Journal of General Virology (2012), 93, 2546–2557
DOI 10.1099/vir.0.043091-0
Typing of viral hemorrhagic septicemia virus by
monoclonal antibodies
Takafumi Ito,1 Jun Kurita,13 Motohiko Sano,24 Helle Frank Skall,3
Niels Lorenzen,3 Katja Einer-Jensen3 and Niels Jørgen Olesen3
Correspondence
Takafumi Ito
takafumi@fra.affrc.go.jp
1
Tamaki Station, Aquatic Animal Health Division, National Research Institute of Aquaculture,
Fisheries Research Agency, 224-1 Hiruta, Tamaki, Mie 519-0423, Japan
2
Aquatic Animal Health Division, National Research Institute of Aquaculture,
Fisheries Research Agency, Minami-Ise, Mie 516-0193, Japan
3
National Veterinary Institute, Technical University of Denmark, Hangøvej 2, DK-8200 Århus N,
Denmark
Received 7 May 2012
Accepted 9 September 2012
Seven mAbs with specific reaction patterns against each of the four genotypes and eight
subtypes of viral hemorrhagic septicemia virus (VHSV) were produced, aiming to establish an
immunoassay for typing VHSV isolates according to their genotype. Among the mAbs, VHS-1.24
reacted with all genotypes except genotype Ie, whilst mAb VHS-9.23 reacted with all genotypes
except genotype III. mAb VHS-3.80 reacted with genotypes Ib, Ic, Id and II. mAb VHS-7.57
reacted with genotypes II and IVa, and mAb VHS-5.18 with genotype Ib only. Interestingly, mAb
VHS-3.75 reacted with all of the genotype III isolates except a rainbow trout-pathogenic isolate
from the west coast of Norway, and reacted in addition with the IVb isolate, CA-NB00-01, from
the east coast of the USA. Finally, mAb VHS-1.88 reacted with all genotype IVb isolates from the
Great Lakes, but not with CA-NB00-01. In conclusion, we can distinguish between all four
genotypes and between five of eight subtypes of VHSV by testing isolates in immunoassay using
a panel of nine mAbs. By Western blotting and transfection of cell cultures, it was shown that mAb
VHS-1.24 recognized an epitope on the viral phosphoprotein (P), whilst all others recognized
antigenic determinants on the nucleoprotein (N). From amino acid alignments of the various
genotypes and subtypes of VHSV isolates, it was possible to determine the epitope specificity of
mAb VHS-1.24 to be aa 32–34 in the P-protein; the specificities of mAbs VHS-3.80, VHS-7.57
and VHS-3.75 were found to be aa 43 and 45–48, aa 117 and 121, and aa 103, 118 and 121 of
the N-protein, respectively.
INTRODUCTION
Viral hemorrhagic septicemia (VHS) is a serious disease
occurring in wild and farmed fish in the northern
hemisphere. Until the 1980s, the disease was believed to
cause severe mortality only in farmed rainbow trout in
Europe. In recent decades, however, the causative agent VHS
virus (VHSV) has been isolated from more than 80 fresh- and
3Present address: Headquarters, Fisheries Research Agency, 15F
Queen’s Tower B, 2-3-3 Minato Mirai, Nishi-ku, Yokohama, Kanagawa
220-6115, Japan.
4Present address: Research Center for Aquatic Genomics, National
Research Institute of Fisheries Science, Fisheries Research Agency,
2-12-4 Fukuura, Kanazawa, Yokohama, Kanagawa 236-8648, Japan.
The GenBank/EMBL/DDBJ accession numbers for the VHSV isolate
sequences included in the study are AB672614–AB672621 and
AB675945.
A supplementary figure, three supplementary tables and supplementary
references are available with the online version of this paper.
2546
seawater fish species in North America, North-East Asia and
Europe (Skall et al., 2005). Some of the findings were linked
to severe die-offs, especially in the pacific North America and
in the Great Lakes in the USA and Canada.
VHSV belongs to the family Rhabdoviridae and is placed in
the genus Novirhabdovirus (Tordo et al., 2005). The virus is
enveloped and consists of an 11.1 kb genome encoding five
structural proteins and one non-structural NV protein.
VHSV isolates can be divided into four major genotypes
and a number of subtypes with almost-distinct geographical distributions (Einer-Jensen et al., 2004, 2005a, b). The
host range and the pathogenicity appear, at least to some
extent, to be linked to the genotype. The genotypes were
identified based on sequencing of full-length and/or
truncated sequences from the N-gene (Einer-Jensen et al.,
2005a; Snow et al., 1999, 2004) and the G-gene (EinerJensen et al., 2004, 2005a, b). Genotype I consists of
sublineages Ia–Ie and a number of unclassified isolates,
and includes all the European freshwater VHSV isolates,
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Typing of VHSV by mAbs
isolates from the Black Sea area and a group of marine
isolates from the Baltic Sea, Kattegat, Skagerrak, the North
Sea and the English Channel. In addition, one isolation of
sublineage Ib was made from Japan (Isshiki et al., 2001).
Genotype II consists of a group of marine isolates from wild
fish in the Baltic Sea. Genotype III isolates originate from
wild and farmed fish in the North Atlantic Ocean from the
Flemish Cap (López-Vázquez et al., 2006) to the Norwegian
coast (Dale et al., 2009), the North Sea, around the British
Islands, Skagerrak and Kattegat, whilst genotype IV consists
of at least two lineages, IVa and IVb, with IVa in western
North America and in Japan and Korea (Batts et al., 1993;
Takano et al., 2000; Nishizawa et al., 2002; Skall et al., 2005)
and IVb isolates in and around the Great Lakes (Elsayed
et al., 2006; Lumsden et al., 2007; Groocock et al., 2007) and
the eastern American coastline (Gagné et al., 2007).
