Drake C. Stenger

Revision of taxonomic criteria for species demarcation in the family Geminiviridae, and an updated list of begomovirus species.

Members of the family Geminiviridae characteristically have circular single-stranded DNAgenomes packaged within twinned (so-called geminate) particles. Geminiviruses are currentlydivided into four genera on the basis of their genome organizations and biological properties[2,20].Thosethathaveamonopartitegenomeandaretransmittedbyleafhoppervectors,primarilyto monocotyledonous plants, are included in the genus Mastrevirus, of which Maize streak virus isthe type species. Viruses that have monopartite genomes distinct from those of the mastrevirusesand that are transmitted by leafhopper vectors to dicotyledonous plants are included in thegenus Curtovirus, with Beet curly top virus as the type species. The genus Topocuvirus, recentlyrecognized by the International Committee on Taxonomy of Viruses (ICTV) [18], has only onemember (also the type species), Tomato pseudo-curly top virus, which has a monopartite genomeandistransmittedbyatreehoppervectortodicotyledonousplants.ThegenusBegomoviruscontainsviruses that are transmitted by the whitefly Bemisia tabaci (Gennadius) to dicotyledonous plants,with Bean golden yellow mosaic virus (originally Bean golden mosaic virus – Puerto Rico)asthetype species. Many begomoviruses have bipartite genomes (DNA A and DNA B components),although numerous begomoviruses with a monopartite genome occur in the Old World, and thereare some for which a single component is not infectious yet no DNA B component has been found.Geminiviruses cause significant yield losses to many crop plants throughout the world [5, 7].Because of their economic importance and the relative ease with which their DNA genomescan be cloned, many geminiviruses have been isolated and characterized. Guidelines for naming

Horseradish curly top virus is a distinct subgroup II geminivirus species with rep and C4 genes derived from a subgroup III ancestor

The complete nucleotide sequence (3080 nt) of an infectious DNA clone derived from the geminivirus horseradish curly top virus (HrCTV) has been determined. The relationship of HrCTV to other geminiviruses was examined using dot matrix plots of nucleotide sequence similarities, and by phylogeny of predicted amino acid sequences of individual ORFs based upon parsimony or neighbour-joining methods. These analyses indicate that the V1 and V2 virion sense ORFs of HrCTV are most closely related to, yet distinct from, the corresponding ORFs of the subgroup II geminivirus beet curly top virus (BCTV). HrCTV also encodes a third virion sense ORF (V3) which is similar (72-74 percent amino acid identity) to the BCTV V3 ORF; however, the HrCTV V3 ORF has diverged in sequence to a greater extent relative to that observed among isolates of BCTV (98-100% amino acid identity). The HrCTV genome encodes only three complementary sense ORFs (Cl, C2 and C4) and lacks a C3 ORF which is conserved among all other subgroup II and III geminiviruses characterized to date. Although the neighbour-joining analysis indicated that the HrCTV C2 ORF was distantly related to the C2 ORF of BCTV, the predicted amino acid sequence deduced from the HrCTV C2 ORF lacks the characteristic zinc-finger domain present in the transcriptional activating protein (TrAP) encoded by the subgroup III ORF AC2, which is also retained within the TrAP-related product of the BCTV C2 ORF. Surprisingly, the rep and C4 proteins encoded by HrCTV share a closer phylogenetic relationship to the corresponding proteins of the subgroup III geminivirus squash leaf curl virus (SLCV) than to BCTV. These results suggest that the HrCTV genome may have arisen by a recombination event between a BCTV-like subgroup II virus ancestor and an SLCV-like subgroup III virus ancestor. Possible mechanisms that may explain recombination events among geminiviruses are discussed.

Emergence of a New Cucurbit-Infecting Begomovirus Species Capable of Forming Viable Reassortants with Related Viruses in the Squash leaf curl virus Cluster.

