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Proposal for a fourth aquabirnavirus group

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DOI: 10.1007/s00705-008-0192-9 · Source: PubMed

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Arch Virol (2008) 153:1937–1941
DOI 10.1007/s00705-008-0192-9
BRIEF REPORT
Proposal for a fourth aquabirnavirus serogroup
P. F. Dixon Æ G.-H. Ngoh Æ D. M. Stone Æ S. F. Chang Æ
K. Way Æ S. L. F. Kueh
Received: 11 October 2007 / Accepted: 29 July 2008 / Published online: 17 September 2008
Ó Springer-Verlag 2008
Abstract Four putative aquabirnaviruses, based on mor-
phology, nucleic acid type and partial RNA-dependent
RNA polymerase gene (VP1) sequence, isolated from three
tropical freshwater fish species were not neutralised by
antisera against type members of the Aquabirnavirus genus
serogroups A, B or C. Antisera produced against two of
the isolates neutralised the homologous and heterologous
isolates, but not any type member of Aquabirnavirus
serogroups A, B or C. The serological comparisons suggest
that the four isolates should be regarded as members of a
fourth Aquabirnavirus serogroup, D.
Members of the family Birnaviridae are icosahedral viru-
ses of approximately 60 nm diameter composed of five
polypeptides and containing two strands of double-stranded
(ds) RNA [10]. There are three birnavirus genera,
Avibirnavirus, Entomobirnavirus and Aquabirnavirus.
Aquabirnaviruses only infect fish, aquatic molluscs and
crustaceans, and the genus comprises three serogroups, A,
B and C [7, 8]; Serogroup A contains nine serotypes (A1–
A9) and Serogroup B comprises a single serotype, B1 [7].
Serogroup C initially comprised one virus, blotched
snakehead virus (BSNV), isolated from a blotched snake-
head cell line [8]. Subsequently, we reported the isolation
P. F. Dixon (&)
D. M. Stone
K. Way
CEFAS Weymouth Laboratory, Barrack Road, The Nothe,
Weymouth, Dorset DT4 8UB, UK
e-mail: peter.dixon@cefas.co.uk
G.-H. Ngoh
S. F. Chang
S. L. F. Kueh
Agrifood and Veterinary Authority, 60 Seng Kang East Way,
Singapore 548596, Singapore
of further birnaviruses belonging to Serogroup C from the
giant snakehead, Channa micropeltes, and marble goby,
Oxyeleotris marmorata, from several south-east Asian
countries [2]. This communication reports serological
comparisons and partial sequence analysis of four bir-
navirus isolates which do not belong to Serogroup A, B or
C, and we propose that they form two serotypes within a
fourth Aquabirnavirus serogroup, D.
The viruses were isolated from three species of fish
imported into Singapore from Malaysia (although the fish
are not native to Malaysia) and were coded M2, M3, M4
and M5. After importation, the fish generally became
moribund and showed some clinical signs of disease. M2
was isolated in 1995 from freshwater angelfish (Ptero-
phyllum scalare) showing behavioural changes (abnormal
‘‘fluttering’’ of fins and abnormal schooling behaviour) and
10% mortality in the population. M3 was isolated in 1997
from dwarf gourami (Colisa lalia) showing exophthalmia
and ascites with 50–80% mortality in affected populations;
the fish from which M2 and M3 were isolated came from
the same farm site. M4 and M5 were isolated in 1997 and
1998, respectively, from ram cichlid (Microgeophagus
ramirezi) with unquantified mortalities in the populations,
but the fish exhibited either no abnormal signs or only
darkening of body colour. The tissues from five to ten
fish were combined as one pool of brain and eye, and a
second pool of liver, spleen and kidney, then homo-
genised in transport medium (Eagle’s minimal essential
medium [EMEM] containing 2% foetal calf serum
(FCS), 3000 units penicillin, 3000 lg streptomycin,
150 lg kanamycin and 50 lg nystatin ml-1). The tissues
were diluted to 10% (w/v) suspensions, clarified at
2,5009g for 30 min at 4°C and filtered through 0.45-lm
filters. The filtrates were further diluted 1:10 (i.e. 1:100
final dilution) with maintenance medium (MM) (EMEM
123
1938
containing 2% FCS, 100 units penicillin, 100 lg strepto-
mycin ml-1). Both sample dilutions were adsorbed onto
each of four wells of monolayers of bluegill fibroblast
(BF-2) (ATCC CCL 91), Epithelioma papulosum cyprini
[5] and Chinook salmon embryo (CHSE-214) (ATCC CRL
1681) cells on 24-well plates. After 30 min, 1 ml of MM
was added. The BF-2 and EPC cells were incubated at 25°C,
the CHSE-214 cells were incubated at 18°C and all were
observed for cytopathic effect (CPE) for up to 10 days, after
which blind passages were done. Further cell line specificity
testing was later conducted using rainbow trout gonad
(RTG-2) (ATCC CCL 55) and fathead minnow (FHM)
(ATCC CCL 42) cells. The test viruses were inoculated at
1:100 and 1:1000 dilutions, the cells were incubated at 25°C
for 7 days, and then, if no CPE was observed, blind pas-
sages were done at 1:10 and 1:100 dilutions.
