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Serology has been the main, or at least an important, tool for differentiating potyviruses that affect solanaceous crops. At present, analysis of the genome by hybridization techniques has supported the differentiation of viruses demonstrated by serology. Phylogenetic groupings, based on nucleic acid sequences, should be combined with serological detection to make the groupings more usable.
At first potyviruses were easily distinguished by biological and serological properties because only a few were known and information on their host ranges was limited. The first evidence of serological cross reaction between two of these viruses was reported in 1951 and was further corroborated for three obviously distinct members of the group in 1960. In 1968 attention was drawn to the fact that some legume and non-legume potyviruses have much wider host ranges than previously known and that within the potyvirus group there is as much biological variation within viruses and overlap between viruses as there is in serology. The concept of continuity within the group was soon supported by others and became known as the "continuum hypothesis." Results with highly sensitive serological methods using polyclonal antisera were conflicting, and nucleic acid hybridization techniques did not unambiguously discriminate between potyviruses. Recent results, obtained with antibodies directed toward epitopes located in the N-termini of the coat proteins of potyviruses, suggest that there are ways to more definitely group strains of one potyvirus and distinguish them from other potyviruses. However, there are exceptions to this rule, as in the case of bean yellow mosaic virus and clover yellow vein virus which are clearly distinct in host range, inclusion bodies, and migration velocity of coat protein, but which still react with antibodies to the N-terminal epitopes of one virus. So the question remains of whether coat-protein properties, especially the serological reactivity of N-termini, which do not alter overall virus integrity when lost, sufficiently represent the genome of a pathogenic virus entity as a single criterion for classification.
The interrelationship of a number of potyviruses infecting legumes has been investigated by comparing molecular properties of their coat proteins. Comparison of the coat proteins by the techniques of amino acid analysis and PAGE was inadequate to distinguish strains from distinct potyviruses. However, high-performance liquid chromatographic peptide profiles of tryptic digests of coat proteins of these legume-infecting potyviruses enabled such assignments to be made. These data indicate that amino acid sequences of coat proteins of azuki bean mosaic virus, the Type and W strains of blackeye cowpea mosaic virus, three isolates (74, PM, PN) of a potyvirus obtained from soybean in Taiwan, and the Blotch and Mild Mottle strains of peanut stripe virus (PStV) may be very similar to the known sequence of PStV Stripe coat protein. In contrast, peptide profiles of coat proteins from soybean mosaic virus, clover yellow vein virus, bean yellow mosaic virus, potato virus Y, and tobacco etch virus were dissimilar to each other and to the profile of PStV Stripe, suggesting that their coat protein sequences were also quite different. Based on observations of the coat protein structure of many potyviruses, the results suggest that the potyvirus isolates with similar coat proteins are strains of the same potyvirus.
Nucleic acid hybridization with the 3' non-coding region of the potyvirus genome as the probe was shown to be a relatively simple means of distinguishing between distinct potyviruses and their strains. Comparisons of the nucleotide sequences of potyvirus genomes (ignoring gaps) showed that the degree of identity between equivalent genes of strains was greater than 96%, while between distinct potyviruses the identity ranged from 42% to 65%, suggesting that any extended sequence could be considered representative of the whole genome and be suitable as a diagnostic probe. The comparisons however, also revealed that some parts of the genome, but not the 3' non-coding region, had local regions of high sequence identity that could lead to cross-hybridization between distinct potyviruses. For this reason, and because its location immediately upstream of the poly(A) tail makes it the most accessible region for the purpose of cloning and sequencing, the 3' non-coding sequence should be most suitable for use as a diagnostic probe. Successful hybridizations (using radiolabeled, polymerase chain reaction-amplified 3' non-coding sequences) have been achieved by probing recombinant clones, purified potyviral RNA, partially purified total RNA from infected plants, and a crude extract of infected plant tissue. The method has been used to support the proposals that watermelon mosaic virus 2 and soybean mosaic virus-N are both strains of the same virus, and to discriminate between several isolates previously believed to be strains of sugarcane mosaic virus. The method should have wide application as a means of differentiating distinct potyviruses from strains.
