Potato Virus S Isolates Research Articles

GENOME SEQUENCE ANALYSIS, RT-PCR DIAGNOSTICS AND CONSTRUCTION OF VIRAL PROTEIN-EXPRESSION CASSETES FOR KAZAKH ISOLATE OF ORDINARY AND ANDEAN STRAINS OF POTATO VIRUS S

Potato virus S (PVS) belongs to the genus Carlavirus of family Betaflexiviridae with a positive-sense single-stranded RNA genome of 8.5 Kb. PVS is one of the most prevalent viruses of cultivated potatoes in Kazakhstan. Here we report phylogenetic analysis of complete genome sequences of Kazakh isolates of PVS from potato cultivars Fortuna and Ushkonyr, design of PVS strain-specific diagnostic PCR and construction of expression cassettes for conserved viral proteins from both isolates. The Fortuna and Ushkonyr isolates were found to share 79.9% nucleotide identity with each other and belong respectively to previously-defined ordinary and Andean strains of PVS. Based on analysis of conserved and variable regions of available PVS isolates, we designed primers for reverse transcription (RT)-duplex PCR to detect both strains in single and mixed infections and established their prevalence in Almaty region of Kazakhstan. Coding sequences of triple gene block proteins (25K, 12K, 7K), coat protein (34K) and cysteine-rich protein (11K) as well as methyltransferase, peptidase, helicase and RNA-dependent RNA polymerase domains of viral replicase were subcloned from both Fortuna and Ushkonyr genomic RNAs into the binary vector pBIN19 under the control of CaMV 35S promoter and nopaline synthetase terminator. These expression cassettes will be used to further investigate the biological properties and strain characteristics of viral proteins by their transient expression in plant cells and tissues or their stable expression in transgenic plants.

Pest categorisation of potato virus S (non-EU isolates).

Following a request from the EU Commission, the Panel on Plant Health has addressed the pest categorisation of non‐EU isolates of potato virus S (PVS). The information currently available on geographical distribution, biology, epidemiology, potential entry pathways, potential additional impact compared to the current situation in the EU, and availability of control measures of non‐EU isolates of PVS has been evaluated with regard to the criteria to qualify as potential Union quarantine pest. Because non‐EU isolates of PVS are absent from the EU, they do not meet one of the requirements to be regulated as an RNQP (presence in the EU); as a consequence, the Panel decided not to evaluate the other RNQP criteria for these isolates. Populations of PVS can be subdivided into two strains: the ordinary strain (PVS‐O) with a worldwide distribution (including the EU), and the Andean strain (PVS‐A) which is absent from the EU or considered to have at most a limited distribution in the EU. Two additional divergent isolates (PVS‐A/PVS‐O recombinants and PVS‐arracacha) have also been categorised. Non‐EU isolates of PVS‐A are expected to have an additional impact as compared to the PVS isolates currently present in the EU, and therefore meet all the criteria to qualify as potential Union quarantine pests; the magnitude of the additional impact is, however, unknown. Non‐EU isolates of PVS‐A/PVS‐O recombinants and of PVS‐arracacha also meet these criteria, with the exception of the criterion regarding the potential additional consequences in the EU territory for which the Panel was unable to conclude. Non‐EU PVS‐O isolates are not expected to have an additional impact in the EU as compared to EU isolates and therefore do not meet the corresponding criterion.

Andean and Ordinary Strains of Potato Virus S Infecting Potatoes in Southern Kazakhstan

