Molecular epidemiological study on Infectious Pancreatic Necrosis Virus isolates from aquafarms in Scotland over three decades

Abstract

Introduction RNA viruses are economically important pathogens of fish, and among these viruses, infectious pancreatic necrosis virus (IPNV) is of particular concern for the aquaculture industry, especially for farmed rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). This non-enveloped aquatic virus, which was first isolated in the UK in 1971, belongs to the family of Birnaviridae and has a bi-segmented dsRNA genome of about 6kb. IPNV is classified in 6 genogroups with correspondence to 10 known serotypes and an additional proposed genogroup of marine aquabirnaviruses (MABV). IPNV causes high mortality in fry and a reduced mortality in adult fish, respectively. Fish, which survive, can become carriers and this can lead to a clinical outbreak by releasing infective material into water or by vertical transmission via oocytes, milt and seminal fluids.

Methods This project aimed at determining the phylogeny and genomic changes of IPNV in Scotland by whole genome sequence analysis of IPNV isolates (diagnostic TCID50 supernatants) spanning 3 decades since 1982, using next generation sequencing technology. Viral RNA of IPNV culture supernatant (CHSE-214 and TO cell culture) was processed for next generation sequencing on an Illumina MiSeq platform. Library preparation was performed using the Nextera XT DNA Library Kit, prior to sequencing according to the manufacturer’s MiSeq Reagent Kit v3 (150cycles) protocol. To optimize whole genome next generation sequencing for IPNV, we compared two RNA processing protocols, the Glasgow (GLAP) and the Goettingen protocol (GOEP) with focus on missing terminal nucleotides after a de novo genome assembly. Sequences were used to determine the phylogeny and selection pressure on the genome as well as a possible virus-host adaptation.

Results The results showed that both protocols were able to give full length genomes as well as genomes with missing terminal nucleotides. The phylogenetic analysis of 57 sequenced IPVN isolates shows that 78.95 % of the isolates group within genogroup V, which includes serogroup Sp and 5.26 % within genogroup I which includes serogroup Ja. Segment A of 15.79 % of the isolate grouped within genogroup III, which includes serotype Ca1 and Te but only 7.02 % of the segment B isolates grouped in the genogroup III. The remaining 8.77 % of segment B groups within genogroup II, containing the Ab serotype. Previous research has shown that residue substitutions at positions 217 and 221 in the major capsid protein VP2 have an impact on the virulence of the virus, leading to different virulence types: virulent (T217, A221), low virulence (P217, A221), avirulent (T217, T221) and persistent (P217, T221). Whole genome sequence results show that 58.93 % of the sequenced isolates belong to the persistent, 32.14 % to the low virulent type, only one isolate was of a virulent type and 7.15 % had not virulence assigned amino acid compositions in positions 217 and 221. The selection pressure analysis showed that especially VP2 is experiencing selection pressure in the variable region. In the VP1 protein we see two sites under positive selection pressure within specific motifs. VP5 showed positive selected sites mostly within the truncated region of the protein. Other proteins showed no particular interesting sites of selection. The codon adaptation analysis showed highest adaptation index for VP2. Besides VP5, which had an CAI index below one, therefore showing negative adaptation, other IPNV proteins had an CAI of barely above the value of 1. The dinucleotide abundance, focussing on CpG, showed that CpG is underrepresented in segment A and B.

Discussion Phylogenetic analysis of the sequenced IPNV strains shows separate clustering of different genogroups. Genetic reassortment is observed in segment B showing a grouping within genogroup III and II although the segment A of these isolates was grouping exclusively within III. We found that over 50 % of the isolates belong to the persistent and over 30 % to the low virulent type, assuming that due to not sterilising vaccination these types were selected in the vaccinated population. The results from the CAI calculations indicate an adaptation of IPNV to its host. Together with the findings that CpG is underrepresented in IPNV it suggests that this leads to an immune escape. Especially since the selection pressure analysis showed positive selection in VP2 within the virulence determination sites of the protein, indicating that IPNV “tries” to downregulate immune recognition. The prevalence of mostly persistent type of isolates indicates together with the assumption of adaptation and immune escape that IPNV is evolving with the host in order to ensure survival.