As VHSV pathogenicity towards various fish species varies
from one genotype to another, it is very important to
prevent the spreading of VHSV from region to region, even
if the receiving region is not approved to be free of VHS; if
new VHSV genotypes are introduced into new areas, they
may cause severe VHS outbreaks in susceptible fish.
According to the World Organisation for Animal Health
(OIE) Aquatic Animal Health Code, even if the same disease
agent is present in both the importing and the exporting
country, the importing country can demand health
certification from the exporting country for imports when
the pathogenicity or host range of the strain in the exporting
country is significantly higher or larger than that in the
importing country. In order to prevent spread or introduction of a new VHSV genotype and in order to issue health
certificates and to implement quarantine and diseasecontrol programmes, a quick and simple detection method
for discrimination between the genotypes is desired. Usually,
genotyping has been undertaken by sequence analyses, but
this is slow and expensive, as full-length G-gene typing
(.1500 bp) is required for proper subtyping (Einer-Jensen
et al., 2005a). Access to other tests for VHSV typing, such as
a reliable immunoassay that is more convenient than
molecular techniques, would be an improvement. mAbs
against VHSV have been established in several laboratories
(Lorenzen et al., 1988, 1990; Mourton et al., 1990, 1992; Sanz
& Coll, 1992; Sanz et al., 1993; Fernandez-Alonso et al., 1998;
Ito et al., 2010). Some are widely used as diagnostic reagents,
such as IP5B11, which reacts with all known VHSV isolates
(Lorenzen et al., 1988); others are neutralizing and are used
for serological discrimination into serotypes by seroneutralization test (Olesen et al., 1993). Recently, a mAb
reacting specifically against VHSV genotype IVa, mAb VHS10, was reported (Ito et al., 2010). This work prompted us to
immunize BALB/c mice with various genotypes of VHSV
and to develop a range of mAbs, enabling us to develop a
quick immunochemical test for discriminating between the
genotypes and subtypes of VHSV. This work describes the
development and validation of methods for discriminating
between four genotypes and eight subtypes of VHSV using a
panel of nine mAbs against VHSV isolates.
http://vir.sgmjournals.org
RESULTS
mAb production and Ig classification
In order to produce mAbs for discriminating between the
VHSV genotypes, BALB/c mice were immunized with
purified preparations of VHSV isolates of seven different
genotypes (I, DK-F1; Ia, DK-3592B; Ib, KRRV9601 and
1p40; II, 1p52; III, 4p168; IVa, JF00Ehi1; IVb, Goby 1-5).
About 12 days after fusion, .70 % of the seeded wells
contained hybridomas in each fusion, and .70 hybridoma
clones secreting mAbs were produced for each virus strain.
These established mAbs were at first selected based on
their reactions in the indirect fluorescent antibody technique (IFAT) and ELISA, using a small panel of isolates
representing most genotypes of VHSV (DK-F1, I; 1p8, Ib;
DK-2835, Ic; GE-1.2, Ie; 1p52, II; 4p168, III; JF00Ehi1, IVa;
Goby 1-5, IVb). Finally, we obtained seven mAbs that
showed a specific reaction against one or a few genotypes.
The name, the homologous isolate and the Ig class of these
mAbs are shown in Table 1. The seven established mAbs
were obtained from mice immunized with VHSV genotypes IVa, Ib, II, III and IVb. None were obtained from
mice immunized with VHSV types I or Ia. Five mAbs
belong to the IgG class and two to IgM.
Reactivity of the selected mAbs against a large
panel of VHSV isolates in ELISA and IFAT
The seven mAbs selected as described above were tested,
together with the mAbs IP5B11 and VHS-10, against a
large panel of 79 VHSV isolates, including all known
genotypes and subtypes. The selection of these isolates aim
to cover a large variation of isolates with respect to their
geographical origin, geno- and serotypes, fish species and
year of isolation. The reactivity of the mAbs in ELISA and
IFAT against this VHSV panel is shown in Table 2. In
addition, a summary of mAb reactions in ELISA for all of
the VHSV genotypes is shown in Fig. S1 (available in JGV
Online). mAb IP5B11 (Lorenzen et al., 1988), known to
react with all VHSV isolates, was used as positive control in
the mAb panel. mAb VHS-1.24 (from a mouse immunized
with the Japanese VHSV IVa isolate JF00Ehi1) reacted with
Table 1. Homologous virus and Ig subclass of established
mAbs
mAb
VHS-1.24
VHS-9.23
VHS-3.80
VHS-7.57
VHS-5.18
VHS-3.75
VHS-1.88
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Homologous isolate
(genotype)
Ig class, light chain
JF00Ehi1 (IVa)
JF00Ehi1 (IVa)
KRRV9601 (Ib)
1p52 (II)
1p40 (Ib)
4p168 (III)
Goby 1-5 (IVb)
IgG1, k
IgM, u
IgG3, k
IgG2a, k
IgG1, k
IgM, u
IgG1, k
2547
T. Ito and others
Table 2. ELISA and IFAT results of mAbs against various genotypes of VHSV
Values are shown as ELISA/IFAT. ELISA: ++++, absorbance value of sample was .150 % of the value with IP5B11 for each virus isolate;
+++, 150 %.absorbance value of sample¢75 %; ++, 75 %.absorbance value of sample¢35 %; +, 35 %.absorbance value of sample¢15 %;
2, negative, absorbance value of sample ,15 %. IFAT: +, positive; 2, negative.
