Phylogeography and molecular evolution of potato virus Y

Potato virus Y (PVY) is an important plant pathogen, whose host range includes economically important crops such as potato, tobacco, tomato, and pepper. PVY presents three main strains (PVY(O), PVY(N) and PVY(C)) and several recombinant forms. PVY has a worldwide distribution, yet the mechanisms that promote and maintain its population structure and genetic diversity are still unclear. In this study, we used a pool of 77 complete PVY genomes from isolates collected worldwide. After removing the effect of recombination in our data set, we used bayesian techniques to study the influence of geography and host species in both PVY population structure and dynamics. We have also performed selection and covariation analyses to identify evolutionarily relevant amino acid residues. Our results show that both geographic and host-driven adaptations explain PVY diversification. Furthermore, purifying selection is the main force driving PVY evolution, although some indications of positive selection accounted for the diversification of the different strains. Interestingly, the analysis of P3N-PIPO, a recently described gene in potyviruses, seems to show a variable length among the isolates analyzed, and this variability is explained, in part, by host-driven adaptation.     

Occurrence and distribution of viruses infecting potato in Russia

Potato viral disease has been a major problem in potato production worldwide including Russia. Here, we detected Potato Virus M (PVM), P (PVP), S (PVS), Y (PVY), and X (PVX) and Potato Leaf Roll Virus (PLRV) by RT-PCR on potato leaves and tubers from the Northwestern (NW), Volga (VF), and Far Eastern (FE) federal districts of Russia. Each sample was co-infected with up to five viruses. RT-PCR disclosed all six viruses in NW, three in VF, and five in FE. Phylogenetic analyses of PVM and PVS strains resolved all PVM isolates in Group O (ordinary) and all PVS isolates in Group O. Seven PVY strains were detected, and they included only recombinants. PVY recombinants were thus the dominant potato virus strains in Russia, although they widely varied among the regions. Our research provides insights into the geographical distribution and genetic variability of potato viruses in Russia.     

Molecular characterisation and differential diagnosis of a necrotic PVY isolate in tomato

A PVY isolate which causes strong necrotic lesions in tomato was isolated and characterised. It did not infect potato and did not react with antiserum specific to the N-strain of PVY. Restriction endonuclease cleavage patterns of PCR products derived from the 5′-end of the virus genome (Hinc II and Rsa I) clearly distinguished the tomato, pepper and potato strains. Additional sequence analysis indicated that the tomato and pepper isolates were closely related, while both markedly differed from the necrotic strains of potato. Hence, it was concluded that the necrotic tomato isolate is uniquely specific for tomato.     

Potato virus Y Strain Spectrum in New Zealand – Absence of Recombinant N:O Strains

Potato virus Y (PVY) is a serious plant pathogen, causing severe yield losses worldwide on members of the Solanaceae, including potato, pepper, tomato and tobacco. During the last two decades new virus strains have been detected, including those representing recombinants between N- and O-strains, now designated PVY^NTN and PVY^N-Wilga, respectively. The question of whether recombination is easily induced in nature by mixed infections of potato might be answered by an investigation of strains appearing under isolated conditions such as those in New Zealand. More than 30 PVY isolates collected during the last 20 years were characterized biologically, serologically and using molecular biological approaches. The New Zealand population of PVY isolates was mainly composed of PVY^N and PVY^O. To date no recombinant strains have been found among the isolates tested. Similarly, experiments performed with these isolates on potatoes under greenhouse conditions with mixed infection PVY^N/PVY^O did not result in signs of recombination. This raises the question as to the driving force for the appearance of recombinant strains. It also demonstrates the efficacy of plant quarantine measures in New Zealand over the past 20 years.     

