Molecular Footprints of Potato Virus Y Isolate Infecting Potatoes (Solanum tuberosum) in Kenya

Potato virus Y (PVY) is a highly diverse and genetically variable virus with various strains. Differential evolutionary routes have been reported in the genus Potyvirus, caused by natural selection pressure, mutation, and recombination, with their virulence being dependent on different environmental conditions. Despite its significance and economic impact on Solanaceous species, the understanding of PVY's phylogeography in Kenya remains limited and inadequately documented. The study centers on the molecular characterization of a Kenyan PVY isolate, GenBank accession number PP069009. In-depth phylogenetic analysis unveiled a strong evolutionary association between the Kenyan isolate and isolate [JQ924287] from the United States of America, supported by a robust 92% probability. Recombinant analyses exposed a mosaic-like genetic architecture within the Kenyan isolate, indicating multiple gene recombination events. Selection pressure scrutiny identified specific sites under selective pressure, with evidence of positive/diversifying and negative/purifying selection. Population genetics analysis revealed a calculated nucleotide diversity (π) of 0.00354881, while analysis of molecular variance (AMOVA) unveiled a structured genetic landscape with an øST value of 0.45224. The extensive haplotype network depicted the possibility of diverse PVY strains occurring across continents. This analysis provides valuable insights into the genetic diversity and distribution of PVY globally, highlighting the importance of understanding evolutionary dynamics for effective management and control strategies of PVY on a global scale.


Background
Irish potato ranks fourth among the most crucial food crops globally, after maize, rice, and wheat.In Kenya, potato is the second most important food crop after maize.It plays a crucial role in addressing food security and alleviating poverty.Sustainable solutions are required to combat various biotic and abiotic stresses afecting its production, to ensure a stable food supply for the growing population [1,2].Among the challenges facing solanaceous crops, Potato Virus Y is a signifcant and economically impactful threat.Te various strains of PVY afect potato production in multiple ways, making it a key focus in the study of potato plant pathogens.Spread by aphids in a nonpersistent manner, PVY poses a threat not only to potatoes (Solanum tuberosum) but also to other solanaceous species including tomatoes (S. lycopersicum), tobacco (Nicotiana tabacum), and nonsolanaceous hosts, such as smooth pigweed (Amaranthus hybridus) [3,4].PVY genome is a singlestranded positive-sense RNA of approximately 9.7 kb to 11 kb and encodes ten functional proteins, each playing a crucial role in the infection process [5,6].Te evolution of PVY strains results in new variants with diferent biological properties that impact potato production globally [4,7].Phylogenetic analyses provide signifcant pieces of evidence information that unveil the degree of evolutionary relationship among genera, species, and phylotypes, the diversity of geographical isolates, and the origin and evolution of diferent plant viruses [8][9][10].Diferential evolutionary routes have been reported in the genus Potyvirus, caused by natural selection pressure, mutation, and recombination [11].
Potyviruses often cause diverse infections, their virulence being dependent on diferent environmental conditions [12,13].Tis stems from the parental origin of resistance genes of the host plant and modulation of the profciency of RNA interference (RNAi) by diferent environmental conditions.Negative selection is strongly evident in the genes HC-Pro, coat protein (CP), and Nia and Nib genes, while the PIPO gene is the least selected [14].Mutations and recombination play a signifcant role in the genetic variation of various PVY strains, potentially leading to more severe symptoms in different crops.Previously, the classifcation of PVY strains was based on the plant species from which the samples were obtained.Tis method identifed four PVY strains associated with specifc hosts: potato, tobacco, tomato, and pepper.However, this classifcation has become less relevant over time, as some strains have demonstrated the ability to infect multiple host species.In major potato-growing regions worldwide, at least nine distinct strain families of PVY have been identifed.Tis includes nonrecombinant parental strains PVY O (ordinary strain), PVY C (common strain), and PVY N (necrotic strain), which all difer in their biological properties [15,16].Several studies have been conducted to highlight the various processes by which diferent strains induce infections in diferent hosts [17][18][19].
In many developing countries where the utilization of certifed potato seed systems is limited, there is evidence of seed degeneration and a steady, long-term decrease in yield attributed to viral infections [20][21][22].Potato virus Y is incurable under normal feld conditions; therefore, known prophylactic measures used in Kenya are focused on preventing or slowing down the viral spread in felds using resistant varieties [23].Te attainment of virus-free propagating material has been realized through molecular procedures.To implement efective control strategies, it is important to obtain molecular-level data on specifc viral strains.Tis is crucial as they may symptomatically reveal epidemiological properties that can aid in accurately diagnosing viral diseases [24].
To efectively control viruses of high economic signifcance to crop production, efcient detection methods that ofer insights into the genomic architecture of the target viruses have to be employed [25].Numerous strategies have repeatedly endured the test of time as initial screening tools for the presence and absence of viral infections.Tese include polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and restriction enzyme analysis.Tese rely on prior knowledge of viral genomes [26].Next-generation sequencing (NGS) technologies have proven benefcial for the detection and characterization of viruses in potato plants.Tey ofer insights into viral-host interactions at a molecular level, illuminating both novel and existing pathogenic viruses [27,28].In Kenya, next-generation sequencing (NGS) is gaining importance and is being applied in the felds of public health [29][30][31] and agriculture [32,33] to mention a few.RNA-Seq is the greatest analytical tool for functional genomics studies, such as diferential gene expression, alternative splicing, and variant fndings.mRNA sequencing (mRNA-Seq) investigates the transcriptome status, short small RNA sequencing (smRNA-Seq) enables genome-wide profling and analysis of both novel and known miRNA variants, and whole transcriptome sequencing (WTS) detects coding and multiple forms of noncoding RNA.Tey are all categorized based on read length [28].Te process of completing cDNA involves three key steps: total RNA isolation, target RNA enrichment, and reverse transcription of RNA into complementary DNA (cDNA).RNA sequencing processes consist of several main components, including RNA stabilization, RNA separation, enrichment, library creation, library controls, barcoding, and RNA sequencing.All the PVY sequences reported to GenBank come from all continents except Antarctica [34] with a vast number of reports signifying a wide range of changing dynamics of viral genome with varying host ranges toward host adaptation.
Tere is a lack of data on preexisting genomic analysis of Potato Virus Y afecting the potato crop in Africa and more so in Kenya.Genome sequencing is crucial because it provides information on the status of diferent genes along genomes.Tis method enables the creation of introgression libraries by crossing a cultivated parent with a wild donor, then backcrossing the F1 generation with the cultivated parent three to four times, enhancing resilience against PVY.Te primary objective of this study was to unveil the molecular phylogeography of Kenyan PVY isolate, providing a foundational analysis of its molecular profle and relative signifcance.

