First report of pepper virus A infecting potato in Egypt

Despite the importance of potatoes to the Egyptian economy, which comprise 14% of Egypt's agricultural exports, (Elawady & Abdulkheir, 2015), virus diversity studies in the main production area, the Delta region, remain limited (Elwan et al., 2017), with only one study using high-throughput sequencing (HTS) exploring the threats more broadly (Elwan et al., 2023). This study aimed to use HTS to evaluate the presence of previously undiscovered viruses in most of the potato-producing areas in the Delta region. Diseased potato samples (three per location) were collected from 33 locations in the 2022 and 2023 cropping seasons, pooled (0.1g from each sample) and the total RNA was extracted (NucleoSpin® RNA Plant isolation kit; Macherey-Nagel, Germany). Illumina MiSeq libraries were prepared using the TruSeq® Stranded Total RNA Library Prep Plant kit (Illumina, USA). Sequence data is recorded under NCBI Bioproject: PRJNA1180791. A new virus was detected only in Mansoura City (Sample 26, Dec 2022); in this field, the potate leaves were observed to have a mosaic pattern (Fig. 1). For this sample, Illumina sequencing generated 1,442,884,300 nt pair-end reads (NCBI, SRA: SRR30598214). Using the Angua pipeline, three viruses were identified in a mixed infection: alfalfa mosaic virus (AMV), pepper virus A (PepVA) and potato virus Y (PVY). Reanalysis in Geneious Prime® 2022.2.2 (medium sensitivity) revealed that 24 reads aligned with 91.4% identity to the AMV RNA3 segment of isolate FRE (GenBank Accession No. KY549685), 66 reads aligned with 83.5% identity to PepVA, isolate IND-SHM (MH725809) and 49 reads aligned with 93% identity to PVY, isolate Egypt7 (KY863549). While AMV and PVY aligned closely with Egyptian isolates (Elwan et al., 2017), this is the first record of PepVA in Egypt and the first in potatoes globally. For PepVA confirmation, SuperScript™ VILO™ cDNA Synthesis Kit (Thermo Fisher Scientific, USA) was used for cDNA synthesis and MyTaq™ Red Mix (SLS Ltd., UK) for PCR amplification of the coat protein gene (ORF4) and putative nucleic acid binding protein gene (ORF5) using primers 18F: 5′-TGTGTGTATGCCTTAGGTTAAGTGGCCGTG-3′, 18R: 5′-CGCTGGGTCTTTCTCAAGTAATCACGCAGG-3′, 19F: 5′-AAACAACATGGCTAGTGCAGAAGAGATGGC-3′, 19R: 5′-GCAATACGCTTGTAAGTGTTGTGCGCTACC-3′, 20F: 5′-GGCACAGAGGTAACTGGAGGTAGACTGGGT-3′ and anchored Oligo dT (IDT Inc., USA). PCR products were purified (QIAquick® PCR Purification & Gel Extraction Kits; Qiagen, Germany) and Sanger sequenced (Eurofins Scientific, Luxembourg). Assembled Illumina and Sanger sequences were mapped to a PepVA reference genome (MH725809) using Geneious Prime® and the full gene sequences were submitted to GenBank (PQ607733, Egy-Mn isolate). Pairwise alignment with the four published PepVA isolates showed that the Egy-Mn isolate had 94.9% identity with PQ360725 and MH725809 (India, aubergine) and 83.9% with KU923763 and KU726694 (India, chilli pepper). A Maximum Likelihood phylogenetic tree was built based on the four ORFs for all published PepVA isolates using MEGA11 (Tamura et al., 2021) (Fig. 2). PepVA (genus Carlavirus) has been reported to infect three solanaceous crops, aubergine, pepper and tomato (Jo et al., 2017; Karnawar et al., 2021), causing mosaic and mottling symptoms in some aubergine varieties, as well as mosaic and leaf rolling in tomatoes. This is the first detection of PepVA in potatoes and in the Middle East indicating a larger than previously reported host range and highlighing the importance of further studies to understand the impact on this important crop. This work was funded by the Ministry of Higher Education of the Arab Republic of Egypt as a full PhD scholarship [Bureau ID: MM11/21] to Omnia T. Elshorbagy at Newcastle University.