Complete genome sequence of a novel marafivirus infecting pearl millet in Burkina Faso.

Pearl millet (Pennisetum glaucum (L.) R. Br.) is a staple food that is widely cultivated in sub-Saharan Africa. In August 2018, a survey was conducted in the main producing regions of Burkina Faso, and leaf samples were analyzed using virion-associated nucleic acid (VANA)-based metagenomic approach and Illumina sequencing. A new virus, tentatively named "Pennisetum glaucum marafivirus" (PGMV), was detected, and its complete nucleotide sequence of 6364 nucleotides was determined. The sequence contains a large open reading frame (ORF) encoding a polyprotein of 224.2 kDa with five domains (methyltransferase, papain-like protease, helicase, RNA-dependent RNA polymerase, and coat proteins), typical of marafiviruses. Additionally, a characteristic conserved marafibox domain was detected in the genome. The nucleotide sequence of the complete PGMV genome shares 68.5% identity with that of sorghum bicolor marafivirus, and its coat protein shares 58.5% identity with that of oat blue dwarf virus. Phylogenetic analysis confirmed that the pearl millet virus is unambiguously grouped with members of the genus Marafivirus in the family Tymoviridae. This is the first report on the occurrence of a marafivirus in pearl millet.

Novel Ampeloviruses Infecting Cassava in Central Africa and the South-West Indian Ocean Islands.

Cassava is one of the most important staple crops in Africa and its production is seriously damaged by viral diseases. In this study, we identify for the first time and characterize the genome organization of novel ampeloviruses infecting cassava plants in diverse geographical locations using three high-throughput sequencing protocols [Virion-Associated Nucleotide Acid (VANA), dsRNA and total RNA], and we provide a first analysis of the diversity of these agents and of the evolutionary forces acting on them. Thirteen new Closteroviridae isolates were characterized in field-grown cassava plants from the Democratic Republic of Congo (DR Congo), Madagascar, Mayotte, and Reunion islands. The analysis of the sequences of the corresponding contigs (ranging between 10,417 and 13,752 nucleotides in length) revealed seven open reading frames. The replication-associated polyproteins have three expected functional domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). Additional open reading frames code for a small transmembrane protein, a heat-shock protein 70 homolog (HSP70h), a heat shock protein 90 homolog (HSP90h), and a major and a minor coat protein (CP and CPd respectively). Defective genomic variants were also identified in some cassava accessions originating from Madagascar and Reunion. The isolates were found to belong to two species tentatively named Manihot esculenta-associated virus 1 and 2 (MEaV-1 and MEaV-2). Phylogenetic analyses showed that MEaV-1 and MEaV-2 belong to the genus Ampelovirus, in particular to its subgroup II. MEaV-1 was found in all of the countries of study, while MEaV-2 was only detected in Madagascar and Mayotte. Recombination analysis provided evidence of intraspecies recombination occurring between the isolates from Madagascar and Mayotte. No clear association with visual symptoms in the cassava host could be identified.

Identification, molecular and biological characterization of two novel secovirids in wild grass species in Belgium.

High throughput sequencing was performed on virion-associated nucleic acids (VANA) from a pool of fifty asymptomatic rough bluegrasses (Poa trivialis L.) collected in a Belgian grazed pasture. Bioinformatics analyses produced some contigs presenting similarities with secovirid genomes, in particular nepoviruses and waikaviruses. Three distinct positive-sense single-stranded RNAs including 5' and 3' UTR were reconstructed and they represented two novel viruses infecting rough bluegrass, for which the provisional names poaceae Liege nepovirus A (PoLNVA, 7298 nt for RNA1 and 4263 nt for RNA2) and poaceae Liege virus 1 (PoLV1, 11,623 nt) were proposed. Compared to other Secoviridae members, the highest amino acid identity reached 90.7 % and 66.7 % between PoLNVA and nepoviruses for the Pro-Pol and CP regions respectively, while PoLV1 presented the highest amino acid identity with waikaviruses but with lower identities, i.e. 41.2 % for Pro-Pol and 25.8 % for CP regions, far below the ICTV demarcation criteria for novel secovirid. Based on sequence identity and phylogenetic analyses, PoLNVA was proposed to belong to the genus Nepovirus and PoLV1 as an unclassified secovirids. Detection of the two novel viruses was confirmed in high prevalence in rough bluegrass and ten other wild Poaceae species (Agropyron repens, Agrostis capillaris, Apera spica-venti, Anthoxanthum odoratum, Cynosorus cristatus, Festuca rubra, Holcus lanatus, Lolium perenne, Phleum bertolini and Phleum pratense) by RT-PCR and Sanger sequencing, revealing a diverse host range within Poaceae for these novel secovirids. Seed transmission was evaluated and confirmed for PoLNVA.

A Novel Divergent Geminivirus Identified in Asymptomatic New World Cactaceae Plants.

Cactaceae comprise a diverse and iconic group of flowering plants which are almost exclusively indigenous to the New World. The wide variety of growth forms found amongst the cacti have led to the trafficking of many species throughout the world as ornamentals. Despite the evolution and physiological properties of these plants having been extensively studied, little research has focused on cactus-associated viral communities. While only single-stranded RNA viruses had ever been reported in cacti, here we report the discovery of cactus-infecting single-stranded DNA viruses. These viruses all apparently belong to a single divergent species of the family Geminiviridae and have been tentatively named Opuntia virus 1 (OpV1). A total of 79 apparently complete OpV1 genomes were recovered from 31 different cactus plants (belonging to 20 different cactus species from both the Cactoideae and Opuntioideae clades) and from nine cactus-feeding cochineal insects (Dactylopius sp.) sampled in the USA and Mexico. These 79 OpV1 genomes all share > 78.4% nucleotide identity with one another and < 64.9% identity with previously characterized geminiviruses. Collectively, the OpV1 genomes display evidence of frequent recombination, with some genomes displaying up to five recombinant regions. In one case, recombinant regions span ~40% of the genome. We demonstrate that an infectious clone of an OpV1 genome can replicate in Nicotiana benthamiana and Opuntia microdasys. In addition to expanding the inventory of viruses that are known to infect cacti, the OpV1 group is so distantly related to other known geminiviruses that it likely represents a new geminivirus genus. It remains to be determined whether, like its cactus hosts, its geographical distribution spans the globe.

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

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.

The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed.

Endogenous viral sequences are essentially 'fossil records' that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus. These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21-24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum clade species.