Potato Virus Y Strain Type Sample Collection.
A survey conducted in 2018-2019 in Kenya's potato main growing areas, namely, Tigoni, Meru, Molo, Bomet, Uasin Gishu, West Pokot, Trans-Nzoia, and Mount Elgon, provided symptomatic potato leaf samples that were subjected to serological tests.It was done during the long rainy season (March-June rains).Potato felds were selected randomly across several administrative levels: two subcounties per county and two to four wards in each subcounty, depending on the availability of the potato crop on the farm.Phenotypic symptoms keenly observed for sample collection included stunted growth, chlorotic mosaics on leaves, crinkling of leaves, and deepening venation (Figure 1).Te potato leaf samples phenotypically identifed were carefully collected and immediately placed in a sterile bag and placed in an ice-flled cool box in the feld then transferred to −20 °for further laboratory analysis.

Serological Characterization of PVY Samples from
Farmer Fields.Te collected samples underwent initial testing for the presence of PVY using a double-antibody sandwich ELISA, following the manufacturer's instructions.Negative controls in the form of virus-free plant samples of potatoes were included, while a confrmed PVY-infected potato sample obtained from the feld served as the positive control.RNA extraction was performed on infected plants using the Spectrum Plant Total RNA Kit obtained from Sigma-Aldrich, following the manufacturer's instructions, with slight modifcations.Frozen potato leaves were fnely ground using a sterile pestle and mortar.Te ground tissue was carefully transferred to precooled RNasefree microcentrifuge tubes.Lysis bufer was added to each sample, with a volume of 1.5 ml per 0.25 g of tissue powder.Te lysate was homogenized by vortexing to thoroughly disperse the sample, followed by incubation for 3 minutes at room temperature.Subsequently, a volume of 350 µl was transferred to a clean homogenization tube and centrifuged at 13,000 rpm for 5 minutes.To each homogenate, one volume of 70% ethanol was added.Te samples were vortexed thoroughly and transferred to a spin cartridge, which was centrifuged at 13,000 rpm for 15 seconds, and subsequently, the fow-through was discarded.Te spin cartridge was reinstated into the same collection tube following the previous steps.A repeat of the process was conducted using bufer II, followed by a fnal spin at 13,000 rpm for 2 minutes to dry the membrane with bound RNA.Te purifed RNA was then stored at −20 °C.To assess the quality of the extracted RNA, one μl of the RNA extract was run on a 1.2% agarose gel.Additionally, the RNA concentration was measured using a NanoDrop (Table 1).

Molecular Detection of PVY
Te reverse transcription (RT) was synthesized in a 25 µl reaction mix utilizing M-MLV reverse transcriptase from Termo Scientifc.Te reaction mix comprised of 1 µl cDNA template, 0.5 µl of forward and reverse primers (Table 2) that were designed, 0.5 µl of 10 mM dNTP, 5 µl of 10X Taq polymerase bufer, 0.25 µl of Taq DNA polymerase (5 U/l), 1.5 µl of MgCl2, and 15.75 µl ddH 2 O composed the PCR Master mix.Te PCR cycle was confgured to start with a 2minute denaturation step at 95 °C, followed by 30 cycles consisting of denaturation at 95 °C for 30 seconds, annealing at 58 °C for 1 minute, and extension at 72 °C for 2 minutes.Finally, there was a single extension step of 5 minutes at 72 °C.Te amplifcation results were separated on a 1.2% agarose gel in 1X TAE bufer (as shown in Figure 2), stained with 0.75 μl of GelRed (from Biotium, USA).

Cloning and Sequencing of the PCR Fragments for PVY
Strain Identifcation.High-fdelity polymerases cloned for sequencing were used to create multiplex-PCR fragments.Te Termo Scientifc GeneJET PCR Purifcation Kit was utilized to purify PCR products, and the cDNA content was quantifed using a NanoDrop.For further validation of purity and concentration, 5 µl of purifed cDNA was subjected to electrophoresis on a 1.2% agarose gel.Following ligation, the purifed PCR product was inserted into the pGEM-T cloning vector using the CloneJET PCR Cloning Kit from Termo Scientifc.Subsequently, the resulting construct was transformed into competent cells of Escherichia coli strain DH5 obtained from Life Technologies, following the manufacturer's instructions.
Plasmid DNA was then extracted from overnight cultures of selected E. coli colonies using the Termo Scientifc GeneJET Plasmid Miniprep Kit.Te extracted plasmid DNA was digested with FastDigest BgIII enzyme from Termo Scientifc, and the digests were examined using agarose gel electrophoresis.Colonies exhibiting the expected insert sizes were selected for sequencing using the Sanger sequencing