ABSTRACT Cucurbit leaf curl virus (CuLCV), a whitefly-transmitted geminivirus previously partially characterized from the southwestern United States and northern Mexico, was identified as a distinct bipartite begomovirus species. This virus has near sequence identity with the previously partially characterized Cucurbit leaf crumple virus from California. Experimental and natural host range studies indicated that CuLCV has a relatively broad host range within the family Cucurbitaceae and also infects bean and tobacco. The genome of an Arizona isolate, designated CuLCV-AZ, was cloned and completely sequenced. Cloned CuLCV-AZ DNA A and B components were infectious by biolistic inoculation to pumpkin and progeny virus was transmissible by the whitefly vector, Bemisia tabaci, thereby completing Kochs postulates. CuLCV-AZ DNA A shared highest nucleotide sequence identity with Squash leaf curl virus-R (SLCV-R), SLCV-E, and Bean calico mosaic virus (BCaMV) at 84, 83, and 80%, respectively. The CuLCV DNA B component shared highest nucleotide sequence identity with BCaMV, SLCV-R, and SLCV-E at 71, 70, and 68%, respectively. The cis-acting begomovirus replication specificity element, GGTGTCCTGGTG, in the CuLCV-AZ origin of replication is identical to that of SLCV-R, SLCV-E, and BCaMV, suggesting that reassortants among components of CuLCV-AZ and these begomoviruses may be possible. Reassortment experiments in pumpkin demonstrated that both reassortants of CuLCV-AZ and SLCV-E A and B components were viable. However, for CuLCV-AZ and SLCV-R, only one reassortant (SLCV-R DNA A/CuLCV-AZ DNA B) was viable on pumpkin, even though the cognate component pairs of both viruses infect pumpkin. These results demonstrate that reassortment among sympatric begomovirus species infecting cucurbits are possible, and that, if generated in nature, could result in begomoviruses bearing distinct biological properties.

Phylogenetic relationships, strain diversity and biogeography of tritimoviruses

North American and Eurasian isolates of Wheat streak mosaic virus (WSMV; genus Tritimovirus) and Oat necrotic mottle virus (ONMV; genus Rymovirus) were examined. Nine WSMV isolates differentially infected oat, barley, inbred maize line SDp2 and sorghum line KS56. The WSMV isolates clustered into groups based on phylogenetic analyses of the capsid protein (CP) cistron and flanking regions. WSMV isolates from the United States (US) and Turkey were closely related, suggesting recent movement between continents. Although more divergent, WSMV from Iran (WSMV-I) also shared a most recent common ancestor with the US and Turkish isolates. Another group of WSMV isolates from central Europe and Russia may represent a distinct Eurasian population. Complete genome sequences of WSMV from the Czech Republic (WSMV-CZ) and Turkey (WSMV-TK1) were determined and comparisons based on complete sequences yielded relationships similar to those based on partial sequences. ONMV-Pp recovered from blue grass (Poa pratensis L.) in Germany displayed the same narrow host range as ONMV-Type from Canada. Western blots revealed a heterologous relationship among CP of WSMV and ONMV. Phylogenetic analyses of the capsid protein cistron and flanking genomic regions indicated that WSMV and ONMV are related species sharing 74.2-76.2% (nucleotide) and 79.2-81.0% (amino acid) identity. Thus, ONMV should be removed from the genus Rymovirus and designated a definitive member of the genus Tritimovirus. Phylogenetic analyses further suggest that Sugarcane streak mosaic virus is not a tritimovirus, and may represent a new genus within the family Potyviridae.

Structure and Temporal Dynamics of Populations within Wheat Streak Mosaic Virus Isolates

