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Identification and classification of potyvirus on the basis of coat-protein sequence data and serology
Abstract
Summary The 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.
... The structure of the BBrMV genome is composed of a monopartite linear single-stranded positive sense (+ssRNA) genome which forms a flexuous filamentous rod with a length of 720 nm to 850 nm and a diameter of 12 mm-15 mm. Although the virion is not enveloped, the single-stranded RNA genome is encapsidated by 2000 copies of the coat protein (CP) (Hollings and Brunt 1981;Shukla and Ward 1989), a gene encoded in the BBrMV genome. The + ssRNA genome encodes for a single long polyprotein that is processed by viral proteinases encoded by BBrMV into 10 mature proteins. ...
... This unique N-terminal region of the CP is immunodominant and thus contains virus-specific epitopes. The remaining two-thirds of the C-terminal region is highly similar to the studied potyvirus sequence (Shukla and Ward 1989). Aside from encapsidating the viral RNA genome, the CP is also responsible for vector transmission (Atreya et al. 1991, Shukla et al. 1991), cell-to-cell and systemic movement (Urcuqui-Inchima et al. 2001Hofius et al. 2007), and regulation of viral RNA amplification (Urcuqui-Inchima et al. 2001). ...
... RNA viruses are characterised by their ability to rapidly adapt to new or resistant hosts (Tsompana et al. 2005;Gibbs and Ohshima 2010) thus studies on genetic variability of populations of plant RNA viruses are important not only for understanding the evolution of viruses but also how these sequence variabilities affect virus-host interactions. Comparison of CP genes can be applied for the identification and differentiation of distinct potyviruses and their strains (Shukla and Ward 1989). PDR is observed to be specific towards the virus or viral strain from which the transgene was derived (Lomonossoff 1995). ...
... Previously, there was no protein-related information in the yam bean crop and thus there has been no work done on functional and structural protein characterization of YBMV globally. In 1988, Shukla and Ward investigated the structural characterization of the potyvirus (coat proteins) CP and observed that the amino acid sequences of CP are sufficient to identify and differentiate the individual potyvirus and its strains [30][31][32]. The coat protein is considered to be the major gene product in the virion [33]. ...
... Thus, the capsid protein and 3' NCR sequences are the most essential parts of the potyviral genome for taxonomic study, and the 3'terminal area may be utilized to identify virus strains from other potyviruses, according to all molecular data [62,65]. According to Shukla and Ward (1989), the nucleotide sequence of the CP gene among strains of a certain potyvirus species was more than 90% similar [31], whereas the degree of similarity in the nucleotide sequences of the 3' UTR across strains of specific potyvirus species ranged from 83-99 percent, whereas different virus species had identities ranging from 39-53 percent, according to Frenkel et al (1989) [66]. Adams et al 2005 looked at several potyvirus sequences and determined that the greatest possible CP nucleotide sequence identity in the same potyvirus species was 76-77 percent, while the highest possible CP amino acid sequence identity was 82 percent [67]. ...
... Thus, the capsid protein and 3' NCR sequences are the most essential parts of the potyviral genome for taxonomic study, and the 3'terminal area may be utilized to identify virus strains from other potyviruses, according to all molecular data [62,65]. According to Shukla and Ward (1989), the nucleotide sequence of the CP gene among strains of a certain potyvirus species was more than 90% similar [31], whereas the degree of similarity in the nucleotide sequences of the 3' UTR across strains of specific potyvirus species ranged from 83-99 percent, whereas different virus species had identities ranging from 39-53 percent, according to Frenkel et al (1989) [66]. Adams et al 2005 looked at several potyvirus sequences and determined that the greatest possible CP nucleotide sequence identity in the same potyvirus species was 76-77 percent, while the highest possible CP amino acid sequence identity was 82 percent [67]. ...
Plants are constantly threatened by a virus infection, i.e., Potyviruses, the second largest genus of plant viruses which results in several million-dollar losses in various essential crops globally. Yam bean (Pachyrhizus erosus) is considered to be one of the essential tuberous legume crops holding a great potential source of starch. Yam Bean Mosaic Virus (YBMV) of Potyvirus group belonging to the family potyviridae affects Yam bean and several angiosperms both in the tropical and sub-tropical regions causing large economical losses in crops. In this study, we attempted to understand the sequence-structure relationship and mode of RNA binding mechanism in YBMV CP using in silico integrative modeling and all-atoms molecular dynamics (MD) simulations. The assembly of coat protein (CP) subunits from YBMV and the plausible mode of RNA binding were compared with the experimental structure of CP from Watermelon mosaic virus potyvirus (5ODV). The transmembrane helix region is present in the YBMV CP sequence ranging from 76 to 91 amino acids. Like the close structural-homolog, 24 CPs monomeric sub-units formed YBMV a conserved fold. Our computational study showed that ARG¹²⁴, ARG¹⁵⁵, and TYR¹⁵¹ orient towards the inner side of the virion, while, THR¹²², GLN¹²⁵, SER⁹², ASP⁹⁴ reside towards the outer side of the virion. Despite sharing very low sequence similarity with CPs from other plant viruses, the strongly conserved residues Ser, Arg, and Asp within the RNA binding pocket of YBMV CP indicate the presence of a highly conserved RNA binding site in CPs from different families. Using several bioinformatics tools and comprehensive analysis from MD simulation, our study has provided novel insights into the RNA binding mechanism in YBMV CP. Thus, we anticipate that our findings from this study will be useful for the development of new therapeutic agents against the pathogen, paving the way for researchers to better control this destructive plant virus.
... However, Shukla and Ward [9] demonstrated, based on nucleotide and amino acid sequences of the coat protein (CP), that this virus was indeed a member of a new potyvirus species, which was given the name Johnsongrass mosaic virus. Previous reports have demonstrated the occurrence of this virus in Australia [8,10,11], South America [5,12,13], Nigeria [14] and the USA [6,15]. In the USA, JGMV was first isolated in Texas and described as an MDMV isolate capable of infecting oat, johnsongrass and maize [6]. ...
... In the USA, JGMV was first isolated in Texas and described as an MDMV isolate capable of infecting oat, johnsongrass and maize [6]. Now, this strain is designated as JGMV-MDO (maize dwarf oat strain) [15]. JGMV has also been reported in Venezuela infecting several varieties of maize [12,16] and in Colombia infecting Brachiaria spp. ...
... JGMV-CNPGL did not infect Triticum aestivium BRS264 (wheat), Hordeum vulgare L. VCU-CPAC (barley), Crotalaria juncea or Glycine max (soybean) under greenhouse conditions. The JGMV-CNPGL host range was similar to that reported for JGMV-N [14] and JGMV-MDO [15]. More importantly, JGMV-CNPGL infected maize, suggesting that this virus represent a potential threat to this crop in Brazil, one of the largest maize producers. ...
The complete genome sequence (9,865 nucleotides) of a highly divergent johnsongrass mosaic virus isolate (JGMV-CNPGL) was determined using Illumina sequencing. This isolate infected 10 genotypes of gramineous plants including maize. A comparative analysis of the complete genome showed 80 % nucleotide (nt) sequence identity (86 % amino acid (aa) sequence identity) to a johnsongrass mosaic virus isolate from Australia. The coat protein (CP) identity values, however, were lower than those for the whole genome (78 % and 80 % for nt and aa, respectively) and were close to the species demarcation values (77 % nt and 80 % aa). Unexpectedly, the amino-terminal portion of CP of JGMV-CNPGL showed only 38 % sequence identity to other JGMV isolates. The biological implications of this sequence divergence remain to be elucidated.
... The amplified fragment contains approximately 800 bp of CP gene and 3'UTR region of the Potyvirus (Fig. 3). Coat protein gene was chosen for sequence analysis of this virus because it is more useful and more easily applied than other properties for identification and classification of Potyviruses (Shukla and Ward, 1989). After sequencing, the consensus sequence was compared to the sequences registered in the nucleotide databases using the Basic Local Alignment Search Tool (blastn; http: //www.ncbi.nlm.nih.gov/blast/). ...
... According to the currently used criteria for Potyvirus classification, viruses sharing under 76-77% coat protein nucleotide identity would be considered members of distinct species (Adams et al., 2005). The amino acid sequence homology between distinct members of Potyviruses, ranged from 38 to 71% (average 54%) while that inter strains of the one virus ranged from 90 to 99% (average 95%) (Shukla and Ward, 1989 Molecular characterization of ten nucleotide sequence of Potyvirus coat protein from patchouli plants in Indonesia with those related species previously reported in GenBank (Table 3) were further performed. All ten isolates shared high (> 85%) nucleotide sequence identities with TeMV Hanoi, Pangda12.1 and Pangda15.1 and it should be classified as those spesies but different strain (Table 4). ...
This study was carried out to identify the virus associated with mosaic disease on patchouli in Indonesia. The diseased plant samples collected from Garut, Ciamis, Bogor (West Java) and West Pasaman (West Sumatera) strongly reacted with Potyvirus antiserum by enzyme-linked immunosorbent assay, but did not show any signal with antisera to Cucumber mosaic virus, Tobacco mosaic virus, Broad bean wilt virus 1 and Broad bean wilt virus 2; except the samples collected from Brebes (Central Java) which strongly reacted with Broad bean wilt virus 2 (Fabavirus) only. Besides that, the diseased plant samples collected from North Sumatera (Pakpak Bharat) did not react to any antiserum tested. The Potyvirus was then isolated. Positive results were obtained using a reverse transcriptase-polymerase chain reaction (RT-PCR) method to detect and identify Potyvirus from nucleic acid extracts of the symptomatic patchouli plants, using a pair of degenerate primers specific for Potyvirus CP gene. The sequence of this RT-PCR fragment, consisted of 800 bp, confirmed association of a Potyvirus with mosaic disease on patchouli plants in Indonesia. Potyvirus infecting patchouli plants in Indonesia are closely related to Telosma mosaic virus (TeMV) and Passionfruit woodiness virus (PaWP). This paper is the first report that TeMV, PaWP and Fabavirus are associated with mosaic disease on patchouli plants in Indonesia.
