Coat protein of potyviruses

Summary 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.     

Architecture of the potyviruses

X-ray fiber diffraction data were obtained and helical pitch and symmetry were determined for seven members of the family Potyviridae, including representatives from the genera Potyvirus, Rymovirus, and Tritimovirus. The diffraction patterns are similar, as expected. There are, however, significant variations in the symmetries, as previously found among the flexible potexviruses, but not among the rigid tobamoviruses. Wheat streak mosaic virus, the only member of the genus Tritimovirus examined, displayed the largest deviations in diffraction data and helical parameters from the other viruses in the group.     

The complete genome sequence of a Passion fruit woodiness virus isolate from Australia determined using deep sequencing, and its relationship to other potyviruses

The complete genome sequence (9,858 nucleotides) of the Passion fruit woodiness virus isolate MU-2 was determined using Illumina sequencing. The large open reading frame (ORF) encodes a polyprotein containing 3,086 amino acids, with an AUG start codon and UAA stop codon. The polyprotein yielded 11 proteins (P1, HC-Pro, P3, PIPO, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and CP). Putative cleavage sites between them were identified by sequence comparison to those of other known potyviruses. Accuracy of the genome sequence information was provided by 42-1691-fold sequence coverage, and viral RNA accounted for 7.38% of total polyadenylated RNA from the host plant.     

Helper components of two potyviruses are serologically distinct

The specificity of antisera to helper component (HC) from tobacco vein mottling virus (TVMV)- or potato virus Y (PVY)-infected tobacco plants was tested in immunoprecipitation and immunoabsorption chromatography experiments. Treatment with the homologous antiserum abolished or drastically reduced the activity of either TVMV-HC or PVY-HC, as measured by their ability to effect aphid transmission of purified tobacco etch virus, while the heterologous antiserum had little or no effect on HC activity. Loss of TVMV-HC and PVY-HC activity in the immunoabsorption chromatography experiments was associated with the removal of a 53- and a 58-kDa polypeptide, respectively. The results indicate that serologically distinct HC proteins are produced in response to specific potyvirus infection and suggest that HC is virus coded.     

Electroporetic transfection of pepper protoplasts with plant potyviruses

Potyviruses are a persistent threat to bell pepper (Capsicum annuum L.) production worldwide. Much effort has been expended to study the resistance response of pepper cultivars at whole plant levels but with only limited effort at the cellular level using protoplasts. A pepper protoplast isolation procedure is available but an inoculation procedure is needed that provides consistent and highly efficient infection. An electroporation-based procedure for inoculation of potyviruses was developed using a base procedure developed for Cucumber mosaic virus (CMV). The final parameters identified for efficient potyvirus infection of pepper protoplasts involves two 25 ms pulses, 200 V each pulse with a 10 s interval between pulses. Depending on the method of detection, e.g., ELISA versus RT-PCR, potyvirus RNA inoculum ranged from 10 to 40 μg with infection detection occurring with samples of 50,000-100,000 protoplasts.     

Preparation of diagnostic monoclonal antibodies against two potyviruses

Diagnostic monoclonal and polyclonal antibodies against bean yellow mosaic virus (BYMV) and plum pox virus (PPV) were prepared, characterized and used for detection of these viruses in infected plants by enzyme-linked immunosorbent assay (ELISA), immunoblot analysis and tissue print immunoblot assay (TPIBA).     

The complete genome sequence of pepper severe mosaic virus and comparison with other potyviruses

Summary. The complete nucleotide sequence of pepper severe mosaic virus (PepSMV) was determined. The viral genome consisted of 9890 nucleotides, excluding a poly (A) tract at the 3′ end of the genome. The PepSMV RNA genome encoded a single polyprotein of 3085 amino acid residues, resulting in ten functionally distinct potyviral proteins. The lengths of the 5′ nontranslated region (NTR) and the 3′ NTR were 164 and 468 nucleotides, respectively. The genome organization of the virus was typical for members of the genus Potyvirus in the family Potyviridae. The coat protein amino acid sequence identity between PepSMV and the other 45 potyviruses ranged from 53.4 to 79.7%. Sequence alignments and phylogenetic analyses of the potyviral polyprotein sequences revealed that PepSMV was the closest to potato virus Y (PVY) and closely related to members of the PVY subgroup. Our genome sequence data clearly confirmed that PepSMV belongs to a separate species in the genus Potyvirus.     

