Serological Studies Using Polyclonal Antisera Prepared Against the Viral Coat Protein of Four Begomoviruses Expressed in Escherichia coli.

Polyclonal rabbit antisera were produced to the coat protein of Bean golden mosaic virus Brazil isolate (BGMV), Cabbage leaf curl virus (CabLCV), Tomato yellow leaf curl virus (TYLCV), and Tomato mottle virus (ToMoV), all expressed in Escherichia coli by the pETh expression vector. The expressed coat protein of each virus was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for use as an immunogen. The antisera to BGMV, CabLCV, TYLCV, and ToMoV reacted in indirect (plate-trapping) enzyme-linked immunosorbent assay (ELISA) with extracts from begomovirus-infected tissue. The antisera to BGMV, CabLCV, TYLCV, and ToMoV also reacted specifically with the test begomovirus antigens in leaf imprint blots and Western blots. The CabLCV and TYLCV antisera were used to detect Bean golden yellow mosaic virus antigens by immunogold labeling of thin sections of infected bean tissues. In tissue blot immunoassays, the TYLCV antiserum reacted well with TYLCV antigens but not with ToMoV antigens, while CabLCV antiserum reacted well with ToMoV antigens and weakly with TYLCV antigens. The results indicate that polyclonal antisera prepared to expressed begomovirus coat proteins were useful for the detection of begomoviruses in an array of assays.

Molecular characterization and coat protein serology of watermelon leaf mottle virus (Potyvirus).

A cDNA library was generated from purified RNA of watermelon leaf mottle virus (WLMV) (Genus Potyvirus). Two overlapping clones totaling 2,316 nucleotides at the 3' terminus of the virus were identified by immunoscreening with coat protein antiserum. The sequence analyses of the clones indicated an open reading frame (ORF) of 2,050 nucleotides which encoded part of the replicase and the coat protein, a 243-nucleotide non-coding region (3'UTR), and 23 adenine residues of the poly (A) tail. The taxonomic status of WLMV was determined by comparisons of the sequence of the cloned coat protein gene and 3'UTR with potyvirus sequences obtained from GenBank. The nucleotide sequence identities of WLMV compared with 17 other potyviruses ranged from 55.6 to 63.5% for the coat protein, and from 37.2 to 48.3% for the 3'UTR. Phylogenetic analyses of the coat protein region and the 3'UTR indicated that WLMV did not cluster with other potyviruses in a clade with high bootstrap support. The coat protein gene was expressed in Escherichia coli and a polyclonal antiserum was prepared to the expressed coat protein. In immunodiffusion tests, WLMV was found to be serologically distinct from papaya ringspot virus type W, watermelon mosaic virus 2, zucchini yellow mosaic virus, and Moroccan watermelon mosaic virus. In Western blots and ELISA, serological cross-reactivity with other cucurbit potyviruses was observed. Serological and sequence comparisons indicated that watermelon leaf mottle virus is a distinct member of the Potyvirus genus.

The nucleotide sequence of the 3'-terminal region of dasheen mosaic virus (Caladium isolate) and expression of its coat protein in Escherichia coli for antiserum production.

A caladium isolate of dasheen mosaic virus (DsMV-Ch) was cloned as cDNA from genomic RNA. The sequence of the 3'-terminal 3158 nucleotides, which consisted of the 3'-terminus of the NIa gene, the NIb gene, the coat protein (CP) gene, and a 246-nucleotide non-coding region, was between 57-68% similar at the nucleotide level and 72-82% similar at the amino acid level when compared with other potyviruses. Phylogenetic analysis of aligned, selected potyviral CP sequences indicate that DsMV-Ch is similar to DsMV isolates infecting taro and closely related to the bean common mosaic virus subgroup in the genus Potyvirus. A recombinant DsMV-Ch CP (approximately 39 kDa) expressed in E. coli was used as an immunogen and the resulting antiserum reacted with DsMV and several other potyviruses in Western blots and indirect ELISA.

Partial Characterization of a Distinct Potyvirus Isolated from Watermelon in Florida.

