Nanopore sequencing of IPNV vp2 gene in Peruvian Andean trout (Oncorhynchus mykiss) cultures

ABSTRACT Nanopore sequencing of the infectious pancreatic necrosis virus (IPNV) vp2 gene from Andean trout cultures in Peru reveals genogroups 1 and 5. This insight aids in understanding strain diversity and pathogenicity, vital for effective disease surveillance, and control measures in aquaculture.

Samples were collected from rainbow trout alevin specimens with clinical signs compatible with IPNV in December 2021 in the Apurimac region, and during 2022 in Puno and Huancavelica regions.The organs collected were the liver, kidney, and spleen (2) by specimen and each sample consisted of a pool of five specimens.These samples were confirmed to IPNV (vp1 gene) with reverse transcription quantitative real-time PCR (RT-qPCR) and then selected for later genotyping with the vp2 gene (6).Amplicon sequencing of the IPNV vp2 gene was amplified by conventional PCR and sequenced using Oxford Nanopore Technologies (ONT, UK).RNA extraction from 20 mg tissue samples used the ReliaPrep RNA Tissue Miniprep System kit (Promega, USA).RNA quantification relied on a Qubit 4 fluorometer (Invitrogen, USA), while cDNA synthesis employed the RevertAid First Strand Kit (Thermo Scientific, USA) with random hexamers.
The phylogenetic analysis involved 13 sequences of the vp2 gene from Peruvian IPNV strains and 49 vp2 gene sequences of IPNV obtained from GenBank.These strains represent all IPNV genogroups (6,16,17).Multiple sequence alignments used the MUSCLE algorithm in MEGA 11 (18).Phylogenetic tree construction employed the neighbor-joining method in MEGA with 1,000 bootstrap replications, and substitution models were determined using the maximum composite likelihood method (Fig. 1).The samples evaluated belong to genogroups 1 and 5.
This study yields critical insights into the pathogenic strains endangering trout farming in Peru, facilitating disease surveillance and IPNV control through vaccine development.

FIG 1
FIG 1Phylogenetic tree based on nucleotide sequence comparisons of the vp2 gene, showing relationships between IPNV samples analyzed in this study (blue and green circles) and reference strains.Analysis was performed in the MEGA 11 program using the neighbor-joining method; confidence in tree construction was assessed using 1,000 bootstrap replicates.Bootstrap values greater than 50% are shown.The evolutionary distances were computed using the maximum composite likelihood method.

TABLE 1
Nanopore data sequencing from vp2 gene of IPNV samples