Plant virus movement proteins originated from jelly-roll capsid proteins.

Numerous, diverse plant viruses encode movement proteins (MPs) that aid the virus movement through plasmodesmata, the plant intercellular channels. MPs are essential for virus spread and propagation in distal tissues, and several unrelated MPs have been identified. The 30K superfamily of MPs (named after the molecular mass of tobacco mosaic virus MP, the classical model of plant virology) is the largest and most diverse MP variety, represented in 16 virus families, but its evolutionary origin remained obscure. Here, we show that the core structural domain of the 30K MPs is homologous to the jelly-roll domain of the capsid proteins (CPs) of small RNA and DNA viruses, in particular, those infecting plants. The closest similarity was observed between the 30K MPs and the CPs of the viruses in the families Bromoviridae and Geminiviridae. We hypothesize that the MPs evolved via duplication or horizontal acquisition of the CP gene in a virus that infected an ancestor of vascular plants, followed by neofunctionalization of one of the paralogous CPs, potentially through the acquisition of unique N- and C-terminal regions. During the subsequent coevolution of viruses with diversifying vascular plants, the 30K MP genes underwent explosive horizontal spread among emergent RNA and DNA viruses, likely permitting viruses of insects and fungi that coinfected plants to expand their host ranges, molding the contemporary plant virome.

Iranian Strawberry crinkle cytorhabdovirus variation assessed using its movement protein (P3) gene.

BACKGROUND: Strawberry crinkle virus (SCV) is a member of the genus Cytorhabdovirus, family Rhabdovirida, and order Mononegavirales. SCV affects the production of various strawberry cultivars. In this study we investigated the genetic diversity of SCV in strawberry fields based on P3 (movement protein) gene. METHODS AND RESULTS: The samples were collected from strawberry fields in the Kurdistan Province, Iran. P3 gene from 20 SCV isolates, representing 18 nucleic acid haplotypes, is composed of 729 nucleotides, encoding a protein with 243 amino acids. SCV-P3 sequences shared 98.77%-99.86% nucleotide and 97.5%-100% amino acid sequence identity. Phylogenetic analyses of the new P3 sequences with two previously published SCV-P3 sequences from the Czech Republic showed that there are two major phylogroups (I and II) and three minor phylogroups in the body of the phylogeny, I-1, I-2, II-1. Comparisons of P3 gene sequences revealed a mutational bias, with more differences being transitions than transversions. The ratio of non-synonymous/synonymous nucleotide changes was < 1, indicating that SCV-P3 gene is under predominantly negative selection. CONCLUSIONS: Phylogenetic and sequence identity analyses showed that SCV isolates from Iran are closely related and have not diverged more than 2% based on P3 gene despite geographical separation and strawberry cultivar. This is the first report of the genetic diversity of SCV worldwide.

The 28 Ser Amino Acid of Cucumber Mosaic Virus Movement Protein Has a Role in Symptom Formation and Plasmodesmata Localization.

Cucumber mosaic virus (CMV, Cucumovirus, Bromoviridae) is an economically significant virus infecting important horticultural and field crops. Current knowledge regarding the specific functions of its movement protein (MP) is still incomplete. In the present study, potential post-translational modification sites of its MP were assayed with mutant viruses: MP/S28A, MP/S28D, MP/S120A and MP/S120D. Ser28 was identified as an important factor in viral pathogenicity on Nicotiana tabacum cv. Xanthi, Cucumis sativus and Chenopodium murale. The subcellular localization of GFP-tagged movement proteins was determined with confocal laser-scanning microscopy. The wild type movement protein fused to green fluorescent protein (GFP) (MP-eGFP) greatly colocalized with callose at plasmodesmata, while MP/S28A-eGFP and MP/S28D-eGFP were detected as punctate spots along the cell membrane without callose colocalization. These results underline the importance of phosphorylatable amino acids in symptom formation and provide data regarding the essential factors for plasmodesmata localization of CMV MP.

Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein.

