difference in virulence of marine and freshwater isolates...

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Difference in virulence of marine and freshwater isolates of viral

hemorrhagic septicemia virus in vivo correlates with in vitro ability to

infect gill epithelial cells and macrophages of rainbow trout

(Oncorhynchus mykiss)

1Bjørn E. Brudeseth,

2Helle F. Skall,

3*Øystein Evensen

1PHARMAQ AS, PO Box 267, N-0213 Oslo, Norway

2Technical University of Denmark, National Veterinary Institute, Hangøvej 2, DK-

8200 Aarhus N, Denmark

3Norwegian School of Veterinary Science, PO Box 8146 Dep, N-0033 Oslo Norway

* Corresponding author

Running title: Virulence traits of VHS virus ACCEPTED

Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.01009-08 JVI Accepts, published online ahead of print on 27 August 2008

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ABSTRACT

Two strains of viral hemorrhagic septicemia (VHS) virus with known different

virulence characteristics in vivo were studied (time-course) for their ability to infect

and translocate across a primary culture of gill epithelial cells (GEC) of rainbow trout

(RBT; Oncorhynchus mykiss). The strains included one low-virulent marine strain

(ma-VHSV; ma-1p8) and a highly pathogenic freshwater strain (fw-VHSV; fw-DK-

3592B). Infectivity to trout head kidney macrophages was also studied (time-course)

and difference in in vivo virulence was reconfirmed, the aim being to determine any

correlation between in vivo virulence with in vitro infectivity. The in vitro studies

showed that the fw-VHSV isolate infected and caused cytotoxic effect in monolayers

of GEC (virulence) at early time (2h) and the same virus strain translocated over a

confluent, polarized GEC-layer by 2h post inoculation. The marine isolate did not

infect monolayers of GEC and a delayed translocation across polarized GEC was seen

by 48h post inoculation. Primary cultures of head kidney macrophages were also

infected with fw-VHSV, with a maximum of 9.5% virus positive cells by 3 days post

infection, while for the ma-VHSV strain only 0.5% of the macrophages were positive

by 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 VHS. The

ma-VHSV showed very low virulence (only one pool of the sampled dead fish was

VHSV positive). This study has shown that the difference in virulence between ma-

and fw-strains of VHSV following in vivo infection in rainbow trout correlates with in

vitro ability to infect primary cultures of gill epithelial cells and head kidney

macrophages of the same species.

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INTRODUCTION

Viral hemorrhagic septicemia (VHS) virus belongs to the Novirhabdovirus genus of

the family Rhabdoviridae. European VHS virus of freshwater origin causes disease

primarily in rainbow trout (RBT; Oncorhynchus mykiss) with high mortality.

Outbreaks of VHS have also been recorded in marine fish species like turbot

(Scophthalmus maximus; 17, 22, 23), and Japanese flounder (Paralichthys olivaceus;

9). VHSV has been implicated in mortality of Pacific herring (Clupea pallasi; 15) and

from later studies it was shown that this species is highly susceptible to experimental

challenge (11). Infection trials have shown that VHSV isolates originating from

marine fish (ma-VHSV) have low pathogenicity in RBT compared with freshwater

VHSV strains (fw-VHSV; 24). However, no studies have been carried 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

suggestively the skin and/or gills. This is based on observations where early virus

replication has been demonstrated in gill epithelial cells in situ of rainbow trout (1,

10; 18). Further, studies show that epithelial cells from skin and gills are capable of

supporting replication of VHSV (31) and the viral replication in excised fins

correlates with resistance to waterborne challenge (21). The progression of an IHNV

infection was suggested to occur from the gills into circulation, and/or from the oral

region to the gastrointestinal tract, with subsequent distribution to circulation (4).

Replication of IHNV at early stage in internal organs was proposed to take place in

the kidney (4). This is concordant with findings reported for both VHSV and IHNV in

RBT (1) and for ma-VHSV in turbot (2) using immunohistochemistry (2,6). In these

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previous studies we showed that VHS and IHN viruses were detected in macrophages

and melanomacrophages at early time post infection, indicating that these cells

support virus replication in vivo. Replication of VHS and IHN viruses in macrophages

cultured in vitro has also been demonstrated (3, 5). In a recent study the fin base was

identified as a possible port of entry for IHNV (8).

