Detection of Cymbidium mosaic virus in Protocorm-Like Bodies in Dendrobium Sonia using One-Step RT-PCR

Dendrobium orchids are clonally mass propagated by tissue culture techniques. However, Cymbidium mosaic virus is prevalent in vegetatively propagated Dendrobium spp. Therefore, a sensitive virus detection method is an important requirement for the production of CymMV-free orchid plants and control of virus spreading in the orchid industry. The presence of CymMV was screened in in vitro PLBs of Dendrobium Sonia using a one-step RT-PCR with specific primers to amplify a 858 bp fragment of a CymMV coat protein gene and flanking regions. Results were compared with those obtained from a conventional indirect ELISA. Only 22% samples showed the presence of CymMV in indirect-ELISA as compared to 78% in RT-PCR. Thus, the one-step RT-PCR appears to be a more sensitive method than indirect-ELISA for detecting CymMV in PLBs.     

小滴玻璃化法脱除蝴蝶兰种苗建兰花叶病毒的研究

以感染建兰花叶病毒(Cymbidium mosaic virus,CymMV)的蝴蝶兰(Phalaenopsis aphrodite)品种‘满天红’为试材,通过筛选蔗糖预培养浓度、预培养时间、PVS2(Plant vitrification solution 2,PVS2)处理时间三个关键因素,建立蝴蝶兰茎尖小滴玻璃化超低温脱毒体系,将再生的茎尖诱导类原球茎,再分化成苗,经RT-PCR检测CymMV的脱除情况,阴性结果的再生植株进行增殖和诱导生根。结果显示:最佳预培养为:BM+0.6 mol·L-1蔗糖处理1~2 d,超低温茎尖的成活率为70%~76.7%,再生率为53.3%~56.7%;PVS2最佳处理时间为60~90 min,超低温茎尖的成活率为73.3%~76.7%,再生率为50.0%~56.7%。再生植株经RT-PCR检测,CymMV的脱除率为50%。该研究为兰科植物脱除CymMV提供了理论和技术基础。     

Genome-wide analysis of small RNAs from Odontoglossum ringspot virus and Cymbidium mosaic virus synergistically infecting Phalaenopsis

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the two most prevalent viruses infecting orchids and causing economic losses worldwide. Mixed infection of CymMV and ORSV could induce intensified symptoms as early at 10 days post-inoculation in inoculated Phalaenopsis amabilis, where CymMV pathogenesis was unilaterally enhanced by ORSV. To reveal the antiviral RNA silencing activity in orchids, we characterized the viral small-interfering RNAs (vsiRNAs) from CymMV and ORSV singly or synergistically infecting P. amabilis. We also temporally classified the inoculated leaf-tip tissues and noninoculated adjacent tissues as late and early stages of infection, respectively. Regardless of early or late stage with single or double infection, CymMV and ORSV vsiRNAs were predominant in 21- and 22-nt sizes, with excess positive polarity and under-represented 5ʹ-guanine. While CymMV vsiRNAs mainly derived from RNA-dependent RNA polymerase-coding regions, ORSV vsiRNAs encompassed the coat protein gene and 3ʹ-untranslated region, with a specific hotspot residing in the 3ʹ-terminal pseudoknot. With double infection, CymMV vsiRNAs increased more than 5-fold in number with increasing virus titres. Most vsiRNA features remained unchanged with double inoculation, but additional ORSV vsiRNA hotspot peaks were prominent. The potential vsiRNA-mediated regulation of the novel targets in double-infected tissues thereby provides a different view of CymMV and ORSV synergism. Hence, temporally profiled vsiRNAs from taxonomically distinct CymMV and ORSV illustrate active antiviral RNA silencing in their natural host, Phalaenopsis, during both early and late stages of infection. Our findings provide insights into offence–defence interactions among CymMV, ORSV and orchids.     