Conspicuous, unusual nuclear inclusions in stained epidermal strips of leaves implicated a virus (designated isolate 2932) as the cause of foliar mosaic in a watermelon plant (Citrullus lanatus) received for analysis from South Florida in 1990. In greenhouse tests, mechanically inoculated plants of Cucurbita pepo (Small Sugar pumpkin and Early Prolific Straightneck squash) and watermelon (Crimson Sweet) developed mosaic or mottle symptoms. Isolate 2932 caused foliar symptoms in 16 cultivars of Cucurbita pepo, including Freedom II and Prelude II, and in six cultivars of watermelon. None of five cultivars of melon (Cucumis melo) or 11 cultivars of cucumber (Cucumis sativus) developed consistent, distinctive symptoms, but all of these cultivars were systemically infected based on back-inoculations to squash. No systemic infection of mechanically inoculated plants of 25 species representing 13 noncucurbitaceous plant families was detected. Crystalline nuclear inclusions, cytoplasmic amorphous inclusions, and cytoplasmic cylindrical inclusions were detected by light and electron microscopy in leaf tissues of infected squash and watermelon. Electron microscopy of squash leaf extracts revealed filamentous particles, and 86% of 159 particles measured ranged from 800 to 890 nm in length. The virus was transmitted in a nonpersistent manner by Myzus persicae from squash to squash in two of three trials. Immunodiffusion tests with polyclonal antisera prepared to partially purified 2932 or its capsid protein showed that the isolate was antigenically different from papaya ringspot virus type W, watermelon mosaic virus 2, and zucchini yellow mosaic virus. In limited testing of field samples of squash and watermelon since 1990, no additional isolates of the 2932 type have been found. The characteristics of isolate 2932 obtained thus far indicate that it is a distinct potyvirus. It is tentatively named watermelon leaf mottle virus to distinguish it from other potyviruses commonly isolated from cucurbits in Florida.

The Association of Severe Epidemics of Cucumber Mosaic in Commercial Fields of Pepper and Tobacco in North Florida with Inoculum in Commelina benghalensis and C. communis.

Since 1995, severe epidemics of cucumber mosaic virus (CMV) have occurred in select fields of tobacco (Nicotiana tabacum) and pepper (Capsicum annuum) in three counties in northern Florida. Yield losses greater than 50% have occurred in both crops. Baker and Zettler (1) identified the presence of CMV in one plant of tropical spiderwort (Commelina benghalensis) in an organic garden on the campus of the University of Florida 10 years ago. In addition, they infected tropical spiderwort and Asiatic dayflower (Commelina communis) with isolates of CMV. Since 1995, in one area of northern Alachua County, Asiatic dayflower has been found in abundance in and around some fields and found to be infected with CMV. Prior to this time, CMV had not been known to be epidemic in any crop in northern Florida. Also, commelinaceous weeds did not occur in such abundance in northern Florida. In Hamilton County, an epidemic of CMV occurred in one field of tobacco in 1997. Tropical spiderwort with viral-like symptoms was growing abundantly in that field. The symptoms in this weed included chlorotic ringspots and chevron-like line patterns. Light microscopy, with Azure A stain, revealed the presence of typical inclusions of CMV in pepper, tobacco, tropical spiderwort, and Asiatic dayflower. Symptomatic samples of the tobacco and the tropical spiderwort reacted in an immunodiffusion test with antiserum to a winged bean isolate of CMV (2). Extracts from tropical spiderwort (isolate 3603) were rubbed on squash. This isolate was thereafter maintained in squash (Cucurbita pepo cvs. Prelude II or Early Prolific Straightneck). Infected plants of both of these cultivars developed strong mosaic symptoms and were stunted. After passage through squash, the 3603 isolate induced mosaic in tobacco (cv. Burley 21). Some plants of the squash cultivars Destiny III and Liberator III, which have transgenic, coat protein-mediated resistance to CMV, developed restricted symptoms after inoculation with this isolate. CMV was recovered by back inoculation from symptomatic plants of these cultivars. Symptomless plants of tropical spiderwort transplanted from the field developed chlorotic ringspots and chevron-like line patterns following inoculation in the greenhouse with isolate 3603. Back inoculations to squash followed by immunodiffusion assays confirmed the presence of CMV in the inoculated tropical spiderwort plants but CMV was not detected in noninoculated control plants. This is the first report of tropical spiderwort being infected with CMV in a commercial situation in the United States. Because commelinaceous plants are well known to be excellent hosts of CMV (1), we believe that the increased presence of perennial, commelinaceous weeds is a factor contributing to the epidemics of CMV in northern Florida. References: (1) C. A. Baker and F. W. Zettler. Plant Dis. 72:513, 1988. (2) C. A. Ku-wite and D. E. Purcifull. Plant Dis. 66:1071, 1982.

Phenotypic variation in transgenic tobacco expressing mutated geminivirus movement/pathogenicity (BC1) proteins.