In studies by Skall et al. (24) 139 ma-VHSV isolates from wild marine fish

and from farmed turbot did not cause mortality in RBT by immersion challenge, but

some isolates caused up to 60 % mortality by injection. In this study we have

investigated if the low virulence of ma-VHSV in RBT can be related to their ability to

translocate over a confluent gill epithelium. Pärt and colleagues (20) have developed a

method for in vitro culturing of gill epithelial cells on filters. The filter cultures of

GEC establish a monolayer firmly attached to glass and plastic supports. These cells

have the appearance of a differentiated epithelium and tight junctions are established

(20). This method has been used in studies of ion transport and acid-base regulations

in rainbow trout (30). In this experiment we used this in vitro model to study the

translocation of VHSV through a confluent gill epithelium and we compared a ma-

VHSV isolate (ma-1p8, genotype 1b) to virulent fw-VHSV reference strain (fw-DK-

3592B, genotype 1a). The marine strain was selected on the basis of being non-lethal

to RBT following immersion challenge, while causing around 40% mortality when

injected intraperitoneally in RBT. The same isolates were also tested for their ability

to infect isolated head kidney macrophages in vitro and the virulence of the isolates

used was again confirmed in experimental challenge of rainbow trout fry.

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MATERIALS AND METHODS

Preparation of GEC. RBT gill epithelial cells were isolated according to the method

described by Pärt et al. (19). RBT with a size of approximately 25-100g cultured in

fresh water were killed by a blow to the head followed by decapitation. The gill

arches were dissected and transferred to a Petri dish and rinsed twice with 10 ml of

PBS (Ca2+

and Mg2+

free) containing 200 µg/ml PEST (Penicillin/Streptomycin from

Gibco), 400 µg/ml gentamycin (Gibco) and 250 µg/ml Fungizone® (Amphotericin B,

Gibco 200 µg/ml.)

Coagulated blood was gently removed from the gills, and the filaments were excised

from the arches and washed twice in 10 ml of the PBS solution. The filaments were

thereafter transported to the lab on ice and transferred to 5 ml trypsin (2.5 % solution

from Gibco) and incubated on a shaker (120-200 rpm) for 20 minutes.

The cell suspension was aspirated from the tubes and filtrated through a 100 µm

nylon filter into a stopping solution; PBS containing 10 % Gibco Fetal bovine serum

(FBS). Remaining filaments were trypsinated in 5 ml 2.5 % trypsin solution for an

additional 20 minutes and filtered into the same stopping solution. The cell

suspension was then centrifuged at 200 x g for 10 minutes at 4ºC, and the cell pellet

was re-suspended in culture medium (Leibowitz L-15 medium supplemented with 2

mM L-glutamine, 5% FBS, 100µg/ml PEST and 200 µg/ml gentamycin). The cells

were then transferred to 75 cm2 Falcon culture flasks with GEC culture medium

(Leibowitz L-15 medium with 2 mM glutamine, 5 % FBS, 100 µg/ml PEST and 200

µg/ml gentamycin), and incubated at 20°C. After 24 hours incubation, the cells were

rinsed twice with PBS to remove non-attached cells. Culture medium was changed

every second day until the experiment started.

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Virus propagation. The virulent freshwater strain fw-DK-3592B (Genotype Ia) of

VHS virus isolated from RBT in Denmark (24), or the marine strain ma-1p8

(Genotype Ib), isolated from herring (Clupea harengus) in the Baltic Sea (16, 24)

were grown on the BF-2 cells in Eagle’s minimum essential medium with Earle’s

balanced salt solution (EBSS) supplemented with 16.4 mM Tris-buffer, 5.3 mM

NaHCO3, 10% fetal bovine serum (FBS), 4mM l-glutamine and 50 og ml-1

gentamicin. A stock of fw-DK-3592B with a dose of 8.66 log TCID50 ml-1

and a

stock of 1p8 with dose of 7.37 log TCID50 ml-1

were frozen in vials at -80 °C.

Infection assay GEC. Adherent gill epithelial cell were trypsinized 5-7 days after

isolation and transferred 400 000 - 500 000 cells/well to 24 well plates covered with

13mm diameter plastic coverslips. The GEC were cultured for 2-3 days. At time of

infection frozen vials with virus were thawed, and GEC were inoculated with 105

TCID50/ml of the fw-DK-3592B strain, or the ma-1p8 strain.