The new isolates were plaque titrated as described [4]
and plaque cloned prior to further work. The viruses were
concentrated using 7% polyethylene glycol 6000 [4] then
applied to carbon-coated Formvar grids, stained with 2%
methylamine tungstate and observed with a JEOL 1210
transmission electron microscope. Resistance to chloroform
and the effect of 50 lg ml-1 5-bromo-20 -deoxyuridine
(BUDR) on replication were determined [14]. Nucleic acid
was extracted from concentrated virus by the SDS-
proteinase K-phenol/chloroform/isoamyl alcohol method
[6]. Electrophoresis of RNA was carried out on 6% poly-
acrylamide gels at 200 V for 1.5 h, and then the gels were
silver stained.
Reference aquabirnaviruses (representing serotypes A1,
A2, A3, A4, A5, A6, A7, A8, A9 and B1) and their
homologous antisera were as previously reported [7].
BSNV [8] (Serogroup C) was a gift from Dr K.R. John, and
other members of Serogroup C (C1, C2, C3, C4, T1, T2,
M1 and V1), isolated from marble gobies, and S1, isolated
from the giant snakehead, have been described previously
[2]. Serogroup C isolate S1 and its homologous antiserum
[2] were used in the serological comparisons. Isolates M2
and M4 were purified, and antisera against them were
produced in rabbits [7]. All viruses were grown in BF-2
cells, either at 15°C (Serogroup A and B) or 25°C (Sero-
group C).
Oligonucleotide primers were designed by alignment of the
RNA polymerase gene sequence of the published sequences
for aquabirnaviruses with the RNA polymerase gene of
infectious bursal disease virus (IBDV) (birnavirus genus
Avibirnaviridae). Two ? sense primers, BirnaGenFext
(AAGACCMGDAACAWHTGG) and BirnaGenFint (TAT
GCNGACAACATMTACAT), and two - sense primers,
BirnaGenRint (GCRTTCCCRCTGCCYTGVCC) and Birn-
aGenRext (TCDATYTTRAAGTTRATSCC) were designed
for use in a nested reverse transcription polymerase chain
reaction (RT-PCR). The first-round primers were predicted
123
P. F. Dixon et al.
to produce a product of 605 bps, and the nested primer set a
product of 266 bps.
Total RNA was extracted from 100 ll of virus super-
natant using TRIzolÒ (Invitrogen Life Technologies) using
the manufacturer’s recommendations. The RNA pellet was
dissolved in 40 ll molecular grade water. Reverse tran-
scription (RT) was performed at 37°C for 1 h in a 20-ll
volume consisting of 19 M-MLV RT reaction buffer
(50 mm Tris [pH 8.3], 75 mM KCl, 10 mM DTT, 3 mM
MgCl2) containing 1 mM dNTP, 100 pmol BirnaGenRext,
20 units M-MLV reverse transcriptase (Promega, South-
ampton, UK) and 1/10 of the total RNA extracted above.
PCR was performed in a 50-ll reaction volume of 19 Go
Taq PCR buffer containing 2.5 mM MgCl2, 200 lM
dNTPs, 100 pmol each of the primers BirnaGenFext and
BirnaGenRext, 2.5 units of Go Taq polymerase (Promega,
Southampton UK) and 2.5 ll RT reaction mix. The reac-
tion mix was overlaid with mineral oil and subjected to 40
temperature cycles of 1 min at 95°C, 1 min at 55°C and
1 min at 72°C, followed by a final extension step of 10 min
at 72°C. The second-round (nested PCR) was performed
under the same conditions, using BirnaGenFint and
BirnaGenRint and 2.5 ll of the first-round product as a
template.