Isolate
DK-F1
DK-Hededam
DK-3592B
DK-3971
DK-3946
DK-5151
DK-6137
DK-7974
DK-9695377
DK-200149
DK-200051
FR-07-71
FR-23-75
FR-02-84
CZ-R5
CZ-2077
DK-5927
AU-8/95
CH-F1 262 BFH
PL-202473
M Rhabdo
1p8
1p40
1p85
1p86
1p93
1p116
1p120
1p121
5p276
SE-SVA-14
SE-SVA-1033
UK-96-43
4p37
DK-2835
DK-5123
DK-5131
FiA01a.00
FiP02b.00
NO-A163-68
EG46
GE-1.2
TR206239-1
1p49
1p52
1p53
1p54
2p51
4p101
4p168
2548
Genotype
mAb
IP5B11
VHS-1.24
I
I
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ia
Ib
Ib
Ib
Ib
Ib
Ib
Ib
Ib
Ib
Ib
Ib
Ib
Ib
Ib
Ic
Ic
Ic
Id
Id
Id
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
++/+
++/+
+++/+
+++/+
+++/+
++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
++/+
+++/+
+++/+
+++/+
++/+
++/+
++/+
++/+
++/+
+++/+
+++/+
+/+
+/+
++/+
++/+
+/+
+++/+
+++/+
+++/+
+/+
+/+
+++/+
Ie
Ie
II
II
II
II
III
III
III
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
2/2
2/2
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
VHS-9.23
VHS-3.80
+++/+
2/2
+++/+
2/2
++++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+
2/2
+++/+ +++/+
+++/+ +++/+
+++/+ +++/+
+++/+ ++++/+
+++/+ +++/+
+++/+ +++/+
+++/+ +++/+
+++/+ +++/+
+++/+ +++/+
+++/+ ++++/+
+++/+
2/2
+++/+ +++/+
+++/+ +++/+
+++/+ ++++/+
+++/+ +++/+
+++/+ +++/+
+++/+ +++/+
+++/+
++/+
+++/+
+/+
+++/+
2/2
++/+
++/+
+++/+
+++/+
+++/+
+++/+
2/2
2/2
2/2
2/2
2/2
++/+
++/+
++/+
++/+
2/2
2/2
2/2
VHS-7.57
VHS-5.18
VHS-3.75
VHS-10
VHS1.88
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
+++/+
+++/+
+++/+
++++/+
+++/+
+++/+
+++/+
+++/+
+++/+
++++/+
+++/+
+++/+
+++/+
++++/+
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
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2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
+/+
+++/+
++/+
+/+
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
+++/+
+++/+
+++/+
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
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Journal of General Virology 93
Typing of VHSV by mAbs
Table 2. cont.
Isolate
4p51
UK-H17/5/93
UK-860/94
UK-H17/2/95
F-L59x
GH30
IR-F13.02.97
NO-2007-50385
USA-Makah
USA-KHV
USA-Elliot Bay
Minter Creek,
WA
Tokul Creek,
WA
Port Angels,
WA
BC’93
CAN-3624
CAN-99-019
Quatsino, BC
JP-Obama 25
JF00Ehi1
BR01Ehi1
JF01Oit1
JSL02Yam1
PM05Ehi1
MI03GL
Goby 1-5
Lake Ontario,
NY
Budd Lake, MI
Skaneateles
Lake
CA-NB00-01
Genotype
mAb
IP5B11
VHS-1.24
VHS-9.23
VHS-3.80
VHS-7.57
VHS-5.18
VHS-3.75
VHS-10
VHS1.88
III
III
III
III
III
III
III
III
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
++/+
+++/+
+++/+
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
+++/+
+++/+
+++/+
+++/+
+++/+
++++/+
+++/+
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
IVa
IVa
IVa
IVa
+++/+
++/+
+++/+ +++/+
+++/+
++/+
+++/+ ++++/+
++++/+
+++/+
+++/+
+++/+
2/2
2/2
2/2
2/2
++/+
+/+
++/+
++++/+
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
++++/+
+++/+
+++/+
++++/+
2/2
2/2
2/2
2/2
IVa
+++/+ ++++/+ ++++/+
2/2
++++/+
2/2
2/2
++++/+ 2/2
IVa
+++/+ ++++/+ ++++/+
2/2
++++/+
2/2
2/2
++++/+ 2/2
IVa
IVa
IVa
IVa
IVa
IVa
IVa
IVa
IVa
IVa
IVb
IVb
IVb
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
+++/+
++++/+
+++/+
+++/+
++++/+
+++/+
+++/+
++++/+
++++/+
+++/+
+++/+
++++/+
+++/+
++++/+
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
++/+
+++/+
+++/+
++++/+
+++/+
+++/+
++/+
++/+
+++/+
+++/+
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
++++/+ 2/2
+++/+ 2/2
+++/+ 2/2
++++/+ 2/2
++++/+ 2/2
+++/+ 2/2
++++/+ 2/2
++++/+ 2/2
+++/+ 2/2
++++/+ 2/2
2/2
+/+
2/2
++/+
2/2
++/+
IVb
IVb
+++/+ ++++/+ ++++/+
+++/+ ++++/+ ++++/+
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
2/2
+/+
+/+
IVb
+++/+
2/2
2/2
2/2
++++/+
2/2
2/2
2/+
+++/+
+/+
++++/+
+/+
++/+
+/+
+/+
+++/+
++/+
++++/+
++++/+
++++/+
+/+
++++/+
all VHSV isolates in the panel except for VHSV Ie isolates,
whilst mAb VHS-9.23, produced from the same mouse,
reacted with all isolates except genotype III. mAb VHS-3.80
(from a mouse immunized with the Japanese VHSV Ib
isolate KRRV9601) reacted with all the genotype Ib, Ic, Id
and II isolates, except the genotype Ib isolate SE-SVA-14
and the genotype Id isolate NO-A163-68 EG46. The SESVA-14 isolate is, however, diagnosed as genotype Ib by
mAb VHS-5.18 (described below). NO-A163-68 EG46 was
isolated in 1968 in Norway, therefore all recent Id isolates
are diagnosed by mAb VHS-3.80 as belonging to genotype
Id. mAb VHS-7.57, from a mouse immunized with the
VHSV genotype II isolate, 1p52 from the Baltic Sea, reacted
only with the genotype II and IVa isolates. mAb VHS-5.18,
http://vir.sgmjournals.org
from a mouse immunized with the VHSV genotype Ib
isolate 1p40 from the Baltic Sea, reacted specifically and
only with Ib isolates. mAb VHS-3.75, from a mouse
immunized with the VHSV genotype III isolate 4p168 from
the North Sea, reacted only with genotype III isolates
except the rainbow trout-pathogenic genotype III NO2007-50-385, recently isolated from diseased sea-farmed
rainbow trout from Norway, and did react with the IVb
isolate from the American Atlantic east coast, the New
Brunswick isolate. mAb VHS-10, from a mouse immunized
with VHSV genotype IVa (JF00Ehi1), was already reported
by Ito et al. (2010) and reacted with all genotype IVa
isolates and none of the others. Finally, mAb VHS-1.88,
from a mouse immunized with the genotype IVb isolate
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T. Ito and others
Goby 1-5 from the Great Lakes, reacted with all genotype
IVb isolates except the east coast New Brunswick isolate.