On each day from day 1 to 6 post inoculation subsets of wells were fixed by adding

1ml of 80% acetone to three wells of both inoculates and left for 2 min. Fixed cell

layers were then washed with Tris buffer saline (TBS) and incubated with IP5B11

(12) against VHSV-N protein for 1 hour at 37ºC. Coverslips were subsequently

washed with TBS and incubated for 30 min with rabbit anti mouse IgG (DAKO),

followed by APAAP (DAKO) mouse monoclonal for 30 min. After washing, Fast red

(1g/l; Sigma, St Louis, MO, USA) and Naphthol AS-MX-phosphate (0,2 g/l; Sigma)

with 1mM levamisole (Sigma) in 0.1 M TBS was added to develop for 20 min.

Counterstained with hematoxylin and mounted with Aquamount (BDH Laboratory).

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The numbers of infected cells were estimated from 200 counted cells in each of three

parallel coverslips at 1, 2, 3 or 6 days post inoculation of virus.

Viability assay GEC. After 3-7 days of culturing the adherent GEC were trypsinized

and transferred to a 96 well plates at a concentration of 40.000 – 50.000 cells/well in

200 µl GEC culture medium. The GEC were sub-cultured for an additional 2-3 days

before removing media and adding 100 µl of a 106

TCID50/ml of fw-DK-3592B or

ma-1p8, respectively. After 0 to 7 days of inoculation, 20 µl of CellTiter AQueous One

solution reagent (Promega) was added to each well containing 100 µl of medium. The

cells were incubated for an additional 24 hours at 15ºC. The quantity of formazan

product in each well was measured at 490nm using a 96 well absorbance plate reader

and is directly proportional with the number of living cells in the culture well.

Transepithelial passage in GEC. GECs were grown in culture flasks for about 5-9

days, after which the cells were trypsinated and resuspended in medium. A sample

was counted by using KOVA Glasstic®

slide 10 with grids. 200 000 cells/insert was

added per filter (FALCON 0.4 µm pore size PET-track-etched membrane Cell culture

insert). The cells were cultured on cell culture chamber inserts with porous bottom

dishes providing virus access to pass through the membrane surface (Fig. 1). The

medium was frequently changed ensuring good cell growth. The transepithelial

resistance was measured daily by use of Millicell®

ERS meter with chopstick

electrodes (Millipore Co., Bedford, MA, USA). Result was expressed as K ohms x

cm2. Infection of cells was initiated when resistance exceeded 1 K ohms x cm

2

(usually after 24-48h of incubation). GEC inserts were inoculated with, 105

TCID50/ml of ma-1p8 or fw-DK-3592B. At 2, 4, 8, 24 and 48 hours, a medium vial of

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100 µl of 3 replicate cell culture chambers was sampled from the basal side of the

filter (Fig. 1). The samples were transferred to a sub-confluent layer of BF-2 cells

cultured on 24 wells plates (20-48 hours old) at 1:100 dilution. The inoculated cells

were incubated at 15flC and inspected for up to 7 days after inoculation for the

occurrence of CPE following primary incubation. After 7 days of culture, cell culture

medium (supernatant) was passed onto fresh (20-48 hours old) BF-2 cells cultured on

24 well plates (2nd

passage). Inoculated cells were inspected for the occurrence of

CPE during the secondary incubation period lasting up to 7 days after inoculation.

Cytopathic effects (CPE) consisted of cell rounding of focal appearance, followed by

detachment of cells from the plastic surface. Identification of virus was performed by

use of a standard ELISA technique using mab IP5B11 as primary antibody (19).

In vitro infection of RBT macrophages. Disease-free RBT (no history of disease

during the life-cycle of the fish) of a size of 30-80g, cultured in fresh water were

anaesthetized with chlorbutanol (1g/L) and killed by decapitation. The head kidney

was dissected out and transferred to incubation buffer (HEPES-buffered (20mM, pH

7.4) saline solution). The head kidney was torn into pieces with two sterile needles

and gently pressed through a nylon mesh (pores 0.3 mm) with a glass rod. The cell

fraction was concentrated by centrifugation and resuspended in incubation buffer.