Southern Kazakhstan comprises Turkistan, Zhambyl, and Almaty administrative regions; it is the most densely populated territory of the country and one of the biggest potato (Solanum tuberosum L.) producing areas. Around 1.17 million metric tons, 33% of the whole country’s production, are produced here annually. Potato virus S belongs to the genus Carlavirus (Betaflexiviridae); it has a single-stranded positive-sense RNA genome (Mackenzie et al. 1989). Based on biological and molecular properties, two strains of potato virus S (PVS) can be differentiated, ordinary (PVSᴼ) and Andean (PVSᴬ) (Dolby and Jones 1987). PVS was reported in the Eastern region (Khassanov and Vologin 2018); however, there is still no information about strain composition for PVS in southern Kazakhstan. One hundred eighty potato tubers of 47 cultivars grown in the commercial fields of Almaty region were collected randomly in September 2015 and 2016. The tubers were kept in the dark until sprouting and then planted in pots in the greenhouse. Four to 6 weeks after emergence, leaves of the grown-out plants were analyzed by double-antibody sandwich ELISA using commercially available polyclonal antibody (BIOREBA AG, Switzerland). Total RNA was extracted from leaves of all 67 PVS-ELISA positive plants using TRIzol (Sigma-Aldrich, U.S.A.), and cDNA was prepared by reverse transcription (RT) using oligo(dT)₁₈. For PCR amplifications, two pairs of CP-specific primers were used: (i) PVS-S, 5′-TGGCGAACACCGAGCAAATG-3′, and PVS-AS, 5′-ACTGCGCCTGTTGGGAACTCCACAG-3′; and (ii) CP634Fw, 5′-TTTCAATGGAATGCACGCTT-3′, and CP-Rv, 5′-TCATTGGTTTGCTGCATTCC-3′. These primers were designed according to the available genome sequences of isolates Yunnan NN (GenBank accession no. KC430335) and RL5 (GenBank accession no. JX683388) representing, respectively, the strains PVSᴼ and PVSᴬ (Vallejo et al. 2016). The resulting PCR products of the expected sizes, specific to PVSᴬ (252 base pair, bp) and PVSᴼ (169 bp), were detected in 20 and 44 PVS-ELISA-positive samples, respectively. In three samples, both strains were detected. Then the full-length CP gene sequences were amplified from 12 PVSᴼ- and four PVSᴬ-positive samples selected randomly. The resulting PCR products of the expected sizes (890 bp) were sequenced directly using conventional Sanger methodology. BLASTn analysis for all samples revealed 96 to 100% nucleotide identity with CP sequences of several known PVS isolates. Pairwise identity and phylogenetic analyses using MegaX confirmed that the 12 isolates belonged to the PVSᴼ and the other four isolates to the PVSᴬ lineage. The full genomes of two isolates from the potato cultivars Fortune (PVSᴼ) and Ushkonyr (PVSᴬ) were then sequenced using the approach described above with overlapping RT-PCR amplicons obtained with specific primers, and the complete nucleotide sequences were deposited in NCBI GenBank database under accession numbers MK442089 and MN095414, respectively. The nucleotide sequence for the Fortune isolate was found to be 96% identical to Qld1 isolate (MF375506, Australia), and the Ushkonyr was found to be 99% identical to BY isolate (MF033144, China). Nucleotide identity between the sequences of the Fortune and Ushkonyr isolates was only 79%. The data indicate that both PVSᴼ and PVSᴬ strains occur in southern Kazakhstan, and regulatory agencies need to monitor the movement and prevalence of this virus to minimize disease losses. In the Eastern region, PVSᴬ seems to dominate seed potato fields (Khassanov and Vologin 2018), and data generated in this work suggest that PVSᴼ is more prevalent in southern Kazakhstan.

Construction and characterization of an infectious cDNA clone of potato virus S developed from selected populations that survived genetic bottlenecks

BackgroundInfectious cDNA clones are a powerful tool for studies on RNA viruses using reverse genetics. Potato virus S (PVS) is a carlavirus with a worldwide distribution. Although the complete genome sequences of many PVS isolates have been reported, the construction of an infectious cDNA clone of PVS is yet to be reported. The aim of this study is the development and molecular characterization of an infectious cDNA clone of PVS.MethodsA full-length cDNA clone pPVS-H-FL-AB was constructed by connecting eight cDNA clones of PVS isolate H95. Capped RNA transcripts from pPVS-H-FL-AB and a modified clone pPVS-H-FL-H, containing the consensus genome sequence of PVS-H95, proved to be non-infectious. Therefore, a full-length cDNA clone pPVS-H-FL-β was reconstructed from PVS-H00, isolated from PVS-H95 populations by repeating a single local lesion isolation in Chenopodium quinoa three times; PVS-H00 appeared to be a selected variant that survived genetic bottlenecks. The sequence of cDNA clone pPVS-H-FL-β was determined as the genome sequence of PVS-H00 and compared with the consensus sequence of PVS-H95 genome.ResultsAll Nicotiana occidentalis plants inoculated with ≥0.2 μg capped RNA transcripts from pPVS-H-FL-β developed symptoms on upper leaves, as observed with PVS-H00 inoculation. Similar levels of viral genomic and subgenomic RNAs and coat protein were detected in systemically infected leaves. Sequence comparison of PVS-H95 and PVS-H00 revealed 370 nucleotide polymorphisms (4.4% of the entire genome sequence), causing 91 amino acid substitutions in six open reading frames (ORFs). The infectivity of chimeric RNAs derived from recombinants between the two cDNA clones revealed that the lack of infectivity of pPVS-H-FL-H transcripts was due to ORF1, which encodes replicase and harbors 80 amino acid substitutions compared with pPVS-H-FL-β. Approximately 71.3% amino acid substitutions in replicase were located within the variable region of unknown function between the putative methyltransferase and ovarian tumor-like protease domains.ConclusionsThis is the first report of the development of an infectious cDNA clone of PVS. Our analyses suggest that PVS population within a plant exists as quasispecies and the replicase sequence diversity of PVS obstruct the construction of a full-length infectious cDNA clone.

