Analysis of Iranian <Emphasis Type="Italic">Potato virus S</Emphasis> isolates

Two hundred forty potato samples with one or more symptoms of leaf mosaic, distortion, mottling and yellowing were collected between 2005 and 2008 from seven Iranian provinces. Forty-four of these samples tested positive with double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) using a Potato virus S (PVS) polyclonal antibody. Of these 12 isolates of PVS were selected based on the geographical location for biological and molecular characterization. The full coat protein (CP) and 11K genes from 12 PVS isolates were PCR amplified, cloned and sequenced. All 12 PVS isolates showed mosaic symptoms on Nicotiana debneyii and N. tabacum cv. Whiteburly and local lesion on Chenopodium amaranticolor, C. quinoa and C. album. The Iranian isolates share between 93 and 100% pairwise nucleotide identity with other PVS^O isolates. Based on maximum likelihood phylogenetic analysis coupled with pairwise identity analysis, we propose 15 genotypes for the PVS^O strain and 3 genotypes for the PVS^A strain.     

Molecular characterization of domestic and exotic potato virus S isolates and a global analysis of genomic sequences

Five potato virus S (PVS) isolates from the USA and three isolates from Chile were characterized based on biological and molecular properties to delineate these PVS isolates into either ordinary (PVS^O) or Andean (PVS^A) strains. Five isolates – 41956, Cosimar, Galaxy, ND2492-2R, and Q1 – were considered ordinary strains, as they induced local lesions on the inoculated leaves of Chenopodium quinoa, whereas the remaining three (FL206-1D, Q3, and Q5) failed to induce symptoms. Considerable variability of symptom expression and severity was observed among these isolates when tested on additional indicator plants and potato cv. Defender. Additionally, all eight isolates were characterized by determining the nucleotide sequences of their coat protein (CP) genes. Based on their biological and genetic properties, the 41956, Cosimar, Galaxy, ND2492-2R, and Q1 isolates were identified as PVS^O. PVS-FL206-1D and the two Chilean isolates (PVS-Q3 and PVS-Q5) could not be identified based on phenotype alone; however, based on sequence comparisons, PVS-FL206-1D was identified as PVS^O, while Q3 and Q5 clustered with known PVS^A strains. C. quinoa may not be a reliable indicator for distinguishing PVS strains. Sequences of the CP gene should be used as an additional criterion for delineating PVS strains. A global genetic analysis of known PVS sequences from GenBank was carried out to investigate nucleotide substitution, population selection, and genetic recombination and to assess the genetic diversity and evolution of PVS. A higher degree of nucleotide diversity (π value) of the CP gene compared to that of the 11K gene suggested greater variation in the CP gene. When comparing PVS^A and PVS^O strains, a higher π value was found for PVS^A. Statistical tests of the neutrality hypothesis indicated a negative selection pressure on both the CP and 11K proteins of PVS^O, whereas a balancing selection pressure was found on PVS^A.     

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.     

Genetic variability in the coat protein gene of Potato virus S isolates and distinguishing its biologically distinct strains

The complete coat protein (CP) nucleotide sequences of 13 Potato virus S (PVS) isolates from Australia and three from Europe were compared to those of 37 others. On phylogenetic analysis, the Australian sequences were in PVS^O sub-clades III and IV, and the European isolates were in sub-clades I and VII. The European isolates invaded Chenopodium spp. systemically, but eight Australian isolates did not. Amino acid sequence differences at the N-terminal ends of the CPs were unrelated to the ability to invade Chenopodium spp. systemically. The acronym PVS^O–CS is suggested for isolates that invade Chenopodium spp. systemically but are not within clade PVS^A.     

A proposal to help resolve the disagreement between naming of potato virus Y strain groups defined by resistance phenotypes and those defined by sequencing

The complete coat protein (CP) nucleotide sequences of nine historical (1943-1984) potato virus Y (PVY) isolates belonging to resistance strain groupings Y^C, Y^Z or Y^O, and nine new Australian isolates from potato and tomato were compared with those of 85 others. New potato isolate BL was in resistance group Y^O. On phylogenetic analysis, the historical potato isolates fitted within phylogenetic groups C1 or C2 (Y^C), ‘Y^O’ (Y^Z, Y^O) or N-Wi (Y^Z), while the new isolates were in phylogenetic groups C1 (tomato) or ‘Y^O’ (potato, tomato). Substitution of the designation (Y^Q) for the current phylogenetic ‘Y^O’ grouping is proposed for consideration.     

