Differences in Virulence of Marine and Freshwater Isolates of Viral Hemorrhagic Septicemia Virus In Vivo Correlate with In Vitro Ability To Infect Gill Epithelial Cells and Macrophages of Rainbow Trout ( Oncorhynchus mykiss )

Abstract

Two strains of viral hemorrhagic septicemia virus (VHSV) with known different virulence characteristics in vivo were studied (by a time course approach) for their abilities to infect and translocate across a primary culture of gill epithelial cells (GEC) of rainbow trout (RBT; Oncorhynchus mykiss). The strains included one low-virulence marine VHSV (ma-VHSV) strain, ma-1p8, and a highly pathogenic freshwater VHSV (fw-VHSV) strain, fw-DK-3592B. Infectivities toward trout head kidney macrophages were also studied (by a time course method), and differences in in vivo virulence were reconfirmed, the aim being to determine any correlation between in vivo virulence and in vitro infectivity. The in vitro studies showed that the fw-VHSV isolate infected and caused a cytotoxic effect in monolayers of GEC (demonstrating virulence) at an early time point (2 h postinoculation) and that the same virus strain had translocated over a confluent, polarized GEC layer by 2 h postinoculation. The marine isolate did not infect monolayers of GEC, and delayed translocation across polarized GEC was seen by 48 h postinoculation. Primary cultures of head kidney macrophages were also infected with fw-VHSV, with a maximum of 9.5% virus-positive cells by 3 days postinfection, while for the ma-VHSV strain, only 0.5% of the macrophages were positive after 3 days of culture. In vivo studies showed that the fw-VHSV strain was highly virulent for RBT fry and caused high mortality, with classical features of viral hemorrhagic septicemia. The ma-VHSV showed a very low level of virulence (only one pool of samples from the dead fish was VHSV positive). This study has shown that the differences in virulence between marine and freshwater strains of VHSV following the in vivo infection of RBT correlate with in vitro abilities to infect primary cultures of GEC and head kidney macrophages of the same species. Viral hemorrhagic septicemia virus (VHSV) belongs to theNovirhabdovirus genus of the family Rhabdoviridae. European VHSV of freshwater origin causes disease primarily in rainbow trout (RBT; Oncorhynchus mykiss), with high mortality. Outbreaks of viral hemorrhagic septicemia (VHS) in marine fish species like turbot (Scophthalmus maximus) (17,22,23) and Japanese flounder (Paralichthys olivaceus) (10) have also been recorded previously. VHSV has been implicated in the mortality of Pacific herring (Clupea pallasi) (15), and in later studies it was shown that this species of fish is highly susceptible to experimental challenge (11). Infection trials have shown previously that marine VHSV (ma-VHSV) isolates originating from marine fish have low levels of pathogenicity in RBT compared with freshwater VHSV (fw-VHSV) strains (24). However, no studies have been carried out to determine the mechanisms for these differences at the level of host-pathogen interactions.Fw-VHSV strains infect through waterborne exposure, and the prime port of entry is suggested to be the skin and/or gills. This supposition is based on previous observations in which early virus repl...

The Major Portal of Entry of Koi Herpesvirus in Cyprinus carpio Is the Skin

“…It has also been suggested that Viral hemorrhagic septicemia virus, another important rhabdovirus of salmonids, enters fish through the skin, but this hypothesis has not been tested (3,23). While the present study demonstrates that intact and healthy skin is the portal of entry for KHV in carp, further experiments are in progress to determine if ectoparasite infestations and/or rough handling of fish enhances the entry of KHV through the skin.…”

A Single Amino Acid Mutation (I1012F) of the RNA Polymerase of Marine Viral Hemorrhagic Septicemia Virus Changes In Vitro Virulence to Rainbow Trout Gill Epithelial Cells

“…VHSV isolates designated DK-3592B (genotype Ia), 1p8 (genotype Ib), NO/650/07 (genotype III), and JF-09 (genotype IVa) were used in this study and propagated in EPC cells (ATCC CRL-2872) in L15 cell medium (Invitrogen) containing 10% fetal bovine serum (FBS; Sigma-Aldrich) at 15°C (15). Strains DK-3592B and 1p8 were a kind gift of Niels Jørgen Olesen, Danish Veterinary Institute, Århus, Denmark, and NO/650/07 was kindly provided by Renate Johansen, Norwegian Veterinary Institute, Oslo, Norway.…”

“…Titration was performed on EPC cells by the 50% tissue culture infective dose (TCID 50 ) method as described previously (17). In addition, we used primary cultured rainbow trout GECs isolated from fry of rainbow trout (18,19) as modified by Brudeseth et al (15).…”

“…One approach has been to identify pathogenicity markers of these new variants through genome sequencing (10,13), but so far it has not been possible to tie such variation or specific amino acid motifs of the viral proteins to virulence (14). We have shown that the ability of defined VHSV strains to infect and translocate across primary cultures of rainbow trout gill epithelial cells (GECs) correlate with in vivo virulence by using a bath challenge of trout fry (15). Isolates not able to infect (flat) cultures of GECs and/or translocate across polarized GECs also did not cause death postchallenge.…”

Rainbow trout Oncorhynchus mykiss ladderlectin, but not intelectin, binds viral hemorrhagic septicemia virus IVb

“…Salmon serum lectin (SSL), very closely related to rainbow trout ladderlectin, not only directly binds to Vibrio anguillarum and Aeromonas salmonicidia, but SSL increases phagocytosis and killing by macrophages when co-incubated with those bacteria , Ottinger et al 1999. As VHSV enters rainbow trout by binding to gill epithelial cells and spreads in part by macrophage phagocytosis (Brudeseth et al 2008), ladderlectin's presence in gill epithelial cells and leucocytes, as well as in plasma (Russell et al 2008a), ideally situate it for interaction with VHSV. Rainbow trout ladderlectin is known to have binding specificities for glucose, N-acetyl-glucosamine, mannose, fucose, and maltose (Jensen et al 1997), which suggests that one or more of these sugars are present in carbohydrates on the glycoprotein outer layer of VHSV.…”