Homogenous cell fractions were obtained by discontinuous Percoll (Pharmacia)

gradient density centrifugation (G/rpm). Cells concentrated in the bands between the

fraction densities 37% and 54% Percoll were diluted in L-15 medium containing

penicillin 100 U/ml, streptomycin 100 たg/ml and kanamycin 100 たg/ml (all from

Gibco) to give a final cell concentration of 106 cells/ml. One ml of this solution was

seeded onto 12 mm Ø coverslips in culture wells, and incubated at 14ºC in air. The

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cell medium was changed and the wells washed twice with PBS to remove the non-

adherent cell fraction. The following day (after 24 hours of incubation) the wells were

inoculated with identical concentrations, 3.9x107

infective particles, of the fw-DK-

3592B strain or the ma-1p8 strain and the number of infected cells was estimated

from 200 counted cells in two parallel coverslips incubated for 1, 2, 3 or 6 days post

infection. The cell layers were fixed in 80% acetone, washed with Tris buffer saline

(TBS) and incubated with mab IP5B11 for 1 hour at 37ºC. Coverslips were then

washed with TBS and incubated for 30 min with rabbit anti mouse IgG (DAKO),

followed by APAAP (DAKO) mouse monoclonal for 30 min. After washing, Fast red

(1g/l; Sigma, St Louis, MO, USA) and Naphthol AS-MX-phosphate (0,2 g/l; Sigma)

with 1mM levamisole (Sigma) in 0.1 M TBS was added to develop for 20 min.

Counterstained with hematoxylin and mounted with an aqueous mounting medium

(Aquamount, BDH Laboratory).

Viability assay M . The wells were added 200 ol of 8 oM ethidium homodimer (red

fluorescent visualizes dead cells) and 2 oM calcein AM (green fluorescent visualizes

live cells; Molecular Probes) and diluted in phosphate buffered saline (PBS) in each

well. Incubation was for 30-120 minutes before the coverslips were evaluated in an

inverted fluorescence microscope (Leica). At 6 days post inoculation the number of

live and dead cells was estimated from 100 counted cells in eight parallel coverslips.

In vivo virulence study in RBT. The fw-DK-3592B and ma-1p8 strains were tested

for their in vivo virulence in rainbow trout fry. Both isolates were propagated and

titrated on the bluegill fry caudal trunk cell line (BF-2; 28) according to standard

procedures (12). Both isolates used were of low passage numbers (maximum 5

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passages). RBT, SPF fish reared at the laboratory, of an average weight of

approximately 0.2g (average of 15 fish) were used in the trial. Approximately 200 fish

were transferred to each of the small tanks (no. 1-4) containing 8 liter of softened tap

water, with temperature maintained at 9-11ºC throughout the experiment. The

challenge dose was 1.6 x 105 TCID50/ml for ma-1p8 (tank 4) and of 2.9 x 10

3

TCID50/ml for fw-DK-3592B (tank 3) and virus free medium for the negative control

(tank 1). The fish were challenged for 2 hours in aerated water by immersion, after

which water flow was resumed. Mortality was recorded daily up to 42 days post

infection. Fish that died during the trial and the survivors were sampled for virus

isolation including the negative control group. From the negative control group and

the group infected with ma-1p8, fish dying on the same day were all pooled (up to 7

fish/pool). The pooled sample was frozen at ÷25flC or ÷80flC until examination. At

termination 25 fish from each group were examined for virus in 3 pools and examined

as described (16).

Virus distribution and quantification. From tank 1-4 sequential sampling of 6 fish

from each group/tank, were collected at day 0 (before onset), 1, 2, 3, 5, 7, 10, 14, and

21 post infection (p.i.). Fish were culled for immunohistochemistry (3 fish) and virus

quantification (3 fish). Immunohistochemistry was performed on slides of paraffin

embedded specimens (14) and examined as described previously (7). Fish were

thawed in cold water for virus quantification, weighed and EMEM was added to give

a dilution of 1:10 (w/v). The fish were grounded with mortar, pestle and sterile sand.

Hexamicin was added and the samples were kept at 4ºC overnight and titrated on BF-

2 cells in 96-well plates (NUNC) with calculation of titer after 7 days. Samples that

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did not produce cytopathogen effect (CPE) upon 1st passage were sub-cultured by 1

additional passage.