Complete nucleotide sequence analysis of <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> Indian isolate: further evidence for natural recombination among potexviruses

The complete nucleotide sequence of an Indian strain of Cymbidium mosaic virus (CymMV) was determined and compared with other potexviruses. Phylogenetic analyses on the basis of RNA-dependent RNA polymerase (RdRp), triple gene block protein and coat protein (CP) amino acid sequences revealed that CymMV is closely related to the Narcissus mosaic virus (NMV), Scallion virus X (SVX), Pepino mosaic virus (PepMV) and Potato aucuba mosaic virus (PAMV). Different sets of primers were used for the amplification of different regions of the genome through RT-PCR and the amplified genes were cloned in a suitable vector. The full genome of the Indian isolate of CymMV from Phaius tankervilliae shares 96–97% similarity with isolates reported from other countries. It was found that the CP gene of CymMV shares a high similarity with each other and other potexviruses. One of the Indian isolates seems to be a recombinant formed by the intermolecular recombination of two other CymMV isolates. The phylogenetic analyses, Recombination Detection Program (RDP2) analyses and sequence alignment survey provided evidence for the occurrence of a recombination between an Indian isolate (AM055720) as the major parent, and a Korean type-2 isolate (AF016914) as the minor parent. Recombination was also observed between a Singapore isolate (U62963) as the major parent, and a Taiwan CymMV (AY571289) as the minor parent.     

建兰花叶病毒单克隆抗体的制备及检测应用

用建兰花叶病毒(Cymbidium mosaic virus,CymMV)免疫的BALB/C鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得3株能稳定传代并分泌抗CymMV单克隆抗体(McAb)的杂交瘤细胞(2C6、5B7和12G9),分别制备它们的单抗腹水。其中5B7和12G92株单克隆抗体腹水间接ELISA效价达10-6,3株单抗的抗体类型及亚类均为IgG1,轻链均为κ链。利用单克隆抗体建立了抗原包被间接ELISA(ACP-ELISA)检测CymMV的方法。蝴蝶兰病叶作1∶10240倍稀释、提纯CymMV病毒浓度为4.87ng/mL(每孔的病毒绝对量为0.487ng)时,该方法仍能检测到病毒。利用ACP-ELISA方法检测了田间样品,发现CymMV在兰花上发病很普遍。     

Development and evaluation of a broadly reactive reverse transcription recombinase polymerase amplification assay for rapid detection of murine norovirus

Background

Murine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. The rapid detection assay for MNV would be a useful tool for monitoring and preventing MNV infection. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV.

Results

The detection limit of the RT-RPA assay for the detection of MNV was 1?×?10² copies of RNA molecules per reaction. The assay was specific since there was no cross-reaction with other common murine viruses. In addition, the broad reactivity of the RT-RPA assay was validated using the synthesized template carrying seven point mutations among several MNV strains. The MNV RT-RPA assay could detect as few as 1?×?10² copies of the mutant per reaction, suggesting the assay could be broadly reactive against a large diversity of MNV strains. Forty eight clinical samples including 16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens were tested for the validation of the new developed RT-RPA assay. The detection results of RT-RPA and RT-qPCR for clinical samples were very similar, except that a gastric tissue sample which was positive by RT-qPCR, with a RNA titer of 27 copies, was negative by RT-RPA.

Conclusions

A broadly reactive RT-RPA assay was successfully established for MNV detection.

     

Argonaute 5 family proteins play crucial roles in the defence against Cymbidium mosaic virus and Odontoglossum ringspot virus in Phalaenopsis aphrodite subsp. formosana

The orchid industry faces severe threats from diseases caused by viruses. Argonaute proteins (AGOs) have been shown to be the major components in the antiviral defence systems through RNA silencing in many model plants. However, the roles of AGOs in orchids against viral infections have not been analysed comprehensively. In this study, Phalaenopsis aphrodite subsp. formosana was chosen as the representative to analyse the AGOs (PaAGOs) involved in the defence against two major viruses of orchids, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). A total of 11 PaAGOs were identified from the expression profile analyses of these PaAGOs in P. aphrodite subsp. formosana singly or doubly infected with CymMV and/or ORSV. PaAGO5b was found to be the only one highly induced. Results from overexpression of individual PaAGO5 family genes revealed that PaAGO5a and PaAGO5b play central roles in the antiviral defence mechanisms of P. aphrodite subsp. formosana. Furthermore, a virus-induced gene silencing vector based on Foxtail mosaic virus was developed to corroborate the function of PaAGO5s. The results confirmed their importance in the defences against CymMV and ORSV. Our findings may provide useful information for the breeding of traits for resistance or tolerance to CymMV or ORSV infections in Phalaenopsis orchids.