Source of virus material/isolate collection.
We collected leaf samples from 118 papaya plants with virus-like symptoms from December 2016 to January 2017. We surveyed locations in eight districts of Bangladesh: Pabna (24.070032°N, 89.113417°E); Rajshahi (24.366346°N, 88.642614°E); Chapai Nawabganj (24.607635°N, 88.290519°E); Chandpur (23.348015°N, 90.708153°E); Munshiganj (23.550215°N, 90.534806°E); Gazipur (23.998997°N, 90.419802°E); Tangail (24.643032°N, 90.055994°E); and Mymensingh (24.746026°N, 90.373906°E) (Supplementary). Disease incidence was visually assessed in each of these locations. The collected leaves were immediately stored in RNAlater solution (Qiagen, Valencia, CA) and brought to the plant virology laboratory at the University of Hawaii at Manoa under USDA PPQ 526 permits P526-160413-009 and P526P-16-03662. After arrival at the plant virology laboratory, all samples were stored at −80°C until total RNA extractions were conducted.
Preliminary detection of PRSV.
The 118 papaya samples with virus-like symptoms were tested with PRSV-specific single-tube nested PCR (STNP) and enzyme-linked immunosorbent assay (ELISA) (Agdia, Inc.), as previously described. Total RNAs were extracted from leaf samples using the RNeasyPlant Mini Kit (Qiagen, Valencia, CA), and cDNAs were synthesized according to the method described previously. RNA purity as assessed by A260/A280 ratios ranged from 1.9 to 2.2. Previously identified PRSV-infected and PRSV-free leaves were included as positive and negative controls, respectively.
HTS of total RNAs from PRSV-infected composite samples.
Eleven leaf samples with virus-like symptoms were randomly selected and divided into two separate composite samples for HTS conducted at Foundation Plant Services (Davis, CA). Composite sample pap-1 consisted of six different papaya samples with virus-like symptoms: CD-141, Gaz-30, Gaz-52, Tang-62, Tang-66, and Tang-72. Composite sample pap-2 consisted of five different papaya samples with virus-like symptoms: CD-112, CD-125, CD-129, Gaz 34, and Gaz-39. The composite samples were prepared using 10-µg aliquots of total RNA from each sample that were then combined. The composite RNA samples were subjected to ribosomal RNA (rRNA) depletion, and a cDNA library was constructed from the RNA template using TruSeq Stranded Total RNA with a Ribo-Zero Plant Kit (Illumina). Sequencing was performed at Foundation Plant Services using the Illumina NextSeq 500 platform, and raw HTS reads were analyzed as described by Al Rwahnih et al. (2018).
RT-PCR sequencing to validate the HTS results.
To validate the results of HTS, specific primers (Supplementary Tables S1 and S2) were designed to amplify DNA fragments of the virus genome by RT-PCR. cDNA templates were produced from the isolated total RNA from the HTS samples using Qiagen RNeasy Kits (Qiagen, Germany). Total RNAs extracted from each sample were quantified as described, and cDNAs were synthesized as described previously. The conditions for PCR amplification were as follows: 4 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 50 to 65°C depending on the primers, and 2 min at 72°C; and final elongation at 72°C for 7 min. The PCR products were resolved on 1% agarose gel in 1X TAE buffer for 60 min at 60 V. Gels were stained with ethidium bromide and visualized in a UVP transilluminator. PCR products of the predicted sizes were purified, cloned into the PGEM-T Easy cloning vector (Promega, USA), and sequenced at the University of Hawaii’s Advanced Genomic and Sequencing Services and at Genwiz in California. Sequences were assembled with the Cap3 sequence genome assembly program. Complete genome sequences of all PRSV strains were deposited in GenBank.