Nucleotide sequence of the capsid protein gene of potato virus Y (PVY)

The nucleotide sequence of the 3 terminal region of potato virus Y (PVY) was determined. Starting with a poly(A) tail of 18 residues a non-coding region of 335 nucleotides precedes the region encoding for the virus coat protein (cp) 801 nucleotides long ending with a TGA. This region was located by comparing the predicted amino acid sequence with the one determined for the PVY capsid protein by Shukla et al. (1). Both sequences contained 267 amino acids sharing about 94% homology. They differ, however, at several positions presumably due to base transitions within their respective nucleotide sequences. Restriction endonuclease sites in and around the cp coding region were identified.     

Nucleotide sequence of the coat protein gene of a necrotic strain of potato virus Y from New Zealand

Summary The sequence of the 3-terminal 1,134 nucleotides of the genome of a New Zealand isolate of a necrotic strain of potato virus Y (PVY^N) has been determined. This sequence contains one large open reading frame of 796 nucleotides, the start of which was not identified, which is capable of encoding a protein of 264 amino acid residues with a molecular weight of 29,631. Comparison of the amino acid sequence with a published coat protein sequence of another strain, PVY-D, and with the amino acid sequence deduced from PeMV cDNA sequence data, confirms that the 3 cistron encodes the viral coat protein in PVY^N. Adjacent to the 3 end of the coding region there is an untranslatable sequence of 326 nucleotides terminating in a polyadenylate tract. An alignment of the PVY^N amino acid sequence with the coat protein sequences of six other potyviruses revealed significant sequence similarities in the internal and carboxy terminal regions. Much amino acid sequence similarity was found between PVY^N, PVY-D, and PeMV (91–93%), suggesting that PeMV should be regarded as a PVY strain. An analysis of the 3-untranslated region of the six potyviruses revealed PVY^N and PeMV as the only viruses displaying sequence similarity in this region. The 3-untranslated sequences of PVY^N and PeMV were further examined for secondary structure.     

Immunoprecipitation analysis of potyviral in vitro translation products using antisera to helper component of tobacco vein mottling virus and potato virus Y

The serological relationships of the products of in vitro translation of the RNA of various potyviruses were analyzed by using antisera to helper component (HC) from tobacco plants infected with either tobacco vein mottling virus (TVMV) or potato virus Y (PVY). The PVY-HC antiserum immunoprecipitated a specific PVY-RNA translation product; this product was not reactive with antisera to PVY-induced cylindrical inclusion protein or capsid protein or to the two tobacco etch virus nuclear inclusion proteins. The antiserum to PVY-HC did not immunoprecipitate significant amounts of any translation products of 16 other potyviruses including TVMV. In contrast the antiserum to TVMV-HC efficiently immunoprecipitated a specific product(s) of four different potyviruses, some isolates of which are poorly transmitted or nontransmissible by aphids, and less efficiently a product(s) from 12 other potyviruses, including PVY. Distinct serotypes were resolved among the major in vitro translation products of 17 different potyviral RNAs by the antisera to TVMV-HC and PVY-HC. There appears not to be a correlation between the serological reactivities of HC-related polypeptides and the ability of different HC-virus combinations to effect aphid transmission of the virus.     

Engineered resistance in potato against potato leafroll virus, potato virus A and potato virus Y

Transgenic potato plants of Solanum tuberosum cultivar Vales Sovereign were generated that expressed fused, tandem, 200 bp segments derived from the capsid protein coding sequences of potato virus Y (PVY strain O) and potato leafroll virus (PLRV), as well as the cylindrical inclusion body coding sequences of potato virus A (PVA), as inverted repeat double-stranded RNAs, separated by an intron. The orientation of the expressed double-stranded RNAs was either sense–intron–antisense or antisense–intron–sense RNAs, and the double-stranded RNAs were processed into small RNAs. Four lines of such transgenic potato plants were assessed for resistance to infection by PVY-O, PLRV, or PVA, all transmitted by a natural vector, the green-peach aphid, Myzus persicae. Resistance was assessed by the absence of detectable virus accumulation in the foliage. All four transgenic potato lines tested showed 100 % resistance to infection by either PVY-O or PVA, but variable resistance to infection by PLRV, ranging from 72 to 96 % in different lines. This was regardless of the orientation of the viral inserts in the construct used to generate the transgenic plants and the gene copy number of the transgene. This demonstrates the potential for using tandem, fused viral segments and the inverted-repeat expression system to achieve multiple virus resistance to viruses transmitted by aphids in potato.     