ABSTRACT Variation within the Type and Sidney 81 strains of wheat streak mosaic virus was assessed by single-strand conformation polymorphism (SSCP) analysis and confirmed by nucleotide sequencing. Limiting-dilution subisolates (LDSIs) of each strain were evaluated for polymorphism in the P1, P3, NIa, and CP cistrons. Different SSCP patterns among LDSIs of a strain were associated with single-nucleotide substitutions. Sidney 81 LDSI-S10 was used as founding inoculum to establish three lineages each in wheat, corn, and barley. The P1, HC-Pro, P3, CI, NIa, NIb, and CP cistrons of LDSI-S10 and each lineage at passages 1, 3, 6, and 9 were evaluated for polymorphism. By passage 9, each lineage differed in consensus sequence from LDSI-S10. The majority of substitutions occurred within NIa and CP, although at least one change occurred in each cistron except HC-Pro and P3. Most consensus sequence changes among lineages were independent, with substitutions accumulating over time. However, LDSI-S10 bore a variant nucleotide (G6016) in NIa that was restored to A6016 in eight of nine lineages by passage 6. This near-global reversion is most easily explained by selection. Examination of nonconsensus variation revealed a pool of unique substitutions (singletons) that remained constant in frequency during passage, regardless of the host species examined. These results suggest that mutations arising by viral polymerase error are generated at a constant rate but that most newly generated mutants are sequestered in virions and do not serve as replication templates. Thus, a substantial fraction of variation generated is static and has yet to be tested for relative fitness. In contrast, nonsingleton variation increased upon passage, suggesting that some mutants do serve as replication templates and may become established in a population. Replicated mutants may or may not rise to prominence to become the consensus sequence in a lineage, with the fate of any particular mutant subject to selection and stochastic processes such as genetic drift and population growth factors.

Genotypic Diversity of Beet Curly Top Virus Populations in the Western United States

ABSTRACT The genotypic diversity of beet curly top virus (BCTV) present in the western United States has been examined by the analysis of 58 field isolates and eight laboratory or nursery isolates of the virus. Full-length clones for each isolate have been characterized for genotype by restriction endonuclease mapping. The results indicate that most of the genotypes examined may be classified as variants of the CFH, Worland, or Cal/Logan strains of BCTV. Two genotypes were recovered that appear to share certain genotypic markers of both Worland and CFH strains. Genotypic variants of the CFH and Worland strains and the two genotypes sharing markers of both strains were recovered from field isolates collected during 1994 and 1995. In contrast, the Cal/Logan strain was recovered only from isolates maintained in laboratories or nurseries. Comparisons of restriction endonuclease maps of cloned BCTV genomes revealed considerable variability both within and between strains. Although a total of 43 distinct genotypes of BCTV were identified, only 36 (84%) were recovered from field isolates. Of 37 field isolates for which more than a single clone was recovered, 16 (43%) contained more than a single genotype of one strain, whereas 4 (11%) harbored mixed infections of the CFH and Worland strains. A phylogenetic analysis using 43 characters derived from restriction endonuclease mapping data supported the grouping of 41 genotypes into three taxa consistent with the three currently recognized strains of BCTV. The relationships of the two genotypes sharing genotypic markers of both the Worland and CFH strains to other BCTV genotypes was unresolved in the phylogenetic analysis. Based on the mild symptom phenotype of the isolates from which these two genotypes were recovered and the presence of Worland genotypic markers in portions of the genome containing both cis- and trans-acting elements determining replication specificity, these two genotypes were tentatively considered as variants of the Worland strain.

Complete Deletion of Wheat Streak Mosaic Virus HC-Pro: a Null Mutant Is Viable for Systemic Infection

ABSTRACT A Wheat streak mosaic virus (WSMV) genome lacking HC-Pro was constructed and confirmed by reverse transcription-PCR to systemically infect wheat, oat, and corn. Coupled in vitro transcription/translation reactions indicated that WSMV P1 proteinase cleaved the polyprotein at the P1/P3 junction of the HC-Pro null mutant. The WSMV HC-Pro null mutant was competent for virion formation, but the virus titer was reduced 4.5-fold relative to that of the wild type. Collectively, these results indicate that WSMV HC-Pro is dispensable for replication and movement, two essential processes that are disrupted by point and small-insertion mutations introduced into potyvirus HC-Pro.