Amplified products were electrophoresed in a 2% (w/v)
agarose/TAE (40 mM Tris-acetate, pH 7.2, 1 mM EDTA)
gel containing 1.0 lg/ml ethidium bromide and visualised
by UV irradiation. PCR products were purified using the
Freeze’n Squeeze DNA purification system (Anachem,
Luton, UK), and both DNA strands were sequenced using
BirnaGenFint and BirnaGenRint and the ABI PRISMTM
dye terminator cycle sequencing system (Applied Biosys-
tems, Warrington, UK).
In the diagnostic investigation, viruses were only iso-
lated in BF-2 cells. All four isolates appeared as isometric
viruses with a diameter of approximately 60 nm by
electron microscopy. Replication of the isolates was not
affected by treatment with chloroform or BUDR, indicating
a non-enveloped virus with an RNA genome. Those
properties, and the virion morphology, suggested that the
isolates might be members of the family Birnaviridae. By
comparison with reference aquabirnaviruses following
acrylamide gel electrophoresis, the extracted nucleic acid
of the new virus isolates was compatible with being two
strands of double-stranded (ds) RNA (Fig. 1), adding
further weight to their identity as aquabirnaviruses. The
electrophoretic mobility of the RNA segments of the four
isolates were similar to each other but differed from ref-
erence aquabirnaviruses representing serotypes A1 (West
Buxton), A2 (Sp), B1 (Tellina virus 1, TV-1) and C1
(BSNV). The electrophoretic mobility of the two RNA
segments of the isolates was different from the other
aquabirnaviruses compared, but similar to each other. The
Proposal for a fourth aquabirnavirus serogroup
Fig. 1 Comparison of the purified nucleic acid segments of isolates
M2, M3, M4 and M5 with reference aquabirnaviruses on silver-
stained 6% polyacrylamide gels. Lane 1 serotype A1 (West Buxton),
lane 2 serotype A2 (Sp), lane 3 serotype B1 (TV-1), lane 4 serotype
C1 (BSNV), lane 5 isolate M2, lane 6 isolate M3, lane 7 isolate M4,
lane 8 isolate M5
relative difference in mobility between the two genome
segments was greater than that of BSNV, which was
reported to have the largest genome size difference of all
the aquabirnaviruses [8].
In the initial report [8] on the identification of BSNV as
the first member of Aquabirnavirus Serogroup C, the virus
was described as phenotypically different from Serogroup
A viruses in its cell line specificity. The cell line specificity
of the new isolates was further investigated. Although on
initial isolation from the fish the isolates did not grow in
EPC and CHSE-214 cells, virus cultured in BF-2 cells was
inoculated onto those cells as well as onto FHM and RTG-
2 cells to confirm the cell line specificity. Reference viruses
(as above) representing Serotypes A2, B1 and C1 were
included in the study for comparison. Apart from BF-2
cells, the new isolates only grew in RTG-2 cells. BSNV
grew in RTG-2 and FHM cells, with variable growth in
EPC and CHSE-214 cells; i.e. sometimes the virus pro-
duced CPE in the cells, but it did not always passage. TV-1
did not grow in CHSE-214 or RTG-2 cells and grew slowly
without reaching full CPE in EPC and FHM cells. Sp grew
in all the cell lines. BSNV was previously reported not to
grow in CHSE-214, EPC, RTG-2 or FHM cells [8],
whereas in this study it did. This variance in the results
may be a result of differences in the sensitivity of the
respective cell lines held at different laboratories, which
has been reported previously for fish cell lines [11, 12].
What is apparent is that Serogroup A viruses can infect a
broader range of cell lines than those of Serogroup B,
Serogroup C and the new isolates. However, a common
1939
feature of the new isolates and viruses from all of the
serogroups was their ability to grow in BF-2 cells.
The serological relationships of the four new isolates to
the aquabirnavirus serogroups were initially investigated
by plaque neutralisation tests using antisera against all nine
reference serotypes of Serogroup A and against reference
Serogroup B and C viruses at 1:100 and 1:1000 (final)
dilutions of antisera. The four isolates were not neutralised
by 1:100 dilutions of any of the antisera, which had
homologous 50% plaque neutralisation titres C 10,000.
Neutralisation tests of all Serogroup A, B and C viruses
using antisera produced against isolates M2 and M4 were
done by the constant serum (1:100):virus dilution (tenfold
step) method (alpha neutralisation, [14]). The antisera did
not neutralise any of the nine Serogroup A serotypes,
serotype B1 or serotype C1.