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RESULTS

Virus infectivity and cytopathic effects in GEC. The infectivity of strain ma-1p8

was assessed in GEC by use of immunohistochemistry and any cytopathic effects

were assessed on the basis of morphological changes (CPE) as defined previously.

GECs were infected with a dose of 105 TCID50/ml per well and over a period of 7

days cells were negative by immunocytochemistry and no cytopathic effects were

observed. For fw-DK-3592B, 23% of the GECs were found infected by

immunocytochemical staining by day 1 post infection (Fig. 2), and 64% were infected

by day 2. At day 3 and 4, counting was not possible as no cells were found attached to

the coverslips because of virus-induced cytopathic effect.

Viability assay GEC. The virulence of the ma-1p8 isolate was also evaluated by

assessing the viability of ma-1p8-infected GEC using CellTiter 96® AQueous One

Solution Cell Proliferation Assay, used for determining the number of viable cells in

cytotoxicity assays. As this is considered a more sensitive method than morphological

assessment it was included to document cytotoxicity (CPE) induced by ma-1p8

infection. GECs inoculated with isolate ma-1p8 was not different from non-infected

cells while GECs infected with fw-DK-3592B showed a rapid decline in viability and

at three days post infection viability was close to zero, indicating full cytolysis (Fig.

3).

GEC transepithelial passage of VHSV. In an attempt to mimic the in vivo infection

of virus across the gill epithelial barrier (Fig. 1), GECs were grown on filters where a

polarization of the cells is established (19, 29) and the filter inserts were subsequently

inoculated with ma-1p8 on the apical side (Fig. 1). Media vials collected from 3

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replicate chambers at each time point from the basal side of GECs inoculated with

ma-1p8 were virus negative by culture on BF-2 cells and by use of ELISA at 2, 4, 8

and 24 hours p.i.. By 48 hours p.i. 2 out of 3 replicate media vials were virus positive

by 48 hours p.i. by culture on BF-2 (CPE) and confirmed by ELISA (Table 1).

Conversely, GECs infected with fw-DK-3592B were virus positive (1 of 3 replicates)

in the basal medium at 2 hours after virus was added to the apical medium. Two of

three replicates were positive by 4, 8, and 24 and by 48 hours p.i. all three replicates

were virus positive in the basal medium (Table 1). These findings indicate a rapid

translocation of the fw-DK-3592B strain across the polarized epithelial cells.

In vitro infection of RBT macrophages. As previous studies (5, 28) have implicated

that macrophages possibly play role in primary replication of VHS virus after the

virus bypass the primary barriers (skin, mucus), head kidney macrophages isolated by

gradient centrifugation were infected with the ma-VHSV and fw-VHSV isolates.

Interestingly, few macrophages were found infected with the fw-DK-3592B strain,

averaging from 4% by day 1 in two parallel wells and peaking at 8.75% positive cells

by culture day 3 (Table 2). By 6 day of culture, the percentage positive cells had

declined to 3.5%. Positive staining for virus in infected macrophages was seen in

confined structures in the cell cytoplasm (Fig. 4). By this time and later, a few

macrophages (viability assessment described below) were found with a condensed

nucleus or in lysis, possibly as a result of virus replication. In macrophage cultures

infected with the ma-1p8 strain, an average of 0.5% virus-positive cells were found by

day 1 and there was no increase in the number of infected macrophages over the

culture period (Table 2). By day 6, no cells were found positive.

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Viability assays of M . Since detection of VHS virus by immunoenzyme methods

has limited sensitivity (7) and there is a possibility of underestimating the number of

virus-infected cells using this method, evaluation of cell viability of macrophages kept

in culture was also included, both in infected and non-infected parallels. The viability

of macrophages was assessed by staining the cultures using a combination of ethidium

homodimer (red fluorescence; dead cells) and calcein AM (green fluorescence; live

cells) to visualize live versus dead macrophages in culture.

The viability assay (counting of 100 cells in 8 parallels) showed that 78% of non-

infected macrophages were viable by day 6 in culture. This was not different from

what was found for cultures infected with strain ma-1p8, while for strain fw-DK-

3592B, only 55% of the cells were viable by day 6 post infection (Fig. 5). By visual

examination in a fluorescence microscope the condensed cells were identified as dead

cells (Fig. 6).