The serological relationships among members of the family Potyviridae are extremely complex and inconsistent. Variable cross-reactivity of polyclonal antisera, unexpected paired relationships between distinct viruses, and lack of cross-reactions between some strains are the major problems associated with the serology of potyviruses. Recent biochemical and immunochemical investigations of coat proteins have established the molecular basis for potyvirus serology and provided explanations for most of the problems with serology of potyviruses. Information from these studies has also formed the basis for the development of several novel approaches to the accurate detection and identification of potyviruses. However, even these novel approaches are not without drawbacks and some of them cannot be applied easily in plant virus laboratories, since they require prior sequence information and facilities for peptide synthesis. These findings suggest that serology is an imperfect criterion for the identification and classification of potyviruses.
The application of protein and nucleic acid sequence analysis in evolutionary and phylogenetic studies is well established. Available sequence information for the 5' untranslated region of potyviruses including the fungus-transmitted barley yellow mosaic virus (BaYMV) RNA-1 suggests that a 12-nucleotide conserved sequence, the "potybox" is unique to this group. Various non-structural proteins of potyviruses share considerable "signature" sequence homology across a broad spectrum of unrelated viruses, which makes their value limited to "supergroup" or "superfamily" identity. However, in potyviruses, the coat-protein N-terminal sequences and 3' noncoding regions are variable among viruses, but similar among strains of the same virus. This suggests that these sequences may be an accurate marker of genetic relatedness. Until complete genome sequences from a large number of potyviruses become available and their value in systematics is tested, coat protein and 3' noncoding regions remain as the choice of taxonomic indicators. The reason being, that cloning and sequencing of the coat-protein gene and 3' noncoding regions are less complicated and time consuming and the sequences show significant differences among the virus species within the family Potyviridae.
Recent knowledge of the structure of the potyvirus particle and its components appears to have resolved what was thought to be an intractable problem of plant virology. This review describes how coat-protein and gene sequence data can be used to provide an hierarchical classification of potyviruses. This classification puts the aphid and non-aphid-transmitted potyviruses into a single family, divides this family into four genera that correspond to the four modes of vector transmission, discriminates distinct potyvirus species from strains, and provides a basis for the formation of subgroups composed of closely related species within a genus.
Bean common mosaic virus (BCMV) consists of a large number of pathotypes and strains which have largely been identified by their characteristic interactions with a selected number of differential bean cultivars. The relationships among these strains and other potyviruses that infect legumes are complex, with indications that BCMV, blackeye cowpea mosaic virus (BlCMV) and azuki bean mosaic virus (AzMV) may be strains of the one virus. Using high performance liquid chromatographic peptide profiles of coat-protein digests, the NL3 and NY15 strains of BCMV were compared with each other, with the Type and W strains of BlCMV and with the mild mottle strain of peanut stripe virus (PStV). The results suggest that BCMV-NL3 and BCMV-NY15 are distinct potyviruses, not strains of the one virus, and that BCMV-NY15 is a strain of the same potyvirus that includes BlCMV, PStV, AzMV and three potyvirus isolates (74, PM, PN) from soybeans.
Eight peptides corresponding to conserved regions of the coat protein of potyviruses were synthesized. All the peptides were recognized by anti-virus or anti-core-virus. Antisera raised to the synthetic peptides were tested with purified viruses and viral antigens present in plant sap. In many cases, the extent of cross-reactivity between different potyviruses was not correlated with the degree of sequence homology between the peptide used for immunization and the corresponding region in the coat protein of the potyvirus tested. An antiserum raised to a peptide of 18 residues containing a highly conserved region was found to react with all seven potyviruses tested.
