Molecular characterization of Potato virus Y (PVY) and Potato virus V (PVV) isolates naturally infecting cape gooseberry (Physalis peruviana) in Antioquia, Colombia Received for publication: 17 May, 2017. Accepted for publication: 16 February, 2018 Doi: 10.15446/agron.colomb.v36n1.65051 1 Laboratory of Industrial Microbiology, Faculty of Sciences, Universidad Nacional de Colombia. Medellín (Colombia) 2 Faculty of Medicine, Universidad CES, Medellín (Colombia) 3 Laboratory of Cellular and Molecular Biology, Faculty of Sciences, Universidad Nacional de Colombia. Medellín (Colombia). * Corresponding author: mamarinm@unal.edu.co Agronomía Colombiana 36(1), 13-23, 2018 Molecular characterization of Potato virus Y (PVY) and Potato virus V (PVV) isolates naturally infecting cape gooseberry (Physalis peruviana) in Antioquia, Colombia Caracterización molecular de aislamientos del virus Y (PVY) y virus V (PVV) de la papa infectando naturalmente uchuva (Physalis peruviana) en Antioquia, Colombia Natalia Álvarez1, Helena Jaramillo1, Yuliana Gallo1,2, Pablo Andrés Gutiérrez1, and Mauricio Marín3* ABSTRACT RESUMEN Due to the increase of the international demand for functional fruits, cape gooseberry (Physalis peruviana) has become one of the crops of highest expansion in Colombia and the Andean region of South America. Unfortunately, the emergence of fungal and unidentified viral diseases has slowed down the cultivation of cape gooseberry in Colombia and, particularly, in the department of Antioquia. In this work, a next-generation sequencing virome analysis of cape gooseberry plants from eastern Antioquia was performed, using leaves exhibiting symptoms such as mosaics, leaf deformation and greening of veins. The complete genomes of Potato virus Y (PVY) and Potato virus V (PVV) were obtained in the assembled data. The presence of both viruses was confirmed in the samples obtained at two commercial cape gooseberry fields by real time RT-PCR (RT-qPCR) and partial Sanger sequencing of the coat protein (CP). Sequence analysis revealed significant sequence similarity between PVY and PVV isolates infecting P. peruviana to previously identified strains infecting potato (Solanum tuberosum and S. phureja) and tomato (Solanum lycopersicum) in the same geographical region. This study suggests that cape gooseberry could be an alternate host to viruses of other economically important solanaceous crops in the Andean region of South America. Dado el incremento en la demanda internacional por frutas con características funcionales, la uchuva (Physalis peruviana) ha sido uno de los cultivos con mayor expansión en los últimos años en Colombia y otros países suraméricanos. Desafortuna- damente, la emergencia de enfermedades micóticas y virales ha reducido dichos planes de siembra, especialmente en el depar- tamento de Antioquia. En este trabajo, se realizó un análisis de secuenciación de nueva generación del viroma asociado a tejidos foliares de plantas de uchuva del oriente antioqueño, con síntomas de mosaicos, deformación de brotes y verdeamiento de venas. Los genomas completos de los virus Y (PVY) y V (PVV) de la papa fueron obtenidos a partir de las secuencias ensambladas. La presencia de ambos virus fue confirmada en dos cultivos comerciales de uchuva, utilizando RT-PCR en tiempo real y secuenciación Sanger de una porción del gen de la cápside. Los análisis filogenéticos de dichas secuencias revelaron la existencia de altos niveles de similitud entre los aislamientos de PVY y PVV obtenidos en uchuva y cepas previamente identificadas para esta misma región geográfica en cultivos de papa (Solanum tuberosum y S. phureja) y tomate (Solanum lycopersicum). Este estudio sugiere que las plantas de uchuva pueden servir como hospedantes alternos de virus de importancia económica en cultivos de solanáceas de la region Andina de Suramérica. Key words: genomics, potyviruses, Solanaceae, virus diseases. Palabras clave: enfermedades virales, genómica, potyvirus, Solanaceae. properties, which is also a good source of phosphorous, dietary fiber and vitamins A, B and C (Ramadan, 2011). Due to its high content of phenolic acids, f lavonoids and other bioactive compounds with antibacterial, anti-inf lamatory, anti-tumorigenic and antioxidant properties, P. peruviana is also considered to be a functional fruit (Wu et al., 2005; Ramadan, 2011). In Colombia, the cape gooseberry crop comprises a total cultivated area of 952 ha with an estimated Introduction Cape gooseberry (Physalis peruviana L.) is a solanaceous fruit crop native to the South American Andes that has recently become one of the most promising agricultural export trades in Latin American countries such as Co- lombia, Ecuador and Perú (Fisher et al., 2014). The cape gooseberry plant produces a fruit with excellent nutritional http://dx.doi.org/10.15446/agron.colomb.v36n1.65051 14 Agron. Colomb. 