Detection of Viral Hemorrhagic Septicemia Virus

Cell culture and molecular assays are used for the detection and

identification of fish viruses. 

Viral hemorrhagic septicemia virus (VHSV) is 

considered to be one of the most important viral 

pathogens of finfish and is listed as reportable by 

many nations and international organizations (Office 

International des Epizooties 2006). Prior to 1988, 

VHSV was thought to be limited to Europe (Wolf 

1988; Smail 1999). Subsequently, it was shown that 

the virus is endemic among many marine and 

anadromous fish species in both the Pacific and 

Atlantic Oceans (Meyers and Winton 1995; Skall et 

al. 2005). Genetic analysis reveals that isolates of 

VHSV can be divided into four genotypes that 

generally correlate with geographic location with the 

North American isolates generally falling into VHSV 

Genotype IV (Snow et al. 2004). In 2005-2006, 

reports from the Great Lakes region indicated that 

wild fish had experienced disease or, in some cases, 

very large die-offs from VHSV (Elsayed et al. 2006, 

Lumsden et al. 2007). The new strain from the Great 

Lakes, now identified as VHSV Genotype IVb, 

appears most closely related to isolates of VHSV 

from mortalities that occurred during 2000-2004 in 

rivers and near-shore areas of New Brunswick and 

Nova Scotia, Canada (Gagne et al. 2007). The type 

IVb isolate found in the Great Lakes region is the 

only strain outside of Europe that has been associated 

with significant mortality in freshwater species. 

As of mid-2007, VHSV strain IVb has been isolated 

from fish in Lake Michigan, Lake Huron, Lake St. 

Clair, Lake Erie, Lake Ontario, the Saint Lawrence 

River, inland lakes in New York, Michigan and 

Wisconsin as well as the coastal areas of eastern 

Canada. The new strain has an exceptionally broad 

host range and has been isolated from over 25 species 

of finfish to date. Significant mortality has been 

reported in muskellunge, freshwater drum, yellow 

perch, round goby, emerald shiners and gizzard shad. 

Fisheries managers throughout the US and Canada 

are concerned about the further spread of this highly 

virulent virus among populations of native freshwater 

fish and the introduction of VHSV into the private 

aquaculture industry could lead to trade restrictions 

as well as direct losses from disease. As a result, 

agencies in the US and Canada have placed 

restrictions on the movement of fish or fish products 

that could represent a risk for the spread of VHSV to 

regions outside of the currently known geographic 

range. These restrictions include requirements for 

viral examinations by standard methods. The purpose

of this information sheet is to review some important 

factors for the isolation of VHSV Genotype IVb 

using cell culture assays and its identification by the 

polymerase chain reaction (PCR) assay. 

U.S. Department of the Interior USGS FS 2007-3055 

U.S. Geological Survey July 12, 2007 

freshwater drum

muskellunge

yellow perch 

emerald shiner

Wayne Dave EPA, Shedd Aquarium
Table 1. Relative plating efficiency for various 

isolates of VHSV on selected cell lines. The numbers shown 

represent the log10 of the virus titer determined by plaque assay of a stock 

suspension of each virus isolate on each of the lines. The VHSV strains are: F1 

(Denmark); 23-75 (France); KRRV (Japan); Makah (WA), muskellunge (MI) and mummichog (NB). The 

genotype of each isolate is shown in parentheses. n.d. = not done. 

Cell culture for initial isolation:

Both the Manual of Diagnostic Tests for Aquatic 

Animals (OIE 2006) and the Suggested Procedures 

for the Detection and Identification of Certain 

Finfish and Shellfish Pathogens (American 

Fisheries Society 2005 - in revision) specify cell 

culture assays for determination of virus-free 

status. While European strains of VHSV grow well 

on the cell lines recommended by the OIE (e.g. 

RTG-2 and BF-2), isolates of the North American 

genotype are more efficiently isolated using the 

EPC, FHM or BF-2 lines (Table 1). These latter 

cell lines, available from the American Type 

Culture Collection, are acceptable to the OIE and 

specified in the revised version of the AFS 

"Bluebook". 