Production and characteristics of strain common antibodies against a synthetic polypeptide corresponding to the C-terminal region of potato virus Y coat protein

Comparing the predicted amino acid sequence between two Japanese potato virus Y (PVY) strains, necrotic strain and ordinary strain, it was found that the C-terminal regions (H₂N-HTTEDVSPSMHTLLGVKNM-COOH) of the coat proteins in the two strains were completely conserved. The conserved amino acid sequence was also found in the C-terminal region coat protein of PVY-36, a strain which did not react with monoclonal antibodies specific to the necrotic and the ordinary strain respectively. Antibodies were produced against a synthetic polypeptide PVY-C19 consisting of 19 amino acids, which correspond to the C-terminal region of the coat protein, using 4 coupling combinations of polypeptide PVY-C19 to protein carriers. Carrier-free polypeptides and those coupled to ovalbumin with ECDI (ethyl-dimethylaminopropyl carbodiimide) produced high titer of antibodies and detected PVY strains from PVY-infected plants by Western blot analysis and by ELISA.     

Evidence that a component other than the virus particle is needed for aphid transmission of potato virus Y

Aphids (Myzus persicae Sulz) did not transmit potato virus Y after probing into purified virus preparations, but did if these preparations were first mixed with extracts of infected plants from which all potato virus Y particles had been removed by centrifuging. The same centrifuged extracts also ‘helped’ aphid transmission of potato aucuba mosaic virus.     

Serological behavior of cucumber mosaic virus (strain Y) and the virus protein

Antisera with titers of 1/128 were obtained by injecting rabbits intravenously (iv) or intramuscularly (im) with purified cucumber mosaic virus (strain Y) (CMV-Y). Gel-diffusion tests with CMV-Y resulted in the formation of a curved band with the iv antisera and both a curved and a straight band with the im antisera. The curved band results from the combination of high molecular weight virus antigen with specific antibody, and the straight band originates from the combination of a product of virus degradation and its specific antibody. Crude sap from infected tobacco and protein obtained from CMV-Y by KCl degradation did not react with iv antiserum in gel-diffusion tests and formed only the straight band with im antiserum. Both CMV-Y and virus protein precipitated with im antiserum in tube precipitin tests, but no precipitation occurred when iv antiserum was tested with virus protein. However, virus protein possesses the antigenic determinants of CMV-Y, as shown by cross absorption in test tubes. The ability of the protein to bind antibodies specific for the curved band antigenic determinant of CMV-Y is reduced when the protein is precipitated by dialysis against water or used in intragel absorption tests, but its ability to bind straight band antibody is not affected. Exposure of CMV-Y to 37 degrees for 1 hour or to buffers other than 0.005 M borate, pH 9.0, such as 0.05 M borate, pH 9.0, and 0.05 M phosphate, pH 8.0, resulted in reduction of the major virus peak and the appearance of smaller nucleoprotein fragments (shown by density gradient centrifugation).     

Partial antigenic characterization of different potato virus Y NTN strain isolates

Eight isolates of potato virus Y NTN strain (PVY-NTN) of different origin were studied by means of monoclonal antibodies (MAbs) in non-competitive and competitive enzyme-linked immunosorbent assay (ELISA), and by immunoblot analysis of the viral coat protein (CP). As the MAbs reacted with the denatured viral CP, their epitopes must be continuous. The ELISA data demonstrate that the epitopes are topologically different. The epitopes may be located on the N-terminal part of CP as showed its partial amino acid sequencing and the immunoblot analysis.     

Complete nucleotide sequence and genome organization of sweet potato feathery mottle virus (S strain) genomic RNA:the large coding region of the P1 gene

