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

Summary Mouse monoclonal antibodies (MAbs) against the Australian B strain of clover yellow vein (C1YVV-B) and the JG strain of Johnsongrass mosaic (JGMV) potyviruses were produced, characterised and the epitopes with which they reacted were deduced. Using intact particles of C1YVV a total of ten MAbs were obtained which reacted strongly with C1YVV-B in an enzyme-linked immunosorbent assay and Western blots. Four of these MAbs (1, 2, 4, and 13) were found to be ClYVV-specific, as they reacted with all five C1YVV strains from Australia and the U.S.A. but not with 11 strains of bean yellow mosaic (BYMV), pea mosaic (PMV), and white lupin mosaic (WLMV) viruses which, together with C1YVV, form the BYMV subgroup of potyviruses. These MAbs failed to react with eight other potyvirus species, including six which infect legumes like the viruses in the BYMV subgroup. The C1YVV MAb 10 was found to be BYMV subgroup-specific. It reacted strongly with 15 of the 16 strains of viruses in the subgroup and gave no reaction with eight other potyviruses. The other five C1YVV MAbs reacted with varying degrees of specificity with the BYMV subgroup viruses and also with other potyviruses. Eight of the C1YVV MAbs (1, 2, 4, 5, 13, 17, 21, and 22) reacted with the intact coat proteins only and not with the truncated (minus amino terminus) coat protein of C1YVV suggesting that the epitopes for these MAbs are located in the surface-exposed, amino-terminal region of the C1YVV coat protein. Comparison of published coat protein sequences of BYMV and C1YVV isolates indicated that the epitopes for the four ClYVV-specific MAbs may be in the amino-terminal region spanning amino acid residues 18 to 30, whereas those for the other four MAbs may be located in the first 17 amino-terminal amino acid residue region. The epitopes that reacted with BYMV subgroup-specific MAb 10 and MAb 30 which reacted with 20 of the 24 potyvirus isolates, are probably located in the core region of C1YVV coat protein as these MAbs reacted with the intact as well as truncated coat protein of C1YVV. Analysis, in Western blot immunoassay, of 17 MAbs raised against virions of JGMV revealed that only two MAbs (1–25 and 4–30) were JGMV-specific, whereas others displayed varying degrees of specificity to different potyviruses. When these MAbs were screened against the intact and truncated (minus 67 amino-terminal amino acid residues) coat proteins of JGMV, the two JGMV-specific MAbs reacted only with the intact coat protein, whereas the other MAbs reacted with the intact as well as with truncated coat proteins, in Western blots. These results suggest that the epitopes for the two JGMV-specific MAbs are located in the surface-exposed amino-terminal 67 amino acid residue region and those for the cross-reactive MAbs are contained in the conserved core region of the JGMV coat protein. Screening of potyvirus MAbs against intact and truncated coat proteins thus appears to be a simple procedure to select virus-specific MAbs to potyviruses.     

Characterization of a distinct <Emphasis Type="Italic">Johnsongrass mosaic virus</Emphasis> strain isolated from sorghum in Nigeria

Summary. A virus isolated from sorghum in Nigeria has been partially characterized. It was tested by enzyme-linked immunosorbent assay (ELISA) using antisera to Maize dwarf mosaic virus, Johnsongrass mosaic virus (JGMV), Sugarcane mosaic virus strain-MDB, Sorghum mosaic virus, and Zea mosaic virus. A partial host range, symptom phenotypes for selected sorghum lines, and the mass of the coat protein (CP) subunit was analyzed by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and its amino acid (aa) sequence determined by time-of-flight mass spectrometry (TOFMS). The Nigerian isolate was positive in ELISA to only JGMV antiserum. It infected sorghum and smooth brome but not oat or johnsongrass. It caused necrosis in 12 of 13 tested sorghum lines, while the USA JGMV isolate caused necrosis in only one sorghum line. In SDS-PAGE, the mass of the Nigerian virus CP was 3,000Da smaller than that of JGMV-MDO. Moreover, TOFMS analyses showed that, while residues 1–7 of the CP aa sequence were identical to those of JGMV (GenBank #A27631), and residues 57–293 were almost identical to residues 67–303 of JGMV, the intermediate region exhibited significant differences, including a 10aa deletion. These data indicate that the virus should be considered a distinct isolate of JGMV (JGMV-N) and expands the known range of JGMV to Africa.     