36(1) 2018 yield of about 13,260 t per year; the departments of Boyacá, Antioquia and Cundinamarca are the main producers, accounting for 58.4, 17.4 and 17.5 percent of the national production (Agronet, 2016). Recently, the production of cape gooseberry has declined from 13.76 t ha-1 yr-1 in 2010 down to 9.81 t ha-1yr-1 in 2014 (Agronet, 2016). This drop has been attributed to several factors, which include climate change, the increase in the incidence and severity of fungal diseases caused by Fusarium oxysporum and Phoma sp. (Fisher et al., 2014) and the infection by several viruses inducing chlorosis, mosaics, leaf deformation, dwarfism and greening of veins (Zapata et al., 2005; Aguirre et al., 2014; Gutiérrez et al., 2015; Rodríguez et al., 2016). P. peruviana can be host to a wide range of viruses such as tobamovirus (Tobacco mosaic virus, TMV) (Capoor and Sharma, 1965; Gómez et al., 1997), polerovirus (Potato leafroll virus, PLRV) (Natti et al., 1953), cucumovirus (Cu- cumber mosaic virus, CMV) (Chamberlain, 1939; Gupta and Singh, 1996; Daza and Rodríguez, 2006), potexvirus (Potato virus X, PVX) (Horvath, 1970; Zapata et al., 2005; Gutiérrez et al., 2015), crinivirus (Tomato chlorosis virus, ToCV) (Trenado et al., 2007), tospovirus (Tomato chlorotic spot virus, TCSV and Tomato spotted wilt virus, TSWV) (Da-Graça et al., 1985; Eiras et al., 2012), several potyvi- ruses (Peru tomato mosaic virus, PTV; Colombian datura virus, CDV; Potato virus Y, PVY and Bean yellow mosaic virus, BYMV) (Horvath, 1970; Salamon and Palkovics, 2005; Kaur et al., 2014; Kisten et al., 2016; Cutler et al., 2018) and the viroid Potato spindle tuber viroid (PSTVd) (Hadidi et al., 1976; Verhoeven et al., 2010). The capacity of Physalis species to serve as virus hosts was investigated by Horváth (1996), who demonstrated the susceptibility of P. alkekengi to 10 viruses, P. ixocarpa to 14 viruses and P. pubescens to two viruses. This work also showed that P. peruviana is systemically susceptible to Alfalfa mosaic vi- rus (AMV), Potato aucuba mosaic virus (PAMV) and PVY, and locally susceptible to Tobacco rattle virus (TRV). The role of P. peruviana as an alternate host of viruses affect- ing economically important crops has been demonstrated for PVY, PSTVd, ToCV in tomato (Solanum lycopersicum) (Trenado et al., 2007; Verhoeven et al., 2009; Kisten et al., 2016) and CDV in tobacco (Nicotiana tabacum) (Salamon and Palkovics, 2005). In the Andean region of Colombia, cape gooseberry is frequently inter-cultivated with other solanaceous crops, such as tamarillo (S. betaceum), tomato, bell pepper (Cap- sicum annuum) and potato (S. tuberosum and S. phureja) and can also grow as a weed within these crops (Fischer et al., 2014). A recent next generation sequencing (NGS) study suggested that P. peruviana could be a natural reservoir host of PVX (Gutiérrez et al., 2015); in this work, the role of cape gooseberry as an alternative virus host to other solanaceous crops in the municipality of La Unión (Antioquia) was furtherly investigated using NGS and RT- qPCR tests on cape gooseberry plants exhibiting mosaics, leaf deformation and greening of veins. Materials and methods Sample collection Ten samples were collected at two commercial cape goo- seberry plots in the municipality of La Unión (Antioquia) (5°58´22´́ N, 75°21́ 40´́ W and 2500 m a.s.l.), where some plants exhibited typical symptoms of viral infection. In the first plot, rugose mosaic and leaf deformation symp- toms were detected in leaves; in the second plot, rugose mosaics and greening of veins were observed (Fig. 1). Six FIGURE 1. Symptoms of viral infection in the cape gooseberry leaves observed in this work. Rugose mosaics (A and B), greening of veins (C) and leaf deformation (D). A B C D 15Álvarez, Jaramillo, Gallo, Gutiérrez, and Marín: Molecular characterization of Potato virus Y (PVY) and Potato Virus V (PVV) isolates naturally infecting cape gooseberry additional asymptomatic leaf samples were included for the RT-qPCR tests. Next generation sequencing High-throughput sequencing of the P. peruviana transcrip- tome was performed on a bulked sample of