Tobacco veinal necrosis determinants are unlikely to be located within the 5′ and 3′ terminal sequences of the potato virus Y genome

Summary.  Three potato virus Y isolates, representatives of distinct PVY groups, identified in potato fields in northern Poland were submitted to biological and molecular analysis. Phenotypically, two isolates, PVY^N-Ny and PVY^N-Wi, belong to the necrotic strain and the third one (PVY^O-LW) to the common strain. PVY^N-Wi, however, did not react with monoclonal antibodies directed against the necrotic strain isolates which recognise PVY^N-Ny. To characterise the isolates, coat protein genes were sequenced and compared with sequences from databases. The necrotic PVY^N-Wi isolate showed 99% amino acid homology with the common one–PVY^O-LW and significantly differed from the second necrotic isolate (PVY^N-Ny). Sequence based homology matrix and phylogenetic analysis lead to classification of PVY^N-Ny into group I, encompassing solely necrotic strain isolates, whereas PVY^N-Wi falls into a phenotypically heterogeneous group II. The sequence analysis allowed for identification of putative group I–specific epitopes. 3′NTR (non-translated region) sequences were identical for PVY^N-Wi and PVY^O-LW. The 5′NTR, P1 gene, coat protein gene and 3′NTR sequences of the common (PVY^O-LW) and the necrotic (PVY^N-Wi) isolates are 99–100% homologous. This suggests that tobacco veinal necrosis determinants are located outside the 3′ and 5′ terminal sequences of the PVY genome. Received April 11, 1996 Accepted September 17, 1996     

Genetic structure and molecular variability of potato virus M populations

To investigate the genetic diversity of potato virus M (PVM; genus Carlavirus, family Betaflexiviridae), the complete nucleotide sequence of the coat protein gene of 30 PVM isolates from a major potato-growing region in Iran were determined. Phylogenetic analysis of these Iranian PVM isolates together with those available in the GenBank database suggested two divergent evolutionary lineages that did not reflect the origin of the isolates, and these were designated as PVM-o and PVM-d. Examination of the genetic variability of the coat protein of Iranian isolates and their counterparts whose sequences are available in the Genbank database revealed 16 genotype groups in the PVM population. Analysis of the synonymous-to-nonsynonymous ratio showed strong purifying selection in the CP gene in the genotype groups of divergent clades.     

Potential gene products of bean golden mosaic virus have higher sequence homologies to those of tomato golden mosaic virus than to those of cassava latent virus

Comparison of the nucleotide sequences of the DNAs of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and cassava latent virus (CLV) revealed a fairly close relationship between BGMV DNA1, TGMV DNA1, and CLV DNA1 and a comparatively distant relationship between BGMV DNA2, TGMV DNA2, and CLV DNA2. The 200-base region common to the two DNAs of each virus had little sequence homology, except for a highly conserved 33–36 base sequence potentially capable of forming a stable hairpin structure. All the potential coding regions in the BGMV DNAs had counterparts in the TGMV and CLV DNAs suggesting an overall similarity in genome organization but two potential coding regions in the BGMV DNAs had no counterparts in the TGMV DNAs. The most highly conserved ORFs, BGMV 1R1, TGMV 1R1, and CLV 1R1, are the putative genes for the coat proteins of BGMV, TGMV, and CLV. BGMV 1R1 has 91.9% and 71.6% homology with respect to TGMV and CLV. The ORFs (BGMV 1L1; CLV 1L1; TGMV 1L1) and the two smaller overlapping ORFs (BGMV 1L2, 1L3; TGMV 1L2, 1L3; CLV 1L5, 1L3) are conserved in the three viruses. BGMV 2R1 and BGMV 2L1 have higher homology with respect to TGMV but not with respect to 2R1 and 2L1 in CLV. From this study we conclude that BGMV is more closely related to TGMV than CLV.     