Tobacco plants were transformed with the movement protein (pathogenicity) gene (BC1) from tomato mottle geminivirus (TMoV), using Agrobacterium-mediated transformation. Different transgenic tobacco lines that expressed high levels of the BC1 protein had phenotypes ranging from plants with severe stunting and leaf mottling (resembling geminivirus symptoms) to plants with no visible symptoms. The sequence data for the BC1 transgene from the transgenic plants with the different phenotypes indicated an association of spontaneously mutated forms of the BC1 gene in the transformed tobacco with phenotype variations. One mutated transgene associated with an asymptomatic phenotype had a major deletion at the C terminus of 119 amino acid residues with a recombination resulting in the addition of 26 amino acid residues of unidentified origin. This asymptomatic, mutated BC1 attenuated the phenotypic expression of the symptomatic BC1 in a tobacco line containing both copies of the BC1 gene. Another mutated form of the BC1 gene amplified from an asymptomatic, multicopy transgenic tobacco plant did not induce symptoms when transiently expressed in tobacco via a virus vector. The symptom attenuation in the transgenic tobacco by the asymptomatic BC1 may involve trans-dominant negative interference.

Characterization of the P1 protein and coding region of the zucchini yellow mosaic virus.

The nucleotide sequence of the 5'-terminal P1 coding region of an aphid-transmissible isolate of zucchini yellow mosaic virus (ZYMV; strain FL/AT), a mild isolate (strain MD) and a severe isolate (strain SV), all from Florida, were compared with two other ZYMV isolates. The ZYMV MD and SV isolates and an isolate from California (ZYMV CA) had 95-98% sequence similarities to FL/AT, whereas an isolate from Reunion Island (ZYMV RU) had a 60% sequence similarity to FL/AT. ZYMV MD had an 18 nucleotide insert following the start codon of the P1 coding region. The P1 proteins of all ZYMV isolates shared conserved amino acids in areas of the C terminus similar to those reported for other potyviruses. Polyclonal antisera were prepared to the P1 proteins of ZYMV FL/AT and RU expressed in Escherichia coli. The FL/AT and RU P1 antisera showed varying degrees of reactivity in Western blots with extracts of pumpkin (Cucurbita pepo L.) singly infected with a number of distinct ZYMV isolates. The reaction of the FL/AT P1 antiserum with isolate RU-infected tissue extracts was very weak compared to the homologous reaction. Neither antiserum reacted with extracts from plants singly infected with three other potyviruses, a potexvirus, or a cucumovirus. The P1 proteins of ZYMV isolates ranged in molecular mass from 33 kDa to 35 kDa. The P1 protein of strain MD was larger (35 kDa) than that of FL/AT (34 kDa). Indirect immunofluorescence tests with FL/AT P1 antiserum indicated that the P1 protein aggregates in ZYMV-infected tissues. The antisera to the ZYMV P1 proteins have potential as serological probes for identifying ZYMV and for distinguishing ZYMV isolates by immunoblotting.

Biological variability of potyviruses, an example: zucchini yellow mosaic virus.

Potyviruses present an important variability which may affect biological properties such as host range, symptomatology, virulence towards resistance genes, and transmissibility by vectors. A brief account of this potential is presented and illustrated by some aspects of the biological variability of zucchini yellow mosaic virus (ZYMV).

A comparison of pepper mottle virus with potato virus Y and evidence for their distinction.

Pepper mottle virus (PepMOV) was identified as a distinct potyvirus infecting peppers in Arizona and Florida in the 1970's. The distinction of PepMoV from potato virus Y (PVY) has recently been challenged on the basis of sequence comparisons of the coat proteins and of the 3' nontranslated regions of the viral RNAs. We summarize the biological, cytological, serological, and in vitro translational studies which compare the apparent differences, and also similarities, between PepMoV and PVY. We conclude that although PepMoV may be more closely related to PVY than to other known potyvirus, PepMoV should be maintained as a separate virus on the basis of its distinctive characteristics.

Evidence that pepper mottle virus and potato virus Y are distinct viruses: analyses of the coat protein and 3' untranslated sequence of a California isolate of pepper mottle virus.

Pepper mottle virus (PepMoV) is a member of the large and complex genus Potyvirus, and is classically distinguished from other members of the genus by differential host range and cytopathology as well as serology of the coat protein and cytoplasmic inclusion body proteins. Here we report the deduced amino acid sequence of the coat protein of a California potyvirus identified by a variety of classical methods as PepMoV (PepMoV C). Comparison of the 3' untranslated nucleic acid sequence and the deduced coat-protein amino acid sequence of the PepMoV C isolate with those of PVY and other potyviruses indicates that PepMoV C is sufficiently diverged to be considered a distinct virus species. Thus, comparative sequence analyses of the PepMoV C isolate support earlier serological and biological evidence that PepMoV and PVY are distinct viruses.

Serological relationships involving potyviral nonstructural proteins.

