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Biomedical subjects

D D Shukla

Publications and source records attributed to D D Shukla.

At least 19 recordsLinked to original sources
PubMed2003

Infectivity of in vitro transcripts of Johnsongrass mosaic potyvirus full-length cDNA clones in maize and sorghum.

In vitro transcripts of full-length cDNA clones of the Johnsongrass strain of Johnsongrass mosaic potyvirus (JGMV-Jg) were infectious on maize and sorghum when inoculated by mechanical or by biolistic bombardment. Two of the cDNA clones with spontaneous mutations in the coat protein were not infectious. Sequence differences between infectious and non-infectious transcripts revealed that alteration of inferred amino sequences, near or in the N-terminus of the coat protein, profoundly affected the infectivity of transcripts. Transcripts of chimeric full-length cDNA of JGMV-Jg, containing coat protein sequences from the Krish-infecting strain of JGMV, were infectious in Krish resistant sorghums.

Base Sequence↗
PubMed1998

Coat protein sequence of Krish-infecting strain of Johnsongrass mosaic potyvirus.

The morphology of an Australian strain of Johnsongrass mosaic potyvirus (JGMV Krish-infecting strain), capable of infecting sorghums carrying the Krish potyvirus resistance gene, was investigated and the base sequence of the coat protein region determined. Under the electron microscope the virus was indistinguishable from the wild type prevalent in Australia, JGMV-Jg. However, there were some significant changes in the inferred amino acid sequence in both the N-terminus and the core regions of the coat protein. Some of these amino acid changes may be responsible for breaking the resistance of sorghums carrying the Krish virus resistance gene. In the discussion mention is made of a preliminary result with a mutated in vitro transcript which supports this suggestion.

Amino Acid Sequence↗
PubMed1996

Chimeric potyvirus-like particles as vaccine carriers.

Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune responses. Coat protein (CP) monomers of a potyvirus (Johnsongrass mosaic virus) when produced in heterologous host expression systems (Escherichia coli, yeast and insect cells) self-polymerized to produce potyvirus-like particles (PVLPs). The N- and C-terminal regions of potyvirus CP are surface-exposed and are not required for assembly. Hybrid CP monomers containing short peptides fused to their N- and/or C-termini, or large target antigens fused to the N-terminus or replacing most of the N- or C-terminal exposed regions retained the ability to assemble into hybrid PVLPs. Such chimeric PVLPs were highly immunogenic in mice and rabbits even in the absence of any adjuvant. Potyvirus CP is highly versatile in accommodating peptides or large antigens and is able to present antigens exposed on the surface of virus-like particles. This, combined with the efficiency of high level bacterial and insect cell expression systems, makes PVLPs an attractive non-pathogenic and non-replicative vaccine carrier.

Animals↗
PubMed1995

Evaluation of complete genome sequences and sequences of individual gene products for the classification of hepatitis C viruses.

Comparisons of genome and polyprotein sequences of hepatitis C virus (HCV) isolates world-wide has led to the identification of nine major genotypes and many subtypes. This classification is based on either complete genome/polyprotein sequences or sequence data from the 5' noncoding region, core, E1, NS3 or NS5B genes. The relative merit of different gene segments as taxonomic markers and the validity of the resulting assignments is not clear at this stage. To resolve the taxonomy of HCV genotypes and subtypes, we have compared the complete genome and polyprotein sequences of 19 HCV isolates available in the databases as well as sequences of individual genes and gene products of these isolates. Based on the correlation between sequence relationships and taxonomic assignments of other RNA viruses, we show that the nine major genotypes of HCV represent nine distinct virus species and their subtypes subspecies. Our sequence comparison of the 5' noncoding regions and the individual gene products suggests that E2, NS2, NS5B, E1, NS4A, NS4B and NS5A (in that order) are the most appropriate regions for the discrimination between species, subspecies and strains of HCV. The 5' noncoding, core and NS3 regions are less effective in distinguishing between species, subspecies and strains. Based on a comparison of the polymerase sequence identities of HCVs, pestiviruses and flaviviruses as well as the recent information on the size and morphology of HCV virions, we propose that HCVs, pestiviruses and flaviviruses should be classified into three separate families, named Hepciviridae, Pestiviridae and Flaviviridae, respectively rather than three genera of the Flaviviridae as currently classified. We also propose "Hepcivirus" as the genus name for HCVs.

Base Sequence↗
PubMed1994

Nucleotide sequence of the 3'-terminal region of the genome confirms that pea mosaic virus is a strain of bean yellow mosaic potyvirus.

The 1,035 nucleotides at the 3'end of the I strain of pea mosaic potyvirus (PMV-I) genomic RNA, encoding the coat protein, have been cloned and sequenced. A comparison of the derived coat protein sequence with those of the bean yellow mosaic virus (BYMV) strains, CS, S, D and GDD, indicates that PMV-I is a strain of BYMV. Sequence comparisons and hybridisation studies using the 3'-noncoding region support this classification. The nucleotide and protein sequence data also suggest that PMV-I and BYMV-CS form one subset of BYMV strains while the other three strains form another.