No isolates of other genotypes reacted with this antibody.
The same reaction patterns were generally observed by
both ELISA and IFAT, except for the genotype IVa isolate
BC’93 against mAb 1.24, which was positive in IFAT and
negative in ELISA; this could be explained by the fact that
this isolate with mAb 1.24 only gave a reaction of 11 % of
its absorbance value when reacting with mAb IP5B11,
while our threshold absorbance value for a positive reaction was 15 % of the value with mAb IP5B11, indicating
low virus yield in the BC’93 sample.
Reactivity of the mAbs against other non-VHSV
piscine rhabdoviruses
The established mAbs were tested against 18 non-VHSV
piscine rhabdoviruses by IFAT for assessing their specificity
to VHSV. None of the anti-VHSV mAbs reacted with any
of these fish rhabdoviruses, whereas all positive controls
included were demonstrated to be positive (Table S1).
Protein specificity of the established mAbs
The results of Western blotting (WB) performed under
non-reduced conditions are shown in Fig. 1(a). mAb VHS1.24 recognized the phosphoprotein (P) of the genotype
IVa isolate JF00Ehi1, and mAb VHS-5.18 recognized the
nucleoprotein (N) of the genotype Ib isolate 1p8. From
Fig. 1(a), it appears that mAb VHS-5.18 reacted with both
the viral N- and the P-proteins. This is probably due to the
fact that the WB was carried out under non-reduced
conditions. When examining the mAb VHS-5-18 under
reduced conditions, only staining of the N-protein was
observed, demonstrating its specificity to this protein
(results not shown). The remaining five mAbs did not react
with any of the virus proteins under either reduced (results
not shown) or non-reduced conditions, indicating that
they only recognize non-linear epitopes that are destroyed
during detergent treatment.
In order to determine the protein specificity of these five
mAbs, FHM cells were transfected to express the viral
proteins of the respective VHSV isolates. The following
genomes of VHSV isolates were used: JF00Ehi1, genotype
IVa, for testing mAb VHS-9.23 and mAb VHS-7.57; 1p8,
genotype Ib, for testing mAb VHS-3.80; 4p168, genotype
III, for testing mAb VHS-3.75; and Goby 1-5, genotype
IVb, for testing mAb VHS-1.88. The results of transfection
trials are shown in Fig. 1(b). The mAbs used as positive
controls were IP1D11 recognizing the viral G-protein,
IP5B11 recognizing the N-protein, IP1C6 recognizing the
P-protein and IP1C3 recognizing the M-protein (Lorenzen
et al., 1988). Their reaction pattern against the transfected
FHM cells are shown in the insets in Fig. 1(b). In IFAT, the
mAb VHS-9.23 reacted only with FHM cells transfected
with the N-gene of genotype IVa. It was therefore
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concluded that mAb VHS-9.23 recognized the viral Nprotein of all VHSV genotypes except Ie. Similarly, the
transfection trials also showed that mAbs VHS-7.57, VHS3.80, VHS-3.75 and VHS-1.88 all recognized the N-protein
of the respective VHSV isolates (Fig. 1b).
Amino acid alignment for assessing the epitope
specificity of mAbs
As shown in Fig. 1(b), all five mAbs reacted against the Nprotein expressed on the respective transfected cells. The
assessment of the epitope specificity was performed by
combining amino acid sequence data and the unique
binding pattern of each mAb in the large VHSV panel. The
epitope specificity of mAbs VHS-1.24, VHS-3.80, VHS7.57 and VHS-3.75 was assessed from the amino acid
alignments shown in Fig. 2(a–d), respectively.
The antigen determinant of mAb-1.24 was thus set by
aligning the amino acid sequences of the P-protein of the
viral genotypes and subtypes I, Ia, Ib, Ie, III, IVa and IVb.
As mAb VHS-1.24 reacted with all VHSV genotypes except
Ie and the only amino acid motif not present in genotype Ie
isolates was RSA (arginine, serine and alanine) in positions
32–34 on the P-protein, the mAb was concluded to include
these amino acids in its epitope.
Likewise, for mAb VHS 3.80, which reacted with all Ib, Ic,
Id and II isolates except SE-SVA-14 (Ib) and NO-A163-68
EG46 (Id) and is known to react with the N-protein, its
epitope specificity was determined to include amino acid E
(glutamic acid) at position 43 and amino acids DGKV
(aspartic acid, glycine, lysine and valine) at positions 45–48
of the N-protein. When the amino acid sequence of the SESVA-1033 isolate was included in the process of epitope
assessment of VHS-3.80, two genome variants were found in
the N-gene. These variants were obtained by repeated
limiting dilutions of the virus isolate followed by nucleotide
sequencing of the N-gene of various clones. The amino acid
sequence at positions 43–48 of the N-protein of the clone
named SE-SVA-1033-9C was found to be EEDGKV, while
another clone named SE-SVA-1033-3F that did not react
with mAb VHS-3.80 was found to have the motif EEDRKV.
This strongly supports the hypothesis that the specific motif
of E at position 43 and DGKV at positions 45–48 of the Nprotein is the epitope of mAbVHS-3.80.
