USDA-ARS, Molecular Plant Pathology Laboratory, Beltsville MD, United States of America.
INRA, UMR DGIMI, Montpellier, France.
PLoS One. 2018 Sep 4;13(9):e0203477. doi: 10.1371/journal.pone.0203477. eCollection 2018.
Viral infections of alfalfa are widespread in major cultivation areas and their impact on alfalfa production may be underestimated. A new viral species, provisionally named alfalfa virus F (AVF), was identified using a virion-associated nucleic acid (VANA) metagenomics-based approach in alfalfa (Medicago sativa L.) samples collected in Southern France. The nucleotide sequence of the viral genome was determined by de-novo assembly of VANA reads and by 5'/3' RACE with viral RNA extracted from enriched viral particles or with total RNA, respectively. The virus shares the greatest degree of overall sequence identity (~78%) with Medicago sativa marafivirus 1 (MsMV1) recently deduced from alfalfa transcriptomic data. The tentative nucleotide sequence of the AVF coat protein shares ~83% identity with the corresponding region of MsMV1. A sequence search of the predicted single large ORF encoding a polyprotein of 235kDa in the Pfam database resulted in identification of five domains, characteristic of the genus Marafivirus, family Tymoviridae. The AVF genome also contains a conserved "marafibox", a 16-nt consensus sequence present in all known marafiviruses. Phylogenetic analysis of the complete nucleotide sequences of AVF and other viruses of the family Tymoviridae grouped AVF in the same cluster with MsMV1. In addition to 5' and 3' terminal extensions, the identity of the virus was confirmed by RT-PCRs with primers derived from VANA-contigs, transmission electron microscopy with virus-infected tissues and transient expression of the viral coat protein gene using a heterologous virus-based vector. Based on the criteria demarcating species in the genus Marafivirus that include overall sequence identity less than 80% and coat protein identity less than 90%, we propose that AVF represents a distinct viral species in the genus Marafivirus, family Tymoviridae.
紫花苜蓿的病毒感染在主要种植区广泛存在，其对紫花苜蓿生产的影响可能被低估了。通过在法国南部采集的紫花苜蓿（Medicago sativa L.）样本，采用基于病毒相关核酸（VANA）宏基因组的方法，鉴定出了一种新的病毒种，暂命名为苜蓿病毒 F（AVF）。通过对 VANA 读段进行从头组装，并分别用从富集的病毒颗粒中提取的病毒 RNA 或总 RNA 进行 5'/3' RACE，确定了病毒基因组的核苷酸序列。该病毒与最近从紫花苜蓿转录组数据中推导的 Medicago sativa marafivirus 1（MsMV1）具有最高的整体序列同一性（~78%）。AVF 外壳蛋白的暂定核苷酸序列与 MsMV1 的相应区域具有约 83%的同一性。在 Pfam 数据库中对预测的编码 235kDa 多蛋白的单一大型 ORF 的核苷酸序列进行序列搜索，结果鉴定出 5 个结构域，这些结构域是 Tymoviridae 科 Marafivirus 属的特征。AVF 基因组还包含一个保守的“marafibox”，这是所有已知的 marafivirus 中都存在的 16nt 保守序列。AVF 和 Tymoviridae 科其他病毒的完整核苷酸序列的系统发育分析将 AVF 与 MsMV1 聚在同一簇中。除了 5'和 3'末端延伸外，还通过使用源自 VANA 连续序列的引物进行 RT-PCR、用感染组织的透射电子显微镜以及使用异源病毒载体瞬时表达病毒外壳蛋白基因，确认了病毒的同一性。根据在 Marafivirus 属中划分种的标准，包括整体序列同一性小于 80%和外壳蛋白同一性小于 90%，我们提出 AVF 代表 Tymoviridae 科 Marafivirus 属中的一个独特病毒种。