... By the late 1980s, the taxonomy of the potyvirus group was in a very unsatisfactory state because of its size, complexity and variability. It was suggested then that successful resolution of potyvirus detection and identification presents a major challenge for plant virologists Shukla and Ward, 1989b). This unsatisfactory state of potyvirus taxonomy was attributed partly to the large size of this group, since it is the most rapidly growing and is the largest among the 35 different plant virus groups . ...
... It was believed, on the basis of biological properties and inconsistent serology that stains of potyviruses form a continuous array (continuum hypothesis) between two or more viruses in such a way that boundaries separating distinct potyviruses cannot be sharply defined (Hollings and Brunt, 1981). Recently, however, amino acid sequences of potyvirus CPs have been adopted as a basis for the identification and classification of potyviruses into distinct viruses and strains Shukla and Ward, 1989b). This criterion proved to be inconsistent with the "continuum" hypothesis and showed a clear demarcation between distinct viruses and between strains. ...
... The pot viruses are particularly problematic because they are readily transmitted in seeds, so they may move over long distances in this form without being spread in the immediate environment by insect vectors. This leads to difficulties in their management (Galves and Molares, 1989;Shukla and Ward, 1989). Some fungal and bacterial diseases are transmitted in and on seeds, and all may be harboured in plant debris and spread by rainfall splashes. ...
Artificial Intelligence (AI) and deep learning have the capacity to reduce losses in crop production, such as low crop yields, food insecurity, and the negative impacts on a country's economy caused by crop infections. This study aims to find the knowledge and technological gaps associated with the application of AI-based technologies for plant disease detection and pest prediction at an early stage and recommend suitable curative measures. An evidence-based framework known as the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) methodology was used to conduct systematic reviews of the state-of-the-art of AI and deep learning techniques for crop disease identification and pest prediction in developing countries. The results demonstrate that conventional methods for plant disease management face some challenges, such as being costly in terms of labour, having low detection and prediction accuracy, and some are not environmentally friendly. Also, the rapid increase in data-intensive and computational-intensive tasks needed for plant disease classification using traditional machine learning methods poses challenges such as high processing time and storage capacity. Consequently, this paper recommends a deep learning and AI-based strategy to enhance the detection, prediction and prevention of crop diseases. These recommendations will be the starting point for future research.
... Symptoms also aid in roguing of diseased plants as a strategy for preventing virus spread (Naidu and Hughes, 2003). Potyviruses have limited host ranges and can be identified based on the characteristic symptoms they produce in certain host plants (Shukla and Ward, 1989). In this study, the symptoms observed on papaya plants included those that are attributed to PRSV infection (Tripathi et al., 2008;Zhao et al., 2016), although PRSV was not detected in our samples. ...
Carica papaya L. is an important fruit crop grown by small- and large-scale farmers in Kenya for local and export markets. However, its production is constrained by papaya ringspot disease (PRSD). The disease is believed to be caused by papaya ringspot virus (PRSV). Previous attempts to detect PRSV in papaya plants showing PRSD symptoms, using enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) procedures with primers specific to PRSV, have not yielded conclusive results. Therefore, the nature of viruses responsible for PRSD was elucidated in papaya leaves collected from 22 counties through Illumina MiSeq next-generation sequencing (NGS) and validated by RT-PCR and Sanger sequencing. Viruses were detected in 38 out of the 48 leaf samples sequenced. Sequence analysis revealed the presence of four viruses: a Potyvirus named Moroccan watermelon mosaic virus (MWMV) and three viruses belonging to the genus Carlavirus. The Carlaviruses include cowpea mild mottle virus (CpMMV) and two putative Carlaviruses—closely related but distinct from cucumber vein-clearing virus (CuVCV) with amino acid and nucleotide sequence identities of 75.7–78.1 and 63.6–67.6%, respectively, in the coat protein genes. In reference to typical symptoms observed in the infected plants, the two putative Carlaviruses were named papaya mottle-associated virus (PaMV) and papaya mild mottle-associated virus (PaMMV). Surprisingly, and in contrast to previous studies conducted in other parts of world, PRSV was not detected. The majority of the viruses were detected as single viral infections, while a few were found to be infecting alongside another virus (for example, MWMV and PaMV). Furthermore, the NGS and RT-PCR analysis identified MWMV as being strongly associated with ringspot symptoms in infected papaya fruits. This study has provided the first complete genome sequences of these viruses isolated from papaya in Kenya, together with primers for their detection—thus proving to be an important step towards the design of long-term, sustainable disease management strategies.
... A aplicação da técnica de sorologia na identificação de potyvirus apresenta, com frequência, reações cruzadas e resultados não conclusivos, em razão da utilização de anticorpos policlonais (SHUKLA et al., 1994;SOUZA et al., 2012a). Entretanto, isso pode ser contornado quando se utiliza o sequenciamento da região do genoma viral que codifica a proteína capsidial para classificação hierárquica de potyvirus (SHUKLA; WARD, 1989). O objetivo do presente trabalho foi utilizar a técnica de RT-PCR para identificar a estirpe de SCMV que causa mosaico em milho e sorgo no Brasil e testá-la para a possível presença dos demais potyvirus do complexo do mosaico. ...
Detecção Molecular do SCMV Infectando Milho e Sorgo no Brasil Milho (acima) e sorgo (abaixo) com sintomas de mosaico. Ê
... As amostras positivas foram submetidas às reações de PCR com oligonucleotídeos específicos para os seis potyvírus do complexo do mosaico, conforme descrito por Souza e Barros (2017) Zhou, 2002) X PJG_R (desenhado pela autora Barros, B.A.) (Tabela 1) resultou na amplificação da espécie de potyvírus JGMV apresentando amplicons, respectivamente, tamanhos de 813 bp e 414 pb. Estes conjuntos de oligonucleotídeos anelam na porção N-terminal da proteína capsidial, permitindo a identificação dos isolados de JGMV por meio do sequenciamento Ward, 1989). Amplicons foram purificados empregando ExoSAP-IT for PCR Product Cleanup (USB) e sequenciados. ...
Sorghum bicolor (L.) Moench é cultivado em várias regiões tropicais e subtropicais do mundo. Entre as doenças, o mosaico comum causado por potyvírus é uma importante limitação, causando redução na produção de sorgo granífero e forrageiro. No Brasil, apenas o Sugarcane mosaic virus (SCMV) havia sido relatado anteriormente como espécie de potyvírus associada ao mosaico em sorgo e milho. Levantamento para monitorar a ocorrência da virose mosaico comum foi realizado em lavouras de sorgo no Estado de Minas Gerais durante a safra 2014/2015. Amostras de plantas de sorgo que expressavam sintomas de doenças virais foram coletadas para análises moleculares. A caracterização molecular da proteína capsidial (PC) dos potyvírus infectando naturalmente o sorgo permitiu identificar o Johnsongrass mosaic virus (JGMV) como mais um agente causal da doença do mosaico comum no sorgo em Minas Gerais. As sequências dos isolados brasileiros de JGMV identificados infectando o sorgo (JGMV-Sr) foram depositadas no GenBank sob os números de acesso KY952241, KY952242 e KY952243. Comparações das sequências do gene da PC desses isolados brasileiros de JGMV-Sr revelaram altas identidades de sequência de nucleotídeos (nt) e de aminoácidos (aa) com o isolado dos Estados Unidos U07218.1 (JGMVMDKS1). Os isolados JGMV-Sr são distintos dos isolados brasileiros que infectam gramíneas forrageiras (JGMV-Fg) (KT833782 e KT289893).
... Fotos: Isabel R. P. de Souza (Jiang; Zhou, 2002) X PJG_R (desenhado pela autora Barros, B.A.) (Tabela 1) resultou na amplificação da espécie de potyvírus JGMV apresentando amplicons, respectivamente, tamanhos de 813 bp e 414 pb. Estes conjuntos de oligonucleotídeos anelam na porção N-terminal da proteína capsidial, permitindo a identificação dos isolados de JGMV por meio do sequenciamento (Shukla; Ward, 1989). Amplicons foram purificados empregando ExoSAP-IT for PCR Product Cleanup (USB) e sequenciados. ...
... Primers CIFor/CIRev produced an amplicon of approximately 0.7 kb, or approximately 37%, of the potyvirus CI gene. Species designation in the genus Potyvirus is determined in part by the sequence of the CP gene (King et al., 2011;Shukla and Ward, 1989), so for many years the CP gene has been a common target for potyvirus detection and discrimination. However, the nucleotide sequence of the CI gene has been demonstrated to be highly useful for virus identification (Adams et al., 2005). ...
The Potyvirus genus is one of the largest genera of plant viruses and encompasses many economically important pathogens. While a number of degenerate primers for use in broad spectrum RT-PCR assays have been published, it is not clear which of these primers would be the most useful for use by plant diagnostic laboratories. Twelve sets of primers were tested for their ability to detect nine potyviruses in a two-step RT-PCR. Viruses were extracted from different host backgrounds and were selected to represent eight clades plus one species between clades (sensuGibbs and Ohshima, 2010). Results of this study indicated that the primers CIFor/CIRev produced easily detectable amplicons from all nine potyviruses without non-specific amplification, false positives, or false negatives. CIFor/CIRev produced single amplicons from potyvirus-infected tissues which could be sequenced directly without gel purification to identify the virus to species.
... From 57 to 100%. The Nterminal part of the HV region of the coat proteins of potyviruses is highly variable in terms of amino acid sequences [9]- [10], [15] and the difference in the amino acid sequences of this immune dominant HV region was demonstrated by phylogenetic analysis and similarity analysis in this study. ...
... The primers set combinations PJG_F X PSR (Jiang and Zhou, 2002), and PJG_F (Jiang and Zhou, 2002) X PJG_R (design by the author Barros, B.A.) (Table 1) resulted in amplification of the JGMV in the samples with fragment sizes of the 813 bp and 414 bp, respectively. These primer sets amplify the N-terminal portion of the CP, allowing the identification of the JGMV isolates by sequencing (Shukla et al., 1989). Amplicons were purified using ExoSAP-IT for PCR Product Cleanup (USB) and sequenced. ...
