Laurent Glais

Genomic variability in Potato potyvirus Y (PVY): evidence that PVYNW and PVYNTN variants are single to multiple recombinants between PVYO and PVYN isolates

Summary. Fourteen Potato virus Y (PVY) isolates representative of PVYO, PVYN, PVYNTN and PVYNW groups were characterised at genomic level. Restriction fragment length polymorphism study (RFLP) of each gene of these isolates and sequencing of the first 2700 nucleotides of two PVYNW isolates were performed. A mosaic structure was revealed in PVYNW and PVYNTN genomes, which showed either PVYO or PVYN-like sequences, depending on the particular gene. Indeed, starting from the 5′-end, these isolates showed a switching, from PVYN- to PVYO-like sequence, in the HC-Pro C-terminal region. Reversion to PVYN-like sequence was also revealed in the NIa N-terminal area of PVYNTN isolates, followed by a switching back to a PVYO-like sequence in the CP gene. Lastly, some PVYNW isolates showed a switching from PVYO- to PVYN-like sequence in the P1 N-terminal part, thus separating our PVYNW isolates into two subgroups. All these apparent recombination events were shown by statistical analysis. Comparison of molecular traits with pathogenic properties of our isolates suggested that the HC-Pro protein is involved in induction of necrosis in tobacco leaves, and the NIa, NIb and/or CP protein in necrosis in potato tubers. Nevertheless, multiple recombination events observed in the PVYNTN genome may play a role in the latter phenomenon.

Molecular characterization of potato virus YN isolates by PCR-RFLP : Differentiation of PVYN isolates by PCR-RFLP

Based on the sequence polymorphism in the 5′ terminal part of the viral genome, a range of PVYN isolates were characterized by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). Three pairs of primers selected in the 5′ non-translated and P1 protein region were tested. Two of them yielded PCR products of about 1Kb from all isolates tested. Restriction analysis of the PCR products gave two distinct electrophoretic patterns, whichever of the three enzymes was used. In this way, the 18 isolates were separated into two easily identifiable subgroups. All tuber necrosing isolates (PVYNTN) were clustered in the same subgroup.

RFLP mapping of the whole genome of ten viral isolates representative of different biological groups of potato virus Y.

SummaryTen PVY isolates representative of four PVY groups (YN, YNTN, YN-W, Y O), differing by their ability to induce reactions of vein necrosis on tobacco and tuber necrosis on potato, were studied in order to research the regions of the viral genome involved in these necrosis phenomena. The whole genome of these isolates was amplified in two fragments (4 063 and 5 670 nucleotides) and was subjected to a restriction fragment length polymorphism (RFLP) study. In the first 4 063 nucleotides of the PVY genome, a phenetic analysis of RFLP data resulted in a clustering of our PVY isolates into three groups: PVYN isolates (group A); PVYNTN and PVYN-W isolates (group B) and PVYO isolates (group C). In the last 5 670 nucleotides, two groups were found: PVYN and PVYNTN isolates (group D) and PVYO and PVYN-W isolates (group E). From this clustering and the necrosing properties known for theseisolates, the tobacco necrosis determinants seem more likely located inthe 5′ than in the 3′ half part of the viral RNA, whereas it would be theopposite situation for the determinants of the necrosis on potato tubers.Moreover a recombination event seemed to have occurred in the genome ofthe PVYN-W isolates.

Fast purification of the filamentous Potato virus Y using monolithic chromatographic supports

Obtaining pure virus suspensions is an essential step in many applications, such as vaccine production, antibody production, sample preparation for procedures requiring enrichment in viruses and other in vitro characterizations. Purification procedures usually consist of complex, long lasting and tedious protocols involving several ultracentrifugation steps. Such complexity is particularly evident in the case of plant viruses, where the virus needs to be isolated from the complex plant tissue matrix. Convective Interaction Media (CIM) monoliths are chromatographic supports that have been successfully utilized for the purification of large bio-molecules such as viruses, virus like particles and plasmids from various matrixes. In this study a CIM monolith based procedure was developed for the fast purification from plant tissue of the filamentous Potato virus Y (PVY) (virion size, 740 nm × 11 nm), which is one of the most important plant viruses causing great economical losses in potato production. Different mobile phases, chemistries and sample preparation strategies were tested. The presence of the virus in the chromatographic fraction was monitored with viral RNA quantification (RT-qPCR), viral protein purity estimation (SDS-PAGE) and viral particle integrity observation (transmission electron microscopy). The optimized procedure involves initial clarification steps, followed by chromatography using CIM quaternary amine (QA) monolithic disk column. In comparison to classical purification procedure involving ultracentrifugation through sucrose and caesium chloride, the developed CIM-QA purification achieved comparable yield, concentration and purity. Plant nucleic acids were successfully removed. Purification showed good reproducibility and moreover it reduced the purification time from four working days required for classic purification to a day and a half. This is the first study where a filamentous virus was purified using CIM monolithic supports. The advantages of this new purification procedure make it an attractive method in serological diagnostic tool production, which requires purified viruses for the immunization step. Moreover, the outcome of this study could serve as starting point for the improvement of the purification methods of other important filamentous viruses.