Similarly, the epitope specificity of mAb VHS-7.57 reacting
with genotypes II and IVa was determined to include the
amino acids Q (glutamine) at position 117 and N (asparagine)
at position 119 of the N-protein.
mAb VHS-3.75 reacts with all genotype III isolates except
NO-2007-50-385 and, in addition, reacts with the New
Brunswick genotype IVb isolate CA-NB00-01; we could
therefore, based on amino acid sequence alignments,
determine its epitope specificity to include the amino acids
G (glycine) at position 103, T (threonine) at position 118
and D (aspartic acid) at position 121 of the viral N-protein.
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Journal of General Virology 93
Typing of VHSV by mAbs
G
G
N
G
G
N
N
N
P
P
P
M
M
P
M
M
Transfected viral genes (positive control, mAb)
mAb (parent
isolate, genotype)
N (IP5B11)
G (IP1D11)
M (IP1C3)
VHS-9.23
(JF00Ehi1, IVa)
VHS-7.57
(JF00Ehi1, IVa)
VHS-3.80
(KRRV9601, Ib)
VHS-3.75
(4p168, III)
VHS-1.88
(Goby 1-5, IVb)
Summary of the reaction patterns, and the protein
and epitope specificities of the established mAbs
A summary is shown in Table 3. The protein specificity of
mAbs VHS-1.24 and VHS-5.18 was determined by WB to
be the P- and N-proteins, respectively. From transfected
cell cultures, it was shown that all mAbs VHS-9.23, VHS3.80, VHS-7.57, VHS-3.75 and VHS-1.88 recognized the
viral N-proteins of their respective homologous virus
isolates. By combining an amino acid alignment of VHSV
isolates with reaction patterns of the respective mAbs, the
http://vir.sgmjournals.org
P (IP1C6)
Fig. 1. Identification of the protein specificity
of established mAbs. (a) Immunoblotting with
mAbs and with a mixture of mAb anti-G
(IP1D11), mAb anti-N (IP5B11), mAb anti-P
(IP1C6) and mAb anti-M (IP1C3) as positive
controls. After transfer, the nitrocellulose
membrane was cut into strips and incubated
with the respective mAbs, then immunostained with HRP-conjugated secondary antibodies. The following purified VHSV isolates
were used as antigens: JF00Ehi1 (genotype
IVa), 1p8 (genotype Ib), 4p168 (genotype III)
and Goby 1-5 (genotype IVb). Strips: 1,
JF00Ehi1 with mixture of positive-control
mAbs; 2, JF00Ehi1 with mAb VHS-1.24; 3,
JF00Ehi1 with mAb VHS-9.23; 4, JF00Ehi1
with mAb VHS-7.57; 5, 1p8 with mixture of
mAbs; 6, 1p8 with mAb VHS-5.18; 7, 1p8
with mAb VHS-3.80; 8, 4p168 with mixture of
mAbs; 9, 4p168 with mAb VHS-3.75; 10,
Goby 1-5 with mixture of mAbs; 11, Goby 1-5
with mAb VHS-1.88. (b) Gene expression of
transfected FHM cells examined by IFAT. The
positive reaction using mAb anti-G (IP1D11),
mAb anti-N (IP5B11), mAb anti-P (IP1C6) or
mAb anti-M (IP1C3), respectively, is shown in
each inset.
antigen determinant of mAb VHS-1.24 was found to be
aa 32–34 in the P-protein. The epitopes of mAb VHS-3.80,
VHS-7.57 and VHS-3.75 were found to include amino
acids at positions 43 and 45–48, 117 and 121, and 103, 118
and 121 of the N-protein, respectively. Unfortunately, it
was not possible in this study to determine the specific
epitopes of mAbs VHS-9.23 (anti-all VHSV except
genotype III), VHS-5.18 (anti-VHSV Ib) or VHS-1.88
(anti-VHSV IVb), as several independent and unique
amino acids are putative candidates for being included in
their antigenic determinants.
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T. Ito and others
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Journal of General Virology 93
Typing of VHSV by mAbs
Fig. 2. Epitope mapping of mAbs VHS-1.24, VHS-3.80, VHS-7.57 and VHS-3.75 by amino acid alignment. (a) Partial
alignment of the amino acid sequences of the P-proteins of nine VHSV isolates for mapping the epitope of mAb VHS-1.24. The
blue-shaded part (aa 32–34 of the full P-protein) is determined as the epitope of mAb VHS-1.24. The isolates shown in blue
font reacted with mAb VHS-1.24. (b) Partial alignment of the amino acid sequences of the N-proteins of 12 VHSV isolates for
mapping mAb VHS-3.80. The red-shaded part (aa 43 and 45–48) is determined as the epitope of mAb VHS-3.80. The isolates
shown in red font reacted with mAb VHS-3.80. (c) Partial alignment of the amino acid sequences of the N-proteins of 12 VHSV
isolates for mapping mAb VHS-7.57. The green-shaded part (aa 117 and 121) is determined as the epitope of mAb VHS-3.80.
The isolates shown in green font reacted with mAb VHS-7.57. (d) Partial alignment of the amino acid sequences of the Nproteins 15 VHSV isolates for mapping mAb VHS-3.75. The brown-shaded part (aa 108, 118 and 121) is determined as the
epitope of mAb VHS-3.75. The isolates shown in brown font reacted with mAb VHS-3.75. All virus isolates included in (a–d)
were also used for IFAT and ELISA in this study. (e) Locations of the epitopes of mAbs VHS-1.24, VHS-3.80, VHS-7.57, VHS3.75 and VHS-10 (Ito et al., 2010) on each viral protein.
DISCUSSION
The objective of this study was to establish a panel of mAbs
able to distinguish between the various genotypes and
subtypes of VHSV. By using a panel of seven new mAbs and
mAb VHS-10 (Ito et al., 2010), reacting specifically against
genotype IVa and with IP5B11 (Lorenzen et al., 1988),
as positive control, we were able to distinguish between
all genotypes and some of the subtypes of VHSV.