Comparison of resistance to potyviruses within Solanaceae: infection of potatoes with tobacco etch potyvirus and peppers with potato A and Y potyviruses

The reaction of several cultivated potato varieties (Solarium tuberosum L.) to three strains of tobacco etch potyvirus (TEV-F, TEV-Mex21 and TEV-ATCC) and the reaction of several pepper lines (Capsicum annuum L. and C. chinense L.) to two strains of potato Y potyvirus (PVY^O and PVY^N) and one strain of potato A potyvirus (PVA-M) was tested. The potato varieties included in this study carried resistance genes against PVY, PVA and potato V potyvirus, but all were susceptible to TEV and developed mottle and mosaic symptoms. TEV was readily transmitted by mechanical inoculation from tobacco and potato to potato, whereas transmission from pepper to potato occurred infrequently. TEV was transmitted through potato tubers, and from pepper to potato plants by aphids. Lack of detectable systemic infection following graft-inoculation indicated extreme resistance to PVY^O and PVA in several pepper lines. No pepper line was systemically infected with PVY^N following mechanical inoculation (graft-inoculation was not carried out with PVY^N). The development of necrotic lesions following mechanical and graft-inoculation indicated hypersensitive response to PVY^O in several pepper lines which resembled the resistance responses to these potyvirus strains in potato. Results of this study together with previous work indicate that C. annuum cv. Avelar is resistant to four potyviruses [PVY, PVA, pepper mottle potyvirus (PepMoV) and some isolates of TEV]; C. annuum cv. Criollo de Morelos and C. chinense PI 152225 and PI 159236 are resistant to three potyviruses (PVY, PepMoV and PVA; and PVY, PepMoV and TEV, respectively); C. annuum 9093–1 and 92016–1 are resistant to PVY and PepMoV; and C. annuum cv. Jupiter and C. annuum cv. RNaky are resistant to PVY^N and PVA.     

Detection of BMYV and BWYV isolates using monoclonal antibodies and radioactive RNA probes, and relationships among luteoviruses

In order to discriminate between sugar beet infecting beet mild yellowing virus (BMYV) and other isolates of beet western yellows virus (BWYV), monoclonal antibodies (MAbs) and radioactive riboprobes were used. With MAbs prepared against BMYV or potato leafroll virus (PLRV) no distinction could be established between BMYV and BWYV. Seven probes were synthesised from a lettuce infecting BWYV isolate; their localisation in the genome is known and they cover almost its entire length. Probes from the '3 part of the genome hybridised with all BMYV and BWYV isolates whereas those from the '5 part did not recognise BMYV isolates, showing that a divergent '5 region exists in the genomes of BMYV and BWYV. Probes also readily detected the virus in single aphids. The relevance of this finding for epidemiological studies is discussed.
MAbs and riboprobes were also tested against other luteoviruses (PLRV; barley yellow dwarf virus (BYDV) MAV, PAV and RPV strains). The serological relationship between BMYV and PLRV was confirmed and an epitope common to PLRV and BYDV-RPV was found. Using probes, PLRV and BYDV-RPV were found to share domains of homology with BWYV. BYDV-PAV showed weak homology with BWYV, while BYDV-MAV showed none.     

Monoclonal antibodies for the detection and differentiation of faba bean necrotic yellows virus isolates

Murine monoclonal antibodies (MAbs) were produced for the detection of faba bean necrotic yellows virus (FBNYV), an isometric ssDNA virus belonging to a new, yet unnamed genus of plant viruses. A total of 19 FBNYV-specific MAbs were obtained from three fusion experiments and characterised by determining their immunoglobulin types and titres as well as their corresponding epitopes. At least six distinct epitopes were revealed on FBNYV particles of different virus isolates. Only two MAbs reacted with SDS-dissociated FBNYV virions in triple antibody sandwich (TAS)-ELISA and with viral capsid protein in Western blots. Almost all MAbs were more sensitive in detecting FBNYV in viruliferous aphids by TAS-ELISA than polyclonal anti-FBNYV IgG by double antibody sandwich ELISA and permitted virus detection in individual aphids even following short acquisition access feeding periods. Coat protein variation among FBNYV isolates and serological relatedness to taxonomically similar viruses was studied by determining the cross reactivity of these MAbs with several field isolates of FBNYV as well as with milk vetch dwarf (MDV), banana bunchy top (BBTV), and subterranean clover stunt (SCSV) viruses. Whereas none of the MAbs reacted with BBTV, only one reacted with SCSV, indicating that FBNYV and SCSV share a common epitope. By contrast, 16 of the 19 MAbs reacted with MDV, suggesting that FBNYV and MDV are serologically closely related and strains of the same virus. When all 19 MAbs produced were tested against a total of 107 samples of FBNYV collected during virus surveys in Egypt, Ethiopia, Jordan, Morocco and Syria, five MAbs showed differential reactions. While the majority of the samples reacted with all 19 MAbs, about 20% of the 107 FBNYV samples did not react with one and/or other of these five MAbs, permitting the differentiation of seven serotypes of FBNYV and suggesting a considerable coat protein variation in FBNYV isolates from the countries surveyed. The MDV isolate from Japan and five FBNYV samples from Ethiopia appeared to be the least closely related to typical FBNYV isolates by not reacting with three and four, respectively, of the five differentiating Mabs.     

Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies

Surface plasmon resonance (SPR)-based biosensors have been widely utilized for measuring interactions of a variety of molecules. Fewer examples include higher biological entities such as bacteria and viruses, and even fewer deal with plant viruses. Here, we describe the optimization of an SPR sensor chip for evaluation of the interaction of the economically relevant filamentous Potato virus Y (PVY) with monoclonal antibodies. Different virus isolates were efficiently and stably bound to a previously immobilized polyclonal antibody surface, which remained stable over subsequent injection regeneration steps. The ability of the biosensor to detect and quantify PVY particles was compared with ELISA and RT-qPCR. Stably captured virus surfaces were successfully used to explore kinetic parameters of the interaction of a panel of monoclonal antibodies with two PVY isolates representing the main viral serotypes N and O. In addition, the optimized biosensor proved to be suitable for evaluating whether two given monoclonal antibodies compete for the same epitope within the viral particle surface. The strategy proposed in this work can help to improve existing serologic diagnostic tools that target PVY and will allow investigation of the inherent serological variability of the virus and exploration for new interactions of PVY particles with other proteins.     

Concurrent evolution of resistance and tolerance to potato virus Y in Capsicum annuum revealed by genome-wide association

We performed a genome-wide association study of pepper (Capsicum annuum) tolerance to potato virus Y (PVY). For 254 pepper accessions, we estimated the tolerance to PVY as the coefficient of regression of the fresh weight (or height) of PVY-infected and mock-inoculated plants against within-plant virus load. Small (strongly negative) coefficients of regression indicate low tolerance because plant biomass or growth decreases sharply as virus load increases. The tolerance level varied largely, with some pepper accessions showing no symptoms or fairly mild mosaics, whereas about half (48%) of the accessions showed necrotic symptoms. We found two adjacent single-nucleotide polymorphisms (SNPs) at one extremity of chromosome 9 that were significantly associated with tolerance to PVY. Similarly, in three biparental pepper progenies, we showed that the induction of necrosis on PVY systemic infection segregated as a monogenic trait determined by a locus on chromosome 9. Our results also demonstrate the existence of a negative correlation between resistance and tolerance among the cultivated pepper accessions at both the phenotypic and genetic levels. By comparing the distributions of the tolerance-associated SNP alleles and previously identified PVY resistance-associated SNP alleles, we showed that cultivated pepper accessions possess favourable alleles for both resistance and tolerance less frequently than expected under random associations, while the minority of wild pepper accessions frequently combined resistance and tolerance alleles. This divergent evolution of PVY resistance and tolerance could be related to pepper domestication or farmer's selection.     

Reactivity of Two Monoclonal Antibodies to the Cylindrical Inclusion Protein of Potato Virus Y

Monoclonal antibodies (mAbs) were produced to the cylindrical inclusion (CI) protein of a necrotic strain of potato virus Y (PVY-N) and their reactivity tested with nine PVY isolates and 12 other potyviruses. mAb 95 showed a reactivity range larger than PVY, unlike mAb 88 which only recognised the PVY isolates. Moreover, immunogold labelling of the Cls of PVY-N, either in situ or in cell extracts, was only obtained with mAb 88, thus showing that the corresponding virus-specific epitope is located at the surface of the Cl polymer.     