symptomatic leaves. The total RNA was extracted by the Trizol method (Chomczynski, 1993) following the manufacturer’s ins- tructions (ThermoFisher Scientific, Waltham, MA, USA) and the integrity was determined with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The ribo- somal RNA was removed with the TruSeq Stranded Total RNA with a Ribo-Zero Plant kit (Illumina, San Diego, CA, USA). The TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) was used for the preparation of cDNA libraries and the ligation of adapters. Sequencing was performed with the Illumina HiSeq 2000 system service provided by Macrogen (South Korea). Once the sequencing data was obtained, adapter sequences and low quality bases (Phred score < 30) were removed from the dataset with SeqTK v.r82 (GitHub, 2015). Sequences were assembled with Trinity (Grabherr et al., 2011) and viral contigs were identified with a local BLASTN (Gish and States, 1993) search against a database containing all plant virus species currently accepted by the International Committee on Taxonomy of Viruses (ICTV). Genome as- semblies were verified by mapping them against the reads with Bowtie2 (Langmead and Salzberg, 2012) and checked for inconsistencies and assembly artifacts with Tablet (Milne et al., 2010). Protein coding regions were annotated using BLASTX (Gish and States, 1993) against reference PVV (NC _004010) and PVY (NC _001616) genomes. Putative protease cleavage sites were confirmed by com- parison to published P1, HC-Pro and NIa-Pro consensus cleavage sites (Adams et al., 2005). The complete genome sequences were deposited in GenBank under accession codes KY711363 and KY711364 with PVY_physalis and PVV_physalis as isolate names, respectively. RT-qPCR tests Primer specifity was first evaluated in bulk samples com- prising either symptomatic (SL1 or SL2) or asymtomatic (AL1 and AL2) leaves collected at each plot. For these bulk samples, the total RNA was extracted from 100 mg of ground tissue with the GeneJET Plant RNA Purification kit following the manufactureŕ s protocol (ThermoFisher Scientific, Waltham, MA, USA) and eluted in 40 μL of DEPC treated water. Purity and concentration were deter- mined by absorbance readings at 260 and 280 nm using a Nanodrop 2000C (ThermoFisher Scientific, Waltham, MA, USA). Synthesis of cDNA and RT-qPCR were performed using the method reported by Muñoz-Escudero et al. (2016a). PVY was detected with the primers PVY-1 FP (5́ -CCAATCGTT- GAGAATGCAAAAC-3´) and PVY-1 RP (5 -́ATATAC- GCTTCTGCAACATCTGAGA-3́ ) (Singh et al., 2013) after amplifying a 74 bp segment of the CP region. The primers PVV_phu_F (5’-ATGCTGGAAAAGATCCAGC-3’) and qPV V_phu _R (5’-CATCCCGCTCCTCA AC-3’) were used to target an 89 bp region of CP for PVV (Álvarez et al., 2016). After primer validation, each leaf sample was tested indi- vidually by Immunocapture Real-Time RT-PCR (IC-RT- qPCR) using an antigen-coated ELISA plate (ACP-ELISA, SRA 27200/0096) containing the PTY 1 monoclonal anti- body for a generic detection of the potyvirus. Positive (LPC 27200) and negative (LNC 27200) controls were purchased from Agdia (Elkhart, IN, USA). Absorbance was measured at 405 nm in a Multiskan plate reader (ThermoFisher Scientific, Waltham, MA, USA). Samples were considered positive for ELISA when the absorbance value has higher than the cut-off value defined by the formula: Cut-off = (average OD405 + 3 s.d.) x 1.1 as recommended by Bioreba (Reinach, Switzerland). For the RT-qPCR step, virus particles were released from the ELISA plate with 70 μl of a 10 mM Tris-HCl buffer (pH 8.0) containing 1% Triton X 100 and incubated at 70°C for 10 min (Wetzel et al., 1992). RT-qPCR reactions included a negative control lacking template cDNA and a positive control containing cDNA from infected potato leaf tissue. Samples were considered positive after exhibiting f luorescence values higher than the threshold before the 35th cycle. Amplicon specificity was verified by High Resolution Melting (HRM) in the 50 and 99°C range and confirmed by Sanger sequencing of three samples plus the positive control. To confirm the phylogenetic affinity of PVY and PVV isolates from P. peruviana, the RT-PCR amplification was performed on three positive RT-qPCR samples using primers to target the CP region. These amplicons were sequenced afterwards. The RT-PCR reaction was per- formed following the procedure reported by Henao-Díaz et al. (2013) with the primers PV YCPF (5´-ACCAT- CAAGSAAATGACACA-3´) and PVYCPR (5́ -CGGAGA- GACACTACATCACA-3´) (Glais et al., 2002) for PVY. For PVV, the primers PVV_phu_F and PVV_phu_R 16 Agron. Colomb. 