Sorghum bicolor (L.) Moench is cultivated in several tropical and subtropical regions in the world. Among the diseases, the mosaic caused by potyvirus is an important constraint for the agricultural production causing reduction in grain and forage sorghum production.In Brazil, only Sugarcane mosaic virus (SCMV) had previously been reported as the potyvirus species causing mosaic in sorghum and maize. A survey was carried out in sorghum plantations of the State of Minas Gerais, Brazil, during the 2014/2015 crop season for monitoring mosaic disease. Samples of sorghum plants expressing virus disease symptoms were collected for molecular analyzes. Molecular characterization of coat protein (CP) of the potyviruses naturally infecting sorghum, allowed us to identify the Johnsongrass mosaic virus (JGMV) as a new causal agent of mosaic disease in sorghum in Brazil. The sequences of the Brazilian JGMV sorghum-infecting (JGMV-Sr) isolates were deposited in the GenBank under the accession numbers KY952241, KY952242, and KY952243. Comparisons of the CP gene sequences of these Brazilian JGMV-Sr isolates revealed high nucleotide (nt) and amino acid (aa) sequence identities, ranging from 97.93 to 98.23%, and 99.12 to 99.20%, respectively, with the U07218.1 (JGMV-MDKS1) isolate. The Brazilian JGMV-Sr isolates were distinct from the Brazilian forage grasses-infecting (JGMV-Fg) isolates (KT833782 and KT289893). Transmission evaluations showed susceptibility of the teosinte, Sorghum verticilliflorum and Sorghum bicolor (L.) Moench, except line QL3.Maize and sugarcane genotypes were not infected by the Brazilian JGMV-Sr isolate. However, it is important to test more genotypes. This is the first report showing the identification and molecular characterization of the JGMV species naturally infecting sorghum at field conditions, expanding the knowledge about the dynamic and range of the mosaic causal agent for this crop in Brazil.
... Then, the positive samples werePCR amplified with primers specific for the CP and a partial sequence of the nuclear inclusion protein (NIb) ( Table 1). These primer sets amplify the N-terminal portion of the CP, which encodes the virus-specific determinants, thus allowing the identification of the SCMV strain by sequencing (Shukla et al., 1989). All PCR reactions were performed according to Souza and Barros, 2016. ...
Sugarcane crops are cultivated in nearly all tropical and sub-tropical regions worldwide andhave a prominent position in the agricultural scenario in Brazil. However, viral diseases can threaten the production of this important commodity leading to large production losses. In the present study, we evaluatedthe reaction of 20 sugarcane genotypes independently inoculated with two different strains of Sugarcane mosaic virus (SCMV) isolated from naturally infected sugarcane (SCMV-SGC) and maize (SCMV-MZ). The maize inbred line L19 was used as a control of susceptibility to the SCMV-MZ strain. Symptoms intensity was evaluated through a visual scale with three levels of severity: weak, intermediate, and intense. The viral infection was confirmed by PCR and DNA sequencing. We observed that, although both strains were able to infect sugarcane genotypes and the maize inbred line, SCMV-SGC was more aggressive, resulting in only four resistant genotypes: IN84-58 (S. spontaneum), RB855536, RB 928064, and SP71-6163. Thirteen genotypes were resistant to SCMV-MZ: IN84-58 (S. spontaneum), NA56-79, CB47-355,CB49-260, RB72454, RB855113, RB855536, RB867515, RB928064, SP70-1143, SP71-1406, SP71-6163, and SP81-3250. This is the first report showing SCMV strains capable of cross-infecting and causing mosaic in sugarcane and maize. Our data emphasize the importance of continuous monitoring and screening for virus resistant genotypes to be used in breeding programs for the development of new resistant cultivars.
... El gen que codifica para la CP es uno de los mejor caracterizados en los potyvirus, debido a su utilidad en la taxonomía, estudios evolutivos y de diagnóstico (Shukla y Ward, 1988;1989a;, así como en la obtención de plantas con resistencia a enfermedades (Fitch et al., 1992;Fermín et al., 2004). La CP se divide en tres dominios: el amino-terminal, región central y carboxilo-terminal. ...
El Virus de la mancha anular de la papaya constituye en muchos países tropicales y subtropicales el
mayor obstáculo en la producción de papaya y es responsable de pérdidas considerables en las cosechas.
El modo de transmisión de este virus impide el éxito de los tratamientos con insecticidas. Además, la
presencia de áfidos vectores durante todo el año en las plantaciones constituye una importante vía para
la distribución de la enfermedad. Una vez infectadas las plantaciones no existe tratamiento eficaz para
su control. En varios países se han desarrollado estrategias mediante la ingeniería genética, donde se
ha logrado obtener buenos resultados, lo que implica el estudio biológico y molecular de los posibles
aislados del PRSV que se pueden presentar en una región o país. En Cuba, se han realizado
investigaciones encaminadas al desarrollo de estrategias para el manejo de esta enfermedad viral. Este
trabajo tuvo como objetivo relacionar los principales aspectos biológicos y epifitiológicos del PRSV, con
resultados de Cuba y el mundo, como herramienta para el manejo de esta enfermedad mediante el uso
de la biotecnología vegetal.
... In virology it is common knowledge that virus strains can be characterized by comparing their symptomatology in differential hosts (Shukla and Ward, 1989). Here the strains are an expression of the variants in a virus species or type (Matthews, 1985). ...
Protein misfolding and aggregation is a key event in diseases like Alzheimer's disease (AD) or Parkinson's disease (PD) and is associated with neurodegeneration. Factors that initiate protein misfolding and the role of protein aggregation in the pathophysiology of disease pose major challenges to the neuroscientific community. Interestingly, although the accumulation of the same misfolded protein, e.g., a-synuclein is detectable in all idiopathic PD patients, the disease spectrum covers a variety of different clinical presentations and disease courses. In a more recent attempt this clinical variance is being explained in analogy to prion diseases by different protein aggregate conformations. In prion diseases a relationship between protein aggregate conformation properties and the clinical disease course was shown by relating different prion types to a dementia and an ataxic disease course in Creutzfeldt-Jakob patients. This principle is currently transferred to AD, PD and other neurodegenerative diseases with protein aggregation. However, differences in protein aggregate conformation are frequently addressed as disease strains. The term "strain" also derives from prion research and evolved by adopting the virus terminology at a time when transmissible spongiform encephalopathies (TSEs; later called prion diseases) were assumed to be caused by a virus. The problem is that in virus taxonomy the term "type" refers to properties of the disease agent itself and the term "strain" refers to host associated factors that interact with the disease agent and may moderately modify the clinical disease presentation. Strain factors can be discovered only after transmission and passaging of the agent in a host of a different species. The incorrect use of the terminology confuses disease agent and host factors and hampers the understanding of the pathophysiology of protein aggregate-associated neurodegenerative diseases. In this review article the discoveries are reviewed that explain how the terms "type" and "strain" emerged for unconventional disease agents. This may help to avoid confusion in the terminology of protein aggregation diseases and to reflect correctly the impact of protein aggregate conformation as well as host factor contribution on different clinical variations of AD, PD and other neurodegenerative diseases.
... To understand the reason for the low capacity for PVA CP dissociation, unlike for the CP of PVY, PVBV, we compared their primary structures. The Shukla's group (Shukla &Ward, 1989) showed that the sequence homology within the different members of the Potyvirus genus be ranged from 38 to 71% (average 54%). Clustal Omega (Sievers et al., 2011) was used for multiple sequence alignments of CPs of three potyviruses -PVA, PVY, and PVBV ( Figure S2). ...
In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency of to self-associate into various multimeric forms containing some fractions of cross-β-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF Mass Spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20-30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.
... These results indicated that the virus under study might be either a new isolate of henbane mosaic virus (HMV) as showed by biological and morphological studies or might be a new isolate of potatao virus Y (PVY) since the nucleotide sequence similarity is relatively high (88%) with PVY Foggia isolate. Shukla andWard (1989a &1989b) found that the N terminus was the only large region in the entire coat protein that is unique to potyviruses and contains virus -specific epitopes. Gambino et al. (2008) reported the importance of validating RNA extraction procedure for different sample matrixes and the ability of the extraction method to provide a suitable nucleic acid free of PCR inhibitors from each sample matrix because plants are known to contain a lot of possible PCR inhibitors such as polysaccharides. ...
... The 10 kb single stranded RNA genome of SCMV encodes a single polypeptide which is cleaved either co-or post translationally into ten mature proteins (P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb, and CP) [2,3]. C-terminus of the polyprotein encodes the coat protein (CP) which encapsidates the viral genome through helical arrangement of its multiple subunits [4]. Hc-Pro is a multifunctional protein and a proteinase, which is responsible for the multiplication of virus genome, systemic virus movement [5], and suppression of plant RNA silencing machinery [6] by interacting with numerous host factors. ...
Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. Sugarcane mosaic virus (SCMV) is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA), targeting simultaneously CP (Coat Protein) and Hc-Pro (helper component-proteinase) genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine) and KITC (Lycine-Isoleucine-Threonine-Cysteine) motifs, derived from SCMV CP and Hc-Pro genes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with the β -glucuronidase gene (GUS) at the 3′ end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21–24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane.
... The sequence information for the CP genes is very important criterion for the taxonomy of potyviruses (Shukla and Ward, 1989b). Consequently, universal primers of Potyviridae (Chen et al., 2001) were used and successfully amplified to 969 bp of NIb gene of FMV in the infected tissue. ...