The genomic sequence of Wisteria vein mosaic virus and its similarities with other potyviruses

Summary. The complete nucleotide sequence of a Beijing isolate of Wisteria vein mosaic virus was determined to be 9695 nucleotides in length excluding the poly(A) tail. Sequence analysis predicted a single large open reading frame of 9279 nucleotides potentially encodes a polyprotein of 3092 amino acids. Phylogenetic analysis based on the genomic and deduced amino acid sequences support the current status of Wisteria vein mosaic virus (WVMV) as a distinct virus of the genus Potyvirus and a member of the Bean common mosaic virus (BCMV) subgroup. Sequence comparisons of WVMV and other members of the BCMV subgroup showed that WVMV is most closely related to both soybean mosaic virus and watermelon mosaic virus.     

Characterisation of potyviruses from sugarcane and maize in China

Sugarcane or maize leaves with mosaic virus symptoms were collected from 13 sites in China. Sequence data showed that all 8 samples from maize contained Sugarcane mosaic virus (SCMV); complete sequences were determined from 2 samples and partial sequences (the CI coding region and the 3'-part of the genome) from the others. The 5 sugarcane samples all contained a virus tentatively described as Sorghum mosaic virus (SrMV) and in three of them SCMV was also detected; 2 SrMV sequences and the 3 SCMV ones were completely determined and partial SrMV sequences were obtained from the remaining 3 samples. The features of the complete sequences of SCMV and SrMV are described for the first time. Sequence comparisons and phylogenetic analysis showed three distinct groups of Chinese SCMV sequences (sugarcane isolates from Zhejiang province, a maize isolate from Guangdong and maize isolates from other provinces). The SrMV sequences were similar to one another (> 93% identical nucleotides); they resembled published sequences in the coat protein but were less similar in the 3'-UTR. The complete sequences of SCMV and SrMV had about 70% nucleotides identical to one another and to Maize dwarf mosaic virus (MDMV). MDMV was not detected in any of the samples.     

The effect of helper component on the uptake and localization of potyviruses in Myzus persicae

125I-labeled virions were used to determine whether helper component (HC) affected the uptake or distribution of potyviruses in aphids. Aphids were allowed to acquire purified, 125I-labeled tobacco etch virus or potato virus Y mixed with HC or with inactivated HC. Helper component had no effect upon uptake of labeled virus, as measured by gamma counting. Autoradiography of freeze-sectioned aphids revealed, however, that in the presence of HC, label was associated with the maxillary stylets and with portions of the alimentary canal anterior to the gut, as well as with the gut region. Label accumulated only in the gut in control aphids. This selective localization of virus acquired in the presence of HC supports a binding mechanism for the mode of action of HC.     

Phylogenetic analysis of the Potyviridae with emphasis on legume-infecting potyviruses

Summary.  The 3′-terminal nucleotide sequences of thirteen authenticated strains of bean common mosaic virus (BCMV) and one strain of bean common mosaic necrosis virus (BCMNV) were obtained. The regions sequenced included the coat protein coding sequence and 3′-end non-coding region. These data, combined with sequence information from other legume-infecting potyviruses and the Potyviridae were used for phylogenetic analysis. Evidence is provided for delineation of BCMNV as distinct from BCMV and the inclusion of azuki mosaic, dendrobium mosaic, blackeye cowpea mosaic, and peanut stripe viruses as strains of BCMV. This relationship defines the members of the BCMV and BCMNV subgroups. These data also provide a basis upon which to define virus strains, in combination with biological data. Other aspects and implications of legume-infecting potyvirus phylogenetics are discussed. Received December 24, 1996 Accepted June 3, 1997     