36(1) 2018 (5’-TGAAAGTGGGCTTTGCG-3’) were used instead (Álvarez et al., 2016). In each case, amplicons of the ex- pected size were obtained (PVY: 801 pb; PVV: 459 pb). Samples were gel purified using the QIAquick Gel Ex- traction kit (Qiagen, Hilden, Germany) and sequenced at Macrogen using an ABI Prism 3730xl sequencer (PE Ap- plied Biosystems, Foster City, CA, EEUU). PVY and PVV CP partial sequences were deposited in GenBank under accesion codes KY711356-62. Phylogenetic analyses Phylogenetic trees using the polyprotein coding segments of PVY and PVV were inferred by the Maximum Likeli- hood method using the General Time Reversible model (Nei and Kumar, 2000) and were modelled with a discrete Gamma distribution with 5 categories plus invariable sites and a gamma parameter of 1.02. The phylogenetic analy- sis using the polyprotein coding segments of PVY and PVV was inferred by the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993) with a discrete Gamma distribution of 5 categories plus invariable sites and a gamma parameter of 1.42. Positions with less than 95% site coverage were eliminated in each case. Nucleotide substitution models were selected with MODELTEST (Posada and Crandall, 1998) and sequences were aligned with MUSCLE (Edgar, 2004). Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). Results and discussion Next generation sequencing Sequencing of the P. peruviana transcriptome resulted in a paired-end library of 13,420,698 reads (101nt/read) for a total of 1,355,490,498 sequenced nucleotides. Two poty- viruses, Potato virus Y (PVY) and Potato virus V (PVV) were identified in the assembled data. The PVY contig (PVY_physalis) comprised 9,675 nt and had an average sequence depth of 1,683x (Fig. 2A). A total of 219,155 reads were mapped after the PVY assembly for an abundance of 1,679 reads per kilobase per million reads (RPKM). Forty one polymorphic sites (39 transitions and 2 transversions) were identified in the assembled genome. The sequence identified as PVV (PVV_physalis) corresponded to a contig of 9,832 nucleotides assembled from 108,629 reads (823 RPKM) and an average sequence coverage of 1,091x (Fig. 3A). In contrast to PVY_physalis, the PVV_physalis assembly did not contain any polymorphic sites. Characterization of the PVY_physalis isolate The ORF encoding the potyviral polyprotein in PVY_ physalis was identified at the nucleotide positions 169-9,354, and corresponds to a protein of 3,061 a.a. (Fig. 2A). The PVY_physalis polyprotein is cleaved into a ten mature proteins product by the action of potyviral proteases. The P1 and HC-Pro cleavage sites were identified at positions 284 (RRMVQF/S) and 740 (KHYRVG/G), respectively. P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP are cleaved by NIa-pro at the positions 1,105, 1,157, 1,791, 1,843, 2,031, 2,275 and 2,794, respectively, These are the sites with the consensus sequence V-x-[HE]-[QE]/[AGSR] (Revers and García, 2015). The GA7T sequence inducing frameshift protein product P3N-PIPO (247 a.a.) was identified at the nucleotide position 2,899 within the P3 segment (Chung et al., 2008) (Fig. 2A). Six nucleotide polymorphisms in the PVY_physalis assembly were translated into amino acid changes within the HC-Pro (T326A, I335M), 6K1 (V1118I), VPg (G1944S, H1964N), and CP (A2809E) segments. A BLASTN search against the complete nucleotide collection at GenBank revealed that PVY_physalis is closely related to the isolates LaUnionT (99.3%, KX531041), mar7 (99.1%, KR270797), VarA (98.7%, KT290511) and VarB (96.7%, KT290512) that are infecting S. tuberosum and S. lycoper- sicum in the department of Antioquia (Muñoz-Baena et al., 2016; Muñoz-Escudero et al., 2016a, b). A phylogenetic analysis of complete PVY genomes clus- tered the sequences into well-defined clades corresponding to the strains PVYN, PVYC, PVYO and PVYNP plus the recombinant strains PVYNTN and PVYN:O/N-Wi (Fig. 2B). PVY_physalis was part of a clade that includes some of the Colombian isolates identified using BLAST. This clade is sister to a group comprising isolates RRA-1, SASA-61, NTNHO90, NTND6 and NTNO92, which are non- recombinant PVY strains, but some of them have shown to induce the tuber necrotic ringspot disease in potato (Lorenzen et al., 2006; Ogawa et al., 2008). PVY_physalis is clearly different from recombinant PVY isolates identified in potato crops in northern (Yarumal_varB) and eastern