Summary. The complete nucleotide sequence of a sweet potato feathery mottle virus severe strain (SPFMV-S) genomic RNA was determined from overlapping cDNA clones and by directly sequencing viral RNA. The viral RNA genome is 10 820 nucleotides long, excluding the poly(A) tail and contains one open reading frame (ORF) starting at nucleotide 118 and ending at 10 599, potentially encoding a polyprotein of 3 493 amino acids (Mr 393 800). The ORF was followed by a 3 untranslated region of 221 nucleotides. The deduced polyprotein includes P1 (74K), HC-Pro (52K), P3 (46K), 6K1, CI (72K), 6K2, NIa-VPg (22K), NIa-Pro (28K), NIb (60K) and coat (35K) proteins, after an analysis of protein cleavage sites analogous to other potyvirus polyproteins. The polyprotein had a high level of amino acid identity with those of other potyviruses, except in the regions of P1 and P3. The P1 of SPFMV-S RNA has 664 amino acid residues, and is the largest and least similar to those of other potyviruses. HC-Pro and CI show high identity with those of other potyviruses. P3 has relatively low identity, however, the length of P3 was within the range of variability among other potyviruses. The 6K1 protein between P3 and C1 is also highly similar to those of other potyviruses. This is the first report on the complete nucleotide sequence of the sweet potato-infecting virus. Received October 28, 1996 Accepted April 3, 1997     

Coat protein sequence of a resistance-breaking strain of potato virus X isolated in Argentina

The PVX coat protein (CP) is involved in many aspects of plant-virus interaction (virion morphology, plant symptoms, viral pathogenesis and virulence, and genomic RNA accumulation). Different virus strains have been distinguished according to their compatibility with the host resistance genesNx, Nb, andRx. Substitution of the Thr 122 on the CP with a Lys in PVX strain HB has been shown to affect the response of potato cultivars with theRx resistance gene. In PVX DX the avirulence determinant for theNx gene has been localized in the Gln 78 of the coat. PVX strain MS, like PVX HB, is able to overcome theRx, Nx, andNb genes. Sequencing of the CP gene of PVX MS (EMBL accession number Z34261) shows that it has a Thr in codon 122 and a Gln in codon 78. These results suggest that, in addition to the coat protein gene, other regions of the viral genome are involved in the pathogenicity.     

Nucleotide sequence of the 3'-terminal region of potato virus A RNA.

The sequence of the 3'-terminal region of the genome of the potato virus A (PVA) was obtained from two independent cDNA clones. This sequence is 1383 nucleotides long and contains an open reading frame of 1178 nucleotides, ending with the translation termination codon TAA and followed by untranslated region of 205 nucleotides. Since the N-terminal amino acid of the coat protein of PVA was blocked, the position of the putative coat protein cleavage site has been deduced by searching for consensus sequences and by the analogy to other potyviruses. The resulting coat protein is 269 amino acids long and has a calculated MW of 30257. Two independently sequenced cDNA clones show sequence heterogeneity at four nucleotide positions: C422/A422, G432/A432, G446/A446 and T706/C706. Three first nucleotide differences are located at the PVA coat protein N-terminal region and led to the change of the amino acid. The coat protein of PVA displayed significant (73-78%) sequence homology to the coat proteins of six other potyviruses: papaya ringspot virus (PRV), pepper mottle virus (PeMV), plum pox virus (PPV), potato virus Y (PVY), sugarcane mosaic virus (SCMV) and tobacco vein mottling virus (TVMV). Even higher sequence homology (82%) was detected with a coat protein of a seventh potyvirus, tobacco etch virus (TEV). Major differences among the coat protein of PVA and of other potyviruses are located at the N-terminal region of the coat protein.     

Complete genome sequence of a novel potato virus S strain infecting Solanum phureja in Colombia

Potato virus S (PVS) (genus Carlavirus, family Betaflexiviridae) is one of the most prevalent viruses in potato crops (Solanum tuberosum and S. phureja) around the world, causing reductions in crop yields between 10 and 20 %. Symptoms of PVS infection may include leaf mottling, rugosity of leaves, deepening of the veins and reductions in crop yields between 10 and 20 %. Virions are flexuous rods of 610-710 nm with a positive-sense ssRNA genome of approximately 8500 nt comprising six ORFs, a 5′CAP and a 3′poly-A tail. PVS has been classified into two groups: PVS^O (Ordinary) and PVS^A (Andean). PVSA induces severe symptoms in infected plants, such as premature senescence and defoliation, and is more efficiently transmitted by aphids than PVS^O. To date, only five PVS genomes have been completely sequenced, including those of three PVS^O and two PVS^A strains. Currently, there are no reports of complete PVS genome sequences from Andean South America. In this work, we present the complete genomic sequence of a novel PVS strain infecting S. phureja that is clearly distinct from currently known PVS isolates.     