In vivo challenge study in RBT

The in vivo virulence characteristics of isolates fw-DK-3592B and ma-1p8 have been

tested previously (24), but since they had been subject to additional passages in cell

culture, a challenge trial in rainbow trout fry was performed to confirm previous

results. For isolate fw-DK-3592B, the cumulative mortality was 100% by day 13 and

fish died with classical VHS symptoms. Dead fish sampled at day 7 and 12 were all

found VHSV positive by culture and ELISA. For isolate ma-1p8, the cumulative

mortality by day 24 post challenge was 26%, higher than for the non-infected controls

(15% by day 24). No fish died between day 24 and 42 in the ma-1p8 challenged group

or the controls.

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VHS was confirmed by histology and immunohistochemistry with

classical histological changes in the fw-DK-3592-B group by day 3 post

infection in target organs such as kidney (not shown). For ma-1p8

infected fish, histopathological changes were not observed at any

collection times and no virus was detected by immunohistochemistry at

any time points post challenge. By culture fish were positive by day 2

post challenge for fw-DK-3592B with a low titer (7.1 x 102 TCID50/g

tissue) reaching a titer peak by day 7 (1.3 x 108 TCID50/g tissue). In the

ma-1p8 group 10 pools of dead fish (pools of 1-7 fish, 31 fish total), were all negative

for VHSV by culture. At day 42, 3 pools of 8, 8 and 9 fish, respectively were

examined and one pool (of 8 fish) was virus positive. In the controls, all pools

examined were negative for VHSV. Together, this indicates a high non-specific

mortality in the ma-1p8 and the control groups.

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DISCUSSION

In previous studies it has been well documented that marine isolates of VHSV

are non-pathogenic to rainbow trout following bath challenge while variable mortality

(up to 60%) has been found after injection challenge (24). The underlying factors

have not been elucidated in any detail. In this study we confirmed published results

that a marine isolate of VHSV (ma-1p8) is very inefficient at infecting fry of rainbow

trout following bath challenge. These findings correlate with a low ability of the virus

to infect and translocate over polarized, primary gill epithelial cell (GEC) cultures and

the low in vitro infectivity of isolate ma-1p8 in primary cultures of head kidney

macrophages. These results contrast the findings for the highly virulent freshwater

isolate of VHSV fw-DK-3592B.

The portal of entry for VHSV infection of rainbow trout has not been

conclusively proven. We have previously shown that fw-VHSV can be detected in gill

epithelial cells by immunohistochemistry at early time following an experimental

infection, however limited to a few epithelial cells (1). Similar observations have been

made by others (3, 10, 18) but these studies can only serve as indication as to how the

virus gains access to systemic circulation in trout. An interesting observation is the

finding that a low virulent strain of VHSV (Makah) for rainbow trout showed low

replication in fin tissue and not at all in gill tissue ex vivo (31). Our findings are in

concert with this observation.

Several studies have suggested that kidney and spleen macrophages have an

important role as target cells for the initial replication of VHSV (10) and previous

studies have shown that kidney and spleen macrophages are infected in vivo (1,6,7).

In this study we found that the strain fw-DK-3592B infected head kidney

macrophages in culture (Fig. 4) also leading to reduced cell viability, i.e. the virus

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strain was virulent (able to induce cell damage) to isolated head kidney macrophages.

In contrast a very low percentage of the cultured macrophages were infected with ma-

1p8 and the number of viable cells was not different from the non-infected control

(Fig. 5) indicating lack of virulence towards macrophages. Also, the number of cells

that was infected and supported viral replication was limited, below 10% for fw-DK-

3592B and 0.5% for ma-1p8. There is also a possibility that we have underestimated

the number of virus positive cells since infected cells are more easily washed off from

the slide during incubation for immunohistochemical staining and viability staining.