Sample Preparation.
Total RNA extraction was performed using a RNeasy Plant Mini Kit, adhering to the manufacturer's protocol with slight modifcations.
2.3.2.Library Preparation.Library preparation followed the Illumina TruSeq low sample preparation protocol with minor modifcations.Fifteen microliters of Elute Prime Fragment mix, containing random hexamers for RT priming and frst-strand cDNA synthesis bufer, were added to sterile PCR tubes containing ten microliters of 500 ng RNA obtained from the previous step.Te mixture was gently pipetted, mixed thoroughly, and then placed in a preprogrammed thermocycler at 94 °C for eight minutes.Subsequently, the thermocycler was held at 4 °C to allow for elution, fragmentation, and priming of the RNA.Following this step, the thermocycler immediately progressed to synthesize frst-strand cDNA.
50 µl of the SuperScriptII was added to the entire stock (one-microliter SuperScriptII for each 9 µl of the First-Strand Master Mix) to the First-Strand Master Mix tube, mixed gently but thoroughly, and centrifuged at maximum speed for 30 seconds.8 µl of the First-Strand Mix containing SuperScriptII was added then entire volume to 17 µl of the primed mix and gently pipetted up and down six times, spun down, and placed in a preprogrammed thermo cycler at 25 °C for ten minutes, 42 °C for ffty minutes, and 70 °C for ffteen minutes, and held at 4 °C with preheated lid option set to 100 °C.Tis immediately progressed to synthesize the second strand.Te thawed Second-Strand Master Mix was centrifuged at 600xg for fve seconds.25 µl was added to the products of the frst-strand cDNA synthesis, the entire volume pipetted, mixed thoroughly, spun down, and then incubated in a preheated thermo cycler for one hour.
To purify the double-stranded (ds) cDNA, 90 µl of wellmixed AMPure XP beads was added to the PCR tubes containing 50 µl of ds-cDNA at room temperature.Te mixture was thoroughly mixed and then transferred to sterile 1.5 ml labeled microcentrifuge tubes.Subsequently, the tubes were incubated at room temperature for ffteen minutes.After the incubation period, the microcentrifuge tubes and their contents were placed on a magnetic stand at room temperature for fve minutes.Tis allowed all the beads to bind to the side of the tubes due to the magnetic feld.Following magnetic separation, the supernatant containing impurities was carefully removed from each well and discarded, while the microcentrifuge tubes remained on the magnetic stand.Without disturbing the pellet, a hundred microliters of freshly prepared 80% EtOH were added and incubated at room temperature for 30 seconds.Te 80% EtOH washes were repeated twice.
After the supernatant containing impurities was removed and discarded, the microcentrifuge tubes containing the pellet were allowed to stand at room temperature to dry briefy.A thawed and centrifuged resuspension bufer (RSB) was then added to each tube after its removal from the magnetic stand.Te entire volume was gently pipetted ten times (up and down) to ensure thorough resuspension of the pellet.Subsequently, the tubes were incubated at room temperature for two minutes to allow for proper rehydration of the pellet.Te microcentrifuge tube was placed in a magnetic stand for fve minutes with the cap open.Fifty microliters of supernatant (ds-cDNA) was removed from each microcentrifuge tube and transferred to clean empty PCR tubes.Ten microliters of resuspension bufer was added to each tube, followed by 40 microliters of End Repair Mix.Te tubes were then placed in a preheated thermal cycler at 30 °C for thirty minutes.
Additionally, 100 microliters of End Repair Mix was transferred to a 1.5 ml microcentrifuge tube.To this, 160 microliters of well-vortexed AMPure XP beads was added, and the entire volume was gently pipetted and thoroughly mixed.Subsequently, the mixture was incubated at room temperature for ffteen minutes.After the incubation step, the tube was placed on a magnetic stand at room temperature for fve minutes, allowing the liquid to become clear.Subsequently, 127.5 µl of the supernatant was carefully removed and discarded.While the tubes remained on the magnetic stand, 200 µl of freshly prepared 80% EtOH was added over the pellet carefully, without disturbing it.Te tubes were then incubated at room temperature for 30 seconds.Te supernatant was carefully removed and discarded without disturbing the pellet.Te wash step was repeated once more.Carefully, the tubes were air-dried at room temperature for ffteen minutes while still on the magnetic stand.Next, 20 µl of resuspension bufer (RSB) was added into the air-dried tubes and gently pipetted up and down to mix thoroughly.Te entire volume was mixed thoroughly and incubated at room temperature for two minutes.Te tubes were placed back on the magnetic stands at room temperature until the liquid became clear.Twenty microliters of supernatant was transferred to a new sterile PCR tube, followed by the addition of 2.5 µl of RSB and 12.5 µl of thawed A-tailing mix.Te entire volume was gently pipetted up and down ten times to ensure thorough mixing.Te mixture was then spun down and placed on a preprogrammed thermocycler at 37 °C for thirty minutes, followed by 70 °C for fve minutes, and a hold temperature of 40 °C with a preheat lid option.Te libraries were quantifed using Qubit High Sensitivity Kit and quality checked on 2% agarose gel and Agilent Technologies tape station with sensitivity D 1000 screen tape to select libraries.Te libraries were diluted to a suitable concentration in resuspension bufer, with dilutions ranging between 4 nM to 10 nM.Two pools were generated from the 4 nM libraries.A sample sheet was generated for each pool in the Illumina experiment manager.Tese were further diluted to a fnal concentration of seven pM and loaded to an Illumina TruSeq system for sequencing.