Viruses cause many severe plant diseases, resulting in immense losses of crop yield worldwide. Therefore, developing novel approaches to control plant viruses is crucial to meet the demands of a growing world population. Recently, RNA interference (RNAi) has been widely used to develop virus-resistant plants. Once genome replication and assembly of virion particles is completed inside the host plant, mature virions or sometimes naked viral genomes spread cell-to-cell through plasmodesmata by interacting with the virus-encoded movement protein (MP). We used the RNAi approach to suppress MP gene expression, which in turn prevented potato leafroll virus (PLRV) systemic infection in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with MP siRNA constructs exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of MP gene expression is an efficient method for generating PLRV-resistant potato plants. Further, we identified novel ATPase motifs in MP that may be involved in DNA binding and translocation through plasmodesmata. We also showed that the ATPase activity of MP was stimulated in the presence of DNA/RNA. Overall, our findings provide a robust technology to generate PLRV-resistant potato plants, which can be extended to other species. Moreover, this approach also contributes to the study of genome translocation mechanisms of plant viruses.

Identification of two additional plasmodesmata localization domains in the tobacco mosaic virus cell-to-cell-movement protein.

Despite decades of intensive studies, the failure to identify plasmodesmata (PD) localization sequences has constrained our understanding of Tobacco mosaic virus (TMV) movement. Recently, we identified the first PD localization signal (major PLS) in the TMV movement protein (MP), which encompasses the first 50 amino acid residues of the MP. Although the major PLS is sufficient for PD targeting, the efficiency is lower than the full-length TMV MP. To address this efficiency gap, we identified two additional PLS domains encompassing amino acid residues 61 to 80, and 147 to 170 of the MP and showed that these two domains target to PD, but do not transit to adjacent cells. We also demonstrated that the MP61-80 fragment interacts with Arabidopsis synaptotagmin A, which was also shown to interact with the major TMV MP PLS. Therefore, our findings have provided new insights to more fully understand the mechanism underlying plasmodesmal targeting of TMV MP.

Identification of cucurbit chlorotic yellows virus P4.9 as a possible movement protein.

BACKGROUND: Cucurbit chlorotic yellows virus (CCYV) is a bipartite cucurbit-infecting crinivirus within the family Closteroviridae. The crinivirus genome varies among genera. P4.9 is the first protein encoded by CCYV RNA2. P5, which is encoded by LIYV, is necessary for efficient viral infectivity in plants; however, it remains unknown whether CCYV P4.9 is involved in movement. FINDING: In this study, we used green fluorescent protein (GFP) to examine the intracellular distribution of P4.9-GFP in plant cells, and observed fluorescence in the cytoplasm and nucleus. Transient expression of P4.9 was localized to the plasmodesmata. Co-infiltration of agrobacterium carrying binary plasmids of P4.9 and GFP facilitated GFP diffusion between cells. Besides P4.9 was able to spread by itself to neighboring cells, and co-localized with a marker specific to the endoplasmic reticulum, HDEL-mCherry, but not with the Golgi marker Man49-mCherry. CONCLUSIONS: Together, these results demonstrate that CCYV P4.9 is involved in cell-cell movement.

Begomoviral Movement Protein Effects in Human and Plant Cells: Towards New Potential Interaction Partners.

Geminiviral single-stranded circular DNA genomes replicate in nuclei so that the progeny DNA has to cross both the nuclear envelope and the plasmodesmata for systemic spread within plant tissues. For intra- and intercellular transport, two proteins are required: a nuclear shuttle protein (NSP) and a movement protein (MP). New characteristics of ectopically produced Abutilon mosaic virus (AbMV) MP (MPAbMV), either authentically expressed or fused to a yellow fluorescent protein or epitope tags, respectively, were determined by localization studies in mammalian cell lines in comparison to plant cells. Wild-type MPAbMV and the distinct MPAbMV: reporter protein fusions appeared as curled threads throughout mammalian cells. Co-staining with cytoskeleton markers for actin, intermediate filaments, or microtubules identified these threads as re-organized microtubules. These were, however, not stabilized by the viral MP, as demonstrated by nocodazole treatment. The MP of a related bipartite New World begomovirus, Cleome leaf crumple virus (ClLCrV), resulted in the same intensified microtubule bundling, whereas that of a nanovirus did not. The C-terminal section of MPAbMV, i.e., the protein's oligomerization domain, was dispensable for the effect. However, MP expression in plant cells did not affect the microtubules network. Since plant epidermal cells are quiescent whilst mammalian cells are proliferating, the replication-associated protein RepAbMV protein was then co-expressed with MPAbMV to induce cell progression into S-phase, thereby inducing distinct microtubule bundling without MP recruitment to the newly formed threads. Co-immunoprecipitation of MPAbMV in the presence of RepAbMV, followed by mass spectrometry identified potential novel MPAbMV-host interaction partners: the peptidyl-prolyl cis-trans isomerase NIMA-interacting 4 (Pin4) and stomatal cytokinesis defective 2 (SCD2) proteins. Possible roles of these putative interaction partners in the begomoviral life cycle and cytoskeletal association modes are discussed.