Sequencing and characterization of the coat protein and 3′ non-coding region of a newsweet potato potyvirus

Summary.  A cDNA library was constructed from viral genomic RNA purified from sweet potato plants affected by “Sweet Potato Chlorotic Dwarf disease” in an attempt to clarify the etiology of this viral complex in Argentina. By sequence analysis, some of the obtained clones were found to belong to sweet potato feathery mottle potyvirus (SPFMV), to a closterovirus and to a new potyvirus. A cDNA clone of 1,103 bp representing the coat protein cistron and 3^′ non-coding region of the newly identified potyvirus was further characterized. The sequence contained an ORF of 855 nucleotides with a coding capacity of 285 amino acids, followed by a 3^′ untranslated tail of 248 nucleotides. The core and C-terminal regions have sequences well conserved among potyviruses. Furthermore, amino acid sequence comparisons of the capsid protein with those of other described potyviruses showed 63% homology with SPFMV, 68 to 70% with two different isolates of sweet potato latent potyvirus (SPLV), 57% with sweet potato G potyvirus (SPGV) and 73% with potato virus Y (PVY). These data allowed us to propose the inclusion of this virus as a new member of the family Potyviridae, genus Potyvirus with the designation sweet potato mild speckling potyvirus (SPMSV). Received January 16, 1997 Accepted March 4, 1997     

Discussion paper: The naming of Potato virus Y strains infecting potato

Summary Potato virus Y (PVY) strain groups are based on host response and resistance gene interactions. The strain groups PVY^O, PVY^C and PVY^N are well established for the isolates infecting potato in the field. A switch in the emphasis from host response to nucleotide sequence differences in the virus genomes, detection of isolates recombining sequences of different strains, and the need to recognize isolates that cause necrotic symptoms in potato tubers have led to the assignment of new acronyms, especially to isolates of the PVY^N strain group. This discussion paper proposes that any newly found isolates should be described within the context of the original strain groups based on the original methods of distinguishing strains (i.e., tobacco and potato assays involving use of ‘differential’ potato cultivars). Additionally, sequence characterization of the complete genomes of isolates is highly recommended. However, it is acceptable to amend the names of PVY isolates with additional, specific codes to show that the isolate differs at the molecular, serological or phenotypic level from the typical strains within a strain group. The new isolates should preferably not be named using geographical, cultivar, or place-association designations. Since many new variants of PVY are being discovered, any new static classification system will be meaningless for the time being. A more systematic investigation and characterization of PVY from potato at the biological and molecular levels should eventually result in a biologically meaningful genetic strain concept. Correspondence: R. P. Singh, Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, Fredericton, New Brunswick, Canada E3B 4Z7     

Evolution of North American PVYNTN Strain Tu 660 from Local PVYN by Mutation rather than Recombination

A North American (NA) isolate of tobacco veinal necrotic strain of Potato virus Y (PVY^N) (N-Jg) and a NA isolate of potato tuber necrotic strain of Potato virus Y (PVY^NTN) (Tu 660) were tested for their phenotypes by inoculation to potato plants of three potato cultivars. Upon inoculation with Tu 660, tubers of the cultivars Norchip and Ranger Russet developed potato tuber necrotic ringspot disease (PTNRD) but not the tubers of Russet Burbank. N-Jg failed to induce PTNRD in the tested cultivars. The genomic RNAs of both strains were completely sequenced and analysed. High homology (98% and 99% identity on nucleotide and polyprotein, respectively) was found between Tu 660 and N-Jg. When polyproteins were compared with other isolates, high identity was observed between Tu 660 and an European (Eu) PVY^N-605 (98%) and with an Eu-PVY^NTN-H (96%). However, when individual mature proteins were compared, much lower identities (86.5–94%) were found between Tu 660 and PVY^NTN-H compared to 98–99.5% between Tu 660 and PVY^N-605 in the P3, 6K1 and CI regions. Further sequence analysis indicated that the PVY^NTN-H is a hybrid molecule of the genomic RNA recombination of PVY^O and Eu-PVY^N as shown by Glais et al. (Arch Virol 147, 363–378), whereas NA-PVY^NTN Tu 660 is free of recombination points. Phylogenetic analysis confirmed this observation, and suggested that, in light of high homology, the Tu 660 might have evolved from NA-PVY^N by mutations rather than the genome recombinations. The non-recombinant nature of NA-PVY^NTN Tu 660 strongly suggests that the recombinant structure of genome is not a necessary prerequisite for the PTNRD phenotype.     

Simultaneous detection and identification of four viruses infecting pepino by multiplex RT-PCR

Potato virus M (PVM), pepino mosaic virus (PepMV), tomato mosaic virus (ToMV), and potato virus S (PVS) infect pepino and cause serious crop losses. In this study, a multiplex RT-PCR method was developed for simultaneous detection and differentiation of PVS, ToMV, PepMV and PVM. The method was highly reliable and sensitive; validation was accomplished by testing pepino samples collected from different regions of China. In this survey, PVM, ToMV and PVS were detected in 37.0 %, 31.0 % and 5.5 % of samples tested, respectively, confirming the widespread occurrence of these three viruses in China. PepMV was not detected in any of the samples, which indicated that this virus may not be prevalent in China. The results suggest that the new multiplex RT-PCR method has potential to be used routinely for surveys of pepino for virus infection.     