Antioquia (La_Union) (Muñoz-Escudero et al., 2016a, b). A phylogenetic analysis using partial CP sequences revealed a similar topology for the complete genome tree with some clades collapsing as a result of recombination as shown in a previous research (Karasev and Gray, 2013). The PVY_physalis is different to isolate PVY-KZNU from South Africa, and it was identified in P. peruviana plants exhibiting mottling, mosaic, and chlorosis symptoms on a tomato farm moderately infested with cape gooseberry weeds (Kisten et al., 2016). The PVY-KZNU was identified as a recombinant PVYC  strain with spliced PVYO-type RNA fragments in the coat protein region (Kisten et al., 2016), a result that agreed with the phylogenetic analysis performed in this research (Fig. 2C). The identification of 17Álvarez, Jaramillo, Gallo, Gutiérrez, and Marín: Molecular characterization of Potato virus Y (PVY) and Potato Virus V (PVV) isolates naturally infecting cape gooseberry C ov er ag e Position (nt) 3000 1000 2000 0 0 600040002000 8000 10000 A B C QFVHHQ/A DVVVEQ/A 2389 YEVHHQ/G 8551 9675 9354 3´ 66946262569855423640 HEVRHQ/SDSMVQF/S P3N/PIPO YDVRHQ/R 3484 ETVSHQ/G QEVEHE/A KHYRVG/G 1021169 5´ CPNlbP1 Nla-ProVPg6K 1 P3HC-Pro Cl 6K 2 PVY_phureja PVY_physalis (KY711363) LaUnionT (KX531041) VarA (KT290511) varB (KT290512) RRA-1 (AY884984) NTNO92 (AB331519) SASA-61 (AJ585198) NTNHO90 (AB331517) mar7 (KR270797) NTND6 (AB331515) 11439 (KC634005) ALF-VI (JQ924287) PVY yarumal (KT336551) Foliar La Union (KR149260) Yarumal varB (KT336552) HR1 (FJ204166) HC-2 quan (HM590406) PB209 (EF026076) LW (AJ890349) 261-4 (AM113988) PRI-509 (EU563512) N (X12456) N-Egypt (AF522296) P07 (U09509) Wilga (EF558545) Oz (EF026074) JVW-186 (KF770835) Adgen (AJ890348) Mb-112 (AY745491) 0.02 SL11 (KY711362): P. peruviana VarA (KT290511): S. lycopersicum SL2 (KY711360): P. peruviana LaUnionT (KX531041): S. tuberosum PVY_physalis (KY711363): P. peruviana AL21 (KY711361): P. peruviana mar7 (KR270797): S. lycopersicum NTNHO92, Japan (AB331519) RRA-1, Canada (AY884984) NTND6, Japan (AB331515) NTNHO90, Japan (AB331517) SASA-61, UK (AJ585198) varB (KT290512) La Unión 1 (KR902698) C+ (KY711359) Yarumal (KT336551) La Unión (KR149260) Yarumal varB (KT336552) La Unión 2 (KR902699) La Unión 4 (KR902701) La Unión 6 (KR902703) 40MY (HQ335260) HR1, USA (FJ204166) HC-2 quan, China (HM590406) ALF-VI, Brazil (JQ924287) La Unión 5 (KR902702) 11439, UK (KC634005) JVW-186, South Africa (KF770835): C. annuum KZNU, South Africa (KU695256): P. peruviana PRI-509, Netherlands (EU563512) Adgen, France (AJ890348) LW, Poland (AJ890349) P07, Canada (U09509) N-Egypt, Egypt (AF522296) (N)W, Poland (EF558545) N, France (X12456) Oz, USA (EF026074) 261-4, Germany (AM113988) Mb-112, Canada (AY745491) PB209, USA (EF026076) La Unión 3, Colombia (KR902700) N /N TN N P C ol om bi a S . t ub er os um C ol om bi a S . t ub er os um O C N :O /N -W i 95 99 100 100 100 100 66 72 100 66 97 52 94 99 99 100 100 85 70 98 0.01 83 51 8757 70 90 99 89 89 98 85 52 97 100 52 FIGURE 2. Genome annotation of the PVY_phureja genome and the phylogenetic relationship to other PVY isolates. A) Sequence coverage of PVY_ phureja assembly and location of some important molecular features. The relative position of each mature protein with their corresponding protease cleavage sites is shown. Phylogenetic analysis of PVY isolates using complete (B) and partial CP (C) sequences confirms the close relationship between PVY_physalis and non-recombinat PVY isolates infecting S. lycopersicum and S. tuberosum in eastern Antioquia. Trees are drawn to scale with branch lengths in units of number of base substitutions per site as indicated in the bar at the bottom. Bootstrap values are shown above the branches. 18 Agron. Colomb. 36(1) 2018 these different PVY variants clearly demonstrates that P. peruviana can serve as a reservoir to PVY strains infecting other solanaceous crops of economic importance such as tomato and potato. So far, and according to different sources, this genome represents the first complete PVY sequence naturally infecting P. peruviana. Characterization of PVV_physalis The PVV_physalis polyprotein (3,065 a.a.) was encoded at nucleotide positions 186-9,383. P1 and HC-Pro cleavage sites were identified at positions 289 (RRMVQF/S) and 745 (IKHRVG/G), respectively. NIa-Pro cleavage sites contained the same V-x-[HE]-[QE]/[AGSD] motif obser- ved in PVY_physalis and were located at the amino acid positions: 1102 (P3), 1,154 (6K1), 1,788 (CI), 1,840 (6K2), 2,028 (VPg), 2,274 (NIa-Pro) and 2,793 (NIb). P3N-PIPO (231 a.a.) is predicted to result from the frameshifting at the GA7C sequence at the nucleotide position 2,907 with the P3 coding region (Chung et al., 2008) (Fig. 3A). The closest homologs to PVV_physalis in GenBank are the PVV isolates La Union_varA (99.9%, KT985458), phureja (99.8%, KP849483) and La Union_varB (99.6%, KT985459) infec- ting S. phureja in Antioquia (Álvarez et al., 2016; Gutiérrez et al., 2016), isolate KER.LAL.P (83.6%, KC433411) from Iran and isolate DV 42 (83.0%, AJ243766) from Scotland (Oruetxebarria et al., 2000; Shamsadden-Saeed et al., 2014). A comparison of PVV polyproteins reveals two aminoacid changes unique to PVV_ physalis. In the first change, at the position 1,393 within the CI, a serine residue replaced a threonine/alanine observed in the PVV isolates infecting potato; this was followed by a second change at the position 2,903 within the CP, where a glutamic acid observed in the PVV strains infecting potato was replaced by lysine in the PVV_physalis. A phylogenetic analysis of PVV genomes confirms that the PVV_physalis isolate has higher affinity to the PVVphu lineage infecting S. phureja in eastern Antioquia and, more distantly, to the PVV isolates DV42 and KER.LAL.P infecting S. tuberosum in Eurasia (Scotland and Iran). Both clades are cleary differentiated and supported by a 100% bootstrap (Fig. 3B). The phylogenetic analysis of the partial CP sequences was in agreement with the complete genome analysis. Again, the CP sequences isolated from P. peruviana (PVV_physalis, SL1 and SL2) formed a dis- tinct clade, along with the sequences infecting S. phureja (PVV_phureja) (Fig. 3C). All these sequences have been iso- lated in the municipality of la Unión in eastern Antioquia. The S. phureja / P. peruviana group is sister to a divergent PVV isolate identified in S. tuberosum cv. Papa Amarillo, which has distinct serological properties from other isolates infecting S. tuberosum (Shiel et al., 2004). The PVVphu  genetic lineage was first identified in a 454 GS-FLX pyrosequencing study of S. phureja root tissue in Colombia (Gutiérrez et al., 2014) and its existence was con- firmed with follow up genome sequencing studies (Álvarez et al., 2016; Gutiérrez et al., 2016). The PVVphu lineage was originally believed to be exclusive to S. phureja, but its detection here in P. peruviana suggests that this lineage might be present in a wider range of hosts. Finally, a recently discovered PVV strain, TamarilloEc, was found to infect tamarillo (S. betaceum) in Ecuador (Insuasti et al., 2016) and proposed to be the first PVV isolate infecting a host different from potato; our analysis contradicts this claim as PVV_TamarilloEc seems to be more closely related to the Ecuatorian rocoto virus than to any member of the PVV group. We believe that this strain has been misclasified and should be renamed. Detection of PVV and PVY by RT-qPCR Infection of P. peruviana by PVY and/or PVV was confir- med by RT-qPCR using specific primers for each species. In a preliminary experiment, the amplification reaction was performed on total RNA extracted from bulks containing either symptomatic (SL1 and SL2) or asymptomatic (AL1 and AL2) samples from each plot (L1 and L2) (Tab. 1). PVY was detected in all four samples with Ct values between 9.97 and 27.12 and very similar melting tempe- ratures (77.5±0.5°C). A Ct of 13.39 was observed in the potato sample used as positive control with slightly lower Tm (76.48), (Fig. 4A). Individual amplification reactions using IC-RT-PCR confirmed the PVY results using the bulked samples. In this case, all samples tested positive, with higher Ct values (26.55-32.47) but with the same distribution of Tm (77.5±0.5°C). Regarding symptoms, leaves exhibiting mosaics (S1-L1 and S1-L2) had the lowest Ct values (26.55-27.52), followed by the samples with mottling (S2-L1, Ct=28.60) and greening of veins (S2-L2, Ct= 28.75). As expected, the highest Ct values were observed in the majority of asymptomatic samples (28.30-32.47) (Tab. 1). Previous work on PVY infecting potato (Medina et al., 2016) and tomato (Muñoz-Baena et al., 2016) in Colombia by RT-qPCR using the same primers reported in this research also resulted in similar Tm values (77.5°C±0.5°C). The identity of RT-qPCR amplification product was confirmed by the Sanger sequencing of four samples which were identical to the CP region of PVY iso- lates from Colombia and Cuba isolated from potato, tama- rillo, tomato and pepper (KT290511, JF939837, HQ335262, HQ335245, KY050811). 