Unfortunately, the mAb panel cannot distinguish between
genotypes I, Ia, Ic and Id, as almost-identical reaction
patterns are observed with isolates of these subtypes (Table
2). Genotype I and Ic have, however, not been isolated since
1970 and 1997, respectively (http://www.fishpathogens.eu).
In conclusion, we can distinguish all four genotypes and five
of eight subtypes of VHSV by testing isolates by IFAT or
ELISA using a panel of nine mAbs. Although five of the
seven recently developed mAbs did not react with any of the
virus proteins in WB, these did react in IFAT on VHSVinfected cells that had been fixed in 80 % acetone and in
ELISA using virus that had been treated with the detergent
Triton X-100. Based on these results, we suggest that the
epitope structures of the five mAbs are destroyed by the
strong detergents used in WB. Actually, in ELISA no binding
of these mAbs was observed if the virus suspension was
mixed with 0.1 % SDS before incubation (results not shown).
The protein specificities of the seven newly established mAbs
were determined and the amino acids included in the epitopes were determined for four of the seven mAbs by
combining reaction patterns with amino acid alignments
(Table 3). The locations of the epitopes of mAbs VHS-1.24,
VHS-3.80, VHS-7.57, VHS-10 and VHS-3.75 are shown
in Fig. 2(e). In general, it seems that the epitopes are
concentrated at two sites on the N-protein with putatively higher antigenicity [aa 38–48 and aa 103–121 in the
Table 3. Summary of protein specificities and epitope sites of mAbs established for genotyping of VHSV isolates
NA,
Not possible to assess in this study;
mAb
ND,
not detected; aa, amino acid.
Reaction with
VHSV isolates
IP5B11
VHS-1.24
VHS-9.23
VHS-3.80
All VHSV isolates
All except Ie
All except III
Ib, Ic, Id and II
VHS-7.57
II and IVa
VHS-5.18
VHS-3.75
Ib only
III and IVb New
Brunswick isolate
except the rainbow troutpathogenic isolate
(NO-2007-50-385)
IVa only
VHS-10
VHS-1.88
IVb only except the
New Brunswick isolate
http://vir.sgmjournals.org
Protein specificity
by WB
Protein specificity
by transfection studies
N (Lorenzen et al., 1988)
P
ND
2
2
N
N
ND
N
N
2
N
ND
ND
ND
(Ito et al., 2010)
N (Ito et al., 2010)
ND
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N
Epitope specificity
by aa alignment
NA
P aa 32–34 (RSA)
NA
N aa 43 (E)
and 45–48 (DGKV)
N aa 117 (Q)
and 121 (N)
NA
N aa 103 (G),
118 (T) and 121 (D)
N aa 38–44
(AGPFGTD)
NA
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T. Ito and others
N-terminal-core domain of the N-protein (Albertini et al.,
2006)]. We also deduced in a previous study (Ito et al.,
2010) that aa 38–46 of the N-protein forms a variable
region in VHSV, due to the presence of many substitutions
in this region. The epitopic domains on the N-protein of the
mAbs are concentrated in two regions with high antigenicity for a mouse. Therefore, if the domain is easy for
mouse antibodies to recognize, it is likely that it will be easy
for fish antibodies as well. Thus, these regions might be the
domains responsible for some of the phenotypic differences
of virus isolates, such as their host range and pathogenicity,
as well as high antigenicity.
All mAbs produced reacted with the viral N- or P-protein,
whereas none reacted with the G-protein responsible for
inducing neutralizing antibodies (Lorenzen et al., 1999). A
major reason for this is probably the immunization procedure used, as it is believed that the G-protein will misfold
significantly when injected into mice with body temperatures
above 37 uC. Olesen et al. (1999) were thus only able to
produce neutralizing antibodies in mammals by multiple
intravenous injections. In addition, the N-protein is the most
abundant protein in the virus particle and thereby more
prone to be target for a humoral immune response in mice.
Overall, there was a significant concordance between the
genotypes and the reaction patterns with the mAb panel,
but some virus isolates did not follow the patterns as
expected (Table 2). Interestingly, most of these isolates
had unique phenotypic appearances distinguishing them
from the other isolates in the same genotype/subtype. For
example, mAb VHS-3.75 reacted with all genotype III
isolates included in the large virus panel except the rainbow
trout-pathogenic isolate from Norway, NO-2007-50-385
(Table 2). Dale et al. (2009) reported that this Norwegian
isolate produced a cumulative mortality of 70 % and nearly
100 % in rainbow trout after experimental infection by
immersion and intraperitoneal injection, respectively,
while the other genotype III isolates were either non- or
low-pathogenic in rainbow trout (Skall et al., 2004). The
differences in reaction pattern of mAb VHS-3.75 between
the NO-2007-50-385 isolate and the other genotype III
viruses might indicate that this mAb recognizes an epitope
of importance for pathogenicity in rainbow trout.
In addition, mAb VHS-3.75, which was established using a
purified genotype III VHSV isolate, did not react with any
of the genotype IVb isolates from the Great Lakes, but did
react with the New Brunswick genotype IVb CA-NB00-01
isolate (Table 2). In contrast, mAb VHS-1.88 reacted with
all the genotype IVb isolates from the Great Lakes, but not
with the New Brunswick isolate. This New Brunswick
isolate was isolated in 2000 from the the Atlantic east coast
and was, based on partial G-gene sequencing (Gagné et al.,
2007), classified into a new subgroup, IVb, together with
the Great Lakes isolates included later. In 2003, genotype
IVb VHSV invaded the Great Lakes regions and caused
mass mortalities in several different fish species throughout
the regions (Elsayed et al., 2006; Groocock et al., 2007;
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Lumsden et al., 2007). Thus, it appears likely that the
isolates of the Atlantic east coast might be considered as the
origin or ancestors of the VHSV strains in the Great Lakes.