A novel monopartite begomovirus infecting sweet potato in Brazil

The complete genome sequences of two monopartite begomovirus isolates (genus Begomovirus, family Geminiviridae) present in a single sweet potato (Ipomoea batatas) plant collected in S?o Paulo, Brazil, are presented. Based on the current taxonomic criteria for the genus Begomovirus, one of the isolates was shown to represent a novel species, tentatively named Sweet potato leaf curl Sao Paulo virus (SPLCSPV). The other isolate represented a new strain of sweet potato leaf curl virus, named sweet potato leaf curl virus-Sao Paulo (SPLCV-SP). The full genome sequence of the SPLCSPV isolate shared the highest nucleotide identity (87.6%) with isolates of sweet potato leaf curl Spain virus (SPLCESV). Phylogenetic and recombination analyses were used to investigate the relationships of these isolates to other monopartite Ipomoea-infecting begomoviruses.     

Phylogenetic and recombination analysis of the homing protein domain of grapevine fanleaf virus (GFLV) isolates associated with ‘yellow mosaic’ and ‘infectious malformation’ syndromes in grapevine

The RNA2 of seven grapevine fanleaf virus (GFLV) isolates from vines with yellow mosaic (YM) symptoms from different origin were sequenced. These sequences showed a high variability in the homing protein (2A^HP) and, in five of them, a putative recombination with arabis mosaic virus (ArMV) was detected. To investigate recombination frequency, the partial sequences of the 2A^HP of 28 additional GFLV isolates from nine different countries, showing either YM or infectious malformations (MF) symptoms, were obtained and compared with those of GFLV isolates from GenBank. The analysis confirmed the high level of sequence variability (up to 41 % at the nucleotide level) among isolates. In phylogenetic trees constructed using different approaches, the sequenced isolates always clustered in four conserved groups, three of which comprised YM strains (groups 1, 2 and 3), and one (group 4) the MF strains. Potential interspecific recombination sites between GFLV and ArMV were predicted in the 2A^HP gene of several isolates, all of which were associated with YM symptoms.     

On the origin of the human influenza virus subtypes H2N2 and H3N2

The RNA of the human influenza virus Singapore (H2N2) strain has been labeled in vivo by phosphorus-32 and separated by polyacrylamide gel electrophoresis into eight segments, which were correlated to the corresponding gene functions and/or proteins. The base sequence homology between the individual genes (segments) of the H2N2 virus and those of different influenza A strains has been determined by molecular hybridization. Segments 1, 5, 7, and 8 of the Singapore strain exhibit a base sequence homology of almost 100% as compared to the FM1 strain (HlNl), while the homology between the other segments is significantly lower (24–76%). For the Singapore and Hong Kong (H3N2) strains all segments except that coding for the hemagglutinin (HA, 24%) exhibit a homology close to 100%. The ³²P-labeled segment 4 (HA-gene) of the avian influenza A strain duck Ukraine (Hav7Neg2) shows a homology of 92% to Hong Kong, while the homology of at least two other segments is significantly lower. These results are taken as an indication that the H2N2 subtype is derived from the HlN1 subtype by a recombination event retaining four H1N1 segments, while the other four segments are gained from another yet unknown strain. The H3N2 subtype is presumably derived from a H2N2 subtype, retaining seven segments of the H2N2 subtype, while the gene coding for the HA is obtained from the duck Ukraine or another highly related strain.     

Complete genome sequence of the first Andean strain of potato virus S from Brazil and evidence of recombination between PVS strains

An isolate of the Andean strain of potato virus S (PVS), named BB-AND, was detected for the first time in a Brazilian potato crop, fully sequenced and analyzed. A comparison of BB-AND with other PVS isolates (Andean and Ordinary) showed that BB-AND is quite distinct. The lowest amino acid sequence identity to the only other fully sequenced Andean isolate was found in ORF 1 (82%) and ORF 6 (87%). Recombination analysis showed that the isolate Vltava (AJ863510), from Germany, is a recombinant between PVS(O) and PVS(A) isolates, with the recombination event located between nucleotides 6125 and 8324.     

