D. D. Shukla

Identification and classification of potyviruses on the basis of coat protein sequence data and serology

SummaryThe identification and classification of potyviruses has been in a very unsatisfactory state due to the large size of the group, the apparent vast variation among the members and the lack satisfactory taxonomic parameters that will distinguish distinct viruses from strains. In the past, use of classical methods, such as host range and symptomatology, cross-protection, morphology of cytoplasmic inclusions and conventional serology, revealed a “continuum” implying that the “species” and “strain” concepts cannot be applied to potyvirses. In contrast nucleic acid and amino acid sequence data of coat proteins has clearly demonstrated that potyviruses can be divided into distinct members and strains. This sequence data in combination with information of the structure of the potyvirus particle has been used to develop simple techniques such as HPLC peptide profiling, serology (using polyclonal antibody probes obtained by cross-adsorption with core protein from trypsin treated particles) and cDNA hybridization. These findings, along with immunochemical analyses of over-lapping synthetic peptides have established the molecular basis for potyvirus serology; explained many of the problems associated with the application of conventional serology; and provided a sound basis for the identification and classification of potyviruses. As a result, the virus/strain status of some potyviruses has been redefined, requiring a change in the potyvirus nomenclature. These new developments necessiate a re-evaluation of the earlier literature on symptomatology, cross-protection, cytoplasmic inclusion body morphology and serology.

Coat protein of Potyviruses

SummaryFour strains of potato virus Y, PVY-D, PVY-10, PVY-18, and PVY-43, obtained from different Australian sources were compared on the basis of their biological, serological and coat protein structural properties. Each of the strains could be distinguished on the basis of their reactions on selected test plant species. Two of the PVY strains, PVY-D and PVY-10, induced symptoms similar to those produced by the PVYO strain group. The reactions of PVY-18 and PVY-43, although comparable to PVYN in some hosts, did not completely match the description of the PVYN strain group. In contrast to the other three strains, PVY-18 could not be transmitted byMyzus persicae in repeated tests. No difference was observed in the serological properties of the four PVY strains in different assay systems, using polyclonal antisera.The amino acid sequences of the coat proteins of PVY-10, PVY-18, and PVY-43 were obtained and compared with the coat protein sequences of pepper mottle virus (PeMV) [Dougherty WG, Allison RF, Parks TD, Johnston RE, Feild MJ, Armstrong FB (1985) Virology 146: 282–292] and PVY-D [Shukla DD, Inglis AS, McKern NM, Gough KH (1986) Virology 152: 118–125]. The homology between the PVY strains ranged from 96.3 to 99.3% and with the PeMV sequence, 91.4 to 92.9%. Based on this high sequence homology, and the previous observation that coat protein sequences of potyvirus strains are always greater than 90% identical, PeMV could be considered a strain of PVY. However, PVY and PeMV are reported to be only distantly serologically related and on this basis PeMV is currently considered to be an independent member of the Potyvirus group.

Coat protein phylogeny and systematics of potyviruses

The feasibility of applying molecular phylogenetic methods of analysis to aligned coat-protein sequences and other molecular data derived from coat proteins or genomic sequences of members of the proposed taxonomic family of Potyviridae, is discussed. We show that comparative sequence analysis of whole coat-protein sequences may be used reliably to differentiate between sequences of closely related strains, and to show groupings of more distantly related viruses; that coat proteins of putative Potyviridae cluster according to the proposed generic divisions, and, even if some are only very distantly related, the members of the family form a cluster distinct from coat proteins of other filamentous and rod-shaped viruses. Taxonomic revisions based on perceived evolutionary relationships, and the lack of feasibility of erecting higher taxa for these viruses, are discussed.

Differentiation of sugarcane, maize dwarf, Johnsongrass, and sorghum mosaic viruses based on reactions of oat and some sorghum cultivars.

(...) These viruses are separable based on symptom expression in sorghum and oat. JGMV, alone infects oat. Strains within MDMV and JGMV react uniformly in sorghum. SCMV strains differ in reactions on some sorghum cultivars. SrMV strains differ on two sorghum cultivars. These infectivity results clearly support the new taxonomy suggesting that, although helpful, it is not essential to have biochemical and serological laboratory capabilities to classify these 15 virus strains

Possible members of the potyvirus group transmitted by mites or whiteflies share epitopes with aphid-transmitted definitive members of the group.

SummaryThere are at least ten viruses identified in the literature that resemble definitive potyviruses in having flexuous filamentous particles and inducing the formation of “pinwheel” cytoplasmic inclusions in infected cells but that are transmitted by eriophyid mites, whiteflies or soil fungi and not by aphids, the vectors of the definitive potyviruses. The taxonomic status of these viruses is uncertain at present. Using a broadly cross-reactive antiserum raised against the dissociated coat protein core (residues 68–285) of a definitive potyvirus (Johnsongrass mosaic virus), we have shown that wheat streak mosaic virus which is transmitted by mite and sweet potato mild mottle virus which is transmitted by whitefly have coat proteins that share epitopes with definitive potyviruses. This finding further supports their classification as definitive members of the potyvirus group. The cross-reactive antiserum used here had been shown previously to react with coat proteins of fifteen different definitive potyviruses. The antiserum did not react with coat proteins of potexviruses and tobamoviruses.

Transient expression of the coat protein of sugarcane mosaic virus in sugarcane protoplasts and expression inEscherichia coli

SummaryThe coat protein (CP) of strain SC of sugarcane mosaic virus (SCMV-SC) was expressed transiently in sugarcane protoplasts after electroporation with one of two plasmids encoding the CP gene. The CP gene was fused with either the cauliflower mosaic virus 35S promoter or the synthetic monocotyledon promoter “Emu”. The coat protein gene was also inducibly expressed inEscherichia coli when fused to the trc promoter. The protein expressed in both systems had the same electrophoretic mobility and antigenic specificity as purified SCMV-SC coat protein. Transient expression of the 35S-CP gene in protoplasts could only be demonstrated in Western blots developed with the chemiluminescence enzyme substrate luminol.

Cross-protection studies between strains of sugarcane mosaic, maize dwarf mosaic, Johnsongrass mosaic, and sorghum mosaic potyviruses

Cross-protection was studied between strains of viruses comprising the sugarcane mosaic virus (SCMV) subgroup, namely Johnsongrass mosaic, maize dwarf mosaic, sorghum mosaic, and SCMV, in 53 different combinations using differential hosts and Western blot immunoassays, Cross-protection occurred only between SCMV-MDB and SCMV-BC when the former was inoculated first and the latter used as the challenge strain, but neither vice versa nor in any other combinations including the 19 that involved recognized strains of the one virus, SCMV. The unidirectional protection between SCMV-MDB and SCMV-BC and the negative cross-protection results between other strains of SCMV appear to correlate with different sequence motifs present in the hypervariable region of the coat protein N-terminus of the SCMV strains