensitivity up to six orders of magnitude (106) higher than an ELISA-based method of detection PNRSV and GFLV.     

三重RT-PCR同步检测马铃薯多种病毒影响因素

根据病毒外壳蛋白区序列设计PVX、PVS特异性引物对,根据P1基因区序列设计PVA特异性引物对,应用三重RT-PCR同步检测马铃薯X病毒,马铃薯A病毒及马铃薯S病毒,分别得到562bp、255bp、182bp大小的扩增片段。试验从反转录反应、PCR反应及循环条件3方面讨论了试剂和循环条件对三重RT-PCR同步检测3种病毒的影响。结果表明反转录反应中dNTPs浓度、3种病毒下游引物浓度比例对整个反应影响较大;其次是PCR反应中MgC12浓度和退火温度;反转录时间,循环条件对RT-PCR影响较小。     

High-Sensitivity Detection of Fruit Tree Viruses Using Bacterial Magnetic Particles

Prunus necrotic ring spot virus （PNRSV） and grapevine fanleaf virus （GFLV） were detected by fluoroimmunoassay using bacterial magnetic particles （BMPs）, and a double antibody sandwich enzyme linked immunosorbent assay （DAS-ELISA）. For the fluoroimmunoassay, fluorescein isothiocyanate labeled anti-PNRSV antibody or anti-GFLV antibody was conjugated onto BMPs of Magnetospirillum gryphiswaldense MSR-1. With this method, a very low minimum antigen concentration （1×10^6 dilution of the original sample concentration） could be detected. Using DAS-ELISA, the minimum antigen detection concentration was the original sample concentration. Thus, comparing these two methods, a BMP-based method could increase the sensitivity up to six orders of magnitude （10^6） higher than an ELISA-based method of detection PNRSV and GFLV.     

Detection of Grapevine Viruses in Poland

During a 3‐year study, grapevines from 23 vineyards in Poland were surveyed for virus diseases and tested to determine the prevalence of the most economically important viruses by RT‐PCR. The rate of positive samples was 2.2% for grapevine leafroll‐associated virus 1 (GLRaV‐1), 1.9% for grapevine leafroll‐associated virus 2 (GLRaV‐2), 1.5% grapevine leafroll‐associated virus 3 (GLRaV‐3), 1.9% for grapevine virus A (GVA), 0.2% for grapevine virus B (GVB), 0.2% for grapevine virus E (GVE), 0.65% for grapevine fanleaf virus (GFLV), 20.4% for grapevine fleck virus (GFkV) and 71.9% for grapevine rupestris stem pitting‐associated virus (GRSPaV). These viruses were found to occur as single or mixed infections of different combinations in individual grapevines. The overall viral infection rate in the surveyed grapevines was 82.6%. GRSPaV is the most widely distributed virus of all the viruses currently detected in the region. DNA sequencing confirmed the identification of the viruses in selected samples, and analysis indicated that the Polish isolates shared a close molecular identity with the corresponding isolates in GenBank. To our knowledge, this is the first detection of GLRaV‐1, ‐2, ‐3, GVA, GVB, GVE, GFLV, GFkV and GRSPaV in Poland.     

Nematode vectors of plant viruses in New Zealand

Abstract

A survey of virus vector nematodes based on a total of 320 published and recent records revealed that 3 Longidorus, 1 Paralongidorus, 6 Xiphinema, 2 Trichodoms, and 3 Paratrichodorus species are present in New Zealand. Xiphinema diversicaudatum and Paratrichodorus minor are the most common and widespread species. Longidorus orongorongensis, an undescribed Paralongidorus species and Trichodoms cottieri appear to be endemic species, and Longidorus taniwha, Paratrichodorus lobatus and probably one (still unidentified) species of the X. americanum group are also indigenous to New Zealand. Most of the other species (e.g. Longidorus elongatus, X. diversicaudatum, and Trichodoms primitivus) were probably introduced from Europe. Maps showing the distribution of the 15 species in New Zealand, and illustrations and tables of the morphological characters for species identification, are presented. Several species are of economic importance through direct damage they cause to cultivated plants, and all can be considered potential vectors of plant viruses.     

Use of Optimised PCR Methods for the Detection of GLRaV3: A Closterovirus Associated with Grapevine Leafroll in Tunisian Grapevine Plants

We report a modification and optimisation of a previously published procedure (Minafra and Hadidi, 1994) for the detection of GLRaV3 in infected grapevine plants. GLRaV3 RNA was successfully detected not only in total crude nucleic acid extracts of infected grapevine tissues but also in viruliferous mealybug extracts by IC-RT-PCR. This detection was rapid, sensitive and specific without occurrence of any background. A comparative ELISA, RT-PCR and IC-RT-PCR assays were carried out and revealed the greater sensitivity and specificity of PCR techniques.     

五种主要上呼吸道病毒多重RT-PCR 检测方法的建立

目的:建立甲型、乙型流感病毒、呼吸道合胞病毒A型、B型(RSV-A、RSV-B)和腺病毒(ADV)五种主要上呼吸道病毒的多重RT-PCR检测方法。方法:利用Primer premier5.0分别针对甲型流感病毒的M基因、乙型流感病毒的PB1基因、RSV-A和RSV-B的F基因及ADV的hexon基因设计五对特异性引物,对Mg2+、dNTP、引物浓度及退火温度等进行优化,建立同时检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV的多重RT-PCR方法,并验证该检测方法的灵敏性。结果:所建立的五种病毒的多重RT-PCR方法可以同时或者分别扩增甲型、乙型流感病毒、RSV-A、RSV-B及ADV的141bp、635bp、525bp、377b和283bp基因片段,敏感度分别达到770PFU/ml、800PFU/ml、680PFU/ml、970PFU/ml和850PFU/ml,且五种病毒间无交叉反应。结论:所建立的多重RT-PCR方法可以迅速准确地检测甲型、乙型流感病毒、RSV-A、RSV-B和ADV,为五种病毒的检测提供了一种方便易行的方法。     

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA — including poly(rA) carrier and ribosomal RNA — from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based ''tagmentation'' step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.     

