Characterization of alfalfa virus F, a new member of the genus Marafivirus

Abstract

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.

High-Throughput Sequencing for Deciphering the Virome of Alfalfa (Medicago sativa L.)

“…Encapsidated viral DNA and RNA are then extracted from enriched viral particles, RNA is converted to cDNA using a 26 nt primer (Dodeca Linker), and double-stranded (ds) DNA is synthesized using large (Klenow)-fragment of DNA polymerase ( Figure 2 ). dsDNAs are further PCR amplified, and the amplicon libraries are sequenced on either the 454 GS FLX Titanium platform ( Bernardo et al, 2018 ) or the Illumina MiSeq platform as 2 × 300 bp paired-end reads ( Nemchinov et al, 2018a ). Viral genomes are assembled using Spades ( François et al, 2018 ), CLC Genomics Workbench ( Candresse et al, 2014 ), IDBA-UD ( Peng et al, 2012 ; Ma et al, 2019 ), CAP3 ( Huang and Madan, 1999 ; Bernardo et al, 2018 ), and other assemblers.…”

“…This novel virus, provisionally designated alfalfa virus F (AVF), was identified using a VANA metagenomics-based approach in symptomless alfalfa samples collected in Southern France ( Nemchinov et al, 2018a ). The distribution of AVF is currently unknown, and it is unlikely to be restricted to a single area.…”

“…Many of the viruses infecting alfalfa have long been known ( Babadoost, 1990 ; Samac et al, 2015 ). In recent years, however, a number of new viral pathogens or pathogens that were not previously described in the crop have been discovered ( Bejerman et al, 2011 , 2015 , 2016 , 2019 ; Roumagnac et al, 2015 ; Nemchinov et al, 2017a , b , 2018a ; Kim et al, 2018 ; Gaafar et al, 2019 ; Jiang et al, 2019a , b ; Samarfard et al, 2020 ). This became possible due to revolutionary advances in nucleic acid sequencing that have been leading to the replacement of traditional detection methods in plant virology with comprehensive, large-scale, unbiased, reliable high-throughput sequencing (HTS) technologies ( Wren et al, 2006 ; Roossinck, 2012b , 2017 ; Nagano et al, 2015 ; Roossinck et al, 2015 ; Jones et al, 2017 ; Villamor et al, 2019 ).…”

Viromes of Ten Alfalfa Plants in Australia Reveal Diverse Known Viruses and a Novel RNA Virus

“…HTS of ADD-diseased plants revealed the presence of several RNA viruses, AMV, BLRV, alfalfa dwarf virus (ADV; genus Cytorhabdovirus, family Rhabdoviridae) and alfalfa enamovirus 1 (genus Enamovirus, family Luteoviridae), and the DNA virus alfalfa leaf curl virus (ALCV; genus Capulavirus, family Geminiviridae) [8][9][10][11][12]. More recently, alfalfa-associated nucleorhabdovirus (genus Nucleorhabdovirus, family Rhabdoviridae), alfalfa virus F (genus Marafivirus, family Tymoviridae), alfalfa virus S (genus Allexivirus, family Alphaflexiviridae), Medicago sativa deltapartitivirus (genus Deltpartitivirus, family Partitiviridae), and alphapartitiviruses including Medicago sativa alphapatitivirus 1 and 2 (family Partitiviridae) were identified in alfalfa with HTS [13][14][15][16][17][18].…”

“…It has many advantages over serological and molecular detection methods that require specific antisera or prior knowledge of nucleotide sequences [19]. Metagenomic HTS has significantly influenced the diagnosis of known and unknown plant viruses as well as genome diversity and evolutionary investigations at different taxonomic levels [14][15][16][17][18][19][20][21][22][23][24][25][26]. Recent advances in HTS technology and improved bioinformatics pipelines have appreciably reduced sequencing costs and time.…”