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.

中文翻译：

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对协同感染蝴蝶兰的齿兰环斑病毒和蕙兰花叶病毒的小 RNA 进行全基因组分析。

蕙兰花叶病毒（CymMV）和齿兰环斑病毒（ORSV）是感染兰花并在全球范围内造成经济损失的两种最流行的病毒。CymMV 和 ORSV 的混合感染早在接种后 10 天就可以在接种的蝴蝶兰中引起症状加剧，其中 ORSV 单方面增强了 CymMV 的发病机制。为了揭示兰花的抗病毒 RNA 沉默活性，我们对 CymMV 和 ORSV 单独或协同感染兰花的病毒小干扰 RNA (vsiRNA) 进行了表征。阿玛比利斯。我们还将接种的叶尖组织和未接种的邻近组织分别暂时分类为感染的晚期和早期阶段。无论单次感染还是双重感染的早期或晚期，CymMV 和 ORSV vsiRNA 均以 21 和 22 nt 大小为主，正极性过多，5′-鸟嘌呤代表性不足。CymMV vsiRNA 主要源自 RNA 依赖性 RNA 聚合酶编码区，而 ORSV vsiRNA 则包含外壳蛋白基因和 3′非翻译区，且特定热点位于 3′末端假结中。通过双重感染，随着病毒滴度的增加，CymMV vsiRNA 的数量增加了 5 倍以上。两次接种后大多数 vsiRNA 特征保持不变，但额外的 ORSV vsiRNA 热点峰很突出。因此，双重感染组织中新靶标的潜在 vsiRNA 介导调节提供了 CymMV 和 ORSV 协同作用的不同观点。因此，来自分类学上不同的 CymMV 和 ORSV 的 vsiRNA 的时间分析表明，在感染的早期和晚期阶段，它们的天然宿主蝴蝶兰中存在活性抗病毒 RNA 沉默。我们的研究结果提供了对 CymMV、ORSV 和兰花之间攻防相互作用的见解。