Incidence of potato viruses and characterisation of Potato virus Y variability in late season planted potato crops in Northern Tunisia

Surveys were conducted of symptomatic potato plants in late season crops, from the major potato production regions in Northern Tunisia, for infection with six common potato viruses. The presence of Potato leafroll virus (PLRV), Potato virus Y (PVY), Potato virus X (PVX), Potato virus A (PVA), Potato virus S (PVS) and Potato virus M (PVM) was confirmed serologically with virus infection levels up to 5.4, 90.2, 4.3, 3.8, 7.1 and 4.8%, respectively. As PVY was prevalent in all seven surveyed regions, further biological, serological and molecular typing of 32 PVY isolates was undertaken. Only one isolate was shown to induce PVYO-type symptoms following transmission to tobacco and to react only against anti-PVYO-C antibodies. Typical vein necrosis symptoms were obtained from 31 samples, six of which reacted against both anti-PVYN and anti-PVYO-C antibodies showing they contained mixed isolates, while 25 of them reacted only with anti-PVYN antibodies. An immunocapture RT-PCR molecular test using a PVYNTN specific primer pair set in the 5’NTR/P1 genomic region and examination of recombinant points in three genomic regions (HC-Pro/P3, CI/NIa and CP/3’NTR) showed that all 25 serotype-N PVY isolates were PVYNTN variants with similar recombinations to the standard PVYNTN-H isolate. This is the first report of the occurrence of the PVYNTN variant and its high incidence in late season potatoes in Tunisia.

Effect of passage of a Potato virus Y isolate on a line of tobacco containing the recessive resistance gene va2 on the development of isolates capable of overcoming alleles 0 and 2

Control strategies developed for plant viral diseases through breeding programs can be impaired by adaptive response of pathogens. A few years after the deployment in France of improved tobacco genotypes introgressed with alleles of the Potato virus Y (PVY) recessive resistance gene va, necrotic symptoms associated with PVY infection have been reported on these cultivars. Due to the lack of efficient alternative methods to control PVY, the va resistance sources have to be managed according to viral parameters, such as the dynamic of emergence of virulent variants. The effects of va alleles on the evolution of PVY was tested in experimental conditions using a PVY infectious clone and two couples of resistant tobacco near isogenic lines BVA/Bva0 and FVA/Fva2 both allelic for the va gene. Infection efficiencies data showed that a single passage on Fva2 line led to the selection of virulent viral populations able to overcome both va0 and va2 alleles. Sequence analyses of va2 derived PVY variants revealed that the acquisition of the capacity to overcome va2 resistance is associated with single point mutations at two different nucleotide positions in the central part of the VPg. The described PVY adaptation process to overcome resistance mediated by different va alleles should be considered for the future development of durable and integrated strategies of management of PVY infections in tobacco crops.

Fluorescently Tagged Potato virus Y: A Versatile Tool for Functional Analysis of Plant-Virus Interactions

Potato virus Y (PVY) is an economically important plant virus that infects Solanaceous crops such as tobacco and potato. To date, studies into the localization and movement of PVY in plants have been limited to detection of viral RNA or proteins ex vivo. Here, a PVY N605 isolate was tagged with green fluorescent protein (GFP), characterized and used for in vivo tracking. In Nicotiana tabacum cv. Xanthi, PVY N605-GFP was biologically comparable to nontagged PVY N605, stable through three plant-to-plant passages and persisted for four months in infected plants. GFP was detected before symptoms and fluorescence intensity correlated with PVY RNA concentrations. PVY N605-GFP provided in vivo tracking of long-distance movement, allowing estimation of the cell-to-cell movement rate of PVY in N. tabacum cv. Xanthi (7.1 ± 1.5 cells per hour). PVY N605-GFP was adequately stable in Solanum tuberosum cvs. Désirée and NahG-Désirée and able to infect S. tuberosum cvs. Bintje and Bea, Nicotiana benthamiana, and wild potato relatives. PVY N605-GFP is therefore a powerful tool for future studies of PVY-host interactions, such as functional analysis of viral and plant genes involved in viral movement.Potato virus Y (PVY) is an economically important plant virus that infects Solanaceous crops such as tobacco and potato. To date, studies into the localization and movement of PVY in plants have been limited to detection of viral RNA or proteins ex vivo. Here, a PVY N605 isolate was tagged with green fluorescent protein (GFP), characterized and used for in vivo tracking. In Nicotiana tabacum cv. Xanthi, PVY N605-GFP was biologically comparable to nontagged PVY N605, stable through three plant-to-plant passages and persisted for four months in infected plants. GFP was detected before symptoms and fluorescence intensity correlated with PVY RNA concentrations. PVY N605-GFP provided in vivo tracking of long-distance movement, allowing estimation of the cell-to-cell movement rate of PVY in N. tabacum cv. Xanthi (7.1 ± 1.5 cells per hour). PVY N605-GFP was adequately stable in Solanum tuberosum cvs. Desiree and NahG-Desiree and able to infect S. tuberosum cvs. Bintje and Bea, Nicotiana benthamiana, and wild pot...

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.

The amino acid 419 in HC-Pro is involved in the ability of PVY isolate N605 to induce necrotic symptoms on potato tubers

The ability to induce the potato tuber necrosis ringspot disease (PTNRD) is a property shared by PVY isolates belonging to different groups (e.g. PVY(N) and PVY(O)) and variants (e.g. PVY(NTN) and PVY(N)-W). The identification of viral molecular determinant(s) involved in the expression of PTNRD symptoms is essential for (i) an easier detection of tuber necrosis isolates and (ii) an improvement of our knowledge on the epidemiology of this potato disease. A reverse genetic approach associated with a biological typing of a collection of PVY chimeras and mutants indicated that residue E419 of the HC-Pro protein is linked to the ability of PVY to induce tuber necrosis on four PTNRD-susceptible potato cultivars. Indeed, the substitution of the N-type glutamic acid (E) in O-type aspartic acid (D) at position 419 in the HC-Pro cistron prevents the expression of tuber necrosis on infected tubers without reducing the virulence of the corresponding E/D419 mutant. This result opens opportunities for the future studies on potato/PVY interactions.

Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays

Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.