Recombination Analysis.
To investigate the role of recombination in PVY evolution, two diferent approaches were employed for recombination analyses.Tese approaches were utilized to identify potential recombination events in PVY sequences concerning the Kenyan isolate PP069009.We identifed potential recombinant and parental sequences using seven diferent algorithms, including RDP, GENECONV, BOOTSCAN, MAXCHI, CHIMAERA, SISCAN, and 3SEQ.Default settings were used throughout.RDP (recombination detection program) applies several recombination detection and analysis methods expanding an array of recombination event detection, recombination breakpoint demarcation, and recombinant sequence identifcation methods, all applied in unison, to yield detailed descriptions of how recombination may have impacted the evolution of any given set of aligned nucleotide sequences [35].GENECONV examines an alignment of multiple sequences pairwise and scans for abnormally long regions of high identity between the focal pair, contingent on the variable site pattern in other sequences.GENECONV employs codon polymorphisms rather than site polymorphisms, as silent sites within the same codon position are expected to be correlated [36].Te BOOTSCAN algorithm investigates the depths of recombinant regions within the alignment, as determined by crossover points identifed through boot scanning.Tese regions are subsequently analyzed separately through phylogenetic analysis [37].Te MAXCHI function implements the maximum chi-square (MaxChi) method, which is utilized for detecting recombination breakpoints [38].CHI-MAERA is an algorithm designed for the accurate detection and estimation of subclone frequencies.It utilizes whole-exome sequencing and whole-genome sequence data obtained from multiarea biopsies, enabling subclones to be sorted in an evolutionary tree structure [39].SIS-CAN assesses phylogenetic and compositional signals in various patterns of identity that occur between four nucleotide sequences [40].3SEQ is a command-line program designed to read in a nucleotide sequence fle, typically in Philip or aligned FASTA format.It tests all sequence triplets in the fle to detect a mosaic recombination signal, indicating that one of the three sequences (the child) is a recombinant of the other two (the parents).Te statistical test employed is a nonparametric test for mosaicism, and p values are precomputed once using the 3SEQ executable [41].

Phylogenetic Analyses of PVY Strains of Kenya.
Phylogenetic analysis was conducted using partial genome sequences that included complete P1 and CP (coat protein) nucleotide sequences enough to distinguish between the PVY strain types collected from farmer potato felds in Kenya.Tese isolates are represented by the accession numbers OR571473, OR571477, OR571474, OR571476, OR571478, OR571479, and OR571475 (refer to Table 3).Additionally, the Kenyan PVY whole-genome isolate with accession number PP069009 was included in a further analysis with a selection of sequences representing diverse countries and continents, sourced from the frst 100 sequences obtained from the NCBI search.Tese sequences were aligned using MAFFT software [42], and the phylogenetic tree (Figures 3 and 4) was constructed using MrBayes [43].Subsequently, the phylogenetic tree was visualized using the Figtree program.

Selection Pressure of the Kenyan PVY Whole-Genome Isolate
2.6.1.Branch Selection.Te notable focus on identifying instances of robust selection has led to a methodological gap concerning biologically intriguing cases where the absence or reduction in the efectiveness of natural selection is signifcant.In this study, the examination of selection pressures within the Potato Virus Y (PVY) phylogeny necessitated a thorough analysis utilizing the aBSREL [44] and RELAX [45] methods.Tis analysis aimed to discern sporadic diversifying and relaxed selection, respectively, with particular emphasis on the PVY_Kenya branches compared to other phylogenetic groups.Employing a stringent signifcance threshold (p ≤ 0.05) and correcting for multiple testing using the likelihood ratio test, the study expected to identify distinct selective pressures acting specifcally within PVY_Kenya branches.

Site Selection Tests.
Te advent of computationally tractable codon-substitution models [46] has sparked increased scientifc interest in natural selection acting on protein-coding genes.Positive selection is inferred when the estimated ratio (ω) of nonsynonymous (β) to synonymous (α) substitution rates signifcantly exceeds one [47,48].Tis phenomenon is more readily observed in smaller alignments.
A mixed-efects model of evolution (MEME) belongs to the broader class of branch-site random-efects phylogenetic methods [49], and it was the most preferred in this study for it allows the distribution to vary from site to site (the fxed efect) and also from branch to branch at a site (ω), reliably capturing the molecular footprints of both episodic and pervasive positive selection [50].It also matched the performance of traditional site methods

6
Advances in Virology Advances in Virology when natural selection is pervasive, reliably identifying episodes of diversifying evolution.In the investigation of pervasive positive/diversifying and negative/purifying selection within the Potato Virus Y (PVY) genome using the FUBAR [51], compelling evidence emerged regarding the distribution and nature of selective pressures across multiple sites.

PVY Strains' Sample Processing.
For the analysis of P1 and CP sequences, RNA extractions were done, and their concentrations were estimated using a NanoDrop spectrophotometer.Most sample extractions yielded satisfactory concentration readings (Table 1).Tese extracted RNA samples were then cloned into pGEM-T vectors to obtain complete P1 sequence data.Before sequencing, PCR products were visualized through gel electrophoresis (Figure 2).
PVY accessions listed in [52] were utilized for the initial identifcation of PVY strains, with the caveat that additional sequence data are necessary for defnitive assignment.