However, our findings are concordant with a previous study (28) where it was shown

that in primary macrophage cultures from rainbow trout 8% of the cells supported

VHSV replication and for turbot (Scophthalmus maximus) 1.7% of the cells were

found positive (a strain of rainbow trout origin was studied). No significant cytopathic

effect was observed in experiments performed by Tafalla et al. (28) in contrast to our

findings while Estepa et al. (5) showed that VHSV lysed macrophages from RBT in

vitro which is more in line with what we observed. It thus seems that macrophages

can be infected with virulent strains of VHSV (of rainbow trout origin). However, the

initial VHSV replication occur in endothelial cells and to a lesser degree in

macrophages. Further there is also a relatively low infectivity in macrophages in

vitro, and taken together it therefore remains to document that macrophages are the

most important target cells for VHS virus at early time of infection.

Concordant with previous findings ma-1p8 infects fry of rainbow trout at a

very low prevalence following in vivo challenge, while the freshwater strain was

highly virulent (10). The mortality developed rapidly in the fw-DK-3592B challenged

fish from day 4 to 10 and with 100% mortality by day 12 p.i. The background

mortality in the controls was higher than normal possibly associated with fry being

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used for challenge. These sized fish are more susceptible to handling and sorting

which can account for some of the background mortality. Fry were used since they are

considered more susceptible to challenge than larger fish which would provide a

sensitive biological test system. Overall from histological examination,

immunohistochemistry and virus re-isolation there is good documentation provided to

state that isolate ma-1p8 was avirulent with no VHS related mortality occurring over

the course of the experiment. At termination three pools were examined and one of

the pools was found positive for VHSV by culture, showing that isolate ma-1p8 can

infect rainbow trout but without clinical signs or pathology in internal organs,

corroborating previous findings by Skall et al. (24).

Over the last 20 years VHSV have been isolated from several marine fish

species (25) and this has created a concern that marine strains of VHSV can be a

potential source of infection for farmed rainbow trout (25). Studies performed by

Skall et al. (24) demonstrate very clearly that rainbow trout has a very low

susceptibility to several VHSV originating from marine fish when tested by

immersion challenge. 139 ma-VHSV isolates from wild marine fish and from farmed

turbot did not cause mortality in RBT by immersion, but some isolates caused

mortality by injection (24). The low virulence of ma-VHSV in RBT can be related to

their limited ability to pass a confluent gill epithelium or the external barriers to

infection. However, one should not rule out the possibility that ma-VHSV can cause

disease in RBT in a commercial setting and studies (26,27) show that the ma-VHSV

isolates are genetically closely related to fw-VHSV isolates. Recently the first known

disease outbreak in sea-farmed rainbow trout caused by genotype 3 of VHSV was

documented in Norway (13). Bath challenge trials in rainbow trout with this isolate

showed high mortality and is the first registration of a marine VHS virus of genotype

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3 being virulent for rainbow trout (13). It is an indication of a possible adaption of a

marine isolates of VHSV to a new host. Future studies including infectivity and

virulence towards RBT gill epithelial cells of this new variant of a marine VHSV

isolate would be an interesting endeavor and should be pursued.

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REFERENCES

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2. Brudeseth, B.E., Raynard, R.S., King, J.A., Evensen, Ø. 2005. Sequential

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3. Chilmonczyk, S., Voccia, I., Monge, D. 1995. Pathogenesis of viral

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4. Drolet, B.S., Rohovec, J.S., Leong, J.A. 2002. The route of entry and

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5. Estepa, A., Frias, D., Coll, J.M. 1992. Susceptibility of trout kidney

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T. 1994. Comparison of immunohistochemistry and virus cultivation for

identification of viral haemorrhagic septicaemia virus in experimentally

infected rainbow trout (Oncorhynchus mykiss). Dis. Aquat. Organ. 20:101-9.

7. Evensen, Ø., Olesen, N.J. 1997. Immunohistochemical detection of VHS virus

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11. Kocan, R., Bradley, M., Elder, N., Meyers, T.R., Batts, W.N. Winton, J.R.

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15. Meyers, T. R. Winton, J. R. 1995. Viral hemorrhagic septicemia virus in North

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16. Mortensen, H.F., Heuer, O.E., Lorenzen, N., Otte, L., Olesen, N.J. 1999.

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17. Munro, A.S. 1996. Report on the first recorded outbreak of viral haemorrhagic

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22. Ross, K., McCarthy, U., Huntly, P., Wood, B., Stuart, D., Rough, E., Smail, D.