The functional analysis of distinct tospovirus movement proteins (NSM) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species.

The lack of infectious tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NSM) of four tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NSM proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NSM of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NSM, but not that of the CSNV, TCSV and TSWV NSM proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NSM mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NSM was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the '30K family'. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent tospovirus species that have a distinct biological behavior.

Bioinformatic and mutational analysis of ophiovirus movement proteins, belonging to the 30K superfamily.

Ophioviridae is a family of segmented, negative-sense, single-stranded RNA plant viruses. We showed that their cell-to-cell movement protein (MP) is an isolated member of the 30K MP superfamily with a unique structural organization. All 30K MPs share a core domain that contains a nearly-invariant signature aspartate. We examined its role in the MP of Citrus psorosis virus (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV). Alanine substitution of this aspartate prevented plasmodesmata accumulation of MP(MiLBVV), while MP(CPsV) was not affected. The capacity of ophiovirus MPs to increase the plasmodesmata size exclusion limit and non-cell autonomous protein feature was abolished in both mutants. To investigate the role of the signature aspartate in cell-to-cell movement, we constructed a new movement-deficient Tobacco mosaic virus vector used for trans-complementation assays. We showed that both ophiovirus MP mutants lack the cell-to-cell movement capacity, confirming that this signature aspartate is essential for viral cell-to-cell movement.

Importin-α-mediated nucleolar localization of potato mop-top virus TRIPLE GENE BLOCK1 (TGB1) protein facilitates virus systemic movement, whereas TGB1 self-interaction is required for cell-to-cell movement in Nicotiana benthamiana.

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.

Evolution of plant virus movement proteins from the 30K superfamily and of their homologs integrated in plant genomes.

Homologs of Tobacco mosaic virus 30K cell-to-cell movement protein are encoded by diverse plant viruses. Mechanisms of action and evolutionary origins of these proteins remain obscure. We expand the picture of conservation and evolution of the 30K proteins, producing sequence alignment of the 30K superfamily with the broadest phylogenetic coverage thus far and illuminating structural features of the core all-beta fold of these proteins. Integrated copies of pararetrovirus 30K movement genes are prevalent in euphyllophytes, with at least one copy intact in nearly every examined species, and mRNAs detected for most of them. Sequence analysis suggests repeated integrations, pseudogenizations, and positive selection in those provirus genes. An unannotated 30K-superfamily gene in Arabidopsis thaliana genome is likely expressed as a fusion with the At1g37113 transcript. This molecular background of endopararetrovirus gene products in plants may change our view of virus infection and pathogenesis, and perhaps of cellular homeostasis in the hosts.

Two basic (hydrophilic) regions in the movement protein of Parietaria mottle virus have RNA binding activity and are required for cell-to-cell transport.

The movement protein (MP) of parietaria mottle virus (PMoV) is required for virus cell-to-cell movement. Bioinformatics analysis identified two hydrophilic non-contiguous regions (R1 and R2) rich in the basic amino acids lysine and arginine and with the predicted secondary structure of an α-helix. Different approaches were used to determine the implication of the R1 and R2 regions in RNA binding, plasmodesmata (PD) targeting and cell-to-cell movement. EMSA (Electrophoretic Mobility Shift Assay) showed that both regions have RNA-binding activity whereas that mutational analysis reported that either deletion of any of these regions, or loss of the basic amino acids, interfered with the viral intercellular movement. Subcellular localization studies showed that PMoV MP locates at PD. Mutants designed to impeded cell-to-cell movement failed to accumulate at PD indicating that basic residues in both R1 and R2 are critical for binding the MP at PD.

Hitching a ride on vesicles: cauliflower mosaic virus movement protein trafficking in the endomembrane system.

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) µA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.

The Tobacco mosaic virus movement protein associates with but does not integrate into biological membranes.