Phylogenetic Analysis of Diferent Potato Virus Y Strains
Sampled from the Farmer Fields.Te core sources accountable for evolutionary changes within positive-strand RNA viruses are recombination, reassortment, and accumulation of mutations.Diferent clades of the tree comprised diferent strains of PVY from diferent parts of the world.Distinctively, four monophyletic groups were observed from the original speciation event.Te Kenyan isolate K4 (PVY n:o recombinant type) shared the oldest common ancestor with samples from MN539908.1,MN380536.1,and KY863549.1 (collectively from Egypt); MK639789.1 (Kazakhstan); and KU757290 (Brazil), illustrating maximum support by 100% probability, suggesting a possible close revolutionary relationship in comparison with the rest of the We found the recombinant PVY strain type PVY N and PVY N:O (Table 3) to be prevalent in the area of study sampled.

Phylogenetic Analysis of the Whole Genome of Potato virus Y Kenyan Isolate.
To delineate the evolutionary relationships and contextualize the genetic divergence of the Potato virus Y (PVY) sample isolated from Kenya, a comprehensive phylogenetic analysis was undertaken with PVY accessions from Europe, Asia, Australia, and Africa, in eforts to further demonstrate the genealogy of the virus on a larger world scale level using MrBayes program (Figure 4), with a pepper mottle virus, accession number M11598 as the root.Advances in Virology PP256240 from Australia shared the oldest common ancestor at the second node.Tis is true for the rest of the clades in this phylogenetic analysis, along with other included accessions.
In the analysis involving accessions from Africa, MF382053 and MF602675 from Zimbabwe were observed to be more closely related to PVY samples from South Africa (KF770835) and Rwanda (ON604844).Tis was determined based on them sharing the most recent common ancestor.Conversely, the Kenyan isolate PP069009 used in this study showed the highest probability of undergoing the most recent speciation event.
PVY_Kenya isolate (GenBank accession number PP069009) demonstrated a close evolutionary relationship with an isolate from the United States of America (JQ924287) with a maximum support at 92% probability.Regarding isolates present in Africa, the phylogenetic analysis revealed a close association between the most recently shared common ancestry with a representative from Tunisia (MG69820) in comparison to isolates from the rest of Africa.

Recombination Analysis. Te recombinant analysis conducted for the phylogeny of Potato virus Y (PVY)
revealed a mosaic-like genetic structure within the PVY Kenya isolate.Employing the GENECONV method, multiple instances of gene recombination events were identifed across the genome, denoted by the specifc gene segments (Start-End) involved.Notably, a signifcant number of these events, spanning various regions, yielded a p value of zero, indicating an exceptionally high statistical signifcance.Furthermore, while most events exhibited a p value of zero, a few instances displayed slightly higher yet still relatively low p values, suggesting potential recombination events with slightly reduced statistical signifcance.Te analysis highlights the complex nature of genetic exchanges within the PVY_Kenya genome, underscoring substantial genetic diversity arising from recombination events with multiple parental sequences.Notably, specifc genomic regions exhibited varied strengths of evidence for recombination, further emphasizing the intricate mosaic pattern characterizing the genetic makeup of the PVY_Kenya isolate.

Branch Selection.
Selective pressures are the factors that infuence the survival and reproductive success of an organism in its environment and are crucial in shaping a population's genetic makeup through natural selection.However, contrary to expectations, neither aBSREL nor RELAX detected statistically signifcant evidence of episodic diversifying selection or relaxed selection within the PVY_Kenya branches or among the other groups in the phylogeny.Tis underscores the complexity of selective forces shaping PVY evolution and highlights the need for further detailed investigations into the dynamics of selection within viral populations.

Site Selection Test.
In the exploration of episodic positive/diversifying selection within the Potato virus Y genome, the MEME [50] method was employed, revealing evidence of selective pressure at a specifc site 2066, with statistical signifcance set at a p value threshold of 0.05, as illustrated in Figure 5. Specifcally, MEME identifed a key parameter, β+, as pivotal in distinguishing between null and alternative models.In the null model, both β+ and β− are constrained, whereas in the alternative model, β+ remains unrestricted.Positive selection at individual sites is inferred when the β+ parameter exceeds α+ and is further validated as signifcant through the likelihood ratio test.Tis signifes that site 2066 exhibited characteristics indicative of positive selection, highlighting its potential importance in the adaptive evolutionary process of PVY.
Te analysis was conducted under the general timereversible (GTR) model, complemented by the specifc model ftting (AICc � 31645.59,log L � −15721.77).FUBAR detected pervasive positive/diversifying selection at fve distinct sites and pervasive negative/purifying selection at 267 sites, each with a posterior probability (Prob [α > β]) exceeding 0.9.Notably, positive selection sites clustered predominantly toward the end of the alignment (Figure 6), while purifying selection was concentrated at the beginning.
Tis result unveiled the complex interplay of selective forces shaping the genetic landscape of this viral population.Te notable clustering of sites under positive selection toward the terminal regions of the alignment and the concentration of sites experiencing purifying selection at the beginning suggests a spatially distinct pattern of evolutionary pressures along the PVY genome.Tis spatial distribution might imply a diferential functional signifcance across the viral genome, where regions toward the end potentially experience more adaptive changes, likely associated with factors, such as host interactions, immune evasion, or adaptation to specifc environmental conditions.Conversely, the regions exhibiting purifying selection at the start might encompass critical genetic elements vital for the virus's replication, structural integrity, or conserved functional domains essential for its life cycle.Such divergent selective pressures across the genome could signify a delicate balance between adaptive evolution, where benefcial mutations confer advantages, and the preservation of essential genomic elements to maintain viral ftness.Tese fndings underscore the dynamic nature of PVY evolution, emphasizing the pivotal role of selective pressures in shaping the genetic diversity and adaptability of this virus, thereby providing valuable insights for understanding its evolutionary strategies and potential implications for disease management and control.

