ABSTRACT
Circular, viral copy DNA (cvcDNA) can reveal the existence of endogenous viral elements (EVE) in shrimp genomic DNA. Here we describe isolation and sequencing of cvcDNA from a breeding stock of the whiteleg shrimp Penaeus vannamei. The stock was developed by onward breeding and selection for white spot syndrome virus (WSSV)-free individual survivors of white spot disease outbreaks. The stock exhibits high tolerance to WSSV. A pool of DNA extracted from 10 shrimp from this stock was subjected to cvcDNA isolation and amplification followed by high throughput sequencing. This revealed DNA fragments corresponding to locations covering much of the 300,000 bp WSSV genome. However, high frequency-read-fragments (HFRF) mapped to a surprisingly small region of approximately 1,400 bp. We hypothesized that the HFRF reflected their selection due to provision of tolerance to WSSV. Four PCR primer sets were designed to cover the 1,365 bp mapped region. One pair (Set 1) targeted the whole 1,365 bp mapped region, while another 3 primer sets (Set 2-4), targeted regions within the 1,365 bp target. All 4 primer sets were used with DNA samples from each of 36 shrimp from the same breeding stock (including the 10 used for cvcDNA preparation). Individual positive PCR results varied widely from shrimp to shrimp, ranging from only 1 primer set up to 4 primer sets. Only 1 specimen gave an amplicon of 1,365 bp, while others gave single to multiple positive amplicons in both continuous and discontinuous regions. This indicated that the amplicons did not arise from contiguous targets and might vary in insertion length. The results also confirmed that none of the shrimp were infected with WSSV. Sequencing of the PCR amplicons of expected sizes revealed sequence identity to extant WSSV genomes. This data will be used to screen the breeding stock for EVE that provide WSSV tolerance.
Competing Interest Statement
The National Aquaculture Group (NAQUA)/ King of Saudi Arabia provided support in the form of salaries for authors; JM, IIU, MBC, MMA, and VAS but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