UNLABELLED: Plant positive-strand RNA viruses require association with plant cell endomembranes for viral translation and replication, as well as for intra- and intercellular movement of the viral progeny. The membrane association and RNA binding of the Tobacco mosaic virus (TMV) movement protein (MP) are vital for orchestrating the macromolecular network required for virus movement. A previously proposed topological model suggests that TMV MP is an integral membrane protein with two putative α-helical transmembrane (TM) segments. Here we tested this model using an experimental system that measured the efficiency with which natural polypeptide segments were inserted into the ER membrane under conditions approximating the in vivo situation, as well as in planta. Our results demonstrated that the two hydrophobic regions (HRs) of TMV MP do not span biological membranes. We further found that mutations to alter the hydrophobicity of the first HR modified membrane association and precluded virus movement. We propose a topological model in which the TMV MP HRs intimately associate with the cellular membranes, allowing maximum exposure of the hydrophilic domains of the MP to the cytoplasmic cellular components. IMPORTANCE: To facilitate plant viral infection and spread, viruses encode one or more movement proteins (MPs) that interact with ER membranes. The present work investigated the membrane association of the 30K MP of Tobacco mosaic virus (TMV), and the results challenge the previous topological model, which predicted that the TMV MP behaves as an integral membrane protein. The current data provide greatly needed clarification of the topological model and provide substantial evidence that TMV MP is membrane associated only at the cytoplasmic face of the membrane and that neither of its domains is integrated into the membrane or translocated into the lumen. Understanding the topology of MPs in the ER is vital for understanding the role of the ER in plant virus transport and for predicting interactions with host factors that mediate resistance to plant viruses.

Identification of sequences required for AL2-mediated activation of the tomato golden mosaic virus-yellow vein BR1 promoter.

A 108 bp sequence has been identified in the tomato golden mosaic virus-yellow vein (yvTGMV) B component that is necessary and sufficient for AL2-mediated activation of the BR1 promoter. The sequence appears to have a bipartite arrangement, with elements located between -144 to -77 and -59 to -36 from the transcription start site, with both being required for activation by AL2. These sequences are located upstream of a TATA box and bind nuclear proteins from spinach, tomato and Arabidopsis. These sequences are also capable of binding Arabidopsis PPD2, which has been shown previously to interact with the yvTGMV coat protein (CP) promoter. We have identified two putative transcription factor-binding sites (CCAAT and GTGANTG10) that are conserved in sequences necessary for activation of the yvTGMV BR1, as well as the yvTGMV and cabbage leaf curl virus (CabLCV) CP promoters, which are all activated by AL2. The yvTGMV BR1 promoter exhibits AL2-independent expression in vascular tissue, similar to the yvTGMV, CabLCV and spinach curly top virus CP promoters. Together, this further confirms a common regulatory mechanism for AL2-mediated activation of bipartite begomovirus promoters.

The tobamovirus Turnip Vein Clearing Virus 30-kilodalton movement protein localizes to novel nuclear filaments to enhance virus infection.

Plant viruses overcome the barrier of the plant cell wall by encoding cell-to-cell movement proteins (MPs), which direct newly replicated viral genomes to, and across, the wall. The paradigm for how a single MP regulates and coordinates these activities is the Tobacco mosaic virus (TMV) 30-kDa protein (MP(TMV)). Detailed studies demonstrate that TMV multiplies exclusively in the cytoplasm and have documented associations of MP(TMV) with endoplasmic reticulum (ER) membrane, microtubules, and plasmodesmata throughout the course of infection. As TMV poorly infects Arabidopsis thaliana, Turnip vein clearing virus (TVCV) is the tobamovirus of choice for studies in this model plant. A key problem, which has contributed to confusion in the field, is the unproven assumption that the TVCV and TMV life cycles are identical. We engineered an infectious TVCV replicon that expressed a functional fluorescence-tagged MP(TVCV) and report here the unexpected discovery that MP(TVCV), beyond localizing to ER membrane and plasmodesmata, targeted to the nucleus in a nuclear localization signal (NLS)-dependent manner, where it localized to novel F-actin-containing filaments that associated with chromatin. The MP(TVCV) NLS appeared to be conserved in the subgroup 3 tobamoviruses, and our mutational analyses showed that nuclear localization of MP(TVCV) was necessary for efficient TVCV cell-to-cell movement and systemic infection in Nicotiana benthamiana and Arabidopsis thaliana. Our studies identify a novel nuclear stage in TVCV infection and suggest that nuclear MP encoded by TVCV and other subgroup 3 tobamoviruses interacts with F-actin and chromatin to modulate host defenses or cellular physiology to favor virus movement and infection.