The antigenic characteristics of the capsid proteins in strains of the potato virus Y]

The antigenic properties of capsid proteins of potato virus Y (PVY) strains have been studied, the most wide antigenic specificity of necrotic group strains has been marked. Several antigen-active strains of necrotic group strains (PVY-N Far East, PVY-N Leningrad, PVY-N Moscow) and common group (PVY-O-3 Moscow, PVY-O Far East) have been revealed. Antisera against these strains reacted with any PVY strain. Virus specific surface epitope, corresponding to position 198-208 of polypeptide chain has been located, which is a group-specific epitope of potyviruses, identified in the Far East.     

A new lineage sheds light on the evolutionary history of Potato virus Y

Potato virus Y (PVY) is one of the rare plant viruses for which some biological traits (host range and symptomatology) are highly correlated with phylogeny, allowing the reconstruction of the evolutionary history of these traits. In this article, a new lineage of PVY isolates from Chile is described, showing unique genomic and biological properties. This lineage was found to be the sister group of all other PVY isolates and helped in the reconstruction of the ancestral traits and evolutionary history of PVY, suggesting that veinal necrosis in tobacco is an ancestral state and that adaptation to pepper ( Capsicum spp.) and potato ( Solanum tuberosum ) has been modified several times during PVY history.     

Comparison of transmission efficiency of various isolates of Potato virus Y among three aphid vectors

Potato virus Y (PVY) strains are transmitted by different aphid species in a non‐persistent, non‐circulative manner. Green peach aphid (GPA), Myzus persicae Sulzer, is the most efficient vector in laboratory studies, but potato aphid (PA), Macrosiphum euphorbiae Thomas (both Hemiptera: Aphididae, Macrosiphini), and bird cherry‐oat aphid (BCOA), Rhopalosiphum padi L. (Hemiptera: Aphididae, Aphidini), also contribute to PVY transmission. Studies were conducted with GPA, PA, and BCOA to assess PVY transmission efficiency for various isolates of the same strain. Treatments included three PVY strains (PVY^O, PVY^N:O, PVY^NTN) and two isolates of each strain (Oz and NY090031 for PVY^O; Alt and NY090004 for PVY^N:O; N4 and NY090029 for PVY^NTN), using each of three aphid species as well as a sham inoculation. Virus‐free tissue‐cultured plantlets of potato cv. Russet Burbank were used as virus source and recipient plants. Five weeks post inoculation, recipient plants were tested with quantitative DAS‐ELISA to assess infection percentage and virus titer. ELISA‐positive recipient plants were assayed with RT‐PCR to confirm presence of the expected strains. Transmission efficiency (percentage infection of plants) was highest for GPA, intermediate for BCOA, and lowest for PA. For all aphid species, transmission efficiency did not differ significantly between isolates within each strain. No correlations were found among source plant titer, infection percentage, and recipient plant titer. For both GPA and BCOA, isolates of PVY^NTN were transmitted with greatest efficiency followed by isolates of PVY^O and PVY^N:O, which might help explain the increasing prevalence of necrotic strains in potato‐growing regions. Bird cherry‐oat aphid transmitted PVY with higher efficiency than previously reported, suggesting that this species is more important to PVY epidemiology than has been considered.     

Molecular Analysis of the Coat Protein of Potato virus Y Isolates in Greece Suggests Multiple Introduction from Different Genetic Pools