The Incidence and Genetic Diversity of Potato virus S in Serbian Seed Potato Crops

It is essential that certified potatoes are free from known viruses which can negatively affect quality and yield. However, very little is known about the distribution and frequency of Potato virus S (PVS) in Serbia. Until 2014, PVS was present sporadically in the domestic seed potato production. The incidence of PVS was studied by a molecular method over 3 years (2014–2016) in four important potato-growing regions (Moravicki, Zlatiborski, Raski and Macvanski) and in different cultivars. The results showed that the incidence of PVS increased steadily over 3 years from 1.52 to 8.84%. The Moravicki region had the highest incidence (13.06%) and Desiree was the most susceptible cultivar with a mean PVS incidence of 8.2% followed by Marabel and Riviera. The highest significant statistical difference was between the cultivars and in the interaction between cultivars and regions. A detailed phylogenic analysis of the tested isolates confirmed that Serbian PVS belongs to PVSO. Of the 18 Serbian PVS isolates included in this study, eight were grouped into the PVSO cluster and formed a subgroup (O-I) with isolates from the USA, Syria, Korea and Chile. Ten Serbian isolates of PVS together with the isolates from Iran were clustered in a branch of subgroup O-VII. This study constitutes the first report of PVS isolates in Serbia which are capable of infecting Chenopodium quinoa and inducing the symptoms of local chlorotic lesions.

The Biology and Phylogenetics of Potato virus S Isolates from the Andean Region of South America.

Biological characteristics of 11 Potato virus S (PVS) isolates from three cultivated potato species (Solanum spp.) growing in five Andean countries and 1 from Scotland differed in virulence depending on isolate and host species. Nine isolates infected Chenopodium quinoa systemically but two others and the Scottish isolate remained restricted to inoculated leaves; therefore, they belonged to biologically defined strains PVSA and PVSO, respectively. When nine wild potato species were inoculated, most developed symptomless systemic infection but Solanum megistacrolobum developed systemic hypersensitive resistance (SHR) with one PVSO and two PVSA isolates. Andean potato cultivars developed mostly asymptomatic primary infection but predominantly symptomatic secondary infection. In both wild and cultivated potato plants, PVSA and PVSO elicited similar foliage symptoms. Following graft inoculation, all except two PVSO isolates were detected in partially PVS-resistant cultivar Saco, while clone Snec 66/139-19 developed SHR with two isolates each of PVSA and PVSO. Myzus persicae transmitted all nine PVSA isolates but none of the three PVSO isolates. All 12 isolates were transmitted by plant-to-plant contact. In infective sap, all isolates had thermal inactivation points of 55 to 60°C. Longevities in vitro were 25 to 40 days with six PVSA isolates but less than 21 days for the three PVSO isolates. Dilution end points were 10-3 for two PVSO isolates but 10-4 to 10-6 with the other isolates. Complete new genome sequences were obtained from seven Andean PVS isolates; seven isolates from Africa, Australia, or Europe; and single isolates from S. muricatum and Arracacia xanthorhiza. These 17 new genomes and 23 from GenBank provided 40 unique sequences; however, 5 from Eurasia were recombinants. Phylogenetic analysis of the 35 nonrecombinants revealed three major lineages, two predominantly South American (SA) and evenly branched and one non-SA with a single long basal branch and many distal subdivisions. Using least squares dating and nucleotide sequences, the two nodes of the basal PVS trifurcation were dated at 1079 and 1055 Common Era (CE), the three midphylogeny nodes of the SA lineages at 1352, 1487, and 1537 CE, and the basal node to the non-SA lineage at 1837 CE. The Potato rough dwarf virus/Potato virus P (PVS/PRDV/PVP) cluster was sister to PVS and diverged 5,000 to 7,000 years ago. The non-SA PVS lineage contained 18 of 19 isolates from S. tuberosum subsp. tuberosum but the two SA lineages contained 6 from S. tuberosum subsp. andigena, 4 from S. phureja, 3 from S. tuberosum subsp. tuberosum, and 1 each from S. muricatum, S. curtilobum, and A. xanthorrhiza. This suggests that a potato-infecting proto-PVS/PRDV/PVP emerged in South America at least 5,000 years ago, became endemic, and diverged into a range of local Solanum spp. and other species, and one early lineage spread worldwide in potato. Preventing establishment of the SA lineages is advised for all countries still without them.