Fig Mosaic Virus (FMV) causes a serious problem for the fig plant (Ficus carica L.) in Egypt and all over the world. Not all of the previous studies agreed on a precise description for viral particle and its genome; in addition, there have been conflicts in those results. A number of samples from naturally infected fig plants exhibiting characteristic fig mosaic virus-associated symptoms were collected from different fields in the north coast of the western desert that extends from west of Alexandria to Marsa Matrouh. The electron microscopy of ultra thin sections of FMV-infected leaves judged by I-ELISA revealed the presence of so-called double membrane bodies (DMBs) in parenchyma cells with two types: Rounded to ovoid, 160~200 nm in size and elongated; straight to slightly flexuous, up to or exceeding 1 µm in length. Also accumulation of starch grain was recorded as ultrastructure change of leaf of F. carica L. affected by FMV-infection. Using potyvirus genus-specific primers, 969 bp of the viral Nuclear Inclusion Body (NIB) gene were amplified. For further confirmation, two degenerate primers were designed based on the amino acids sequences of 69 different FMV coat protein genes; amplicones with 374 bp were obtained. Both the amplicones 969 bp of the NIB and the 374bp of the coat protein genes were sequenced. The sequence and phylogenetic analysis indicated that the Egyptian FMV isolate was closely related to other FMV isolates, especially the Italian and Arkansas isolates with similarity not exceeding 55%.
... The divergence in the amino termini of the CP can account for their differences in host specificity as reported for Sugarcane mosaic virus (Xiao et al., 1993). It is therefore tempting to speculate that variation in the N-terminal region of the protein, which is on the outer surface of the CP (Shukla and Ward, 1989) and is close to the aphid transmission motif (Atreya et al., 1990), can be correlated to the geographical distribution of PRSV. ...
Papaya ring spot virus (PRSV) causes major diseases in papaya and cucurbits in the Indian sub-continent that result in significant yield losses. Molecular characterization of the coat protein (CP) gene of a South Indi-an strain (INP-UAS) of PRSV-P revealed an open reading frame of 849 bp that encoded the putative coat protein of 283 amino acids (GenBank Acc No. AF528190). The DAG triplet associated with aphid transmissibility and the potential protease cleavage site Q/S, located in the N-terminus of the INP-UAS CP, were conserved, as has been reported for other PRSV coat proteins. The sequence had a deletion of 24 nucleotides that corresponded to eight amino acids in the N-terminal region of the CP. A comparison of the amino acid sequence of the INP-UAS CP with those reported for other PRSV isolates showed that the N-termini were variable and suggested that the distinctiveness of INP-UAS was linked to its geographical location. Phylogenetic analysis also showed that the INP-UAS strain coat protein gene was relatively divergent from those of other PRSV-P isolates as it formed a separate and distinct group. The implications of sequence variability for the use of CP-genes in the development of transgenic plants for viral resistance are discussed.
... Although the potexviruses require their CP to potentiate cell-to-cell movement (Baulcombe et al. 1995;Forster et al. 1992), the CP does not have the capacity to mediate its own intercellular transport, nor that of viral RNA, via PD (Lough et al. 1998;Santa Cruz et al. 1998). The CP in potexviruses is a threedomain protein containing highly conserved central region that interacts with viral RNA (Lico et al. 2006;Shukla and Ward 1989). The N-and C-terminal regions are quite variable, and exhibit low similarity among potexviruses. ...
Plant viruses utilize plasmodesmata (PD), unique membrane-lined cytoplasmic nanobridges in plants, to spread infection cell-to-cell and long-distance. Such invasion involves a range of regulatory mechanisms to target and modify PD. Exciting discoveries in this field suggest that these mechanisms are executed by the interaction between plant cellular components and viral movement proteins (MPs) or other virus-encoded factors. Striking working analogies exist among endogenous non-cell-autonomous proteins and viral MPs, in which not only do they all use PD to traffic, but also they exploit same regulatory components to exert their functions. Thus, this review discusses on the viral strategies to move via PD and the PD-regulatory mechanisms involved in viral pathogenesis.
In the last three decades, there has been a significant increase in the number of pathogenic virus species affecting pepper (Capsicum spp.) plants in tropical and subtropical regions. These viruses typically induce various symptoms in pepper plants, including chlorotic mosaic, spotting, leaf deformation, narrowing of leaf veins, fruit shape abnormalities, and irregular color formation. These diseases pose a serious threat to pepper cultivation and result in economic losses.
In Turkey, numerous virus species affect pepper plants in production areas. These include common ones such as Cucumber Mosaic Virus (CMV), Potato Y Virus (PVY), Pepper Mild Mottle Virus (PepMoV), Tobacco Etch Virus (TEV), Tomato Spotted Wilt Virus (TSWV), Beet Western Yellows Virus (BWYV), Pepper Vein Yellows Virus (PeVYV), and several others. The effects of these viruses on pepper plants vary widely, typically manifesting as chlorotic mosaic patterns, spotting, leaf deformation, and narrowing of leaf veins. Additionally, these viruses can cause fruit malformations and discoloration. These symptoms hinder plant growth, reduce productivity, and adversely affect the quality of harvested crops. The mechanisms of virus transmission are diverse, with the most common route being transmission from diseased plants to healthy ones through contact, especially facilitated by vector insects. Insects carrying the virus can spread the disease rapidly as they move from plant to plant. The economic impacts of these viruses on pepper cultivation are significant. Decreased plant health and reduced crop quality can lower growers' incomes. Moreover, the additional costs incurred in disease management can negatively impact profit margins.
This study provides comprehensive information on the genetic structures, resistance mechanisms, transmission methods, and control strategies of commonly encountered viruses in pepper production regions in Turkey. Such knowledge can assist farmers in developing more informed and effective approaches to disease management. Furthermore, scientific endeavors of this nature can contribute to more extensive research and the development of solutions for effectively controlling virus diseases in pepper production in the future. Ultimately, enhancing the sustainability and productivity of pepper cultivation can be achieved, thereby ensuring economic and environmental sustainability in the sector.
This reference provides comprehensive insights on the harm inflicted by pests and diseases on leguminous crops. Internationally acclaimed authors provide succinct reviews on breeding and impact of biotic stress factors such as insect pests, microbial pathogens, spiders, and vertebrate pests in legumes like soybean, cowpea, and common bean. The book also contains detailed technical analysis of methods such as the PCR-based detection, next generation sequencing / marker-assisted selections, low cost lethal-non-lethal vertebrate pest control and mechanisms of climate/nutrient induced resistance. The unique feature of this book is its focus on the optimization and development of environmentally friendly methods for pest and disease control in leguminous crops. Other features include structured sections for easy reading and a list of references for advanced readers.
Key themes:
Biotic Stress and Plant Resistance
Biotic Stress in Legumes (Cowpea and Soybean)
Diagnostic and Control Methods for Microbial Plant Pathogens
Viral Diseases of Legumes and Management:
Vertebrate Pests in Legumes and Economic Implications
Spiders in Legume Agroecosystems
Climate-Driven Factors and Insect Pests of Legumes
Sustainable Crop Nutrition for Biotic Stress Alleviation in Legumes
Physiological Responses in Legumes to Combined Stress Factors
The chapter describes about the brief history and background, detection, characterization, and management of viruses infecting gladiolus. It also describes about the methods of elimination of potyviruses from infected gladiolus corm explants through in vitro chemotherapy, thermotherapy, and electrotherapy. In this chapter, the work published by researchers all over the world and by virology group at NBRI, Lucknow, has been summarized.
Bean yellow mosaic virus, a species of the genus Potivirus, has a wide host range and a broad geographical
distribution. BYMV causes high annual economic damage in various legumes such as faba beans in Iran. In this
study, 13 BYMV isolates were collected from faba bean fields of different provinces of Iran (Sistan and
Baluchestan, Hormozgan, Kerman, Khuzestan, Fars, Lorestan, Ilam, Hamadan, Ghazvin, Zanjan, Ardabil, East
Azerbaijan). The coat protein (CP) region of the collected isolates was sequenced and then compared with the CP
sequence of 178 isolates available in GenBank. The selected Iranian sequences showed 86-99% nucleotide
sequence identities with other BYMV isolates. Phylogenetic relationships based on CP nucleotide sequences were
estimated using the Maximum Likelihood method, after removing all recombinant sequences. Accordingly, all
isolates excluding three isolates, AI38, PAC-1, BYMV-W were placed in eight monophyletic groups. Iranian
isolates were located in two distinct groups, along with broadbean, lentil, bean, gladiolus and sunflower isolates
from Japan, Australia, Iraq and Spain. According to the results there is no significant relation among clustering of
BYMV isolates based on phylogenetic analysis of CP sequences and original host and country. The CP structure
analysis of Iranian isolates and other selected isolates from GenBank revealed conservation of the C-terminus and
the central region of the coat protein and the variation of the N-terminus.
Bean yellow mosaic virus, a species of the genus Potivirus, has a wide host range and a broad geographical distribution. BYMV causes high annual economic damage in various legumes such as faba beans in Iran. In this study, 13 BYMV isolates were collected from faba bean fields of different provinces of Iran (Sistan and Baluchestan, Hormozgan, Kerman, Khuzestan, Fars, Lorestan, Ilam, Hamadan, Ghazvin, Zanjan, Ardabil, East Azerbaijan). The coat protein (CP) region of the collected isolates was sequenced and then compared with the CP sequence of 178 isolates available in GenBank. The selected Iranian sequences showed 86-99% nucleotide sequence identities with other BYMV isolates. Phylogenetic relationships based on CP nucleotide sequences were estimated using the Maximum Likelihood method, after removing all recombinant sequences. Accordingly, all isolates excluding three isolates, AI38, PAC-1, BYMV-W were placed in eight monophyletic groups. Iranian isolates were located in two distinct groups, along with broadbean, lentil, bean, gladiolus and sunflower isolates from Japan, Australia, Iraq and Spain. According to the results there is no significant relation among clustering of BYMV isolates based on phylogenetic analysis of CP sequences and original host and country. The CP structure analysis of Iranian isolates and other selected isolates from GenBank revealed conservation of the C-terminus and the central region of the coat protein and the variation of the N-terminus.