Endoplasmic reticulum export and vesicle formation of the movement protein of Chinese wheat mosaic virus are regulated by two transmembrane domains and depend on the secretory pathway.

The 37K protein of Chinese wheat mosaic virus (CWMV) belongs to the 30K superfamily of plant virus movement proteins. CWMV 37K trans-complemented the cell-to-cell spread of a movement-defective Potato virus X. CWMV 37K fused to enhanced green fluorescent protein localized to plasmodesmata and formed endoplasmic reticulum (ER)-derived vesicular and large aggregate structures. CWMV 37K has two putative N-terminal transmembrane domains (TMDs). Mutations disrupting TMD1 or TMD2 impaired 37K movement function; those mutants were unable to form ER-derived structures but instead accumulated in the ER. Treatment with Brefeldin A or overexpression of the dominant negative mutant of Sar1 retained 37K in the ER, indicating that ER export of 37K is dependent on the secretory pathway. Moreover, CWMV 37K interacted with pectin methylesterases and mutations in TMD1 or TMD2 impaired this interaction in planta. The results suggest that the two TMDs regulate the movement function and intracellular transport of 37K.

In vitro phosphorylation of the N-terminal half of hordeivirus movement protein.

The N-terminal half of TGB1 movement protein of poa semilatent hordeivirus, which forms a ribonucleoprotein complex involved in movement of the viral genome in the plant, and its two domains, NTD and ID, are phosphorylated in vitro by a fraction enriched in cell walls from Nicotiana benthamiana. Using a set of protein kinase inhibitors with different specificities, it was found that enzymes possessing activities of casein kinase 1, protein kinase A, and protein kinase C are involved in phosphorylation. Commercial preparations of protein kinases A and C are able to phosphorylate in vitro recombinant proteins corresponding to the N-terminal half of the protein and its domains NTD and ID. Phosphorylation of the NTD has no effect on the efficiency and character of its binding to RNA. However, phosphorylation of the ID leads to a decrease in its RNA-binding activity and in the ability for homological protein-protein interactions.

A conserved C-terminal motif is essential for self-interaction of Barley stripe mosaic virus China strain TGB3 protein.

The triple gene block (TGB) 3 protein is essential for the cell-to-cell movement of Barley stripe mosaic virus (BSMV). Previous studies have shown that TGB3, together with TGB2, facilitates the movement of TGB1 to the plasma membrane. However, the interactions among the three proteins (i.e., TGB3, TGB1, and TGB2) have not been thoroughly understood. The interactions of BSMV China strain (BSMV-CH) TGB3 with itself and with other two TGB proteins were investigated using a Gal4-based yeast two-hybrid system and pull-down assays. The results show that neither TGB1 nor TGB2 interacts with TGB3. However, self-interaction was detected for TGB3. The C-terminal 37 amino acids (amino acids 87-123) containing a conserved C-terminal motif were found required for the self-interaction of TGB3. The roles of the novel property of BSMV-CH TGB3 in virus cell-to-cell movement were discussed.

Population genetic analysis of grapevine fanleaf virus.

Population genetic analysis of grapevine fanleaf virus (GFLV) was done on the basis of the virus movement protein (MP) gene sequences from the isolates detected and identified in this study and those of all previously reported GFLV strains/isolates. These revealed that the GFLV populations of Iran and Slovenia were highly distinct, whereas those of France, Germany, Italy and the USA were composed of multiple lineages. All populations were significantly differentiated from each other. However, two GFLV isolates from Tunisia, the only recorded GFLVs from that country, were not statistically distinct from the French, German and Italian populations. The ratio of non-synonymous nucleotide diversity to synonymous nucleotide diversity (Pi(a)/Pi(s)) was less than 1, suggesting that the MP gene has been under purifying selection. The neutrality tests were indicative of a balancing selection that is operating within Iranian and USA GFLV isolates, but they show a purifying selection within the other populations. Eleven recombination events were detected in a total of 50 isolates from France, Germany, Iran, Italy, Slovenia and the USA. The results from the recombination analysis were in agreement with those of the phylogenetic analysis. This study suggests that diversity among GFLV geographical populations resulted from possible host adaptation, recombination and founder effects.