Comparative genomic sequence analysis between a standard challenge strain and a vaccine strain of duck enteritis virus in China

Here, we present the complete genomic sequence of the Chinese standard challenge strain (CSC) of duck enteritis virus (DEV), which was isolated in China in 1962. The DEV CSC genome is 162,131 bp long and contains 78 predicted open reading frames (ORFs). Comparison of the genomic sequences of DEV CSC and DEV live vaccine strain K at passage 63 (DEV K p63) revealed that the DEV CSC genome is 4,040 bp longer than the DEV K p63 genome, mainly because of 3,513-bp and 528-bp insertions at the 5′ and 3′ ends of the unique long segment, respectively. At the nucleotide level, 63 of the 76 ORFs in the DEV CSC genome were 100 % identical to the ORFs in the DEV K p63 genome. Two ORFs (UL56 and US10) had frameshift mutations in the C-terminal regions, while LORF5 was unique to the DEV K p63 genome. It is difficult to assign attenuated virulence to changes in specific genes. However, the complete DEV CSC genome will further advance our understanding of the genes involved in virulence and evolution. The DEV CSC genome sequence has been deposited in GenBank under accession number JQ673560.     

The complete nucleotide sequence of the Popp (1967) strain of Marburg virus: a comparison with the Musoke (1980) strain

Summary The nucleotide sequence of genomic RNA of Marburg virus strain Popp was determined. Strain Popp was isolated in 1967 during the first filoviral outbreak. The virus was purified from blood of infected guinea pigs in which it had been maintained. The length of the determined sequence was 19112 nucleotides. Amino acid sequences of seven known virion proteins were deduced. Nucleotide and amino acid sequences were compared with those of strain Musoke of Marburg virus isolated in 1980 in Kenya and purified from Vero cells. Homology between nucleotide sequences of two strains was 93.9%. Comparisons revealed conserved and variable regions of the nucleotide and amino acid sequences. The GP, the envelope protein of the virion, was found to be the most variable protein. The greatest differences in the protein were located in the supposedly external part of the molecule. Amino acid substitutions in the L protein, the main component of viral RNA-dependent RNA polymerase, were also distributed extremely non-randomly. It was shown that the non-coding regions of the genome were more variable than the coding ones; 37.6% of nucleotide differences corresponded to the former. 72.6% of nucleotide substitutions located in the coding regions were found to be at the third codon position.Presented in part at the Ninth International Conference on Negative Strand Viruses, Estoril, Portugal, October 2–7, 1994 (Abstract 237), and at IXth International Congress of Virology, Glasgow, Scotland, 8–13 August 1993 (Abstract W52-4).     

Comparative full-length sequence analysis of Marek's disease virus vaccine strain 814

The complete DNA sequence of Marek’s disease virus (MDV) serotype 1 vaccine strain 814 was determined. It consisted of 172,541 bp, with an overall gene organization identical to that of the MDV-1 type strains. Comparative genomic analysis of vaccine strains (814 and CVI988) and other strains (CU-2, Md5, and Md11) showed that 814 was most similar to CVI988. Several unique insertions, deletions, and substitutions were identified in strain 814. Of note, a 177-bp insertion in the overlapping genes encoding the Meq, RLORF6, and 23-kDa proteins of strain 814 was identified, and a 69-bp deletion was also located in the origin of replication site (Ori) in the gene encoding RLORF12. Compared to the CVI988 vaccine strain, a deletion of 510 bp was identified in the UL36 gene. These analyses identified key mutations in the 814 strain and the vaccine strain that could be exploited for future MDV vaccine design.     

Multiplex microsphere immuno-detection of potato virus Y, X and PLRV

To monitor seed potatoes for potato virus X, Y and PLRV, a multiplex microsphere immunoassay (MIA) was developed based on the Luminex xMAP technology, as an alternative to ELISA. The xMAP technology allowed detection of a number of antigens simultaneously whereas ELISA only allowed simplex detection of antigens. The use of paramagnetic beads in the MIA procedure allowed efficient removal of excess sample compounds and reagents. This resulted in lower background values and a higher specificity than a non-wash MIA procedure using conventional beads. In a simplex MIA detection, levels for PVY and PLRV in potato leaf extracts were 10 times lower than ELISA but for PVX 10 timers higher, whereas the specificity was similar. Results of a multiplex assay performed on viruses added to potato leaf extracts were largely similar to those of ELISA for individual viruses. Results of samples infected naturally with PVX, PVY or PLRV were comparable with ELISA.