Genomic characterization of phenotypic variants of beet curly top virus

Full-length infectious DNA clones were constructed for four distinct phenotypic variants of beet curly top virus (BCTV). Southern hybridization assays indicated that each cloned BCTV genome shared sequence homology with pBCT-028, a full-length infectious DNA clone of a California isolate of BCTV previously characterized by others. Restriction endonuclease maps of the cloned BCTV genomes were distinct from one another. Infectivity assays determined that plasmids containing tandem repeats of BCTV genomes were generally more infectious than excised linear DNA inserts. Progeny virus, derived from plants inoculated with cloned DNAs, differed in their ability to infect sugarbeet, Beta vulgaris L., and the severity of symptoms produced in B. vulgaris and other experimental hosts.

Fully biologically active in vitro transcripts of the eriophyid mite-transmitted wheat streak mosaic tritimovirus.

ABSTRACT Infectious RNA of wheat streak mosaic virus (WSMV) has been produced using a full-length cDNA clone as a template for in vitro transcription with SP6 RNA polymerase. Infectivity was dependent on the use of template plasmid DNA that had not undergone spontaneous rearrangement during amplification in Escherichia coli. The presence of WSMV in systemically infected wheat plants inoculated with in vitro transcripts was confirmed by reverse-transcription polymerase chain reaction of the WSMV P3 gene and by accumulation of WSMV coat protein as detected by immunoblotting. Maintenance of the full-length WSMV cDNA in the high copy number plasmid pUC18 was problematic because of spontaneous rearrangement of WSMV sequences during growth in liquid media for more than 8 h or if the clone was subcultured. Stability of the WSMV cDNA clone was improved by the use of the low copy number plasmid pACYC177, and it could be grown in large scale volumes (up to 1 liter) of liquid culture for 14 h without noticeable rearrangements. Both the original WSMV culture and the progeny virus derived from infectious in vitro transcripts were efficiently transmitted by the natural eriophyid mite vector Aceria tosichella. This is the first report of infectious in vitro transcripts for any eriophyid mite-transmitted plant virus and represents the only monopartite member of the family Potyviridae infecting monocotyledonous hosts for which infectious in vitro transcripts are available.

Biotic, molecular, and phylogenetic characterization of bean calico mosaic virus, a distinct Begomovirus species with affiliation in the squash leaf curl virus cluster.

ABSTRACT Bean calico mosaic virus (BCMoV), a whitefly-transmitted geminivirus from Sonora, Mexico, was purified, and the genome components were cloned and sequenced. Purified viral fractions and cloned genome components were infectious by biolistic inoculation to bean, completing Kochs postulates for both. The B biotype of the whitefly Bemisia tabaci efficiently transmitted both native virus and progeny virus derived from cloned DNA inoculum. Host ranges of native virus and of progeny virus derived from cloned DNA were identical based upon whitefly and biolistic mediated transmission, respectively. BCMoV has a relatively wide experimental host range among begomoviruses known to infect bean, encompassing genera and species within the Fabaceae, Malvaceae, and Solanaceae. BCMoV has a bipartite genome, as do other New World begomoviruses. BCMoV DNA-A shared highest nucleotide sequence identities with squash leaf curl virus-E strain (SLCV-E) and cabbage leaf curl virus (CaLCV) at 80.1 and 80.7%, respectively. BCMoV DNA-B shared highest nucleotide sequence identity with SLCV-E at 70.7%. The common region (CR) sequences of BCMoV and SLCV-E are 73 to 76% identical; however, modular cis-acting elements within the CR involved in replication origin function and recognition are 100% conserved. Phy-logenetic analysis indicated that BCMoV DNA-A shares a most recent common ancestor with the DNA-A of two viruses that also occur in the Sonoran Desert, SLCV-E and Texas pepper virus (TPV-TAM), and CaLCV from Florida. In contrast, a phylogenetic analysis indicated that BCMoV DNA-B shares a most recent common ancestor with SLCV-E; whereas DNA-B of CaLCV clustered in a separate clade with pepper hausteco virus. Collectively, biological and molecular characteristics indicate that BCMoV is a distinct begomovirus species with the northernmost distribution of any begomovirus isolated from bean in the Americas. Furthermore, the phylogenetic relationships of begomovirus cognate components are not necessarily identical, suggesting that DNA-A and DNA-B of some begomoviruses may have different evolutionary histories.