F1 

(Ia) 

23-75 

(Ia) 

KRRV

(IVa) 

Makah 

(IVa) 

muskellunge

(IVb) 

mummichog

(IVb) 

EPC-ATCC 6.5 6.8 5.6 8.6 7.3 7.0

EPC-Newport 6.3 7.0 5.2 8.6 7.5 7.3

FHM-J 6.3 6.9 5.4 8.5 7.4 7.4

FHM-Lamar 6.5 n.d. 5.5 8.7 7.5 7.2

CHSE-214 7.0 5.4 5.1 8.1 5.5 6.2

RTG-2 7.2 n.d. <3.7 5.1 3.3 3.7

BF-2 7.9 6.7 7.4 8.3 7.4 7.0

Incubation temperature affects the growth of all fish 

viruses and strains of VHSV are best isolated at 

incubation temperatures between 15-18°C as 

recommended by the Manual of Diagnostic Tests for 

Aquatic Animals (OIE 2006) and the Suggested 

Procedures for the Detection and Identification of 

Certain Finfish and Shellfish Pathogens (American 

Fisheries Society 2005). As shown in Table 2, the 

Genotype IVb isolate of VHSV from the Great Lakes 

grew best at 15°C. Plating efficiency began to decline 

at 20°C, and the isolate did not grow at 25°C. 

Table 2. Plaque assay titers for a North American 

Genotype IVb isolate of VHSV from muskellunge 

plated on three cell lines and incubated at selected 

temperatures. The numbers shown represent the 

log10 of the virus titer detected by each of the 

lines. 

Cells 

EPC-ATCC 

BF-2 

FHM-J 

10°C 

7.04 

6.85 

7.00 

15°C 

7.06 

7.22 

7.34 

20°C 

6.26 

6.98 

6.99 

25°C 

<3.40 

<3.40 

<3.40 
Table 3. Plaque assay titers for a North American 

Genotype IVb isolate of VHSV from muskellunge plated 

on three cell lines and incubated at selected pH levels. 

The numbers shown represent the log10 of the virus titer 

detected by each of the lines. 

More so than for other fish viruses, the pH of the 

cell culture medium is particularly important for

the successful isolation of VHSV. Table 3 shows 

the effect of selected pH levels on the ability of 

three cell lines to detect the Genotype IVb isolate 

of VHSV from the Great Lakes. It is obvious from 

the data that the pH of the culture medium should 

remain at or above pH 7.4 during the assay. 

Sites of VHSV IVb isolation in the Great Lakes as 

of summer 2007. 

Hemorrhagic disease in gizzard shad. Photos courtesy of Mohammed Faisal.

West Coast: VHSV genotype IVa causes significant 

mortality in wild marine forage fish such as herring and 

sardines. These fish are critical to Pacific ecosystems.

Great Lakes: As of mid-2007, VHSV strain IVb has been 

isolated from fish in Lake Michigan, Lake Huron, Lake St. 

Clair, Lake Erie, Lake Ontario, the Saint Lawrence River, 

inland lakes in New York, Michigan andWisconsin as well as 

the coastal areas of eastern Canada. Significant mortality has 

been reported in muskellunge, freshwater drum, goby, burbot, 

yellow perch, gizzard shad, and smallmouth bass. It has been 

isolated from several other species including chinook salmon.

Photos courtesy of Garth Traxler.

VHSV Disease Outbreaks in North America 

Polymerase chain reaction for confirmation: 

The polymerase chain reaction assay has largely 

replaced the serum neutralization assay as a 

confirmatory test for VHSV. For the PCR assay to 

be broadly useful, it is important that the primers 

be located in regions of the virus genome that are 

conserved among all the strains of the virus that 

might be encountered. Following discovery of the 

Great Lakes strain of VHSV, sequence analysis of 

the new isolates showed that the primers 

recommended by the American Fisheries Society 

Fish Health Section Bluebook for PCR 

identification of VHSV were not optimal. The 

revised version of the VHS section of the 

Bluebook (available at no charge on-line at 

http://web.fisheries.org/units/fhs/VHS_inspection.

html) contains new PCR primer sequences that are 

identical to those currently specified by the OIE 

Manual of Diagnostic Tests for Aquatic Animals. 

In addition, the revised VHS section of the 

Bluebook now recommends use of an extraction 

procedure for preparation of viral RNA from cell 

culture fluids rather than a simple heat treatment.

These changes have resulted in the VHS sections 

of the Bluebook and OIE Manual becoming 

essentially equivalent. 

