Investigation of laboratory methods for characterization of aquatic viruses in fish infected experimentally with infectious salmon anemia virus

Abstract

Rapid growth in aquaculture has resulted in high-density production systems in ecologically and geographically novel conditions in which the emergence of diseases is inevitable. Well-characterized methods for detection and surveillance of infectious diseases are vital for rapid identification, response, and recovery to protect economic and food security. We implemented a proof-of-concept approach for virus detection using a known high-consequence fish pathogen, infectious salmon anemia virus (ISAV), as the archetypal pathogen. In fish infected with ISAV, we integrated histopathology, virus isolation, whole-genome sequencing (WGS), electron microscopy (EM), in situ hybridization (ISH), and reverse transcription real-time PCR (RT-rtPCR). Fresh-frozen and formalin-fixed tissues were collected from virus-infected, control, and sham-infected Atlantic salmon (Salmo salar). Microscopic differences were not evident between uninfected and infected fish. Viral cytopathic effect was observed in cell cultures inoculated with fresh-frozen tissue homogenates from 3 of 3 ISAV-infected and 0 of 4 uninfected or sham-infected fish. The ISAV genome was detected by shotgun metagenomics in RNA extracted from the medium from 3 of 3 inoculated cell cultures, 3 of 3 infected fish, and 0 of 4 uninfected or sham-infected fish, yielding sufficient coverage for de novo assembly. An ISH probe against ISAV revealed ISAV genome in multiple organs, with abundance in renal hematopoietic tissue. Virus was detected by RT-rtPCR in gill, heart, kidney, liver, and spleen. EM and metagenomic WGS from tissues were challenging and unsuccessful. Our proof-of-concept methodology has promise for detection and characterization of unknown aquatic pathogens and also highlights some associated methodology challenges that require additional investigation.

Keywords: Atlantic salmon; histopathology; infectious salmon anemia virus; metagenomics; virus discovery.

Figure 1.

Experimental pipeline for detection and characterization of infectious salmon anemia virus (ISAV) in fish. FFPE = formalin-fixed, paraffin-embedded; RT-rtPCR = reverse-transcription real-time PCR; WGS = whole-genome sequencing.

Figure 2.

Electron microscopic image of infectious salmon anemia virus (ISAV) viral particle from negatively stained virus culture medium of a day-11 ISAV-infected fish. Bar = 100 nm.

Figure 3.

Organ histology (A, C, E; H&E) and corresponding distribution of infectious salmon anemia virus (ISAV) genome by in situ hybridization (ISH; B, D, F; hematoxylin counterstain of ISH). ISAV RNA is randomly distributed in the gill filaments (A, B), the interstitial connective tissue of the myocardium (C, D), and cells lining the sinusoids (presumed endothelial cells) of the liver (E, F).

Figure 4.

Organ histology (A, C, E; H&E) and corresponding distribution of infectious salmon anemia virus (ISAV) genome by in situ hybridization (ISH; B, D, F; hematoxylin counterstain of ISH). ISAV RNA is concentrated in the hematopoietic interstitium of the head kidney (A, B), the white pulp of the spleen (C, D), and the lamina propria and interstitial connective tissue of the intestines (E, F).

Figure 5.

Infectious salmon anemia virus (ISAV) in tissues by reverse-transcription real-time PCR (RT-rtPCR) and in situ hybridization (ISH). The Ct values for each day-11 ISAV-infected fish was determined by RT-PCR, as well as amount of viral genome visualized in each of the corresponding tissues by ISH (represented as average dots per 400× magnification field). In both tests, there is an apparent trend that kidney tissue contains a greater amount of virus; however, this was not statistically significant when comparing means (p > 0.05).