Amino Acid Sequence↗
PubMed1994

Cowpea aphid borne mosaic virus-Morocco and South African Passiflora virus are strains of the same potyvirus.

High performance liquid chromatography (HPLC) profiles of tryptic peptides and partial amino acid sequence analysis have been employed to establish the taxonomic status of the Moroccan isolate of cowpea aphid-borne mosaic virus (CABMV). Some previous reports have suggested CABMV to be very closely related to blackeye cowpea mosaic virus (B1CMV) while other reports have concluded that this relationship is distant. In this report a tryptic digest of the coat protein of CABMV-Morocco was compared with those of the coat proteins of B1CMV-Type, B1CMV-W, the mild mottle strain of peanut stripe virus (PStV-MM) and the NY15 strain of bean common mosaic virus (BCMV-NY15), all of which are now recognised as strains of BCMV. The comparisons also included the NL-3 strain of bean necrosis mosaic virus (BNMV-NL3), which had previously been classified as a strain of BCMV. The HPLC peptide profiles indicated that CABMV-Morocco was distinct from BCMV and BNMV. Amino acid sequence analysis of peptides accounting for more than half of the coat protein confirmed that CABMV-Morocco was not a strain of BNMV or BCMV but was a distinct member of the BCMV subset of viruses that previously has been shown to include BCMV, BNMV, soybean mosaic virus, zucchini yellow mosaic virus, passionfruit woodiness virus and South African Passiflora virus (SAPV). Comparison of the partial sequence data with these and other published sequences revealed that the coat protein of CABMV-Morocco is very similar to that of SAPV suggesting that they are strains of the same virus. Since CABMV was described over 25 years earlier than SAPV, the name CABMV should take precedence and SAPV should be renamed CABMV-SAP, the South African Passiflora strain of CABMV.

Amino Acid Sequence↗
PubMed1993

Sequence diversity in the surface-exposed amino-terminal region of the coat proteins of seven strains of sugarcane mosaic virus correlates with their host range.

The N-terminal region of the coat proteins of five strains (Isis, Brisbane, Sabi, Bundaberg, and BC) of sugarcane mosaic virus (SCMV) isolated from four different plant species (sugarcane, sabi grass, wild sorghum, and blue couch grass) have been compared with the previously published data for SCMV-SC and SCMV-MDB, isolated from sugarcane and maize, respectively. The region, beginning at residue 11 and ending 16 residues beyond the second trypsin cleavage site of the coat protein, varied in size from 68 amino acid residues (Bundaberg) to 115 residues (BC) and contained repeat sequence motifs. Comparisons of the sequence identity and the nature of the repeats in the seven sequences showed that there were five different sequence patterns. These could be grouped further into three subsets which appeared to correlate with the host range of the strains. SCMV-Brisbane, SC, and Isis, isolated from sugarcane, showed almost identical sequence patterns and formed one subset. The other four strains had different sequence patterns and could be grouped further into a Sabi and Bundaberg subset (isolated from sabi grass), and a BC and MDB subset.

Amino Acid Sequence↗
PubMed1993

Characterisation and epitope analysis of monoclonal antibodies to virions of clover yellow vein and Johnsongrass mosaic potyviruses.

Mouse monoclonal antibodies (MAbs) against the Australian B strain of clover yellow vein (ClYVV-B) and the JG strain of Johnsongrass mosaic (JGMV) potyviruses were produced, characterised and the epitopes with which they reacted were deduced. Using intact particles of ClYVV a total of ten MAbs were obtained which reacted strongly with ClYVV-B in an enzyme-linked immunosorbent assay and Western blots. Four of these MAbs (1, 2, 4, and 13) were found to be ClYVV-specific, as they reacted with all five ClYVV strains from Australia and the U.S.A. but not with 11 strains of bean yellow mosaic (BYMV), pea mosaic (PMV), and white lupin mosaic (WLMV) viruses which, together with ClYVV, form the BYMV subgroup of potyvirses. These MAbs failed to react with eight other potyvirus species, including six which infect legumes like the viruses in the BYMV subgroup. The ClYVV MAb 10 was found to be BYMV subgroup-specific. It reacted strongly with 15 of the 16 strains of viruses in the subgroup and gave no reaction with eight other potyviruses. The other five ClYVV MAbs reacted with varying degrees of specificity with the BYMV subgroup viruses and also with other potyviruses. Eight of the ClYVV MAbs (1, 2, 4, 5, 13, 17, 21, and 22) reacted with the intact coat proteins only and not with the truncated (minus amino terminus) coat protein of ClYVV suggesting that the epitopes for these MAbs are located in the surface-exposed, amino-terminal region of the ClYVV coat protein. Comparison of published coat protein sequences of BYMV and ClYVV isolates indicated that the epitopes for the four ClYVV-specific MAbs may be in the amino-terminal region spanning amino acid residues 18 to 30, whereas those for the other four MAbs may be located in the first 17 amino-terminal amino acid residue region. The epitopes that reacted with BYMV subgroup-specific MAb 10 and MAb 30 which reacted with 20 of the 24 potyvirus isolates, are probably located in the core region of ClYVV coat protein as these MAbs reacted with the intact as well as truncated coat protein of ClYVV. Analysis, in Western blot immunoassay, of 17 MAbs raised against virions of JGMV revealed that only two MAbs (1-25 and 4-30) were JGMV-specific, whereas others displayed varying degrees of specificity to different potyviruses.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Watermelon mosaic virus-Morocco is a distinct potyvirus.