The "maize common mosaic", caused by potyvirus, is among the major virus diseases of this crop in Brazil. Although there were evidences indicating Sugarcane mosaic virus (SCMV) as the most common potyvirus species in maize (Zea mays L.) in Brazil, information about those species that infect sorghum plants [Sorghum bicolor (L.) Moench] are few. Leaves showing characteristic mosaic symptoms were collected from maize and sorghum and used in serological and sequencing analysis of the coat protein (CP) gene for potyvirus species identification. Amino acid (aa) analysis of the CP N-terminal sequence of our samples showed a different repeated sequence, a higher content of the dipeptide GT, and a 15 aa longer than the majority of the SCMV sequences used for comparisons. The Brazilian maize and sorghum potyviruses formed a monophyletic group, suggesting that they can be classified within a new SCMV strain. Studies using potyvirus CP gene sequencing from Brazilian sorghum potyvirus have been reported for the first time. RESUMO-O "mosaico comum do milho", causado por potyvirus, destaca-se entre as viroses mais importantes desta cultura. Embora existam evidências indicando Sugarcane mosaic virus (SCMV) como a espécie de potyvirus predominante em milho (Zea mays L.) com sintomas dessa virose no Brasil, poucas são as informações sobre as espécies de potyvirus infectando plantas de sorgo [Sorghum bicolor (L.) Moench]. Folhas de milho e de sorgo apresentando sintomas típicos de mosaico foram coletadas e utilizadas em análises sorológicas e de sequenciamento do gene da proteína capsidial para identificação da espécie de potyvirus. Análise da sequência de aminoácidos (aa) da região do N-terminal da proteína capsidial (CP) dessas amostras mostrou uma sequência repetida diferente, com elevado conteúdo do dipeptídeo GT e 15 aa maior que a maioria dos acessos utilizados para comparações. Os potyvirus infectando milho e sorgo, no Brasil, formam um grupo monofilético, sugerindo tratar-se de uma nova estirpe de SCMV. Estudos utilizando sequenciamento do gene da CP de potyvirus infectando sorgo no Brasil estão sendo relatados pela primeira vez.
The Potyviridae is the largest family of RNA plant viruses, members of which have single-stranded, positive-sense RNA genomes and flexuous filamentous particles 680–900nm long and 11–20nm wide. There are eight genera, distinguished by the host range, genomic features and phylogeny of the member viruses. Genomes range from 8.2 to 11.3 kb, with an average size of 9.7 kb. Most genomes are monopartite but those of members of the genus Bymovirus are bipartite. Some members cause serious disease epidemics in cultivated plants. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Potyviridae, which is available at www.ictv.global/report/ potyviridae.
Plant viruses are estimated to cause economic losses world-wide of $15 billion per annum, and the development of effective control strategies is dependent on the availability of reliable methods of detection and identification (Ward and Shukla, 1991). A number of immunological and molecular techniques have been developed over the years for the detection of plant viruses. The sensitivity of some of the recent techniques, viz enzyme-linked immunosorbent assay and nucleic acid hybridization, has reached the level that any known plant virus can now be detected even in a single infected seed or a single virus-carrying insect (Table 1).
Plant viruses cause serious diseases and economic losses in numerous crops. The viruses show a great diversity of particle structure, and of genome organisation and expression, which are reflected in the diversity of interactions with the host and the number of types of resistance mechanisms that plants have evolved to combat them, or that man has invented. Resistance may operate at a number of points in the virus infection and replication cycle. Different types of resistance mechanisms may operate at the species, cultivar and individual plant level. These are considered using a variety of conceptual models, as well as observation and experimental evidence. Non-host resistance involves restriction of virus spread from the initially-infected cell in some cases, but other models are also considered. A study of the genetics of cultivar resistance (as used by plant breeders) indicates that resistance can be variously dominant, semi-dominant or recessive to susceptibility, and that this may be correlated with different types of biochemical mechanisms of resistance. A number of examples of these are described. Many of the resistances considered have been overcome by virulent isolates of the virus, and some quite complex gene-for-gene interactions have been established. Recombinant DNA methods have allowed the mapping of the determinants of virulence/avirulence to a number of different viral functions. Resistance may also be conferred on normally susceptible plants by a variety of treatments, or occur in particular plant parts or at particular times after infection. Examples of mechanisms considered include virus-free green islands, satellite protection, cross protection, genetic transformation and gene silencing. Transgenes derived from various parts of the viral genome, and from numerous other sources, have been shown to confer resistance by diverse mechanisms. The future prospects for exploitation of different types of resistance in crop protection are considered.
Papaya ringspot virus (PRSV) YK isolate used in this study is a local mosaic strain isolated from YungKang, Tainan, and its genome has been cloned and completely sequenced. A NcoI site before the coat protein (CP) reading frame of PRSV YK was generated by oligonucleotide-directed mutagenesis, and then the CP reading frame with the 3' noncoding region of PRSV YK was ligated with the gus leader sequence from the pGEM vector to create the construct pGGCP. To express the CP with a homologous viral translation sequence, the gus leader was replaced by the cDNA sequence corresponding to the 5' region (nt 1-347) of PRSV genome to generate a protein containing 9 kDa polypeptide of PRSV P1 protein fused with the CP, and the construct was designated as pG5'CP. In vitro translation from the transcripts derived from pGGCP and pG5'CP generated protein products of 36 kDa and 45 kDa, respectively. Both proteins reacted with the antiserum to PRSV CP, and the level of 36 kDa protein was higher than that of 45 kDa protein. The CP reading frame with the gus or PRSV 5' leaders was individually subcloned into a Ti binary vector. Transgenic tobacco plants (Nicotiana tabacum L. Havana 423) expressing the PRSV CP gene with the gus leader (GCP lines) or with the viral leader (5'CP lines) were obtained by Agrobacterium-mediated transformation. When the transgenic lines were analyzed by western blotting, the protein products of 36 kDa and 45 kDa reacting to PRSV CP antiserum were detected in the GCP lines and 5'CP lines, respectively. The presence of the CP gene in the transgenic tobacco was also confirmed by polymerase chain reaction (PCR) using primers specific to the CP gene. Analysis of segregation ratios in the R1 plants of four GcP lines and four 5'CP lines indicated that the CP gene in all of them was nuclearly inherited as a single dominant trait. R0 and R1 plants of the four GCP lines and four 5'CP lines were inoculated with tobacco etch virus (TEV), potato virus Y (PVY), or pepper mottle virus (PepMoV). The transgenic lines showed significant delay in symptom development and the severity of symptoms was attenuated. The GCP lines expressing the PRSV CP gene by the gus leader accumulated higher levels of CP and showed higher degrees of resistance than the 5'CP lines with the PRSV 5' leader. Our results indicate that the homologous viral leader does not enhance CP expression either in vitro or in vivo, nor does it provide better resistance in transgenic tobacco.
The potyviruses have long been favored subjects for study by plant virologists. They constitute the most numerous of the three dozen or so groups of plant viruses, and collectively are responsible for more damage to the world’s crop plants than is caused by the viruses of most of if not all the other groups. This was rather dramatically illustrated by a recently conducted international election of “favorite” filamentous plant viruses among several eminent virologists in which the potyviruses emerged with a clear “victory” (Milne, 1988). The ecological and epidemiological aspects of diseases caused by potyviruses, with their many fascinating but varied and complex considerations, have also prompted the major efforts in research that have been directed over many years to this group of viruses.
The C-terminal part of the RNA of Cocksfoot streak potyvirus (CSV) belonging to the Potyviridae was analyzed. CSV is transmissible by aphids and has a narrow host range in the Gramineae. The cloned cDNA fragments represent a single open reading frame (ORF) followed by a '''-non coding region and a poly(A)tail. By comparison with other potyviruses, the ORF includes the CP, the NIb and the NIa gene of CSV. An alignment of the ORF of CSV to the corresponding region of several potyviruses like PPV, PVY, TEV and TVMV shows about 50% identity. In contrast, the comparison of CSV to mite-transmitted as well as fungus-transmitted viruses shows only an identity of 30% and 23%, respectively. This reflects a low homology between CSV and mite- and fungus-transmitted Potyviridae infecting Gramineae. Alignments of the C-terminal potyviral proteins (NIa, NIb, CP) show more homology between CSV and potyviruses than between CSV and the other genera of the Potyviridae. Specific motifs, described for potyviral polyproteins, are all present in the polyprotein of CSV, too. The motif for aphid-transmission in the coat protein is found as well as the polymerase motif in the NIb-protein and the protease motif in the NIa-Pro protein. So far, these results indicate that a potyvirus infecting monocots (Gramineae) is quite similar to potyviruses infecting dicots.
The complete nucleotide sequence of the RNA genome of a Taiwan isolate of Zucchini yellow mosaic virus (ZYMV TW-TN3) was determined from five overlapping cDNA clones 9591 nucleotides in length excluding the poly (A) tail. Computer analysis of the sequence revealed a large open reading frame (ORF) that encodes a polyprotein of 3080 amino acids. Comparison of the gene products of TW-TN3 with those of the reported California (CA), Reunion Island (RU), and Singapore (S) isolates of ZYMV revealed that P1 protein is most variable, with amino acid identities of 59.0-93.2%. The 5′ untranslated region (UTR) of TW-TN3 shares 61.6-83.3% nucleotide identities, and the 3′ UTR shares 90.4-95.7% nucleotide identities, with those of the other isolates. A phylogenetic tree derived from the sequences of P1 proteins of TW-TN3 and the other six reported ZYMV isolates revealed four major genotypes. TW-TN3 was classified in genotype I, and US isolates were in genotype II. The Reunion Island and Singapore isolates were separated into genotypes III and IV, respectively. The distance relationships of P1 protein of genotype I were closer to genotype II, indicating that the Taiwan and US isolates may evolve from the same ancestor. Analyses on the cleavage sites of the C-terminal halves of the polyproteins of TW-TN3, CA, RU, and S isolates revealed that NIa protease cleaves at Q-S, and E-S dipeptide sequences, with a consensus sequence of V-x-x-(Q, E)/(S, A, G). The genetic organization of TW-TN3 was concluded as Vpg/5′ leader/P1 (36 kDa)/HC Pro (52 kDa)/P3 (40 kDa)/6K1 (6 kDa)/CIP (71 kDa)/6K2 (6 kDa)/NIa-Vpg (22 kDa)/NIa-Pro (27 kDa)/NIb (60 kDa)/CP (31 kDa)/3′ UTR-poly(A) tract.