Population Genetics Analysis of Potato virus Y (PVY)
Phylogeny.An alignment of Potato virus Y (PVY) sequences was generated using the Muscle program v5 [53] and subsequently analyzed for haplotype with DnaSP v6 [54].Te program allows the thorough characterization of the levels and designs of DNA sequence disparity at diverse time scales, using polymorphic variants (intraspecifc data), divergence data (interspecifc or interpopulation data), or a combination of both [54].It also allows for the analysis of  4).Tis fnding may imply various evolutionary pressures acting upon PVY populations across continents, potentially leading to an excess of low-frequency polymorphisms or recent population growth within specifc geographical regions.Subsequent analysis using the analysis of molecular variance (AMOVA) unveiled a structured genetic landscape within the PVY population across continents.Te ø ST value of 0.45224, indicative of genetic diferentiation among continents, portrayed a moderate yet discernible level of diferentiation between the PVY strains originating from America, Africa, Asia, and Europe.Te fxation indices (Phi_ST, Phi_SC, and Phi_CT) provided a comprehensive understanding of genetic variance across diferent hierarchical levels.Phi_ST (0.45224) demonstrated substantial genetic diferentiation among continents, while Phi_SC (0.39131) indicated genetic variation among populations within continents, albeit to a lesser extent.However, Phi_CT (0.10010) did not achieve signifcance (p � 0.161), implying limited variation among continents relative to the total genetic diversity observed.Signifcance testing of these indices reafrmed the observed genetic structure, with both Phi_ST (p < 0.001) and Phi_SC (p � 0.006) showing signifcant values, indicating tangible genetic structure among continents and populations.Tese fndings underscore a structured genetic landscape within the PVY population across continents, characterized by moderate diferentiation among continents, low nucleotide diversity, and potential departures from neutrality.Tis multicontinental analysis emphasizes the need for further investigations into the specifc evolutionary dynamics and environmental pressures shaping PVY populations within distinct geographical regions, essential for developing tailored management and control strategies on a global scale.

Haplotype Networks.
Haplotype networks are used in the analysis of population genetic data to visualize genealogical relationships at the intraspecifc level, as well as to make inferences about the biogeogphraphy and history of diferent populations of organisms [55] and thus defning them [56].PopArt embraces the least spanning, medianjoining, and TCS network methods, as well as AMOVA [57] and Tajima's D statistic [58].With the primary function of POPART being to infer and visualize genetic relationships among intraspecifc sequences, an extensive haplotype list was generated from the DnaSP v6 and further analyzed with PopArt 1.7.2 [55] program to generate haplotype networks and maps.Te resulting list depicted a diverse spectrum of haplotypes, each characterized by a unique combination of sequences identifed by accession numbers and country codes, denoting their geographical origins.Te frequencies associated with these haplotypes provided valuable insights into the genetic diversity and distribution of PVY strains across diferent continents-America, Africa, Asia, and Europe.Te diverse array of haplotypes signifes the potential evolutionary divergence or recombination events within the PVY population.Tis information served as