Genetic variability in the coat protein genes of two orchid viruses: Cymbidium mosaic virus and Odontoglossum ringspot virus

The variability in coat protein gene sequences of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) that naturally infect orchids worldwide was investigated. Samples were collected from Korea, Singapore and Taiwan. The sequence data were compared with available published coat protein gene sequences of CymMV and ORSV, including those from Japan and Thailand. Among CymMV isolates, the homology was 89.1%-99.7% and 93.2%-100% at the nucleotide and amino acid levels, respectively. Among the ORSV isolates, the homology was 95.5%-100% and 93%-100% at the nucleotide and amino acid levels, respectively. No particular region of variability could be defined in either of the viruses. In deduced amino acid sequence, the N-terminal was more conserved than the C-terminal in both CymMV and ORSV. By comparing all sequences determined in this study and those that are published in the GenBank databases, we did not find clustering based on geographical distribution or sequence identity. Such high sequence conservation suggests that both CymMV and ORSV coat protein genes are suitable candidates to provide resistance to orchids cultivated in different geographical locations.     

The nucleotide sequence of the coat protein gene and 3′ untranslated region of papaya ringspot virus type W (Aust)

Summary The nucleotide sequence of the 3 terminal region of the Australian isolate of papaya ringspot virus type W [PRSV-W (Aust)] was determined. An open reading frame (864 bp), encoding the putative coat protein gene, occurs upstream of the putative 3 untranslated region (206 bp) and poly(A) tail. A 23 amino acid sequence was obtained from N-terminal analysis of the coat protein from purified virions. This sequence has 100% homology with a region of the amino acid sequence inferred from the nucleic acid sequence of the coat protein gene. However, this region is 13 amino acids downstream from the N terminus predicted for two American isolates of PRSV. The coat protein gene of PRSV-W (Aust) was shown to have 96.8% and 96.4% nucleotide sequence similarity with American isolates of PRSV-W and PRSV-P, respectively.     

Sequence diversity in the NIb coding region of eight sugarcane mosaic potyvirus isolates infecting sugarcane in Australia

Summary We have sequenced the NIb coding region of sugarcane mosaic potyvirus strain SC (SCMV-SC) and eight field isolates of SCMV from Australia. This region comprised 1563 nucleotides and encoded a putative protein of 521 amino acids containing the consensus motif GDD. The protease cleavage sites between the NIa/NIb and the NIb/coat protein were found to be Q/C and Q/A, respectively. The SCMV sequences were most similar to sorghum mosaic potyvirus with identities of 70% and 78% at the nucleotide and amino acid levels, respectively. When the sequences were compared to each other, there was a maximum of 3.3% variation between isolates at the nucleotide level and a maximum of 0.8% at the amino acid level. Phylogenetic analysis of the sequences indicated the field isolates were grouped according to their geographical location. The SCMV sequence with most homology to all other isolates has been selected to generate constructs for replicase-mediated resistance.Sequence data deposited with Genbank, accession number U51455.     

Nucleotide sequence analysis of the coat protein genes of two Korean isolates of sweet potato feathery mottle potyvirus

Summary.  The coat protein (CP) genes of the genomic RNA of two Korean isolates of sweet potato feathery mottle potyvirus (SPFMV), SPFMV-K1 and SPFMV-K2, were cloned and their complete nucleotide sequences were determined. Sequence comparisons of the two Korean isolates showed 97.8% amino acid identity in the CP cistron, and 79.9% to 99.0% identity with those of 6 other known SPFMV strains. Of 74 amino acid changes totally among the SPFMV strains, 39 changes were located at the N-terminal region. Pairwise amino acid sequence comparison revealed sequence similarities of 48.6 to 70.2% between SPFMV and 20 other potyviruses, indicating SPFMV to be a quite distinct species. Multiple alignment of the CP cistrons from other potyviruses showed that most of the conserved amino acid residues of the genus Potyvirus are well preserved in the corresponding locations. Accepted November 13, 1997 Received September 1, 1997     

Molecular characterization of a strain of cucumber mosaic virus based on coat protein and movement protein genes