Cells 

EPC-ATCC 

BF-2 

FHM-J 

pH 6.6 

<3.40 

<3.40 

<3.40 

pH 7.0 

3.40 

<3.40 

<3.40 

pH 7.4 

7.04 

7.26 

7.25 

pH 7.8 

7.13 

6.92 

7.25 

pH 8.2 

6.94 

7.15 

7.38 
References: 

American Fisheries Society. 2005. Suggested 

Procedures for the Detection and Identification of 

Certain Finfish and Shellfish Pathogens. American 

Fisheries Society, Bethesda, MD. This publication 

is currently in revision. For the new section on 

VHSV go to: http://web.fisheries.org/units/fhs/ 

VHS_inspection.html

Elsayed, E., M. Faisal, M. Thomas, G. Whelan, W. 

Batts and J. Winton. 2006. Isolation of viral 

hemorrhagic septicemia virus from muskellunge, 

Esox masquinongy (Mitchill), in Lake St. Clair, 

Michigan, USA reveals a new sub-lineage of the 

North American genotype. Journal of Fish 

Diseases 29:611-619. 

Gagné, N., A.-M. MacKinnon, L. Boston, B. 

Souter, M. Cook-Versloot, S. Griffiths, and G. 

Olivier. 2007. Isolation of viral haemorrhagic 

septicaemia virus from mummichog, stickleback, 

striped bass and brown trout in eastern Canada. 

Journal of Fish Diseases 30:213-223. 

Lumsden, J. S., B. Morrison, C. Yason, S. Russell, 

K. Young, A. Yazdanpanah, P. Huber, L. AlHussinee, D. Stone and K. Way. 2007. Mortality 

event in freshwater drum Aplodinotus grunniens

from Lake Ontario, Canada, associated with viral 

haemorrhagic septicemia virus, Type IV. Diseases 

of Aquatic Organisms 76:99-111. 

Meyers, T.R. and J.R. Winton. 1995. Viral 

hemorrhagic septicemia virus in North America. 

Annual Review of Fish Diseases 5:3-24. 

Office International des Epizooties. 2006. Manual 

of Diagnostic Tests for Aquatic Animals (5th 

Edition). Office International des Epizooties, Paris. 

Skall, H. F., N. J. Olesen, and S. Mellergaard. 

2005. Viral hemorrhagic septicaemia virus in 

marine fish and its implications for fish farming – 

a review. Journal of Fish Diseases 28:509-529. 

Smail, D. A. 1999. Viral haemorrhagic septicaemia.

Pages 123-147 in P. T. K. Woo, and D. W. Bruno, 

editors. Fish Diseases and Disorders, Volume 3: 

Viral, Bacterial and Fungal Infections. CAB 

International, New York, New York. 

Snow, M., N. Bain, J. Black, V. Taupin, C. O. 

Cunningham, J. A. King, H. F. Skall, and R. S. 

Raynard. 2004. Genetic population structure of 

marine viral haemorrhagic septicaemia virus 

(VHSV). Diseases of Aquatic Organisms 61:11-21. 

Winton, J. R., and K. Einer-Jensen. 2002. Molecular 

diagnosis of infectious hematopoietic necrosis and 

viral hemorrhagic septicemia. Pages 49-79 in C. 

Cunningham, editor. Molecular Diagnosis of 

Salmonid Diseases. Kluwer, Dordrecht. 

Wolf, K. 1988. Viral hemorrhagic septicemia. Pages 

217-248 in Fish Viruses and Fish Viral Diseases.

Cornell University Press, Ithaca, NY. 

For additional information, please contact: 

James Winton, Gael Kurath or William Batts 

U.S. Geological Survey

Western Fisheries Research Center 

6505 NE 65th Street, Seattle, WA 98115 

Phone: 206-526-6282; FAX 206-526-6654 

E-mail: jim_winton@usgs.gov; gael_kurath@usgs.gov; 

bill_batts@usgs.gov 

Infectious disease is increasingly recognized as an 

important feature of aquatic ecosystems; however, 

the impact of disease on populations of wild fish 

has been difficult to study. While many of the viral, 

bacterial, protozoan and fungal pathogens of fish 

that were initially discovered in captive animals are 

actually endemic among wild populations, the 

introduction of exotic pathogens into aquatic 

systems can lead to explosive mortality and may be 

especially threatening to native stocks. At the 

WFRC, field and laboratory investigations, aided 

by the tools of molecular biology, have begun to 

provide information on the ecology of infectious 

diseases affecting natural populations of fish in 

freshwater and marine ecosystems.

Further Reading: 

A complete list of WFRC publications may be 

found at: http://wfrc.usgs.gov/pubs/pubs.htm