19Álvarez, Jaramillo, Gallo, Gutiérrez, and Marín: Molecular characterization of Potato virus Y (PVY) and Potato Virus V (PVV) isolates naturally infecting cape gooseberry In contrast to PVY, the incidence of PVV was lower in both plots. PVV was detected in the asymptomatic and symptomatic bulks from the first plot but tested negative in both samples from the second plot. Surprisingly, a lower Ct value was observed in the asymptomatic sample (A-L1, Ct=22.38) than in the symptomatic one (S-L1, Ct=28.69) (Fig. 4B). Tm values were in good agreement with the posi- tive control suggesting a sequence similarity. IC-RT-PCR C ov er ag e Position (nt) 2000 1000 0 0 600040002000 8000 10000 A CB QFVHHQ/G DEVREQ/G 2421 YEVRHQ/A 8565 9832 9383 3´ 70086270570655493648 SERRHQ/SRRMVQF/S P3N/PIPO ENVMHQ/D 3492 NSVVHQ/G ENVVHE/S IKHRVG/G 1053186 5´ CPNlbP1 Nla-ProVPg6K 1 P3HC-Pro Cl 6K 2 PVY_phureja PVV_physalis (KY711364) La Unión VarB (KT985459) La Unión VarA (KT985458) DV42 (AJ243766) KER.LAL.P (KC433411) Wild potato mosaic virus (AJ437279) Peru potato mosaic virus (AJ437280) Brugmansia suaveolens mottle virus (AB551370) Phujera (KP849483) Pepper mottle virus (EU586131) 0.2 C+, Colombia (KY711356): P. phureja SL1, Colombia (KY711357): P. peruviana Sam-06, UK (GU144343) Suomi, Finland (AJ278894) M97, UK (AJ253123) Sam-09, UK (GU144341) DV42, UK (AJ143766) 508 Dutch, Netherlands (AJ253121) M95, UK (AJ253122) R Norwegian, Norway (AJ253124) 506 Dutch, Netherlands (AJ253120) 508 Dutch, Netherlands (AJ253119) Sam-17, UK (GU144339) UK (X61279) PVV_physalis, Colombia (KY711364): P. peruviana P V V S . p hu je ra / P. p er uv ia na S . t ub er os um 98 100 100 100 100 100 99 0.05 60 99 61 73 98 82 95 100 85 Swedish, Sweeden (AJ516024) 136, UK (GU144337) 147, UK (GU144344) KER.LAL.P, Iran (KC433411) KER.ZAR.1, Iran (KC438301) KER.ZAR.3, Iran (KC438303) PA1.3, Peru (AJ516023) 140, UK (GU144333) PA1.0, Peru (AJ516021) PA1.1, Peru (AJ516022) PA-4, USA (AY521595): S. tuberosum cv Papa Amarillo SL2, Colombia (KY711358): P. Peruviana Colombia, Colombia (KP849484): S. phureja La Union varB, Colombia (KT985459): S. phureja Phujera, Colombia (KP849483): S. phureja La Union varA, Colombia (KT985458): S. phureja Ecuadorian rocoto virus, Ecuador (EU495234): C. pubescens PVV Tamarillo Ec, Ecuador (KT803903): S. betaceum 100 100 73 73 FIGURE 3. Genome annotation of the PVV_phureja genome and phylogenetic relationship to other PVV isolates. A) Sequence coverage of PVV_phu- reja assembly and location of some important molecular features. The relative position of each mature protein with their corresponding protease cleavage sites is shown. Phylogenetic analysis of PVV isolates using complete (B) and partial CP (C) sequences confirms the close relationship between PVV_physalis and PVV isolates infecting S. phureja. Trees are drawn to scale with branch lengths in units of number of base substitutions per site as indicated in the bar at the bottom. Bootstrap values are shown above the branches. 20 Agron. Colomb. 36(1) 2018 results differed from the RT-qPCR as only one sample in the first plot (S2-L1, Ct=26.83) tested positive for PVV, contrasting three positive samples in the second plot (A2- L2, A3-L2 and S1-L2) (Fig. 4B). There was no significant correlation between the symptoms and Ct values for PVV. The Tm values of 80±0.5°C are in good agreement with a previous study on PVV isolates infecting S. phureja isolates in Colombia (Álvarez et al., 2016). Sequencing of RT-qPCR amplicons reveals a nucleotide sequence identity between 95 to 99% to PVV GenBank accesions KT985459, KT985458 and KC438304. The natural ocurrence of potyviruses in cape gooseberry was first reported in Hawaii in 1953 (Sakimura, 1953) and later confirmed by serological and electron microscopy studies in India (Prakash et al., 1988). P. peruviana has been thoroughly shown to be an alternate host to several viruses of tomato (Trenado et al., 2007; Verhoeven et al., 2010; Kisten et al., 2016), tobacco (Schubert et al., 2006) and potato (Prakash et al., 1988; Gutiérrez et al., 2015) and there is an increasing number of reports of viruses infecting P. peruviana in comercial plots all over the world such as the tospovirus TCSV in Rio Grande do Sul State of Brazil (Eiras et al., 2012) and the potyvirus BYMV in Barabanki (India) (Kaur et al., 2014). In Colombia, PVY was first identified in the department of Cundinamarca in a study of P. peruviana plants with leaf mosaics