The relationship of the Morocco isolate of watermelon mosaic virus (WMV) to WMV2, soybean mosaic virus (a virus closely related to WMV2) and the W strain of papaya ringspot virus (PRSV-W), formerly WMV1, was examined by comparing tryptic peptide profiles using high performance liquid chromatography. The profiles indicated that the coat protein sequence of WMV-Morocco differed substantially from those of the other potyviruses. This conclusion was supported by sequence data from five tryptic peptides from the coat protein of WMV-Morocco, which showed only 61-68% identity to equivalent sequences in PRSV-W, WMV2 and zucchini yellow mosaic, another potyvirus infecting cucurbits. Based on the above data, and on known correlations between coat protein sequence similarities and potyvirus relationship, it is concluded that WMV-Morocco should be regarded as a distinct potyvirus.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence of Johnsongrass mosaic potyvirus genomic RNA.

The complete nucleotide sequence of the RNA genome of Johnsongrass mosaic virus (JGMV), which infects monocotyledonous plant species, has been determined from cloned viral cDNAs. The JGMV genomic RNA is 9,766 nucleotides in length, excluding the poly (A) tail, and contains a large open reading frame that codes for a polyprotein of 3,052 amino acids. The open reading frame is flanked by a 5' untranslated region of 135 and a 3' untranslated region of 475 nucleotides. A comparison of the JGMV polyprotein with the sequences of other members of the Potyvirus genus allowed the delineation of putative proteolytic cleavage sites. This comparison also showed that JGMV has a genome organization that is similar to other members of the genus Potyvirus. As with other potyviruses, the JGMV P1 and P3 proteins were found to be the most variable and the NIb protein the most conserved when compared with the corresponding proteins of other potyviruses. JGMV differed from other members of the genus Potyvirus in the amino acid at the -1 position of the putative cleavage sites of the NIa proteinase. The putative cleavage site for P3/CI, CI/NIa, NIa/NIb, and NIa-VPg/NIa-Pro in the JGMV polyprotein each contained a glutamate at the -1 position which is glutamine in most other potyviruses. Glutamate at the -1 position has also been observed at the CI/NIa-VPg junction of pea seed borne mosaic virus and turnip mosaic virus and at the NIb/CP junction of papaya ringspot virus polyproteins.

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

Sequence data as the major criterion for potyvirus classification.

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.

Capsid↗
PubMed1992

Differentiation of the four viruses of the sugarcane mosaic virus subgroup based on cytopathology.

A cytological comparison has been made of representative isolates of johnsongrass mosaic (JGMV), maize dwarf mosaic (MDMV), sorghum mosaic (SrMV) and sugarcane mosaic (SCMV) viruses. These four viruses now encompass the complex of virus strains which were formerly considered as strains of sugarcane mosaic and/or maize dwarf mosaic viruses. The structure of the cytoplasmic cylindrical inclusions induced by these viruses, together with other cytological alterations, allow the four viruses to be distinguished. Pinwheels, scrolls and laminated aggregates were produced only by SCMV whereas JGMV, MDMV, and SrMV produced only pinwheels and scrolls. SrMV produced amorphous cytoplasmic inclusions which are not produced by JGMV and MDMV. The latter two were rather similar in cytological effects except that the SCMV-JG (U.S.A.) isolate of MDMV produced aggregates of needle-like structures in the cytoplasm which were not found with JGMV and the other MDMV isolates. The specific cytological effects induced by these viruses thus corroborate the recent classification of these viruses based mainly on the properties of the coat-protein gene, the 3' noncoding nucleotide sequences, and host reactions.

Cytopathogenic Effect, Viral↗
PubMed1992

Present status of the sugarcane mosaic subgroup of potyviruses.

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.

Edible Grain↗
PubMed1992

Strains of bean common mosaic virus consist of at least two distinct potyviruses.

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.

Capsid↗
PubMed1992

Serology of potyviruses: current problems and some solutions.

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.

Animals↗
PubMed1992

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

The 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 in Escherichia 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.

Base Sequence↗