Cucumber mosaic virus (CMV) A strain (CMV-A) isolated from Amaranthus tricolor was partially characterized at molecular level. Complete coat protein (CP) and movement protein (MP) ORFs were cloned and sequenced. The 657 bp region of CP gene and the 840 bp region of MP gene encode 218 and 276 amino acids, respectively. CP, at nucleotide level, showed 90-98% sequence identity with the CMV subgroup I and less than 80% with the CMV subgroup II, it showed at amino acid level 92-96% identity with the subgroup I and 74-87% with the subgroup II. The nucleotide and amino acid sequence identities of MP ranged in 91-94% and 92-96%, respectively with the subgroup I but in 81-83% with the subgroup II. Phylogenetic trees generated from nucleotide and amino acid sequences of both CP and MP genes identified the virus strain as a member of the subgroup IB. CMV-A CP also displayed a remarkably higher homology with Indian strains of CMV than with other CMV strains and formed a separate cluster within the subgroup IB.     

Nucleotide and deduced amino acid sequence of the 3′ end of the BYMV-MI genome

Summary We have cloned and sequenced cDNA transcribed from the 3 1239 nucleotides of the genomic RNA of a Western Australian isolate (MI) of bean yellow mosaic potyvirus (BYMV). This sequence contains 246 nucleotides of the NIb (replicase) gene and 819 nucleotides representing the entire coding region of the viral coat protein gene, followed by a 3 non-coding region of 174 nucleotides. The coding region of the coat protein gene is identical in length (273 amino acids) to that already reported for other isolates of this virus. The sequence identities obtained for BYMV-MI and published sequences of BYMV isolates range between 85% and 92% for the coding region of the coat protein and 90% to 98% for the 3 non-coding region. Likewise, the region of the NIb gene sequenced shows 99% and 97% sequence identity in the deduced amino acid and the nucleotide sequences, respectively.     

Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina

The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host.     

Identification of a Naturally Occurring Recombinant Isolate of <Emphasis Type="Italic">Sugarcane Mosaic Virus</Emphasis> Causing Maize Dwarf Mosaic Disease

The complete nucleotide sequence of a potyvirus causing severe maize dwarf mosaic disease in Shaanxi province, northwestern China was determined (GenBank accession No. AY569692). The full genome is 9596 nucleotides in length excluding the 3 -terminal poly (A) sequence. It contains a large open reading frame (ORF) flanked by a 149 nt 5-untranslated region (UTR) and a 255 nt 3-UTR. The putative polyprotein encoded by this large ORF comprises of 3063 amino acid residues. Sequence comparisons and phylogenetic analyses showed that this potyvirus is an isolate of Sugarcane mosaic virus (SCMV). The entire sequences shared identities of 89.6–97.6 % and 79.3–93.3% with 9 sequenced SCMV isolates at the nucleotide and deduced amino acid levels, respectively. But it showed much lower identities with Maize dwarf mosaic virus (MDMV), Sorghum mosaic virus (SrMV) and Johnsongrass mosaic virus (JGMV) isolates. The putative coat protein sequence is identical to that of a Chinese maize isolate SCMV-HZ. However, partition comparisons and phylogenetic profile analyses of the viral nucleotide sequences indicated that it is a recombinant isolate of SCMV. The recombination sites are located within the 6K1 and CI coding regions.     

2008年甘肃省新分离乙脑病毒的分子生物学特征

目的 对2008年甘肃省新分离乙脑病毒的PrM和E基因区段进行序列测定和分析,明确新分离病毒的基因型别并对E基因序列的分子特征进行分析.方法 对新分离乙脑病毒的PrM和E基因区段进行PCR扩增并测定序列.使用ClustalX2.09、MegAlign和Mega4软件对核苷酸和氨基酸序列进行分析并绘制系统发生树.结果 系统进化分析结果显示6株病毒均为基因Ⅰ型乙脑病毒,并且与2001和2002年越南分离株、2004年日本分离株及2004年我国四川省分离株进化关系较近.新分离株与减毒活疫苗株SA14-14-2相比,E基因核苷酸同源性为87.5%～87.9%,氨基酸同源性为96.8%～97.2%.新分离株与疫苗株在E基因区段存在11处共同位点的氨基酸差异.结论 2008年甘肃省分离的乙脑病毒均为基因Ⅰ型乙脑病毒,新分离株E基因氨基酸序列与疫苗株相比有部分差异,但均不属于决定抗原性的关键位点.     