and mottling in 2006 (Daza and Rodríguez, 2006). In the mentioned research, PVY was detected using a combinantion of serological assays and electron microscopy and it was demonstrated to be transmitted through the aphid Myzus persicae acting as vectors from the cape gooseberry plants and by mechanical C+ (80.04°C) C- AL2 SL2 AL1 (79.87°C) SL1 (79.48°C) C+ (76.48°C) C- AL2 (77.64°C) SL2 (77.38°C) AL1 (77.29°C) SL1 (77.16°C) Temperatures (°C) dF /d T B Temperatures (°C) dF /d T 55 60 65 70 80 85 90 9575 Cycle number Fl uo re sc en ce 0.0 0.1 0.2 0 5 10 15 20 25 30 35 PVV Cycle number Fl uo re sc en ce A 0.0 0.2 0.4 0.6 PVY 0 5 10 15 20 25 30 35 55 60 65 70 80 85 90 9575 C-C+AL2 AL1SL2SL1 FIGURE 4. Molecular detection of Potato virus Y (PVY) and Potato virus V (PVV) in P. peruviana samples from La Unión (Antioquia). RT-qPCR ampli- fication curves of PVY (A) and PVV (B) of symptomatic and asymptomatic leaf samples SL1-2 and AL1-2. Corresponding melting curve profiles are shown to the right with Tm values indicated in parentheses. 21Álvarez, Jaramillo, Gallo, Gutiérrez, and Marín: Molecular characterization of Potato virus Y (PVY) and Potato Virus V (PVV) isolates naturally infecting cape gooseberry infection to indicator plants. A later investigation also detected potyvirus infecting cape gooseberry plants in the municipality of Mosquera (Cundinamarca) using generic antibodies for aphid transmitted potyvirus and confirmed their results by electron microscopy (Aguirre et al., 2014). Based on ELISA tests, PVY was also recently reported in three P. peruviana samples from Cundinamarca and Boyaca (Cutler et al., 2018). The great diversity of viruses shown to infect cape goose- berry highlights the importance to continue the virome research on this host, including different geographical regions and growth conditions such as mixed cropping, crop rotation systems and even considering this species as a weed for other crops. Our results support the notion that mixed cultivation of P. peruviana with other solanaceous plants should be avoided and its presence as weed should be controlled as vectors transmitting potyviruses, such as M. persicae, Aphis gossypii and Macrosiphum euphorbiae are insects frequently associated with P. peruviana (Afsah, 2015). Future work should address the cross pathogenici- ty of PVV and PVY in the South American Andes and other places where there is coexistence between P. peruviana and other solanaceous crops as well as their effect on yield, plant longevity and physicochemical properties of the cape goosberry fruit. Conclusions The analysis of next generation sequencing data from P. pe- ruviana leaf samples and the symptoms of the viral disease revealed an infection caused by the potyviruses PVY and PVV in the municipality of La Unión (Antioquia). These re- sults were confirmed by real time RT-PCR (RT-qPCR) and the Sanger sequencing of the capsid region. Phylogenetic analysis confirmed these potyvirus isolates to be closely related to PVY and PVV isolates identified previously in tomato and potato crops in Antioquia, respectively, which suggests that cape gooseberry could be an alternate host to viruses of other economically important solanaceous crops in the Andean region of South America. Acknowledgments This work was supported by The Universidad Nacional de Colombia (VRI Grant 34579) and Colciencias (706-2015, VRI project 30498). TABlE 1. RT-q PCR detection of Potato virus Y (PVY) and Potato virus V (PVV) in leaf samples from P. peruviana plots in Antioquia (Colombia). RT-qPCR (Total RNA) PVY PVV Sample name Type of sample Ct* Tm** Ct Tm C- >35 C+ Infected Potato leaf 13.39 76.48 80.04 4.66 SL1 Symptomatic bulk 9.97 77.16 28.69 79.48 SL2 Symptomatic bulk 26.37 77.38 >35 AL1 Asymptomatic bulk 27.12 77.29 22.38 79.87 AL2 Asymptomatic bulk 12.49 77.64 >35 RT-qPCR (Immunocapture) PVY PVV Sample name Type of sample Ct* Tm** Ct Tm C- >35 C+ Infected Potato leaf 27.02 76.99 5.02 79.78 A1-L1 Asymptomatic 30.28 77.29 >35 A2-L1 Asymptomatic 31.07 76.56 >35 A3-L1 Asymptomatic 28.30 77.46 >35 S1-L1 Mosaic 26.55 77.35 >35 S2-L1 Mottling 28.60 77.39 26.83 79.27 A1-L2 Asymptomatic 30.48 77.56 >35 A2-L2 Asymptomatic 31.21 77.34 29.97 78.62 A3-L2 Asymptomatic 32.47 77.29 26.82 79.79 S1-L2 Mosaic 27.52 77.51 28.59 79.44 S2-L2 Greening of veins 28.75 77.64 >35 *Threshold cycle. **Melting temperature. 22 Agron. Colomb. 36(1) 2018 literature cited Adams, M.J., J.F. Antoniw, and F. Beaudoin. 2005. 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