Strain differentiation and detection of infectious hematopoietic necrosis virus

The virulence of selected isolants of infectious hematopoietic necrosis virus (IHNV) were differentiated and correlated with electrophoretic mobilities of virion structural proteins by sodium dodecylsulfate polyacrylamide gel electrophoresis. A type 1 electropherotype of IHNV was more virulent for kokanee salmon (Oncorhynchus nerka) and a type 2 strain more virulent for rainbow trout (Oncorhynchus mykiss). Viruses from the Columbia River basin (electropherotypes 2 and 3) were more virulent for steelhead trout (Oncorhynchus mykiss) and IHNV electropherotypes from southern Oregon and California (type 3) were most virulent for chinook salmon (Oncorhynchus tshawytscha). Microscopic pathology induced by type 1 and 3 strains of IHNV in rainbow trout was less severe and involved fewer tissues than that caused by a type 2 providing further evidence that type 2 strains are more pathogenic for rainbow trout. A fluorescent antibody test (FAT) was developed for the rapid detection of IHNV. The FAT was used for the detection of IHNV in blood smears and organ imprints from clinically infected juveniles and IHNV-infected cells in ovarian fluid from adult carriers. The test was equal in sensitivity to the plaque assay method and required less time to obtain a definitive diagnosis. Other sources of virus examined included cells contained in the ovarian fluid (OF) of sexually mature steelhead trout. Ovarian fluid collected from spawning steelhead trout determined to be virus-free tested viruspositive when reexamined post-spawning. Results indicated that cells free in OF allowed replication of IHNV and could be used in diagnostic tests to detect this delayed virus expression. Infectious hematopoietic necrosis virus was also detected in mucus obtained from naturally infected juvenile and adult salmonids. In adult chinook and kokanee salmon, a higher prevalence of virus was detected in mucus collected from the external surface of fish than observed in OF, seminal fluid, or spleen tissue homogenates. Experimental infection of juvenile rainbow trout suggested IHNV detected in mucus was a result of the normal progression of disease and that the integument may have been a site of virus replication and a possible portal of entry

Mucosal immunoglobulins at respiratory surfaces mark an ancient association that predates the emergence of tetrapods

Gas-exchange structures are critical for acquiring oxygen, but they also represent portals for pathogen entry. Local mucosal immunoglobulin responses against pathogens in specialized respiratory organs have only been described in tetrapods. Since fish gills are considered a mucosal surface, we hypothesized that a dedicated mucosal immunoglobulin response would be generated within its mucosa on microbial exposure. Supporting this hypothesis, here we demonstrate that following pathogen exposure, IgT(+) B cells proliferate and generate pathogen-specific IgT within the gills of fish, thus providing the first example of locally induced immunoglobulin in the mucosa of a cold-blooded species. Moreover, we demonstrate that gill microbiota is predominantly coated with IgT, thus providing previously unappreciated evidence that the microbiota present at a respiratory surface of a vertebrate is recognized by a mucosal immunoglobulin. Our findings indicate that respiratory surfaces and mucosal immunoglobulins are part of an ancient association that predates the emergence of tetrapods

Identification of B Cells as a Major Site for Cyprinid Herpesvirus 3 Latency

Cyprinid herpesvirus 3 (CyHV-3), commonly known as koi herpesvirus (KHV), is a member of the Alloherpesviridae, and is a recently discovered emerging herpesvirus that is highly pathogenic for koi and common carp. Our previous study demonstrated that CyHV-3 becomes latent in peripheral white blood cells (WBC). In this study, CyHV-3 latency was further investigated in IgM⁺ WBC. The presence of the CyHV-3 genome in IgM⁺ WBC was about 20-fold greater than in IgM⁻ WBC. To determine whether CyHV-3 expressed genes during latency, transcription from all eight open reading frames (ORFs) in the terminal repeat was investigated in IgM⁺ WBC from koi with latent CyHV-3 infection. Only a spliced ORF6 transcript was found to be abundantly expressed in IgM⁺ WBC from CyHV-3 latently infected koi. The spliced ORF6 transcript was also detected in vitro during productive infection as early as 1 day postinfection. The ORF6 transcript from in vitro infection begins at -127 bp upstream of the ATG codon and ends +188 bp downstream of the stop codon, +20 bp downstream of the polyadenylation signal. The hypothetical protein of ORF6 contains a consensus sequence with homology to a conserved domain of EBNA-3B and ICP4 from Epstein-Barr virus and herpes simplex virus 1, respectively, both members of the Herpesviridae. This is the first report of latent CyHV-3 in B cells and identification of gene transcription during latency for a member of the Alloherpesviridae.This is the publisher’s final pdf. The published article is copyrighted by the American Society for Microbiology and can be found at: http://jvi.asm.org/

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2 days, defining a generally uniform early peak period of shedding from 1 to 4 days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority of fish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur. (C) 2016 Elsevier B.V. All rights reserved

Differential characterization of emerging skin diseases of rainbow trout - a standardized approach to capturing disease characteristics and development of case definitions

Farmed and wild salmonids are affected by a variety of skin conditions, some of which have significant economic and welfare implications. In many cases, the causes are not well understood, and one example is cold water strawberry disease of rainbow trout, also called red mark syndrome, which has been recorded in the UK since 2003. To date, there are no internationally agreed methods for describing these conditions, which has caused confusion for farmers and health professionals, who are often unclear as to whether they are dealing with a new or a previously described condition. This has resulted, inevitably, in delays to both accurate diagnosis and effective treatment regimes. Here, we provide a standardized methodology for the description of skin conditions of rainbow trout of uncertain aetiology. We demonstrate how the approach can be used to develop case definitions, using coldwater strawberry disease as an example