In phylogenetic analysis carried out by Gagné et al. (2007)
and Winton et al. (2008), the New Brunswick isolate did
not cluster with the Great Lakes isolates, although they all
belonged to genotype IVb. These results, together with ours,
may justify a future splitting of IVb into two subgroups (the
Great Lakes isolates and the Atlantic East coast isolates) or
into groups IVb and IVc, respectively. Actually, the genotype of the VHSV isolate CA-NB00-01 was described as
IVb by Gagné et al. (2007), but the sequence of CA-NB00-01
is at present registered in GenBank as belonging to genotype IVc.
Dopazo et al. (2002) and López-Vázquez et al. (2006)
reported that genotype III VHS viruses were isolated from
Greenland halibut caught close to the Flemish Cap in
the western part of the Atlantic Ocean. One of them, the
GH30 isolate, which was included in our panel, did react
with mAb VHS-3.75, which also reacted with the New
Brunswick genotype IVb isolate and the other genotype III
isolates except the Norwegian rainbow trout isolate.
Therefore, the VHSV genotype III strain, which is present
in the western Atlantic Ocean, may relate to the ancestor of
the genotype IVb strain at the American east coast, thereby
linking genotype IV of VHSV over the Atlantic Ocean to
European VHSV genotype III, II and I isolates.
Another interesting unique reaction pattern can be observed
in the genotype Ib group (Table 2), where one (SE-SVA-14)
of the only two rainbow trout-pathogenic isolates did not
react with mAb VHS 3.80, whereas the other (SE-SVA-1033)
did, despite the fact that they originated from the same farm
on the west coast of Sweden close to Göteborg (Nordblom &
Norell, 2000). By a later cloning of the SE-SVA-1033 isolate,
the nucleotide sequencing of the N-gene indicated that two
variants, named SE-SVA-1033-9C and SE-SVA-1033-3F,
showing different reactions to mAb VHS-3.80, were
included in the isolate. SE-SVA-1033 was isolated from a
pooled sample of tissues from five diseased sea-farmed
rainbow trout (http://www.fishpathogens.eu). Thus, we
might have received an isolate containing more than one
variant of an Ib isolate due to the pooling. The difference in
properties between these variants is very interesting for
assessing virulence determinants of VHSV to rainbow trout.
Infection trials with cloned variants of SE-SVA-1033 in
rainbow trout have, therefore, been initiated in a collaborative study involving the National Research Institute of
Aquaculture (Japan) and the OIE reference laboratory for
VHS in Denmark.
METHODS
Cell lines. The BF-2 cell line (Wolf et al., 1966) was used for
propagation of VHSV genotype Ib, Id, II and III isolates, the EPC cell
line (Fijan et al., 1983) for I, Ia, Ic and Ie isolates and the FHM cell
line (Gravell & Malsberger, 1965) for IVa and IVb isolates. The
cell lines were maintained in minimum essential medium (MEM;
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Typing of VHSV by mAbs
Mediatech) supplemented with 10 % FBS (Equitech-Bio) and
antibiotics (100 U penicillin ml21 and 100 mg streptomycin ml21).
The cultivation of cell lines and the propagation of virus were
conducted at 25 and 15 uC, respectively.
Virus. Seven isolates of different genotypes were used to immunize mice:
I, DK-F1 (Jensen; 1965); Ia, DK-5151 (Olesen et al.; 1991); Ib,
KRRV9601 (Isshiki et al.; 2001) and 1p40 (Mortensen et al.; 1999); II,
1p52 (Mortensen et al.; 1999); III, 4p168 (Mortensen et al.; 1999); IVa,
JF00Ehi1 (Nishizawa et al.; 2002) and IVb, Goby 1-5 (Groocock et al.,
2007). The virus isolates were concentrated and sucrose gradientpurified as described by Nishizawa et al. (1991). The purified isolates
were used for immunization of mice, in WB against the various mAbs
and for the RNA extractions used for cloning of each viral gene. Seventynine VHSV isolates representing all geno- and subtypes from throughout
the world were used to characterize the obtained mAbs (Table S2). In
addition, 18 non-VHSV piscine pathogenic rhabdovirus isolates were
used in the assessment of the specificities of the mAbs (Table S3).
Immunization of mice. Immunization of BALB/c mice with purified
VHSV DK-F1, DK-5151, KRRV9601,
Goby 1-5 was performed as described
Immunization of mice with the VHSV
performed as described by Lorenzen et
1p52, 4p168, JF00Ehi1 and
previously (Ito et al., 2010).
genotype Ib isolate 1p40 was
al. (1988).
IP5B11 were used as positive controls against infectious hematopoietic necrosis virus isolates, the spring viremia of carp virus isolate and
carpione rhabdovirus (Bovo et al., 1995), respectively. For the other
non-VHSV rhabdoviruses, rabbit antisera prepared by DTU-Vet were
used as positive controls.
WB. SDS-PAGE was performed according to the method of Laemmli
(1970) using 10 % (w/v) acrylamide gels under non-reducing
conditions. WB was performed according to the principles described
by Lorenzen et al. (1988). As a positive-control reagent for
immunostaining, a mixture of the anti-VHSV mAbs IP1D11 recognizing the viral G-protein, IP5B11 recognizing the N-protein, IP1C6
recognizing the P-protein and IP1C3 recognizing the M-protein
(Lorenzen et al., 1988) was used. As the mAbs were selected for their
reaction with specific genotypes of VHSV, the following purified virus
isolates were used: JF00Ehi1, genotype IVa, for testing mAbs VHS-1.24,
VHS-9.23 and VHS-7.57; 1p8, genotype Ib, for testing mAbs VHS-5.18
and VHS-3.80; 4p168, genotype III, for testing mAb VHS-3.75; and
Goby 1-5, genotype IVb, for testing mAb VHS-1.88. The protein
concentrations of the purified VHSV isolates JF00Ehi1, 1p8, 4p168 and
Goby 1-5were measured by BCA Protein Assay (Pierce), to be 583,
562, 509 and 890 mg ml21, respectively. Each gel had a width of
approximately 5 cm and was loaded with 150 ml purified virus material.