This report represents a compilation of many of the publications on antigenic properties of potyviral-specified nonstructural proteins. Polyclonal antisera have been prepared for use in characterization of six nonstructural proteins. These include antisera to the cylindrical inclusion proteins of at least 28 potyviruses, to small nuclear inclusion protein (protease) of four potyviruses, to large nuclear inclusion protein (putative replicase) of three viruses, helper component-protease or amorphous inclusion protein of at least four viruses, to the P1 protein (located at the N-terminus of the polyprotein) of one virus, and to the P3 protein (located between helper component protease and cylindrical inclusion protein) of one virus. Monoclonal antibodies also have been prepared to several of these nonstructural proteins. The evidence thus far indicates that cylindrical inclusions of different potyviruses have both conserved and unique epitopes. Nuclear inclusion proteins and amorphous inclusion proteins also may have conserved and unique epitopes. Antigenic relationships of potyviral nonstructural proteins have potential for the identification and classification of potyviruses.

Isolation and partial characterization of the amorphous cytoplasmic inclusions associated with infections caused by two potyviruses.

The potyviruses pepper mottle (PeMV) and watermelon mosaic virus-1, a strain of papaya ringspot (PRSV-W), induce the formation of a second type of cytoplasmic inclusion in addition to cylindrical (pinwheel) inclusions. The conspicuous aggregates of electron-dense material with imperfect spherical shapes have been called amorphous inclusions (AI). The AI were isolated from extracts of infected tissue using a combination of clarification with Triton X-100 and low-speed centrifugations through sucrose cushions. Electrophoresis, in sodium dodecyl sulfate-permeated polyacrylamide gels (SDS-PAGE), of the SDS-dissociated AI revealed a single constituent protein with a molecular weight of 51,000. SDS-PAGE-purified AI proteins were immunogenic and serologically unrelated to proteins of host, capsid, cylindrical inclusions, and tobacco etch virus nuclear inclusions. The AI protein of PeMV was distinct from the AI protein of PRSV-W on the basis of peptide analysis and serological tests. The antisera prepared to SDS-PAGE-purified AI proteins reacted specifically with Al in situ in immunofluorescence tests. The characteristics of the AI indicate that they are of viral origin.

Identification of potyviral amorphous inclusion protein as a nonstructural, virus-specific protein related to helper component.

Antisera to amorphous inclusion (AI) proteins associated with infections by pepper mottle virus (PeMV) and the watermelon mosaic virus-1 strain of papaya ringspot virus (PRSV-W) were used to probe in vitro translation products of the viral RNAs. The major translation product of PeMV RNA in the rabbit reticulocyte lysate (RRL) system was a previously reported polypeptide of apparent molecular weight 78,000 (Mr 78K). It reacted with anti-AI serum, whereas the major translation product in the wheat germ (WG) system was a 30K polypeptide that did not react with the antiserum. These results, the Mr values, and analyses of peptides generated by partial digestion with proteinase indicate that the amino acid sequences of the 30K polypeptide and the (Mr) 51K AI protein are distinct subsets of the 78K polypeptide amino acid sequence. Similar results were obtained with PRSV-W except that the Mr values of the corresponding translation products are 110K (RRL) and 60K (WG). Thus the 5'-most region of the PeMV and PRSV-W RNAs (corresponding to 78K and 110K, respectively) appears to encode two proteins rather than one as previously supposed on the basis of RRL translation products. Reciprocal serological tests revealed that the tobacco vein mottling virus aphid transmission helper component protein was related to AI protein. There is direct evidence that the AI represent another potyviral-coded nonstructural protein and the first evidence that a biologically functional protein is related to a component of a potyviral inclusion.

Mapping of the two coat protein genes on the middle RNA component of squash mosaic virus (comovirus group).

Squash mosaic virus, a member of the comovirus group, has a divided genome identified as middle-component RNA (M RNA; MW = 1.4 x 10(6)) and bottom-component RNA (B RNA; MW = 2 x 108). The isometric capsid of squash mosaic virus is composed of two distinct protein monomers with molecular weights of 22,000 (22k) and 42k. The isolated RNA components were translated in a rabbit reticulocyte lysate system. Translation products of the B RNA had estimated molecular weights of 190k, 51k, and 32k, while the M RNA products ranged in estimated molecular weight from 22k to 112k. Products of the B RNA did not react with antisera prepared to the 22k and 42k coat proteins. All of the M RNA products reacted with antiserum to the 22k coat protein and all the products larger than 35k reacted with antiserum to the 42k coat protein. The 22k product of M RNA translation had a V-8 protease peptide pattern identical to that of the 22k coat protein. Protease peptide patterns of the M RNA 64k and 112k-105k translation products showed a number of peptide fragments similar to those produced by the 22k and 42k coat proteins, indicating that the translation products contained the sequences of the two coat proteins. The proposed gene order of translation for squash mosaic virus M RNA is as follows: 5' end-22k coat protein gene-42k coat protein gene-48k unidentified protein gene-untranslated sequence-3' end.