Discussion
Pathogen adaptability is inevitable for better survival to the advanced resistance of hosts through mutations, recombination, or gene fow [59].Loss of genetic purity, gene erosion, and accumulation of pathogens in the seed potato have contributed to fuctuations observed in the global scales of potato production [20,60].Accurate extrapolation of the evolutionary course of pathogens, evaluating population  Te phylogenetic analysis provides crucial insights into the evolutionary relationships among genera, species, and phylotypes, revealing the diversity of geographical isolates and the origin and evolution of plant viruses [8][9][10].Te Incas, the frst South American farmers, initiated potato farming, and the domestication of arable Irish potatoes originated from wild potato species crossings in the Andean region [61].Te introduction of arable Irish potatoes to North America and Asia from South America dates back to the late 15th and 16th centuries, facilitated by seed exchange with Colombia, eventually spreading to other nations [62,63].
Reports from the NCBI GenBank database refect the changing dynamics of viral genomes with varying host ranges and host adaptation.Despite the efcacy of seed certifcation methods in reducing viral infections, Potato virus Y (PVY) remains a signifcant issue in potato seed production, impacting yields and tuber quality.Te spontaneous evolution of new PVY strains poses challenges to certifcation and farm management measures, raising concerns in potato production.
PVY sequences in GenBank span all continents except Antarctica [34], yet information on the molecular footprints of PVY in Africa is scarce.Tis study conducted comprehensive phylogenetic analyses using the NCBI Nucleotide Blast and a curated sequence dataset, including the PVY_Kenya genome and sequences from countries in the continents of Asia, Africa, Europe, North America, and Oceania.Phylogenetic analysis revealed a shared common last ancestor node between the PVY_Kenya isolate (Gen-Bank accession number PP069009) and isolates from the United States of America (JQ924287), with maximal support at 100% probability.Tis positioning suggests a close evolutionary relationship between PVY_Kenya and its United States of America counterpart, potentially tracing back to a common ancestral lineage rooted in Slovakia.Our fndings indicate a migration trail from South America to Europe, possibly introducing PVY to Europe from South America, with subsequent spread to Africa (Figures 7(a Te current study sought to do a comprehensive phylogenetic analysis of Potato virus Y, utilizing the NCBI Nucleotide Blast of curated sequence dataset comprising the partial sequences of the Kenyan PVY, that saw the amplifcation of the P1 (full sequence) and coat protein region of isolates from the farmer felds and the whole genome of PVY_Kenya.Seven potato plants of diferent cultivars collected across the major potato-producing counties that tested positive for PVY exhibiting foliar mosaic, crinkling, and mottle were collected in seed potato production and were sequenced to reveal the existence of diference in strains with the Kenyan PVY strains.Seven diferent algorithms were used in this study, and we identifed potential recombinant and parental sequences (Figure 3).Tere has been an evolutionary shift detected over the years resulting from the genome recombination of two of the traditionally known strains (PVY O and PVY N ) to new recombinant strains, predominantly PVY NTN and PVY N−Wi/N:O [16,64,65] in diferent parts of the world.
Te spatial population structure of plant pathogens difers from one area to another due to diferent biotic factors like host genetics and abiotic factors, for example, the environment.Extreme resistance (ER) in potato is expressed by Rysto and it averts viral replication without causing cell death because it associates substantially with PVY coat protein.However, when the Potato virus Y coat protein is overexpressed, it can prompt macroscopic hypersensitive reactions in plants, such as tobacco [66].Te molecular mechanism of Rysto-triggered recognition and immunity is poorly understood to date.Toughtfulness of the indestructible mechanisms in the control of viral recognition is important for understanding the resilience of plants to disease resistance after the translation to efective crop protection.Rysto associates directly with PVY CP in plants that are conditioned by the presence of a CP central 149 amino acids domain.Each deletion that afects the CP core region impairs the ability of Rysto to trigger defense [66].
High-throughput sequencing has brought new possibilities to the diversity and complexity of mixed viral infections.Response to Potato virus Y infection difers in diferent potato cultivars described as tolerance (no indications are visible, but unobtrusive yield loss may be documented), resistance (no indicators develop), or susceptibility (indicators appear).Te most cost-efective method to combat PVY infections is the use of resistant plant material.
Virus infection is recognized in plants through RNA silencing in potatoes and other plants as a universal manner for defense against viral attacks, interfering with viral replication in the plant cell.Te frst protein of PVY found to suppress RNA silencing was HCPro.Te P1 protein preceding HCPro in the viral polyprotein may stabilize PVY HCPro and its impact on the suppression of RNA silencing [67].Te relative efciency of antiviral RNA silencing in a potato plant and the counterdefensive suppression of RNA silencing by the virus determine the amount of virus accumulated in infected tissues.
Introgression of resistant genes from wild potato varieties into commercial cultivars has been successful [68,69] even though over time there is resistance breakdown noted from the local cultivars, resulting in new PVY infection.Kenyan farmers have been changing potato cultivars over time due to reasons of preference, and the potato farming industry has noted a shift from cultivars Nyayo and Desiree, which were the major cultivars grown in the 1980s and early 1990s [70] to Tigoni, Nyayo, Timathuti, Dutch Robyjn, Asante, and Shangi varieties being more embraced (MoA/GTZ/PSDA 2009).Tis study supports the fndings in [71] of the main PVY strain prevalence in Kenya to be the recombinant strain type PVY N:O .In the future, however, we recommend the use of modern methods that can precisely identify and diferentiate PVY strains beyond the four main strains of PVY such as described in [72].
Phylogenetic analysis of sequences from several parts of the world retrieved from the GenBank vis-a-vis our Kenyan whole-genome isolate GenBank accession number PP069009 and a USA counterpart JQ924287 revealed a shared common last ancestor node with isolates from Slovakia (Figure 4).Tis displayed maximal support at 100% probability.Tis phylogenetic positioning suggests a close evolutionary relationship between the PVY_Kenya isolate and its counterpart from the United States, potentially tracing back to a common ancestral lineage rooted in Slovakia.Notably, the MT522445.1 sequence of the potato yellow vein virus was included as an outgroup for comparative analysis.Employing the MAFFT alignment tool [42] followed by Bayesian phylogenetic inference through MrBayes [43] within the SATO v 0.1.4pipeline [73], a phylogenetic tree was constructed (Figure 4).We established a strong provision for a migration trail from South America to Europe (Figure 4).Tis can be explained by a probable situation, in which PVY was frst introduced into Europe from South America, through gene fow from the global trade of potatoes and further possibly spread to Africa.
A mosaic-like genetic structure within the PVY Kenya isolate was evident, with prominent multiple illustrations of gene recombination events.A study done by Gibbs [74] revealed the fve distinct phylogroups that all PVY isolates for which complete genome sequences were then known were as a result of recombination from distinct parent phylogroup studies [75,76].Recombination is a signifcant driving force in the evolution and divergence of many plant viruses as supported in the fndings of a study in [77] as evident in Figures 3 and 4. Tis is also demonstrated in Figures 7(a) and 7(b) where acumens of genetic diversity and distribution of PVY strains across diferent continents (America, Africa, Asia, and Europe) have been illustrated.Te diverse array of haplotypes signifes the potential evolutionary divergence or recombination events within the PVY population.Te fndings of this study are similarly consistent with that of a study [74], which observed that genetic variations of this pathogen have geographicallydriven adaptation as a signifcant factor of the diversity of Potato virus Y. Previous studies on PVY in diferent parts of the world share parental history with where it was frst domesticated in the Andean region of South America and spread to other regions through similar historical events [4,34], which has a shared evolutionary background with Potato virus V [78].Studies from [9,79] support most of the viruses (e.g., wild potato mosaic virus, sunfower ring blotch virus, Potato virus V, Peru tomato virus, and Bidens mosaic virus) all of which are from the PVY lineage were isolated from plants native to South America.
Recombinant analysis conducted for the phylogeny of Potato virus Y (PVY) revealed a mosaic-like genetic structure within the PVY Kenya isolate.RNA viruses show exciting evolutionary dynamic forces due to large population sizes, short replication cycles, and high mutation rates [80].