Cloning and sequencing of the coat protein gene of tobacco ringspot virus isolates from UK and Iran

The coat protein (CP) gene and the 3' untranslated region (UTR) of genomic RNA 2 of Tobacco ringspot virus (TRSV, the genus Nepovirus, subgroup a) isolates from the UK and Iran were cloned from total viral RNA and sequenced. Comparison of these isolates with an isolate from the USA revealed a high degree of nucleotide and amino acid identity which extends the knowledge of molecular relationships between these three TRSV isolates. The UK isolate shared the highest nucleotide identity (95%) with the US isolate as compared to a lower identity with the Iranian isolate (92%). The highest identity (98%) was found between the UK and US isolates at amino acid level. Comparative analysis of the Iranian, UK and US isolates revealed some differences concerning some members of other subgroups of nepoviruses. For example, the N-terminal FDAYXR and the C-terminal FYGRXS motifs conserved among some nepoviruses, which occur adjacent to each other in folded CP molecules, were not detected in the Iranian, UK or US TSRV isolates. These isolates shared similarity only with Tomato ringspot virus (TomRSV) belonging to the subgroup c of nepoviruses. Another similarity of these isolates with TomRSV and Raspberry ringspot virus (RRSV) was the presence of a 34 nucleotide (nt) long sequence within the 3'-UTR.     

Fusion protein predicted amino acid sequence of the first US avian pneumovirus isolate and lack of heterogeneity among other US isolates

Avian pneumovirus (APV) was first isolated from turkeys in the west-central US following emergence of turkey rhinotracheitis (TRT) during 1996. Subsequently, several APV isolates were obtained from the north-central US. Matrix (M) and fusion (F) protein genes of these isolates were examined for sequence heterogeneity and compared with European APV subtypes A and B. Among US isolates the M gene shared greater than 98% nucleotide sequence identity with only one nonsynonymous change occurring in a single US isolate. Although the F gene among US APV isolates shared 98% nucleotide sequence identity, nine conserved substitutions were detected in the predicted amino acid sequence. The predicted amino acid sequence of the US APV isolate's F protein had 72% sequence identity to the F protein of APV subtype A and 71% sequence identity to the F protein of APV subtype B. This compares with 83% sequence identity between the APV subtype A and B predicted amino acid sequences of the F protein. The US isolates were phylogenetically distinguishable from their European counterparts based on F gene nucleotide or predicted amino acid sequences. Lack of sequence heterogeneity among US APV subtypes indicates these viruses have maintained a relatively stable population since the first outbreak of TRT. Phylogenetic analysis of the F protein among APV isolates supports classification of US isolates as a new APV subtype C.     

Variation in the coat protein sequence of British isolates of <Emphasis Type="Italic">Turnip yellow mosaic virus</Emphasis> and comparison with previously published isolates

Summary. Isolates of Turnip yellow mosaic virus (TYMV) were collected from wild cabbage (Brassica oleracea) on a 400 m stretch of Dorset coastline. The coat protein genes of four isolates showed high homology in nucleotide sequence (0.970–1.000, mean 0.987). Lower levels of homology where found to previously published sequences of Australian isolates [10] (0.725–0.775, mean 0.741). The amino acid composition of the Dorset isolates showed high levels of homology (0.964–1.000, mean 0.986). Numerous amino acid substitutions occurred between the Dorset and Australian isolates (0.705–0.819, mean 0.742). Comparison with other isolates showed large genetic distances between the Dorset isolates and both European and Australian isolates.     

Molecular variability of the coat protein gene of Potato virus Y from tobacco in China

Thirty-three tobacco samples showing typical symptoms of Potato virus Y (PVY) infection were obtained from tobacco fields in various regions of China. The results of indirect ELISA confirmed the infection with PVY. All the isolates had a capacity to infect tobacco systemically in greenhouse, causing either of two main symptoms: veinal necrosis and mosaic. The nucleotide and amino acid sequences of the coat protein (CP) gene and protein, respectively, of the isolates were determined. Comparison of the isolates revealed a high conservation of the CP gene with an identity of 83.2%. A phylogenetic tree of 41 Chinese isolates of PVY, based on complete CP gene, showed 3 groups corresponding to the strains PVYNTN (A group), PVYO (C group) and a putative new strain similar to PVYN (B group). The amino acid sequences of complete CP protein of the isolates showed an identity of 87.6%. The highest identity was observed in the C-terminal half of the CP protein, where only 11 amino acid differences could be observed, in contrast to the N-terminal half with 22 differences.