Identification and sequence analysis of potyviruses infecting crops in Vietnam

Summary Fifty-two virus isolates from 13 distinct potyvirus species infecting crops in Vietnam were identified and the 3′ region of each genome was sequenced. The viruses were: bean common mosaic virus (BCMV), potato virus Y (PVY), sugarcane mosaic virus (SCMV), sorghum mosaic virus (SrMV), chilli veinal mottle virus (ChiVMV), zucchini yellow mosaic virus (ZYMV), leek yellow stripe virus (LYMV), shallot yellow stripe virus (SYSV), onion yellow dwarf virus (OYDV), turnip mosaic virus (TuMV), dasheen mosaic virus (DsMV), sweet potato feathery mottle virus (SPFMV) and a novel potyvirus infecting chilli, tentatively named chilli ringspot virus (ChiRSV). With the exception of BCMV and PVY, this is first report of these viruses in Vietnam. Further, rabbit bell (Crotalaria anagyroides) and typhonia (Typhonium trilobatum) were identified as new natural hosts of the peanut stunt virus (PStV) strain of BCMV and of DsMV, respectively. Sequence and phylogenetic analyses of the entire CP-coding region revealed considerable variability in BCMV, SCMV, PVY, ZYMV and DsMV. Correspondence: A/Prof. Rob M. Harding, Tropical Crops and Biocommodities Domain, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane 4001, Australia     

The complete genome sequence of freesia mosaic virus and its relationship to other potyviruses

We have completed the genomic sequence of a potyvirus, freesia mosaic virus (FreMV), and compared it to those of other known potyviruses. The full-length genome sequence of FreMV consists of 9,489 nucleotides. The large protein contains 3,077 amino acids, with an AUG start codon and UAA stop codon, containing one open reading frame typical of a potyvirus polyprotein. The polyprotein of FreMV-Kr gives rise to eleven proteins (P1, HC-pro, P3, PIPO, 6K1, CI, 6K2, VPg, NIa, NIb and CP), and putative cleavage sites of each protein were identified by sequence comparison to those of other known potyviruses. Phylogenetic analysis of the polyprotein revealed that FreMV-Kr was most closely related to PeMoV and was related to BtMV, BaRMV and PeLMV, which belong to the BCMV subgroup. This is the first information on the complete genome structure of FreMV, and the sequence information clearly supports the status of FreMV as a member of a distinct species in the genus Potyvirus.     

The genomic sequence of cowpea aphid-borne mosaic virus and its similarities with other potyviruses

Summary.  The genomic sequence of a Zimbabwe isolate of Cowpea aphid-borne mosaic virus (CABMV-Z) was determined by sequencing overlapping viral cDNA clones generated by RT-PCR using degenerate and/or specific primers. The sequence is 9465 nucleotides in length excluding the 3′ terminal poly (A) tail and contains a single open reading frame (ORF) of 9159 nucleotides encoding a large polyprotein of 3 053 amino acids and predicted Mr of 348. The size of the genome and the encoded polyprotein is in agreement with other potyviruses and contains nine putative proteolytic cleavage sites and motifs conserved in homologous proteins of other potyviruses. The P1 and P3 were the most variable proteins while CI, NIb and CP were the most conserved. Received August 2, 2001; accepted January 15, 2002     

Different helper factors associated with aphid transmission of some potyviruses

Myzus persicae transmitted watermelon mosaic virus (WMV) after acquiring it through artificial membranes from a solution of purified virus mixed with the soluble fraction from infected leaf extracts or by prefeeding on the soluble fraction before acquiring purified virus. WMV-induced helper factor assisted M. persicae in transmitting purified turnip mosaic virus (TuMV) but not potato virus Y (PVY). TuMV-induced helper factor from infected leaves was ineffective for the transmission of purified WMV or PVY. PVY-induced helper factor from infected leaves was capable of helping the transmission of TuMV but not that of WMV. The results indicate that there are at least three distinct helper factors with different specificity associated with potyviruses.     