本邦のジャガイモから分離されたジャガイモYウイルス(PVY)の普通系統(PVY-O)とえそ系統(PVY-T)の生物学的な病原性の比較を行うと,PVY-OはNicotiana tabacumにえそを示さず,Chenopodium amaranticolor, C. quinoaに局部えそ斑点を示した。一方,PVY-TはN. tabacumの葉肉および葉脈にえそを示したが,C. amaranticolor, C. quinoaには感染しなかった。PVY-OあるいはPVY-Tを接種した植物からそれぞれのウイルスに特異的なモノクローナル抗体7)を用いて検定すると,それぞれのウィルスのみを検出できたことから,両系統の混合感染は認められなかった。これらのウイルスを用いて外被タンパク質遺伝子の塩基配列を決定した。PVY-OおよびPVY-Tの外被タンパク質遺伝子の塩基数はともに801であり,アミノ酸数はともに267と推定され,また,PVY-OとPVY-Tの外被タンパク質のアミノ酸配列の相同性は91.4%であった。本邦で分離された二系統と本邦あるいは諸外国で分離されたPVY分離株間の外被タンパク質のアミノ酸配列を比較すると,PVY-Tは本邦のえそ系統に属するPVY-TH5),オランダのPVYN(N) (tobacco veinal necrosis strain)19),および系統の記載のないドイツのPVY (GO16)20)と98.5%の高い相同性を示し,フランスのえそ系統であるPVYn (F)7)とは93.3%と若干低い相同性を示した。また,アミノ酸の変異はN末端領域に変異が多くみられ,C末端領域にはほとんどみられなかった。普通系統とえそ系統の外被タンパク質のアミノ酸配列の比較からはそれらを区別できるそれぞれに特異的なアミノ酸配列は認められなかった。
Two polyclonal antisera to plum pox virus isolates, PPV-C and PPV-A, raised after a single immunization were collected at different stages after injection and serologically studied using electro-blot-immunoassay and indirect ELISA. Antisera, collected one week after the single injection, reacted strongly with the respective homologous capsid protein, but weakly or not at all with the core capsid protein. Batches of both antisera obtained 3, 4, 5, 6 and 7 weeks after injection reacted as well as with the core capsid proteins, indicating a continuously increase in the proportion of antibodies to the core capsid proteins. Comparing the antisera collected 1 week after immunization and N-termini antibodies to PPV-C and PPV-A, respectively, demonstrated that both types of antisera possess strain specificity and confirmed our previously findings on the serological relationships between the PPV isolates studied.
For several years now, intensive efforts are made in studding of different PPV strains. A range of biological, serological and molecular criteria's are applied for strain delineation of Sharka virus. This review is an attempt to synthesize the results obtained till now by different methods on PPV strain variability and differentiation.
The cross-reactivity of two antisera with various members of the Potyviridae was determined using four different serological methods. An antiserum (no 314) prepared against apparently intact Turnip mosaic virus (TuMV) particles showed a high cross-reactivity with all of 20 aphid-borne potyviruses tested as well as the mite-borne rymovirus Ryegrass mosaic virus (RGMV) and the fungus-borne Barley mild mosaic virus (BaMMV) with two of the four methods used (indirect PTA-ELISA or IEM and Western Blotting). In immunoelectron microscopic decoration, about two thirds of the viruses tested reacted positively whereas in the direct DAS-ELISA only the homologous TuMV reacted postively.
Another antiserum (no. 203) produced by using as immunogen a mixture of the dissociated core proteins of three trypsin-treated potyviruses (Potato virus A and V and Plum pox viruses) reated postively in both indirect ELISA and Western blotting with all aphid-borne potyviruses and RGMV. No reactions were obtained with the two other methods. The causes for these different reactions are discussed. Both antisera are useful for the detection of aphid-borne potyviruses, but one (no 314) is especially suitable for the detection of these viruses under routine conditions by use of a simple indirect PTA-ELISA.
Th e coat protein (CP) genes of the genomic RNA of 2 severe Turkish isolates of Zucchini yellow mosaic virus (ZYMV) from squash and muskmelon [ZYMV-Adana (Ad) and ZYMV-Ahlat (Ah), respectively] were cloned, and their complete nucleotide sequences and deduced amino acids were determined. Th e analysis revealed that both Turkish ZYMV-CP genes contained 837 nucleotides encoded for a CP of about 31.2 kDa. Phylogenetic trees based on nucleic acid sequences were constructed by the neighbor joining and unweighted pair group mean arithmetic (UPGMA) methods with 100 bootstrap replicates. A high degree of homology was detected between the 2 Turkish isolates on the nucleotide sequences (97%). Th e CP sequence of ZYMV-Ad and ZYMV-Ah varied among the 23 isolates with overall identity of 93%-98% and 94%-99%, respectively, at the nucleotide level. Comparison of the nucleotide sequences of 23 isolates from diff erent geographical regions worldwide showed the ZYMV-Ad isolate clustered with isolates from Middle Eastern countries (Israel, Jordan, and Syria); ZYMV-Ah isolate was clustered with isolates from Far Eastern countries (Korea and Taiwan). Th e N-terminal of the Turkish ZYMV-Ah CP contained a distinctive sequence at nucleotide positions 9324-9328, which distinguished the Turkish ZYMV-Ah isolate from all previously reported ZYMV isolates. Th e CP cistron of the ZYMV-Ad and Ah isolates contained 279 amino acid residues. Pairwise nucleotide sequence comparison revealed sequence similarities of 58%-70% between ZYMV Turkish isolates and 22 other potyviruses. Mechanical inoculations showed that ZYMV-Ah produced faster systemic symptom induction than ZYMV-Ad on squash, suggesting that ZYMV-Ah was a more severe isolate than ZYMV-Ad (GenBank accession JF317296-JF317297).
A virus isolated from a tick bean (Vicia /aba L. var. minor Beck) from South Australia has been shown to be closely related to bean yellow mosaic virus (BYMV) by its particle length, by the types of inclusion bodies it induced in cells, and by the serological relationships and amino acid composition of its particles. However, its experimental host range and the symptoms it caused differed from those of typical BYMV, and it is concluded therefore that it is a previously undescribed strain of this virus, BYMV-S. Analyses of the coat protein of BYMV-S confirmed the value of using the amino acid composition as a criterion for assessing the relationships of potyviruses and for identifying new isolates.
SUMMARY The sequence of 1232 nucleotides from the 3' region of the plum pox virus (PPV) RNA has been determined. This sequence contains one long open reading frame (ORF). The sequence of the 17 amino-terminal amino acids of the PPV capsid protein was determined chemically. An identical amino acid sequence was found in the translation product of the large ORF, starting at amino acid - 330, which is alanine. Our data suggest that the PPV capsid protein, like that of other potyviruses, is a product of the maturation of a large polyprotein. The putative cleavage site is at a glutamine-alanine dipeptide. The capsid protein gene consists of 990 nucleotides and corresponds to a region coding for 330 amino acids which have a combined calculated Mr of 36593. The adjacent 3' untranslated region is of 215 nucleotides and ends in a polyadenylate tract. PPV capsid protein molecules are thus larger than those of the other potyviruses that have been characterized. The amino acid sequence of PPV coat protein is 47 to 60~ homologous to other potyvirus coat proteins and most of the variations are in the amino-terminal region.
SUMMARY Mild proteolysis by trypsin of particles of six potyviruses (bean yellow mosaic virus, clover yellow vein virus, Johnson grass mosaic virus, passion-fruit woodiness virus, potato virus Y and watermelon mosaic virus II) revealed that the N- and C-terminal regions of their coat protein are exposed on the particles' surfaces. The enzyme treatment removed the N-terminal region (30 to 67 amino acids long, depending on the virus) and 18 to 20 amino acids from the C terminus of the coat proteins, leaving a fully assembled virus particle composed of coat protein cores consisting of 216 or 218 amino acid residues. These core particles were indistinguishable from untreated native particles in an electron microscope and were still infectious. The core particles lacked the virus-specific surface epitopes that are recognized by the bulk of the polyclonal antibodies raised against the whole virus particles. Epitopes thought to be group- specific were located in the trypsin-resistant core protein region. The implications of these findings are discussed in relation to the similar surface location of the N- and C- terminal regions of the coat protein of other rod-shaped plant viruses and the observed common structural features displayed by isometric plant and animal viruses.
Attempts to identify and classify distinct potyviruses and their strains have frequently been hampered by the presence of variable proportions of cross-reacting antibodies in antisera. Investigations of reactivities in electroblot immunoassays of 11 polyclonal antisera raised by injection of intact particles of potyviruses produced in different laboratories with 12 distinct potyviruses showed that such cross-reacting antibodies were directed towards the homologous core protein region of potyvirus coat proteins. A simple method was developed to obtain virus-specific antibodies using affinity chromatography. It involved removal of the surface-located, virus-specific N-terminal peptide region from particles of one potyvirus using lysyl endopeptidase, coupling of the truncated coat protein to cyanogen bromide-activated Sepharose gel, and passing antisera to different potyviruses through the column. Antibodies that did not bind to the column were found to be highly specific.
SUMMARY Analysis of the 136 possible pairings of the coat protein amino acid sequences from 17 strains of eight distinct potyviruses revealed a bimodal distribution of sequence homology. Distinct members of the group exhibited sequence homologies ranging from 38 to 71 ~ (average 54%) with major differences in the length and sequence of their N termini and high sequence homology in the C-terminal half of the coat proteins. In contrast strains of individual viruses exhibited sequence homologies of 90 to 99% (average 95 9/o) and had very similar N-terminal sequences. These findings cast doubt on the currently held 'continuum' hypothesis proposed to explain the unsatisfactory taxonomy of the potyvirus group. The coat protein sequence data, in combination with information on the nature of the potyvirus particle assembly, can be used to develop rationally designed, simple serological techniques that appear to be more useful and more easily applied than those properties previously used for potyvirus identification and classification.
