First report of pepper vein yellows virus-associated RNA detected in chili pepper in Indonesia

Chili pepper (Capsicum annuum L.) is an economically important vegetable crop in Indonesia (Sativa et al. 2017). During a field survey conducted in August 2024 in Cianjur, Indonesia, virus-like symptoms, including leaf yellowing, vein clearing, and mosaic, were observed in commercial chili pepper fields, with disease incidence estimated at 50-70%. Because mixed infections are common in chili pepper (Kim et al. 2014), these symptoms might not be attributed to a single viral agent. Thus, to analyze the virome of these fields, 20 symptomatic leaf samples (10 from each of two fields) were randomly collected from the fields and pooled for RNA-seq. Total RNA was extracted using the Hybrid-RTM RNA extraction kit (GeneAll, Korea). A cDNA library was prepared using the TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina, USA) and analyzed on an Illumina NovaSeq 6000 platform (Macrogen, Korea). De novo assembly of 78,486,422 quality-filtered reads using the Trinity pipeline generated 84,362 contigs. BLASTn/x analyses identified contigs corresponding to cucumber mosaic virus, chilli veinal mottle virus, pepper yellow leaf curl Indonesia virus, pepper cryptic virus 2, and pepper vein yellows virus 5 (PeVYV-5). In addition, a 2,911-nt contig supported by 491,491 paired-end 150-bp reads showed the highest nucleotide identity (96.5%) to PeVYV-associated RNA (PeVYVaRNA) isolate PRO54353 from South Africa (GenBank accession no. MT321510), with nearly complete sequence coverage. To determine the complete sequence of the Cianjur isolate (PeVYVaRNA-CJ), overlapping RT-PCR fragments covering the assembled contig were amplified and sequenced, and the terminal sequences were determined by 5′ and 3′ rapid amplification of cDNA ends assays. The complete PeVYVaRNA-CJ sequence was 3,001 nt and was deposited in GenBank under accession number PZ459831. The same 20 individual field samples were tested by RT-PCR using PeVYVaRNA-specific primers (5′-TCGCTCGCCACGTTTAGAGTA-3′ and 5′-GAGGTGGGAGTACGAAGATTT-3′). Amplicons of the expected size were obtained from 16 samples, and Sanger sequencing confirmed their identity as PeVYVaRNA-derived sequences. PeVYVaRNA detection was further verified by dot blot hybridization using a specific probe (5'-ACGTTAGGCTGCGCGTTCCTTGGCCTTTATCCGGGGCAA-3'). The same 20 samples were also assayed by RT-PCR using PeVYV-5-specific primers (5′-GTCGAGGTTTCAGGACAACTGGA-3′ and 5′-GTGCGACCACAACGACAGGG-3′). PeVYV-5 was detected only in the 16 PeVYVaRNA-positive samples, indicating consistent co-detection of PeVYV-5 and PeVYVaRNA in the samples tested. PeVYVaRNA was first identified in South Africa in 2020 (Schravesande et al. 2021). Its detection in Indonesia, a geographically distant country, suggests that PeVYVaRNA may be more widely distributed than currently recognized. Because the samples contained mixed viral infections and symptom severity was not quantitatively assessed, no causal relationship between PeVYVaRNA and the observed field symptoms can be inferred from the present study. However, other polerovirus-associated RNAs have been implicated in enhanced symptom development and viral fitness during mixed infections involving helper viruses (Sanger et al. 1994). Therefore, further studies are needed to evaluate the potential epidemiological relevance of PeVYVaRNA in viral disease complexes affecting pepper. To our knowledge, this is the first report of PeVYVaRNA detected in chili pepper in Indonesia.