Characterisation of<Emphasis Type="Italic"> potato virus Y</Emphasis> isolates from Iran

A survey of Potato virus Y (PVY) was conducted in cultivated fields in six Iranian provinces between January 2005 to July 2007. Two hundred samples from potato and tomato were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) for potyviruses. Almost one fourth of the samples were found to be infected by PVY. Analysis of these PVY-positive samples using three monoclonal antibodies (MAbs) facilitating the simultaneous detection of three main strains namely the ordinary (PVY^O), strain (PVY^N) and C (PVY^C) strains. However, the fourth strain (PVY^NTN) and some others recombinant isolates were also identified by molecular methods. Host range and symptoms analysis using sap inoculation of four different strains of PVY onto a range of plants revealed that the four strains showed biological properties that seemed to be consistent with their molecular grouping. Fourteen isolates of PVY were chosen based on the host and geographical location, primer specificity and serology for further biological and molecular characterisation. The coat protein (CP) and P1 genes and 3′-non-translated region (3′NTR) from 14 representative isolates were sequenced and analysed with the sequences available in GenBank. Composite analysis of the P1, CP and 3′-UTR sequences with all full genome sequences of PVY revealed that there are three potential strains of PVY in Iran, PVY^O, PVY^N-W and PVY^NTN. Isolate KER.SA_N was the most divergent of all the 14 isolates reacted with PVY^N specific MAbs but grouped with PVY^O strains in maximum likelihood phylogentic analysis. The PVY^NTN isolates from Iran more closely related to the European than North American PVY^NTN isolates.     

Whole genome characterization of Potato virus Y isolates collected in the western USA and their comparison to isolates from Europe and Canada

Summary. Potato virus Y (PVY) is a serious potato pathogen that affects potato seed and commercial production crops. In recent decades, novel PVY strains have been described that cause necrotic symptoms on tobacco foliage and/or potato tubers. The major PVY strains that affect potato include PVY^O and PVY^N, which have distinct serotypes that can be differentiated by immunoassay. Other economically important strain variants are derived from recombination events, including variants that cause tuber necrotic symptoms (PVY^NTN) and PVY^O serotypes that cause tobacco veinal necrosis (PVY^N-W, PVY^N:O). Although the PVY^NTN and PVY^N-W variants were first reported in Europe, apparently similar strains have been appearing in North America. Confirmation of the existence of these recombinant strains in North America is important, as is whether they spread from a common source or were derived by independent recombination. Whole genome sequencing can be used to positively identify strain variants and begin to address the issue of origins. Symptomology, serology, RT-PCR, and partial sequencing of the coat protein region were used to identify isolates of the PVY^NTN, PVY^N, PVY^NA-N, and PVY^N:O for whole-genome sequencing. Sequencing confirmed the presence of PVY^NTN and PVY^N isolates that were >99% identical to European sequences deposited in GenBank in the 1990’s. Sequences of the PVY^NA-N and PVY^N:O types were 99.0% and 99.5% identical to known sequences, respectively. There was no indication that recombinant strains PVY^NTN or PVY^N:O had different parental origins than recombinant strains previously sequenced. This is the first confirmation by whole-genome sequencing that “European”-type strain variants of PVY^N and PVY^NTN are present in North America, and the first reported full-length sequence of a tuber necrotic isolate of PVY^N:O.     

Genetic analysis of the products of a cross involving a suppressive ‘petite’ mutant of S. cerevisiae

Summary A genetically defined highly suppressive petite yeast strain ( ^–cob⁺A^sE^oC^oO^oP^o) was crossed with a grande strain carrying a multiply marked mitochondrial genome ( ⁺A^rE^rC^rO rp^r). Petite diploid progeny, isolated from individual zygotic clones consisting either of wholly petite or mixtures of grande and petite cells, were characterised genetically by crossing to grande haploids. The diploid petites were found to closely resemble the petite parent and in general not to carry mitochondrial markers from the grande parent. In the petites from the mixed clones recombination was detected, but only within the region of homology between the genomes. These observations are inconsistent with models of suppressiveness based on destructive recombination and suggest that the petite genome eliminates the grande genome from zygotic progeny through being preferentially replicated. The most plausible model to explain the observed pattern of zygotic clones postulates a limited number of mDNA replication sites in zygotes, competition for sites between input mDNA molecules and an advantage in this competition for suppressive ^– mDNA.