Thin sections of leaf tissue infected with 12 rod-shaped viruses varying from 180 mμ to 750 mμ in length were examined in the electron microscope. Neither intranuclear nor cytoplasmic inclusions occurred in healthy tissue. Intranuclear inclusions were observed only in material infected with tobacco etch virus. Several types of cytoplasmic inclusions were induced by the group of viruses varying in length from 730 mμ to 750 mμ; however, only one type of inclusion was common to all seven viruses of this group. It is proposed that this inclusion, which appears as a pin-wheel in cross section and as a bundle in longitudinal section, is diagnostic for infection with viruses of the potato Y group, i.e., rod-shaped viruses whose lengths vary from 730 mμ to 750 mμ.
The original aim of this book was to cover different aspects of the tradi tionally "filamentous" potex-, carla-, poty-, clostero-, and capilloviruses. The title The Filamentous Plant Viruses seemed the only suitable one, but it has led us to discuss also the quite different filamentous viruses of the rice stripe group-recently officially named the tenuivirus group which otherwise, indeed, might not have been conveniently covered in any volume of this series. The question must be asked: What is there new that justifies the presentation of a book of this kind? An outline of the answer may be Among the traditional filamentous viruses, much pro given as follows. gress has been made in elucidating the physical structure of potexvirus particles, and this work serves as an excellent model for discussion of and future experiments on the poty-, carla-, clostero-, and capilloviruses, which have comparable structures, although they are more difficult to manipulate. Work on the structure and strategy of the genomes of poty viruses is, however, relatively advanced and at a very interesting stage. The helper component that assists the aphid transmission of potyviruses has also recently received considerable attention, although the more we know about that, the less seems clear about the aphid transmission of the carlaviruses and closteroviruses, which apparently neither possess nor require a helper component.
Serological techniques are among the most efficient means for the identification and characterization of plant viruses and their associated proteins. Techniques and tools that have proved to be especially useful in studies with filamentous plant viruses are listed in Table I together with the major areas of their present and anticipated applications. Those aspects which are especially important in studies with filamentous viruses will be discussed in Section II. As world-wide efforts continue to improve the sensitivity and resolving power of serological assays, tests which were highly valued at one time may suddenly be outdated; Section III will, therefore, discuss which techniques seem to be most appropriate at the present state of knowledge to solve a certain problem. In Section IV a brief description of the serology of individual groups of viruses will be given.
The host range, transmission, cross-protection, antigenic relationships, and coat proteins of four distinct potyviruses (bean common mosaic virus (BCMV), blackeye cowpea mosaic virus (BlCMV), azuki bean mosaic virus (AzMV), and soybean mosaic virus (SMV)) were compared. BCMV, BlCMV, AzMV and SMV were easily distinguished by the host range and seed transmission. Two BCMV isolates, two BlCMV isolates and AzMV were serologically identical according to examination by agar gel diffusion plates containing 0.5% lithium 3, 5-diiodosalicylate. Five SMV isolates from Japan were serologically identical and distantly related to a SMV isolate from Thailand. BCMV, BlCMV and AzMV were distantly related to SMV. None of these viruses were serologically related to turnip mosaic virus (TuMV). Positive cross-protection was found between BCMV and BlCMV, between BCMV and AzMV, and between BlCMV and AzMV. The molecular weights of the coat proteins of the virus isolates estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 32.8×103(K) for two BCMV isolates, two BlCMV isolates and AzMV, 30.1K for five SMV isolates from Japan, 31.5K for a SMV isolate from Thailand, and 34K for TuMV. The patterns of the peptides of two BCMV isolates, two BlCMV isolates and AzMV after cleavage with Staphylococcus V8 protease appeared nearly identical. Likewise, polypeptide patterns of five SMV isolates from Japan were also very similar. Good correlation was found among serological typing, molecular weights, and peptide mapping data of 12 virus isolate proteins. These findings suggest that SMV is a distinct virus, while BCMV, BlCMV and AzMV are virus strains, not distinct viruses.
The tremendous advances in electronic data processing are likely to result in revolutionary changes in the theories and practices of taxonomy. The process of classification is being removed from speculations regarding the origin of the taxa being classified. A natural classification is one whose taxa share the largest number of properties and which is most useful for a wide range of purposes. The principles of numerical taxonomy are stated briefly and illustrated by means of diagrammatic examples. The relative roles of the taxonomist and computer are discussed and estimates given of computer time and costs involved in numerical taxonomic work. The numerical taxonomic work done in botany so far is discussed and the paper concludes with a brief mention of several problems of numerical taxonomy with regard to botanical work. These are: scarcity of characters, correlations between cytogenetic work and phenetic similarities, and problems raised by hybridization.
ABSTRAK Kedua ahli subkumpulan 'watermelon mosaic virus', WMV-I dan WMV-2 dianggap sebagai virus-virus yang berlainan berdasarkan kepada bidang hos dan serologi. lmmun mikroskop elektron dengan penunjuk pembezaan indeks 2 ke 3 mengsahkan perbezaan serologi ini. Analisis hibridisasi molikul dengan menggunakan DNA berkomplimen yang disediakan oleh transkrzpsi berbalz"k RNA WMV-I berprima rawak, menunjukkan tiada homologi di antara WMV-I dan WMV-2 dan DNA berkomplimen juga menunjukkan tiada homologi dengan RNA dari beberapa 'potyvirus' yang menjangkiti kekacang dan 'potato virus Y' (PVY). Analisa hibridisasi molikul selanjutnya meng-sahkan yang WMV-I dan WMV-2 adalah virus-virus yang nyata berbeza. ABSTRACT The two members of the watermelon mosaic virus subgroup, WMV-I and WMV-2, were considered to be different viruses on the basis of host range and serology. Immune electron microscopy with serological differentiation indices of 2 to 3 confirmed this serological difference. Molecular hybridization analysis using complementary DNA prepared by reverse transcrzption of randomly primed WMV-I RNA showed no homology between WMV-I and WMV-2 and the complementary DNA from a range of legume infecting potyviruses and'potato virus Y (PVY). Molecular hybridization analysisfurther confirmed that WMV-I and WMV-2 are distinct viruses.
Antisera were prepared to one strain of boan yellow mosaie virus (B.Y.lVLV.) and two strains of pea mosaie virus (P.i\LV. strain 1 and P.lII.V. sLraill 2). By utilizing the aggluLinatioll reaetiol1, the serological activit,y of both straills of P.lVLV. is clemollst,rated for the first timo. The results of erose-agglutination experim81lt,s show the strains of P.M. V. and B.Y.M.V. to be serologically related. Cross-protection tests support tho hypothesis that Lhose are strains of one and the same virlls.
Cucurbit crops in South Africa are seriously affected by a flexuous rod‐shaped virus 706 to 770 nm long which causes the plant to be stunted, the leaves to display symptoms of chlorotic mosaic, dark green blisters and malformation, and fruit to be malformed. The virus was purified from infected Cucurbita pepo by extraction in 0.5 M borate buffer, pH 8, containing ethylenediaminotetra‐acetic acid and mercapto‐ethanol, clarification with chloroform, addition of Triton X‐100, sedimentation by ultracentrifugation for which a sucrose cushion was used and centrifugation in 10 to 40 % sucrose gradients. The virus was mechanically transmitted to a limited host range with Chenopodium album, C. amaranticolor, C. quinoa and Gomphrena globosa being the only hosts infected outside the Cucurbitaceae. Luffa cylindrica, Cucumis metuliferus, Coccinia sessilifolia and Citrullus ecirrhosus all members of the Cucurbitaceae, were not infected by the virus. The virus was non‐persistently transmitted by Myzus persicae, produced pinwheel and bundle inclusions in the plant cell cytoplasm and has a single coat protein with a molecular weight of 36,000 daltons and a degraded lighter component of 26,000 daltons. Serological comparisons with antiserum to watermelon mosaic virus 2, Papaya ringspot virus strain W and watermelon mosaic virus Morocco (WMV‐Mor.) identified the virus as an isolate of WMV‐Mor. It was found that WMV‐Mor. is the dominant virus in all the main cucurbit producing areas of South Africa which were surveyed.
Two potyvirus isolates from the eggplant (Solanum melongena L.) cultivars ‘Ex Benin’ and ‘Ex Jos’, respectively, in Nigeria proved to be almost identical in host range, symptomatology and reactivity with antisera to various potyviruses. In eggplant they caused a severe systemic mottle, blistering and malformation of leaves and an abnormal serration of the leaf margins. A potyvirus isolate from tomato showing mosaic symptoms was similar, but not identical to the eggplant isolates. In the slide, precipitin test the serological differentiation indices were between 1 and 3 for the eggplant and tomato isolates. In the immunoelectron microscopical decoration test all three virus isolates showed some reactivity with antisera to the following potyvir, uses: dioscorea green banding mosaic, groundnut eyespot, a mungbean isolate of peanut stripe, pepper veinal mottle, telfairia mosaic and a tomato isolate from Taiwan. No reactions were observed with antisera to other potyviruses. Cytopathogenic effects w,ere similar for all three isolates in the arrangement of virus particles, the structure of the cylindrical inclusions and the occurrence of clusters of small vesicles. However, other cytological alterations like accumulations of rod-shaped aggregates of,granular material, formation of giant mitochondria, degeneration of mitochondria and occurrence of a nucleolar inclusion differentiated the isolates.