Production of avian antibodies to three potyviruses in coturnix quail

Avian antibodies against three potyviruses were produced in a small bird, coturnix quail (Coturnix coturnix japonica Temminck et Schlegel), with 15-60 micrograms of purified virus preparations. Intramuscular injections of immunogen with Freund's incomplete or complete adjuvant into the birds did not result in higher titer of antibody compared to that of control birds given intravenous injections. Quail egg yolk antibody was as useful as hen antibody for indirect-ELISA and allowed virus to be detected in purified preparation (10-50 ng/ml) and in crude extracts (10(-6)-10(-7) dilution). The advantages of using quail to produce avian antibodies are discussed.     

Characterisation of some carla- and potyviruses from bulb crops in China

Summary.  Conserved carla- and potyvirus primers were used in RT-PCR to amplify virus fragments from garlic and other bulb crops in China and the fragments were subsequently sequenced and compared in phylogenetic analyses. Garlic plants from Henan, Hubei, Jiangsu, Shangdong and Yunnan provinces all contained at least one isolate each of Garlic latent virus (genus Carlavirus), Onion yellow dwarf virus (OYDV, genus Potyvirus) and Leek yellow stripe virus (LYSV, genus Potyvirus). The complete sequence of a Zhejiang isolate of LYSV was also determined, providing the first complete sequence of this virus. The genome was 10142 nucleotides long excluding the poly(A) tail and had the typical features of the genus Potyvirus, although some of the amino acids surrounding the polyprotein cleavage sites were unusual. Shallot yellow stripe virus (SYSV) was amplified from bunching onion (Allium fistulosum var. caespitosum) in Zhejiang province, providing the first record of SYSV in China. Lily mottle virus was amplified from dragon-teeth lily (Lilium brownii var. viridulum). Received July 4, 2001 Accepted September 14, 2001     

The bean common mosaic virus lineage of potyviruses: where did it arise and when?

There are more than 30 species in the bean common mosaic virus lineage of the genus Potyvirus. We have used their partial coat protein gene sequences to infer their phylogenies and have compared these with host and provenance information. Members of six species of the lineage have been isolated from crops distributed around the world, but three of these show clear links with South and East Asia. Members of the remaining species have been found in wild plants, minor crop species or ornamentals, and the majority of these have only been found in south-east and East Asia, Oceania or Australia. This phylogeographic pattern suggests that the bean common mosaic virus lineage arose in that region. Maximum-likelihood trees of the sequences were dated using the report that the initial major radiation of all potyviruses was 6,600 years ago. In this way, the bean common mosaic virus lineage was found to have first diverged 3,580 years ago, and one sub-lineage of seven species, found only in Australia, probably diverged there 2005 years ago. We discuss the ways in which the viruses could have moved from south-east Asia to Australia and note that their movement coincided with the spread of the Austronesian sea-faring/farming culture from China/Taiwan throughout the islands of the southern and eastern Pacific Ocean. Our study shows that virus isolates from wild or minimally domesticated plants, and from islands, are probably more useful indicators of the origins of viruses than those from widely grown well-travelled crop species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection of picogram quantities of potyviruses using a dot blot immunobinding assay

Highly sensitive direct visual detection of potyviruses was achieved using a dot blot immunobinding assay (DBIA). The small sample volumes required permit the detection of as little as 0.5 pg virus in purified preparations. The binding of rabbit antibodies could be visualized using goat anti-rabbit IgG (GAR) conjugated to alkaline phosphatase, beta-D-galactosidase, glucose oxidase, or horseradish peroxidase and histochemical substrates. The avidin-biotin system was also useful, but somewhat less sensitive than GAR-enzyme conjugates. Detection of potyviruses in an aphid vector was also attempted, but without success due to endogenous aphid enzymes.