Virus-specific and cross-reactive monoclonal antibodies have been produced to at least 19 different aphid-transmitted potyviruses. This report summarizes the development of these monoclonal antibodies as well as presents information on the delineation of the virus-specific and group-common epitopes defined by these monoclonal antibodies. Virus-specific and group-common antigenic determinants were mapped by a variety of techniques, including analysis of antigen: antibody reactivity patterns, determination of N-terminal vs. trypsin-resistant core peptide-specificity, immunoanalysis of overlapping synthetic peptides, and immunoanalysis of bacterially expressed coat-protein gene products. Of those monoclonal antibodies that have been examined, monoclonal antibody-defined virus-specific epitopes are virion surface-located within the 30+ amino acid amino terminus, whereas the group-common epitopes are found in the trypsin-resistant core protein not usually located on the virion surface, as has been shown previously with certain polyclonal antibodies. New information is presented on the analysis of bean yellow mosaic virus amino terminal epitopes as well as on the identification of amino terminal antigenic determinants shared between strains of bean yellow mosaic virus and pepper mottle virus. A recommendation on the evaluation and use of a panel of potyvirus broad-spectrum reacting monoclonals as reference monoclonal antibodies for the detection and classification of aphid-transmitted potyviruses is also presented.
Amino acid sequences of the cytoplasmic cylindrical inclusion protein (CIP), large nuclear inclusion protein (NIb), and coat protein (CP) of potyviruses were re-examined in light of reported serological relationships, and correlated with known and deduced biological functions. No obvious correlations were observed between either amino acid sequences or epitopes recognized by monoclonal antibodies and the natural host ranges of the potyviruses examined. Whereas the identified sequence motifs of the RNA helicase (CIP) and replicase (NIb) are predicted to be antigenic, most of the conserved sequences and epitopes in the CIP, NIb and CP were presumed to be maintained for structural rather than functional reasons. Three possible potyvirus clusters are proposed on the basis of the length and composition of the virion surface-exposed amino terminal extension of the CP; these clusters do not correlate with overall CP sequence homology, host range, or vectors, but are of potential evolutionary significance and hence of possible taxonomic value.
Until recently, sugarcane mosaic virus (SCMV) was believed to be a single potyvirus consisting of a large number of strains, differing from each other in certain biological and antigenic properties. The use of affinity-purified polyclonal antibodies directed towards the surface-located, virus-specific amino termini of the coat proteins showed that 17 strains from Australia and the United States represented four distinct potyviruses, namely johnsongrass mosaic virus (JGMV), maize dwarf mosaic virus (MDMV), sorghum mosaic virus (SrMV) and SCMV. Comparisons of strains from each of these four viruses on the basis of reactions on differential sorghum and oat cultivars, cell-free translation of RNAs, morphology and serology of cytoplasmic cylindrical inclusions, amino acid sequence and peptide profiling of coat proteins, 3' non-coding nucleotide sequences, and molecular hybridization with probes corresponding to the 3' non-coding regions, resulted in exactly the same taxonomic assignments as obtained using amino-terminal serology. These results further confirm that the former sugarcane mosaic virus actually consists of four distinct viruses and show that MDMV, SrMV, and SCMV are more closely related to each other than they are to JGMV. Because these four viruses are closely related but distinct, formation of a sugarcane mosaic subgroup in the genus Potyvirus would be appropriate.
The determination of relationships among and the identification of potyviruses with polyclonal antibodies does not always lead to a proper conclusion. The potyvirus specific monoclonal PTY 1 does not detect all potyviruses tested.
Infectious RNA transcripts were generated from full-length cDNA clones of the tobacco etch potyvirus genome containing an insertion of the bacterial beta-glucuronidase (GUS) gene between the polyprotein-coding sequences for the N-terminal 35-kDa proteinase and the helper component-proteinase. The recombinant virus was able to spread systemically in plants and accumulated to a level comparable with wild-type tobacco etch potyvirus. Proteolytic processing mediated by the 35-kDa proteinase and helper component-proteinase resulted in production of an enzymatically active GUS-helper component-proteinase fusion protein. A virus passage line that retained the GUS insert after numerous plant-to-plant transfers, as well as a line that sustained a deletion of the GUS sequence, was recovered. Use of an in situ histochemical GUS assay in time-course experiments allowed the visualization of virus activity in single, mechanically inoculated leaf epidermal cells, in neighboring epidermal and mesophyll cells, in phloem-associated cells after long-distance transport, and in cells surrounding vascular tissues of organs above and below the site of inoculation. This system represents a powerful tool to study plant virus replication, short- and long-distance virus movement, and virus-host interactions. Additionally, we show that potyviruses may serve as highly efficient, autonomously replicating vectors for the expression of foreign genes in plants.