The phylogenetic relationships among Potato virus Y (PVY) isolates from northern and southern Greece were investigated. A large part of coat protein gene of 49 tobacco isolates and three from pepper was examined. The analysis showed that all 52 isolates consisted of 34 distinct haplotypes, with only one haplotype found in both northern and southern regions. The southern population was more diverse than that from the north. The phylogenetic analyses of the Greek haplotypes alone or in combination with isolates from other countries using the maximum likelihood method classified unambiguously almost all the haplotypes examined. Nine tobacco haplotypes from the south were classified as C‐like (particularly C1), whereas 22 haplotypes from tobacco and two from pepper from both north and south were classified as N‐like. One tobacco haplotype from the south was found recombinant between N‐like and C1 lineages. The pattern of molecular evolution was examined using the fixed‐effects likelihood and the single‐likelihood ancestor counting methods. The analysis indicated that the evolution of PVY isolates appeared to be conservative (purifying selection and neutral evolution). These findings are discussed in relation to the introduction of PVY in the tobacco crop in Greece and the between region dispersal. A scenario of multiple introductions of PVY isolates in north and south Greece from different genetic pools and low or nil between region spread of the virus isolates was proposed.     

An Ry-mediated resistance response in potato requires the intact active site of the NIa proteinase from potato virus Y

Ry confers extreme resistance to all strains of potato virus Y (PVY). To identify the elicitor of the Ry-mediated resistance against PVY in potato, we expressed each of the PVY-encoded proteins in leaves of PVY-resistant (Ry) and -susceptible (ry) plants. For most of the proteins tested, there was no evident response. However, when the NIa proteinase was expressed in leaves of Ry plants, there was a hypersensitive response (HR). Proteinase active site mutants failed to induce the Ry-mediated response. The HR was also induced by the NIa proteinase from pepper mottle virus (PepMoV), which has the same cleavage specificity as the PVY enzyme, but not by the tobacco etch virus (TEV) or the potato virus A (PVA) proteinases that cleave different peptide motifs. Based on these results, we propose that Ry-mediated resistance requires the intact active site of the NIa proteinase. Although the structure of the active proteinase could have elicitor activity, it is possible that this proteinase releases an elicitor by cleavage of a host-encoded protein. Alternatively, the proteinase could inactivate a negative regulator of the Ry-mediated resistance response.     

Serological detection and antigenic variation of two whitefly-transmitted geminiviruses: tobacco leaf curl and croton yellow vein mosaic viruses

A panel of 25 monoclonal antibodies (MAbs) raised against particles of two heterologous whitefly-transmitted geminiviruses (begomoviruses) was used in triple antibody-sandwich ELISA (TAS-ELISA) to determine the detectability and epitope profiles of 26 Indian isolates of tobacco leaf curl virus (TLCV) and 13 of croton yellow vein mosaic virus (CYVMV). Stock cultures of the two viruses had indistinguishable epitope profiles although they differ in symptomatology and particle stability. Their epitope profiles also strongly resembled those of Indian isolates of bhendi (okra) yellow vein mosaic and Indian cassava mosaic (ICMV) viruses. TLCV isolates from Andhra Pradesh, Gujarat and Karnataka States differed slightly in epitope profile: they reacted with at least eight out of 10 MAbs raised to ICMV but only one to four out of 15 MAbs raised to African cassava mosaic virus (ACMV). Virus isolates serologically indistinguishable from TLCV were detected in symptom-bearing weeds (Acanthospermum hispidum, Ageratum conyzoides, Euphorbia geniculata, Parthenium hysterophorus) found in leaf curl-affected tobacco fields and shown previously to be experimental hosts of TLCV. Indian TLCV isolates had small, consistent differences in epitope profile from Pakistani isolates but large differences from isolates from Burkina Faso, Malawi or Uganda. Isolates from the three African countries reacted with four or five of the ACMV MAbs but only one or two of the ICMV MAbs, and there were small but consistent inter-country differences. CYVMV isolates from three Indian States showed less epitope variation than did Indian isolates of TLCV. TAS-ELISA with MAb SCR 18 was a more sensitive test for detecting Indian TLCV isolates than was double antibody-sandwich ELISA with polyclonal antibodies.     