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 610-710nm 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.

Strain Characterization of Potato virus S Isolates from Tasmania, Australia.

Potato virus S (PVS) is prevalent within potato (Solanum tuberosum) production worldwide. Traditionally, PVS has been split into two strains, Ordinary (PVSO) and Andean (PVSA), based on reaction in herbaceous indicator species such as Chenopodium quinoa. However, recent research has identified further strain designations, such as PVSO-CS (Ordinary and Chenopodium systemic). Forty-four isolates of PVS were collected from potato seed lines in different geographical regions within Tasmania, Australia. Isolates were initially characterized by reactions in C. quinoa. Nineteen isolates were characterized as PVSO, based on the development of local lesions and serological detection in inoculated leaves only. Three isolates were identified as PVSA-like, based on local lesion development in inoculated leaves, mild mottling or chlorotic spots on noninoculated leaves, and serological detection in both inoculated and noninoculated leaves. Thirteen isolates produced no symptoms, and were detected serologically in inoculated leaves only (PVSO-like). Four isolates produced no symptoms but were detected serologically in both inoculated and noninoculated leaves (PVSA-like). Five isolates produced symptoms in inoculated leaves only but were detected serologically in both inoculated and noninoculated leaves (also PVSA-like). The ability of isolates to infect tomato has also been used as a criterion to assist in PVS strain differentiation. A subsample of isolates (n = 16) was unable to infect tomato 'Grosse Lisse'. Seventeen isolates representative of these groupings based on reactions in C. quinoa were also characterized by coat-protein sequencing. Phylogenetic comparisons suggested that all isolates were PVSO rather than PVSA. Therefore, whereas some of these PVS isolates were systemic in C. quinoa, findings from this study suggest that they were not PVSA, and that only PVSO and PVSO-CS isolates are present in Tasmania. The implications of this finding for disease management are discussed.

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.

Analysis of Iranian Potato virus S 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.

The Occurrence of Potato Viruses in Syria and the Molecular Detection and Characterization of Syrian Potato virus S Isolates

Potato virus Y (PVY) is the main potato virus in Syria but no information is available on the prevalence of other potato viruses. A survey was conducted by ELISA using specific antibodies to nine potato viruses. PVY, Potato virus S (PVS), Cucumber mosaic virus and Potato leafroll virus were detected with infection rates of 54.2, 8.4, 3.7 and 0.9%, respectively. The overall virus infection rate was as high as 72.9% in ware potato fields. A preliminary characterization was carried out on PVS isolates. Syrian PVS isolates infected Chenopodium amaranticolor only locally and therefore were classified as PVSO. Phylogenetic analysis of the coat protein gene showed that PVS comprised two main clusters, cluster-O and cluster-A, which contained the ordinary and Andean strains respectively. Cluster-O was separated into two subclusters, O1 and O2. Two Syrian PVS isolates, PVS3-5 and PVS6-2, fell into the O1 subcluster. PVS3-5 coat protein, however, shared the highest nucleotide identity with European isolates of the O1 subcluster, whereas PVS6-2 was closely related to Asian isolates of the same subcluster. Owing to the high incidence of PVY and PVS in Syria, a duplex reverse-transcription PCR was developed to detect these two viruses in a single PCR.

Detection of strains of potato virus S by nucleic acid spot hybridization (NASH)

Two complementary DNA (cDNA) clones (0.9 kb and 1.6 kb) reacting to the ordinary strain of potato virus S (PVS) were compared with single-stranded randomly primed cDNA (prepared to total genomic RNA) as probes for various strains of PVS, using the technique of nucleic acid spot hybridization (NASH). The cDNA clones detected 11 PVS isolates well, including both Andean and ordinary strains, while one of each strain was detected weakly. As little as 5 femtograms of PVS RNA could be detected. A low cross hybridization was observed with cDNA probes to PVS with potato virus M and carnation latent virus both members of the carlavirus group. No cross homology was detected with seven other potato viruses.