Potyviruses occur in nature as a variable population. A number of strains have been reported for many potyviruses. Two or more viruses have been separated from "one virus" isolate. Experimental isolation and/or transmission often results in atypical viral isolates. A virus may be considered as a fuzzy population. We should properly understand the range of variation in one virus. The range of variation as well as typical characteristics of a virus should be included in future descriptions.
In the past vector relationships have been one of the criteria used for delineating plant virus taxa. The proposed family, the Potyviridae, continues that practice. Aphid transmission of viruses within the genus Potyvirus is a useful characteristic in terms of identification, but is of only limited use in terms of taxonomy. This conclusion is based on a greater understanding of the molecular biology of potyviruses. The molecular basis of aphid transmission is not well understood at the present, but these data suggest that, beyond disease diagnosis, virus identification and characterization, and potential identification of genome microheterogeneity, aphid transmission should only be considered as a minor taxonomic criterion.
The serological relationships among strains of bean common mosaic virus (BCMV) (genus Potyvirus, family Potyviridae) were investigated by testing 13 isolates of the 10 known BCMV pathotypes with two monoclonal antibodies and six antisera to BCMV strains. In addition, other properties of serologically distinct BCMV strains were compared. Two groups of BCMV strains were obtained by ELISA and Western blot serology: serotype A contained the BCMV strains NL3, NL5, and NL8 and serotype B contained the BCMV strains NL1, NL2, NL4, NL6, US4, NL7, NY15, and Fla. SDS polyacrylamide gel electrophoresis and Western blotting of freshly purified preparations, and of extracts from leaves infected with eleven BCMV strains showed that the apparent molecular mass of the capsid protein of the serotype A isolates NL3, NL5, and NL8 are lower (about M(r) 33,000) than those of the serotype B isolates (M(r) 34,500 to 35,000). The normal lengths of the particles of the serotype A isolates were shorter (810-818 nm) than those of most isolates (except NL6 and NY15) of serotype B (847-886 nm). All isolates studied induced cytoplasmic pinwheel and scroll inclusions. Cells infected with serotype A isolates contained a specific type of proliferated endoplasmic reticulum which was never found in cells infected with serotype B isolates. The capsid protein gene of a representative member of each serotype was cloned and sequenced. Molecular mass calculations based upon nucleotide sequence-derived amino acid sequences yielded M(r) of 29,662 and 32,489 for the capsid proteins of the serotype A isolate NL8 and the serotype B isolate NL4, respectively. Comparison of the coat-protein sequences showed considerable differences at the N-termini whereas the core regions and the C-termini appeared to be highly conserved. Marked differences were also observed within the 3' non-coding regions of cloned cDNAs of NL 4 and NL 8. The striking differences between the two serotypes of BCMV strongly suggest that they be classified as two distinct potyviruses which naturally infect Phaseolus beans.
The sequence of the 3'-terminal 1492 nucleotides of the genome of a New Zealand isolate of clover yellow vein potyvirus (CYVV) has been determined. This sequence encodes a large open reading frame of 1314 nucleotides, the start of which was not identified, but which encodes a putative 272 amino acid coat protein. Downstream of the coat protein coding region is a 177 nucleotide untranslated sequence terminated by a polyadenylate tract. Comparison of the deduced CYVV-NZ coat protein amino acid sequence with two other strains of CYVV showed 86-93% similarity, suggesting CYVV-NZ should be regarded as a separate CYVV strain. CYVV-NZ shares with other CYVV strains a direct repeat of 14-16 nucleotides that is capable of forming a stem-loop structure. Examination of 35 strains of 15 other potyviruses showed a similar stem-loop structure conserved in all cases. A possible role in replication is hypothesized for the structure.