Advances in Virology
Te current study hypothesizes that the PVY_Kenya isolate derives genetic material from multiple parental sequences, as evidenced by the reference sequences obtained from the NCBI database.Tis helps the virus's adaptability to different geographical positions.Phylogenetic positioning and potential historical transmission patterns across continents have been revealed in this study (Figures 7(a Plant pathogens often undergo strong selective pressures that rapidly change depending not only on the vagaries of the ecosystems they inhabit but also on direct inputs from humans [81].High mutation frequencies determined by mutation and ftness produce quasispecies, which is very common among RNA viral species [82,83].Te direction and strength of selection can be calculated and categorized as purifying selection, neutral evolution, or positive selection, depending on values obtained from a study.RNA viruses can quickly adapt to changing selective pressures and new hosts.Tis study revealed pervasive positive/diversifying selection at fve specifc sites and pervasive negative/purifying selection at 267 sites within the Potato virus Y (PVY) genome, each supported by a robust posterior probability (Prob [α > β] >0.9), unveiling a complex interplay of selective forces shaping the genetic landscape of this viral population.Here, the selection pressures within PVY_Kenya branches suggest a more intricate evolutionary history or potentially diferent, undetectable types of selective pressures acting on PVY evolution and a relatively low overall genetic diversity within the PVY population, despite its multicontinental origins.Similar to studies done by Gao et al. [68] on the evolutionary history and global spatiotemporal dynamics of Potato virus Y in Russia suggest South America was a hub for the domestication of potatoes, which later was spread to the rest of the world (Figures 7(a) and 7(b)).Europe has also played a major role in the spread of many potato viruses.However, due to the rate of recombination scenarios within the PVY, further investigations need to be employed to ofer a complete understanding of the evolution of PVY on a global scale.

Conclusion
Potato virus Y impacts negatively on solanaceous crop yield.Despite this, a substantial gap in knowledge is evident, especially regarding the comprehensive genomic status of PVY in Africa, and notably in East Africa.Limited insights into mutations and recombination events within the Kenyan PVY genome compound the outdated understanding of PVY strains afecting potato production in Kenya.Farmers lack vital information on the pathotypes responsible for losses caused by various PVY strains, highlighting the urgent need for further research.Tis study reveals the dynamic phylogeny of PVY, indicating ongoing recombination activities within the genome that could lead to the emergence of new strains.Molecular tools ofer insights into the genomic dynamics of viruses, providing valuable information on PVY's genomic diversity, especially within the economically important virus genus.Our fndings, which showcase the frst molecular footprint of the Kenyan PVY whole genome (GenBank accession number PP069009), enrich scientifc understanding by revealing the molecular phylogeography and evolutionary connections of the PVY_Kenya isolate.Placed within the broader context of global PVY diversity, our study illuminates phylogenetic positioning, historical transmission patterns across continents, and adaptive selection pressures.Tis underscores its potential contribution to PVY's adaptive evolutionary process.Nevertheless, we emphasize the necessity for further investigations into the precise evolutionary dynamics shaping PVY populations in various geographical regions, particularly in Africa.Such endeavors are crucial for advancing tailored management and control strategies to tackle the challenges posed by PVY on a global scale within the scientifc discourse [84,85].

2
Advances in VirologyAdjustments were made as necessary based on troubleshooting eforts.Te monoclonal PVY O/C , PVY C , and PVY N antibodies were employed for diferentiating PVY strains.

Figure 1 :
Figure 1: (a) (A) Healthy potato plant in a farm feld; (B) potato virus Y-positive potato plant; (C) PVY-positive plant (short with stunted growth) next to a healthy plant; (D) potato virus Y-positive potato plant in the feld.(b) Nonsolanaceous host (black nightshade; Solanum nigrum) infected with PVY-source: survey data, Kiambu County, Kenya.

Table 3 :
Description of the PVY type found among sampled feld accessions from Kiambu, Nyandarua, and Nakuru counties of Kenya.

Figure 3 :
Figure 3: Phylogenetic tree of diferent PVY variants identifed in mixed infection reconstructed from partial coat protein sequences and selected reference genomes using MrBayes program, visualized with Fig Tree program.

Figure 4 :
Figure 4: Genetic relatedness and evolutionary connections of the PVY_Kenya isolate within the broader context of global PVY diversity, providing crucial insights into its phylogenetic positioning and potential historical transmission patterns across continents using the MrBayes program, visualized with the Fig Tree program.

10
Advances in Virology diferent recreations under a wide array of demographic circumstances.Analyzing PVY samples from distinct continents-America, Africa, Asia, and Europe-revealed a complex genetic landscape refective of the virus's worldwide distribution and diversity.Te calculated nucleotide diversity (π) of 0.00354881 underscored a relatively low overall genetic diversity within the PVY population, despite its multicontinental origins.Te identifcation of 290 segregating sites, with 53 being parsimony-informative, highlighted substantial variations across the viral genome, showcasing the diverse genetic makeup of PVY strains originating from various countries within each continent.Tajima's D statistic further illuminated the population dynamics, indicating a value of −2.26205, suggesting potential departures from neutral evolution (Table

Figure 5 :
Figure 5: Demonstration of episodic positive/diversifying selection within the Potato virus Y (PVY) genome exhibiting selective pressure at 2066 position, at a (p < 0.05) using the multiple expectation maximizations for Motif Elicitation (MEME) program.

Table 1 :
RNA concentration of 7 PVY (+ve) samples collected from diferent potato farm felds in Kenya.

Table 2 :
Primers names and sequence in the sequence in 5′-3′.