A new viral disease of chickpea (Cicer arietinum) characterized by filiform leaves was detected in the USDA Cicer germ plasm collection at Central Ferry, WA. Incidence of virus-infected chickpeas was<1%, and the virus, designated chickpea filiform virus (CFV), had a very restricted host range. All Cicer accessions tested (>30), including six wild species, were susceptible and developed filiform leaves. The only other plants infected systemically were lentil (Lens culinaris), fenugreek (Trigonella foenum-graecum), and Nicotiana clevelandii. CFV was transmitted in a styletborne (nonpersistent) manner from virus-infected chickpea to chickpea and fenugreek by Myzus persicae and Acyrthosiphon pisum. Seed yields of chickpea mechanically inoculated in the field with CFV at prebloom and full bloom were reduced 80 and 46%, respectively. Seed size was also adversely affected. No seed transmission of CFV was detected in chickpea or fenugreek. Virus particles typical of the potyvirus group were observed under the electron microscope. A serological relationship with bean yellow mosaic virus (BYMV), blackeye cowpea mosaic virus (BlCMV), and cowpea aphidborne mosaic virus (CAMV) was suggested in indirect enzyme-linked immunosorbent assay (ELISA) with BYMV, BlCMV, and CAMV antisera. A relationship with BYMV was confirmed by serologically specific electron microscopy. Ascitic fluid produced in mice against CFV, however, did not react in ELISA with BYMV, BlCMV, CAMV, or with three other potyviruses. Further research is needed to clarify the relationship of CFV to BYMV, BlCMV, and CAMV.
A virus identified as zucchini yellow mosaic virus (ZYMV-TS2) isolated from diseased squash in Turkey was partially characterized and compared with other ZYMV isolates from Egypt, Italy, and the United States. ZYMV-TS2 was also compared with isolates of watermelon mosaic virus strain 2 (WMV-2) and the WMV-1 strain of papaya ringspot virus (PRSV-W). ZYMV isolates from Turkey and Egypt were similar to the Connecticut strain but different from the Florida strain in timing and severity of symptoms in squash. All ZYMV isolates were distinguished from WMV-2 by giving only local symptoms on Black Turtle 2 bean and from PRSV-W by giving only local reactions on Chenopodium quinoa. Purification of ZYMV-TS2 yielded 522 mg of virus per kilogram of tissue, with a 260/280 nm ratio of 1.231.32. Virus-specific polyclonal antibodies capable of detecting ZYMV were produced to ZYMV-TS2 in mice. Unfractionated ascites fluid reached a maximum titer of 1:1,600,000 in indirect ELISA. Serological comparisons using various antisera to ZYMV, PRSV-W, and WMV-2 revealed that ZYMV and WMV-2 cross-reacted strongly with some antisera and not at all with others. No cross-reactions were observed between PRSV-W and WMV-2 or between PRSV-W and ZYMV.
(...) In this paper we have compared 17 SCMV/MDMV strains from Australia and the United States on the basis of their reactivities, in electro-blot immunoassay, with cross-absorbed polyclonal antibodies directed towards surface-located, virus-specific N-termini of coat proteins. (...)
SUMMARY Immunoaffinity columns were prepared by covalently binding monoclonal antibodies specific for soybean mosaic virus (SMV) to an agarose support matrix. SMV, purified by binding and elution, contained no contaminating proteins detectable by SDS-polyacrylamide gel electrophoresis. Affinity-purified SMV was essentially identical to virus purified by standard procedures in its infectivity, u.v. absorbance spectrum ratio of A 2s0/-4 260, sedimentation coefficient, electrophoretic pattern of coat protein, morphology and antigenicity. This method of purification is rapid, reproducible, and yields highly purified virus preparations. It may therefore be applicable in the purification of a wide variety of plant viruses.
SUMMARY The nucleotide sequence of the 3' terminal 1782 nucleotides of sugarcane mosaic virus (SCMV) genome has been determined. There is an open reading frame, from the 5' end, of 1307 nucleotides upstream from a 475 nucleotide 3' non-coding region that is polyadenylated. The open reading frame encodes a polypeptide of 435 amino acids. The segment of the genome encoding the viral capsid protein (mol. wt. 34200) is adjacent to the 3' non-coding region. The predicted capsid protein is similar in sequence to the capsid protein sequence predicted for tobacco etch virus (TEV). Part of another protein encoded in the same reading frame, similar to the predicted nuclear inclusion protein from TEV, has been identified upstream from the coat protein gene. The results indicate that the genome of SCMV encodes one or more large proteins that are processed to the mature proteins.
SUMMARY Cultures of hybrid cells secreting monoclonal antibodies to potato virus Y (PVY) were produced by fusing a non-secreting FO myeloma cell line with spleen cells from BALB/c mice immunized with an isolate of the tobacco veinal necrosis strain group (PVY N 605). Monoclonal antibodies were obtained which reacted with common antigenic determinants of 24 isolates belonging to the homologous (pVyN), common (PVY °) and stipple streak (PVY c) strain groups of PVY. Other antibodies were either strictly specific to PVY N isolates or of intermediate specificity. In enzyme-linked immunosorbent assay (ELISA) for the detection of PVY viruses, a preparation of monoclonal antibody to a common antigenic determinant gave more Uniform reactions than polyclonal antibodies from rabbit serum. Enzyme conjugates of monoclonal antibody preparations could be used highly diluted without loss of activity in ELISA, thereby decreasing the background caused by non-specific reactions.
SUMMARY Comparison by HPLC of tryptic digests of coat proteins from six biologically and serologically distinct potyviruses, namely bean yellow mosaic virus, Johnson grass mosaic virus, passion-fruit woodiness virus (PWV), potato virus Y (PVY), sugarcane mosaic virus (SCMV) and water-melon mosaic virus II, demonstrated that each potyvirus can be distinguished from the others. HPLC of tryptic peptides from coat proteins of four strains of PVY, two strains of PWV and three strains of SCMV showed that peptide patterns of strains from the same potyvirus were very similar. These findings were supported by amino-terminal amino acid sequence analysis of the peptides. The use of enzymes from different sources and variation in the temperature (35 °C to 40 °C) and time (16 to 20 h) of digestion caused small variations in the profiles but did not change the main features of the peptide patterns of each potyvirus. The results suggest that HPLC profiles of tryptic digests of the coat proteins of potyviruses could be useful criteria for the identification and classification of potyviruses.
A virus reported earlier to cause a green mosaic disease of eggplant in Nigeria was studied in more detail. Its filamentous particles with a normal length of 820 nm reacted in immunoelectron microscopical tests strongly with the homologous antiserum and less strongly with antisera to dioscorea green banding mosaic, groundnut eyespot, zucchini yellow mosaic viruses and to a tomato potyvirus isolate from Taiwan. No reactions were seen with antisera to 25 other potyviruses. Several new host plants were identified. Infected cells contained cylindrical inclusions with scrolls and short curved laminated aggregates and clusters of small vesicles with electron-dense content. Host range and serological reactivities differentiate the virus for which the name eggplant green mosaic virus is suggested from all potyviruses so far known.
Genomic RNA of plum pox virus (PPV) was used as a template for the synthesis of complementary DNA (cDNA). The generated cDNA molecules were subsequently cloned into pBR 322. A physical map covering 9700 bases of the PPV genome was constructed from 8, clones by hybridization and restriction endonuclease digestion. Clone pPPV-NAT 309, starting at the 3′-end, with an 866 bp insert was used in Northern- and Dot-hybridizations for the detection of single-stranded viral RNA in total nucleic acid as well as in sap preparations of PPV infected Nicotiana clevelandii. The nucleotide sequence of this clone was determined, the amino acid sequence of the coat protein C-terminal part was deduced and compared with four other coat proteins of potyviruses.
The amino acid sequence of the coat protein of the Johnsongrass (JG) strain of sugarcane mosaic virus (SCMV) has been determined by protein sequencing techniques. The protein contains 303 amino acid residues corresponding to a molecular weight of 33,510 and when compared to the coat proteins of other potyviruses that have been characterized (263–267 residues) is found to have additional residues at its N-terminus. The N-terminus is acetylated as shown by fast atom bombardment mass spectrometry. Partial amino acid sequences of the coat proteins of the other three Australian SCMV strains, sugarcane (SC), Queensland blue couch grass (BC) and sabi grass (Sabi) have also been obtained. The sequence data and the comparative tryptic peptide HPLC profiles showed that the JG coat protein was substantially different from those of the other three SCMV strains, the sequence homology being around 66 per cent. This is in marked contrast to the high sequence homology between SC, BC and Sabi strains (95–100 per cent) but similar to that (51–62 per cent) found between coat proteins of distinct members of the potyvirus group. On the basis of these structural findings and other information on major differences in serological, biological and biochemical properties we believe that the present JG strain should not be considered a strain of SCMV but should be regarded as an independent member of the potyvirus group. The name Johnsongrass mosaic virus is proposed for this new member.
Three strains of passionfruit woodiness virus, Tip Blight (PWV-TB), Severe (PWV-S) and Mild (PWV-M), 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 but no differences were observed in the serological properties of the three PWV strains. Molecular weight estimates on SDS-PAGE suggest the PWV coat protein contains 275 amino acid residues and sequence data for 269 of these residues is presented. The amino terminal peptide is blocked and has not been sequenced. The coat proteins of PWV-TB and PWV-S, which cause severe symptoms in infected plants, showed only three sequence differences compared to the eleven or twelve sequence changes between their coat proteins and that of the mild strain. The high sequence homology (96–99%) between the three PWV strains is similar to that previously reported for two strains of tobacco etch virus and three strains of sugarcane mosaic virus. Sequence homology between the three strains of PWV and published sequences for the coat proteins of eight distinct potyviruses ranged from 43–71% (average 57%).
The importance and need for standardized procedures in the international identification (description and diagnosis) of legume viruses is pointed out and suggestions are given to aid in this accomplishment. Outlines of techniques for using common methods such as host range studies, symptomatology, physical properties tests, cross-protection tests and insect transmission data are presented. Likewise, suggestions are given for the use of some of the newer approaches including physicochemical property studies, serology, and electron microscopy. The importance of these latter studies for virus characterization is emphasized. Some proposals for the uniform recording of results have been put forward. Most of the procedures may also be useful for the identification of viruses in general.