Incidence of Heat-Resistant Molds in Eastern Orchards and Vineyards

Over 70% of the samples of fruit, vegetation, and soil obtained in surveys of New York orchards and vineyards were contaminated with heat-resistant molds. The counts generally were low, under one per gram. Byssochlamys fulva was the most common isolate. Other isolates were identified as B. nivea, Paecilomyces varioti, Aspergillus fischeri, A. fischeri var. spinosus, A. fumigatus, Penicillium vermiculatum, and P. ochro-chloron.     

Incidence of Prunus necrotic ring spot virus on Malus domestica in India

In surveys of apple (Malus domestica) orchards in various parts of Himachal Pradesh, samples from trees showing necrotic symptoms on the leaves were collected and tested for detection of Prunus necrotic ringspot virus (PNRSV) initially by ELISA followed by RT‐PCR using coat protein gene primers. Positive results were obtained in samples from Kullu and Kalpa regions. The virus gene sequences showed 88–97% similarity to corresponding sequences of other PNRSV isolates deposited in the GenBank database using ncbi.nih.nlm.gov. Although the similarity was high, there were some distinct differences with the Spanish isolate. This is the first report of PNRSV in apple from India.     

Incidence and detection of negative-stranded RNA viruses infecting apple and pear trees in California

Novel negative-stranded RNA (nsRNA) viruses have been recently identified in multiple agronomic crops, including pome fruit trees. Citrus concave gum-associated virus (CCGaV), citrus virus A (CiVA) and apple rubbery wood viruses 1 and 2 (ARWV1 and 2) are examples of such viruses. Given the novelty and lack of information about these pathogens in Californian orchards, in this study, real-time RT-PCR assays for CCGaV, CiVA, ARWV1 and 2 were developed and employed in a field survey. Initially, the new assays were challenged against a comprehensive set of positive and negative samples, previously analysed by high-throughput sequencing (HTS), to determine specificity. Aiming to investigate the presence of nsRNA viruses in California apple and pear orchards, 186 samples were collected from 21 different locations. As a result, 79 (42%) samples were found to be infected by these viruses in single or mixed infections. The incidence of each virus in relation to the total number of samples was 36%, 15%, 11% and 0% for ARWV2, CCGaV, ARWV1 and CiVA respectively. Overall, not considering the no detected CiVA, the other three nsRNA viruses were widely distributed among sampled orchards. To further validate the reliability of the new real-time RT-PCR assays, six samples tested positive during the survey were screened by previously described detection assays and HTS. This is the first detection of these nsRNA viruses in California, which may represent an issue in apple and pear production.     

Viruses infecting sweet potato in Rwanda: occurrence and distribution

A survey of sweet potato virus diseases was conducted in the major sweet potato production areas in low, medium and high altitude zones of Rwanda. A total of 205 symptomatic and 103 asymptomatic samples were collected from 51 sweet potato fields and assayed for Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato latent virus (SwPLV), Sweet potato caulimo‐like virus (SPCaLV) and Cucumber mosaic virus (CMV) using nitrocellulose membrane enzyme‐linked immunosorbent assay. The viruses detected in the samples were SPFMV, SPMMV, SPCSV, SPCFV and SwPLV. Viruses were detected in 83% and 31% of the symptomatic and asymptomatic samples, respectively. SPFMV was detected in 49% of the samples. SPCSV, the second most common virus, was detected in 28% of samples collected from 73% of the fields. About 19% of the samples were tested positive for SPMMV. Thirteen combinations of multiple virus infections were detected in the samples. Viruses were detected in samples from all the fields surveyed, and the frequency of detection was greatest in samples from low altitude zones.     

口蹄疫病毒非结构蛋白3AB 的原核表达及应用

旨在建立一种检测口蹄疫病毒非结构蛋白抗体的敏感、特异的ELISA方法。克隆、表达了口蹄疫病毒非结构蛋白3AB基因,原核表达的重组蛋白经亲和层析法纯化及Western blotting鉴定后作为包被抗原,建立检测口蹄疫病毒非结构蛋白抗体的3AB间接ELISA方法,通过与商品化试剂盒3ABC-ELISA的比对试验对其进行评价。结果显示,重组蛋白3AB以包涵体形式表达;能与口蹄疫病毒感染血清发生特异性反应,而不能与疫苗免疫动物血清发生反应;在检测田间样品时,与3ABC-ELISA具有同样的特异性和敏感性 (P＞     

几种常见花生病毒血清学关系的研究

花生是世界上重要的经济作物,中国也是个花生消费大国,但是常有8种重要的花生病毒危害,包括花生斑驳病毒（PMV）、花生条纹病毒（PStV）、花生丛簇病毒（GRV）、花生丛矮病毒（PCV）、著茄斑萎病毒（TSWV）、花生芽枯病毒（BNN）、花生矮化病毒（PSV）和黄瓜花叶病毒（CMV）。70年代以来在全国各地多次爆发花生病毒病,并引起大面积减产,经济损失巨大,因而花生病毒的检测和转基因花生抗毒株的选育变得尤为重要［‘－’］。本研究应用免疫双扩散和酶联免疫法（ELISA）Z4几种花生病毒的血清学关系进行了研究。三材料与方法…