Discovery of the First Insect Nidovirus, a Missing Evolutionary Link in the Emergence of the Largest RNA Virus Genomes

Nidoviruses with large genomes (26.3–31.7 kb; ‘large nidoviruses’), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7–15.7 kb; ‘small nidoviruses’). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3′-5′exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60–80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3′-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3′-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2′-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that – in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.

基因组大小为26.3~31.7 kb的尼多病毒（Nidoviruses），涵盖冠状病毒科（Coronaviridae）与罗尼病毒科（Roniviridae），是结构最为复杂的正链单链RNA（positive-sense single-stranded RNA, ssRNA+）病毒。基于基因组尺寸，它们与所有其他ssRNA+病毒（基因组均小于19.6 kb）存在显著分化，其中亦包含亲缘关系较远的动脉炎病毒科（Arteriviridae，基因组12.7~15.7 kb，即‘小型尼多病毒’）。作为ssRNA+病毒中的特例，大型尼多病毒编码一种3'-5'外切核糖核酸酶（Exoribonuclease, ExoN），该酶被证实参与调控RNA复制保真度。这类病毒的祖先或许通过获得该酶演化而来，本文借助比较基因组学方法，结合新发现的首株昆虫源性尼多病毒，为这一假说提供了演化生物学层面的支持证据。该病毒被命名为南定病毒（Nam Dinh virus, NDiV），得名于越南的一个省份，从蚊子体内分离得到，目前尚未发现其与任何病理状态相关联。这种包膜病毒的直径为60~80 nm，基因组全长20192 nt，具备尼多病毒典型的多顺反子组构：包含两个大型、部分重叠的开放阅读框（open reading frames, ORF）1a与1b，其后接续数个位于3'端近端的小型开放阅读框。肽段测序结果将三种病毒粒子蛋白分别对应至ORF 2a、2b与3，这些蛋白由两个3'端共线的亚基因组RNA（subgenomic RNAs）表达。NDiV的ORF1a/ORF1b移码信号（frameshifting signal）与多种复制相关蛋白被初步定位至尼多病毒基因组中的经典位置，其中包括6个在所有尼多病毒中保守的复制酶结构域，以及大型尼多病毒特有的ExoN与2'-O-甲基转移酶（2′-O-methyltransferase）。NDiV的ORF1b还编码一种假定的N7-甲基转移酶（N7-methyltransferase），该酶仅在部分大型尼多病毒中被鉴定到；而尿苷酸特异性核酸内切酶（uridylate-specific endonuclease）则未在该病毒中存在——与当前学界的认知范式不同的是，这类酶目前仅见于已报道的脊椎动物尼多病毒。通过贝叶斯法（Bayesian）与最大似然法（Maximum Likelihood）构建的有根系统发育树显示，NDiV与罗尼病毒科聚类为一支，且该分支在大型尼多病毒从小型尼多病毒分化后不久，便从大型尼多病毒的演化谱系中分离出来。综合以上特征，NDiV可被认定为一个新的尼多病毒科的代表毒株，同时也是小型尼多病毒向大型尼多病毒演化过程中的一个缺失环节。