Construction of viral gene expression vector. To determine the
Cell fusion. Cell fusion was performed as described previously (Ito
et al., 2010). Cell fusion of spleen cells from mice immunized with
1p40 was performed as described by Lorenzen et al. (1988).
Ig class determination. The Ig class of the mAbs was determined
using a mouse monoclonal isotyping kit (AbD Serotec) according to
the manufacturer’s instructions.
IFAT for screening of hybridoma cell-culture supernatants. The
binding to VHSV-infected cells of Ig in hybridoma cell-culture
supernatants mAb was examined by IFAT, performed as described
previously (Ito et al., 2010).
ELISA. The reactivity of selected mAbs against the panel of VHSV
(Table S2) was evaluated by the double-sandwich ELISA described by
Olesen & Jørgensen (1991), with the exception that a mixture of protein
A-purified rabbit anti-VHSV strain DK-F1 was used as a first layer. As a
positive control, mAb IP5B11 against VHSV N-protein (Lorenzen et al.,
1988) was used. This mAb has been shown to react with all know VHSV
isolates (tested against .1000 isolates; data not shown). In this study,
when the absorbance of a tested sample was ,15 % of the value
obtained with mAb IP5B11, it is considered as negative, and when the
value was ¢15 %, it was judged as positive. In order to compare the
strength of reaction of each mAb against each virus isolate, all data are
shown by the number of + compared with the reaction value of mAb
IP5B11 to the given virus isolate, as follows: ++++, absorbance
value of sample was .150 % of the IP5B11 value; +++, absorbance
value of sample was between 75 and 150 %; ++, absorbance value of
sample was between 35 and 74 %; +, absorbance value of sample was
between 15 and 34 %; 2, negative, absorbance value of sample was
,15 % of the IP5B11 value. To normalize each ELISA test, the DK-F1
isolate was included as a standard isolate in every test run.
IFAT for mAb characterization. The binding of selected mAbs to
the large panel of VHSV isolates (Table S2) was performed using fixed
VHSV-infected and non-infected EPC cells in black 96-well plates for
IFAT (Corning) using the same procedure as for the hybridoma cellculture supernatants described above. As a positive control, mAb
IP5B11 was used. The cross-reaction of mAbs against 18 non-VHSV
fish rhabdoviruses (Table S3) was examined using fixed VHSVinfected and non-infected EPC cells as described above. mAbs 136-3
(Fregeneda-Grandes et al., 2009), 2E1 (Reschova et al., 2007) and
http://vir.sgmjournals.org
protein specificity of the mAbs, expression vectors expressing the
following VHSV genes were constructed: (i) N-, P-, M- or G-gene of
JF00Ehi1, genotype IVa, for testing mAbs VHS-9.23 and VHS-7.57;
(ii) N-, P-, M- or G-gene of KRRV9601, genotype Ib, for testing mAb
VHS-3.80; (iii) N-, P- or M-gene of 4p168, genotype III, for testing
mAb VHS-3.75; and (iv) N-, P- or M-gene of Goby 1-5, genotype
IVb, for testing mAb VHS-1.88. Each entire viral gene was cloned into
expression vector as follows: viral RNA was extracted from each
purified virus isolates using TRIzol LS Reagent (Life Technologies),
and submitted to RT-PCR amplification with primer sets designed
from the start codon to the stop codon of each gene. After treatment
with the A-attachment mix in a TArget Clone-Plus TA cloning kit
(ToYoBo) for addition of a terminal A residue, each RT-PCR product
was purified and then cloned into the pTARGET mammalian
expression vector (Promega) using Escherichia coli strains DH5a,
JM109, JM105 or ABLE C (Agilent Technologies). The nucleotide
sequence of each inserted gene was confirmed by sequencing of the
extracted plasmid.
Transfection trials. Each constructed expression vector was
extracted and purified by a ChargeSwitch-Pro Filter Plasmid Mini
kit (Life Technologies) from cultured E. coli. FHM cells were
transfected with each purified expression vector at 15 uC, using
Lipofectamine LTX and PLUS Reagent (Life Technologies) according
to the manufacturer’s instructions. Eighteen or thirty-two hours after
transfection, the cells were fixed by 80 % acetone and gene expression
of each was examined by IFAT.
Amino acid alignment for assessing the epitope specificity of
mAbs. Data of amino acid sequences of VHSV P- and N-proteins
were obtained from GenBank. The amino acid data of the isolates
representing each genotype were compared. The determined epitope
specificities of mAbs VHS-1.24, VHS-3.80, VHS-7.57 and VHS-3.75
are shown in Fig. 2(a–d), respectively.
ACKNOWLEDGEMENTS
The authors would like to acknowledge our colleagues in Europe,
North America and Japan who provided viruses for our panel;
especially we wish to thank Dr J. R. Winton, Western Fisheries
Research Center, Seattle, WA, USA, and Dr G. H. Groocock,
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T. Ito and others
Department of Microbiology and Immunology, College of Veterinary
Medicine, Cornell University, New York, NY, USA, for kindly
providing the American genotype IVa and IVb isolates. The authors
are grateful for the technical support and useful advice from
colleagues of the National Veterinary Institute, Technical University
of Denmark, and National Research Institute of Aquaculture,
Fisheries Research Agency in Japan. This work was undertaken while
the first author stayed at the National Veterinary Institute as a Longterm Researcher by Abroad fellowship from the Fisheries Research
Agency. Part of this work was supported by a grant for international
corroborated study from the Fisheries Research Agency of Japan.
Gagné, N., Mackinnon, A. M., Boston, L., Souter, B., Cook-Versloot, M.,
Griffiths, S. & Olivier, G. (2007). Isolation of viral haemorrhagic
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