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24. Skall, H.F., Slierendrecht, W.J., King, J.A., Olesen, N.J. 2004. Experimental

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31. Yamamoto, T., Batts, W.N., Winton, J.R. 1992. In vitro infection of salmonid

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hemorrhagic septicemia virus. J. Aquat. Animal Health 4:231-39. ACCEPTED


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Fig. 1 depicts the layout of the filters on which the GEC was grown. The setup

mirrors the water-to-blood barrier of the gills of rainbow trout.

Fig. 2. Gill epithelial cells (GECs) infected with fw-DK-3592B at 1 day p.i. Single

cells are detected as virus positive in the cytoplasm by alkaline phosphatase (red

color) using mab IP5B11 and APAAP/Fast Red. The slide is counterstained with

Mayer’s hematoxylin (blue nuclei). Bar = 10 たm.

“GILLS” Basolateral

medium

”WATER”

Apical

medium

Samples transferred to BF2.

”BLOOD” Filter

If CPE:

VHSV verified by ELISA

Well

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Fig. 3. GEC cultures were infected with strains fw-DK-3592B or ma-1p8,

respectively and incubated for 7 days. 1 parallel was left non-infected. Mean

absorbance value of GEC incubated with CellTiter AQueous One solution reagent (at

490 nm) is given and each value is based on mean value of 11 wells. Background

absorbance is set to zero.

Fig. 4. Macrophages, infected with fw-DK-3592B at 3 days p.i. cells with cytoplasmic

staining (red coloration). Virus antigen is detected by alkaline phosphatase coupled

antibodies visualized by Fast Red. Counterstained with Mayer’s hematoxylin. Bar =

10 たm.

GEC viability

0

0.05

0.1

0.15

0.2

0.25

1 3 5 7

Days post infection

Absorb

ance 6

00 n

m

fw-DK3592B

ma-1p8

Non-infected

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Fig. 5. The mean percent (+standard error of the mean) live macrophage cells infected

with fw-DK-3592B or ma-1p8 isolates, respectively. Results by 6 days p.i. are given.

Cells were stained with ethidium homodimer and calcein to stain dead and live cells,

respectively. The numbers are based on counting 100 cells in eight parallel wells.

Fig. 6. Viability assay visualizing dead macrophages using ethidium homodimer (red

fluorescence) and calcein AM to visualize live macrophages (green fluorescence).

Bar=10 たm.

Macrophage viability

0

20

40

60

80

100

fw-DK-3592B ma-1p8 Non-infected

% L

ive c

ells

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0 %

10 %

20 %

30 %

40 %

50 %

60 %

70 %

80 %

90 %

100 %

1 4 7 10 13 16 19 22 25 28 31 34 37 40

Days after challenge

% c

um

ula

tive m

ort

ality

fw-DK-3592B

ma-1p8

Control

Fig. 7. The percentage cumulative mortality following VHSV challenge with fw-DK-

3592B and ma-1p8 strains is shown. Each group was challenged in a separate tank. A

separate control tank with non-challenged fish was also included. Fish sampled for

studies of viral distribution are not included in the figure.

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Hours p.i. fw-DK-3592B ma-1p8

2 1 0

4 2 0

8 2 0

24 2 0

48 3 2

Table 1. GEC filter inserts infected with fw-DK-3592B and ma-1p8 on the apical side and

samples collected at the basal side (see Fig. 1 for details) were examined for presence of virus

in BF-2 cells. The number of positive wells (of 3 per time point) is indicated over an

incubation period of 48h after virus was added to the apical side.

% +ve cells infected with fw-DK-3592B % +ve infected with ma-1p8 Days p.i.

C1 C2 C1 C2

1 3,0 5,0 0 1,0

2 4,0 8,0 0 0,25

3 8,0 9,5 0,5 0,5

6 3,5 3,5 0 0

Table 2. Detection of VHSV in cultured macrophages isolated from head kidney of RBT and

subsequently infected with isolate fw-DK-3592B or isolate ma-1p8. The number of infected

cells was estimated from 200 counted cells in each of two parallel coverslips (C1 and C2)

after staining of virus positive cells using mab IP5B11 and developed with APAAP.

Percentage positive cells are indicated.

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difference in virulence of marine and freshwater isolates...

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