Strain specificity and simultaneous transmission of closely related strains of a Potyvirus by Myzus persicae

Potato virus Y (PVY), a Potyvirus, is transmitted by aphids in a nonpersistent manner. PVY severely affects potato production worldwide. Single and mixed infections of PVY strains, namely PVY(O), PVY(NTN), and PVY(N:O) are a common occurrence in potato systems. However, information available on the ability of aphids to simultaneously transmit multiple PVY strains, specificity associated with simultaneous transmission, and factors affecting specificity are limited. Aphid-mediated transmission experiments were conducted to test the ability of individual aphids to transmit multiple strains using a PVY indicator host. Preliminary results revealed that aphids can transmit at least two viral strains simultaneously. Subsequently, aphid-mediated transmission of three dual-strain combinations was tested using potato plants. Individual aphids transmitted two viral strains simultaneously for all three dual-strain combinations. In all aphid-mediated dual-strain infections involving PVY(NTN), the rate of PVY(NTN) infection was greater than the infection rates of the second strain and dual-strain combinations, indicating specificity associated with transmission of PVY strains. Results of aphid-mediated transmission experiments were compared with results obtained through mechanical transmission. In general, PVY infection rates from aphid-mediated transmission were lower than the rates obtained through mechanical transmission. Unlike aphid-mediated transmission, component strains in dual-strain inoculations were not eliminated during mechanical transmission. These results suggest that there may also be interference associated with aphid-mediated transmission of closely related PVY strains. Perhaps, the observed specificity and/or interference may explain the increase in the incidence of PVY(NTN) and other necrotic strains in recent years.     

Synergistic neutralization of human immunodeficiency virus type 1 by a chimpanzee monoclonal antibody against the V2 domain of gp120 in combination with monoclonal antibodies against the V3 loop and the CD4-binding site.

Synergistic neutralization of human immunodeficiency virus type 1 (HIV-1) was observed in studies using a chimpanzee anti-V2 monoclonal antibody (MAb), C108G, in combination with anti-V3 loop and anti-CD4 binding-site (bs) MAbs of different epitope specificities. C108G paired with either of two anti-V3 loop MAbs or either of two anti-CD4 bs MAbs synergistically neutralized both the uncloned IIIB and clonal HXB2 strains of virus in H9 target cells. Synergism was quantitated by calculation of combination indices. Significant synergy with a given MAb pair was seen over a range of MAb ratios, with the optimal effect centering around the ratio at which the MAbs were equipotent for a given HIV-1 strain (on the basis of the 50% neutralization titer). In preliminary experiments with monocytotropic strains of HIV-1 in peripheral blood mononuclear cell targets, significant synergism was also observed between anti-V2-anti-V3 and anti-V2-anti-CD4 bs MAb pairs. Synergism by all MAb pairs tested was greater against heterogeneous isolates of HIV-1 (IIIB and Ba-L) than against clonal isolates (HXB2 and NLHXADA), suggesting that strain broadening may be a component of the synergism observed against the heterogeneous isolates. In addition, conformational changes in gp120 upon binding of one or both MAbs may result in increased affinity or exposure of the epitope of one or both MAbs. Finally, a three-MAb combination of C108G, an anti-V3 MAb, and an anti-CD4 bs MAb was more effective in neutralizing the HXB2 strain of HIV-1 than any of the three two-MAb combinations within this trio, as determined by the dose reduction indices of each MAb required to achieve a given level of neutralization. This is the first report of synergistic neutralization of HIV-1 by a three-MAb combination composed of MAbs directed against the three major neutralization epitope clusters in gp120. Implications for vaccine design and for immunoprophylaxis and immunotherapy with a combination of MAbs are discussed.     

Production of monoclonal antibodies for the detection of potato virus Y

Monoclonal antibodies (McAb) were obtained to potato virus Y (PVY) after immunisation of BALB/c mice with purified PVY, tobacco necrotic strain (PVY_n). Spleen cells from a mouse showing a high serum titre were used for fusion with X63NS1 myeloma cells. Hybridomas were selected in medium containing HAT. Culture supernatants were screened for antibody production against PVY, ordinary strain (PVY₀) and PVY_n using indirect ELISA. Clones of interest were further cross-reacted with 12 isolates each of PVY₀ and PVY_n and two isolates of potato virus A (PVA) and healthy sap. For further trials, two clones which reacted specifically with PVY_n isolates and one which detected all PVY isolates except two of potato virus C (PVC) were selected.