First Report of Pepper Cryptic Virus 2 in Chili Pepper in Indonesia

Chili pepper (Capsicum annuum L.) is an economically important horticultural crop in Indonesia (Sativa et al. 2017). In August 2024, during a survey of a commercial field in Bogor, West Java, approximately 30% of pepper plants exhibited virus-like symptoms, including yellowing, mosaic, and dwarfing. To identify the causal agent(s), leaf samples were collected from ten symptomatic pepper plants, pooled, and subjected to RNA extraction using the PureLink RNA Mini Kit (Invitrogen, USA). A cDNA library was prepared using the Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, USA) and sequenced on an Illumina NovaSeq 6000 system (Macrogen, Korea). De novo assembly of 100,413,852 reads using the Trinity pipeline generated 105,175 contigs. BLASTn and BLASTx analyses against the GenBank viral reference database revealed that eight contigs, ranging from 1,507 to 6,187 bp in size, were of viral origin, corresponding to cucumber mosaic virus (CMV) RNAs 1-3 (1,125,482; 3,244,585; and 8,721,654 reads, respectively), pepper vein yellows virus (59,382 reads), pepper cryptic virus 2 (PCV2) RNA1 (43,170 reads), PCV2 RNA2 (29,549 reads), pepper yellow leaf curl Indonesia virus (PepYLCIV) DNA-A (5,188 reads), and PepYLCIV DNA-B (1,888 reads). Among these, PCV2 (genus Deltapartitivirus; family Partitiviridae) has not previously been reported in Indonesia. To obtain the complete genome sequence of the Indonesian PCV2 isolate (PCV2-IDN), RNA extracted from a leaf sample was used for RT-PCR amplification of the contig sequences corresponding to PCV2 RNAs and for 5'/3' rapid amplification of cDNA ends analysis to obtain the terminal sequences of PCV2-IDN (Kwon et al. 2014). The amplified products were sequenced using an ABI PRISM 3730XL analyzer (Applied Biosystems, USA). The full-length sequences of PCV2-IDN RNA1 and RNA2 were 1,610 and 1,525 nucleotides in length, respectively, and were deposited in GenBank under accession numbers PV939908 and PV939909. BLASTn analysis showed that PCV2-IDN RNA1 and RNA2 shared maximum identities of 99.7% and 99.9% with RNA1 of the PCV-2 isolate PCV21HH (OR353018) and RNA2 of the isolate SC10-2 (KY923704), respectively. To investigate PCV2 distribution in other regions of Indonesia, ten symptomatic pepper leaf samples were collected from each of two fields located in geographically distinct areas: Cianjur, Garut, and Bandung. Ten samples from each field were pooled for RNA extraction and tested by RT-PCR using PCV2-specific primers (5'-GCGACACCAGTTAGTGACACAC-3' and 5'-GTTGTTGTGCGCCTACCTGGTG-3'). RT-PCR followed by de novo sequencing using an ABI PRISM 3730XL analyzer revealed that all pooled samples tested positive for PCV2, which was further confirmed by dot blot hybridization using a PCV2-specific probe (5'-GCGTTATAGGGGGCGTAGCTGCCGGTAATTGGTCAGTCTTGTC-3'). These findings suggest that PCV2 is widespread in pepper-growing regions of Indonesia and highlight the need for further epidemiological studies to assess its impact on pepper production. Although PCV2 infection is typically asymptomatic in pepper (Sabanadzovic et al. 2011), co-infection with other viruses may exacerbate symptom expression and yield loss. Because PCV-2 is not mechanically transmissible, studies on its pathogenicity remain limited. Developing PCV2 infectious cDNA clones will be crucial for elucidating its pathogenic potential in crop plants. This study represents the first report of PCV2 in chili pepper in Indonesia.