The 50% plaque reduction method was used to compare
isolates M2, M3, M4 and M5 using the antisera against M2
and M4. The serological relationship between isolates M2
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
and M4 was determined using the formula r ¼ r1  r2
[1], where
reciprocal of heterologous titre obtained with virus 2
r1 ¼
reciprocal of homologous titre obtained with virus 1
and
reciprocal of heterologous titre obtained with virus 1
r2 ¼
reciprocal of homologous titre obtained with virus 2
The criteria [7] that a 1/r value of \10 would indicate that
isolates were of the same serotype and a value significantly
greater than 10 would indicate a new serotype were used.
The results are shown Table 1; the 1/r value for isolates
M2 and M4 was 555.15, indicating that they are different
serotypes within the same serogroup.
Products of the expected size were obtained for all the
viruses tested following RT-PCR, with the exception of
TV-1. In that case, no product was produced in repeat
extraction and amplifications, even when other primer sets
in addition to those described above were used (data not
shown). It has recently been reported that TV-1 is phylo-
genetically distinct and ‘‘deeply divergent’’ from IPNV and
BSNV [13], which may account for this failure to produce
a product with TV-1. Multiple alignment of the sequences
Table 1 Fifty-percent plaque reduction titres of isolates M2, M3, M4
and M5 with antisera against M2 and M4
Antiserum Virus
M2 M3 M4 M5
Anti-M2 646,200 329,750 99 300
Anti-M4 2,400 2,300 113,250 184,100
Homologous reactions are shown in bold type
123
1940 P. F. Dixon et al.

obtained for new isolates with published sequences showed
that M2 and M3 shared 76 and 75% nucleotide identity,
respectively, with BSNV and that M4 and M5 shared 79
and 78% nucleotide identity, respectively, with BSNV. M2,
M3, M4 and M5 shared 70–73% nucleotide identity with
IBDV. M2, M3, M4 and M5 shared 80–87% deduced
amino acid identity with BSNV and 74% deduced amino
acid identity with IBDV.
Phylogenetic analysis (Fig. 2) assigned isolates A1, A2
and A3, representing different serotypes of Serogroup A, to
a distinct clade. Likewise, isolates C1, C2, C3, C4, C5, M1,
S1, T1, T2 and V1 (from several South-East Asian coun-
tries) were assigned to a clade with the serogroup C type
species, BSNV. The four new isolates M2, M3, M4 and M5
were assigned to a clade that was distinct from both the
serogroup C and serogroup A viruses. There was also a
further separation of M2 and M3 isolates from M4 and M5
into two distinct subclades. The latter was supported by
bootstrap values of 100%. This assignment to a clade and
subclades of isolates M1, M2, M3 and M4 reflects the
serological relationships of those isolates. It is noteworthy
that isolates M2 and M3, isolated in different years from
different species from the same farm site, were closely
related serologically and were assigned to the same
subclade.
The morphology of isolates M2, M3, M4 and M5, their
insensitivity to chloroform and BUDR, their genome
comprising two strands of dsRNA, comparisons of partial
nucleotide sequences, phylogenetic analysis and their
aquatic hosts suggested that they were aquabirnaviruses.
However, serological comparisons showed that the isolates
were distinct from the three established Aquabirnavirus
serogroups. In the original report [8] describing BSNV it
was questioned whether the virus should be regarded as the
reference virus of the new Serogroup C, or whether it
should form a new species in the genus Aquabirnavirus,
based on its biological, biochemical and serological char-
acteristics. It was later suggested that BSNV should be
regarded as a member of a new aquatic birnavirus species
as it was more similar to the avibirnavirus IBDV than to the
aquabirnavirus infectious pancreatic necrosis virus [3].
However, until such a new species is accepted by the
International Committee on Taxonomy of Viruses, it is
prudent to create new serogroups of the genus Aqua-
birnavirus for isolates that do not react in serological
comparisons with members of established serogroups using
homologous and heterologous antisera. Therefore, it is
proposed that isolates M2, M3, M4 and M5 should be
assigned to a fourth Aquabirnavirus serogroup, D. The 1/r
value of 555.15 for isolates M2 and M4 distinguishes them
as different serotypes within Serogroup D; hence, M2
should be reference serotype D1, and M4 should be
reference serotype D2. Although reciprocal cross-
neutralisation tests have not been done, the serological
reactions of isolate M3 suggest that it should be provi-
sionally assigned to serotype D1, and those of isolate M5
suggest that it should be provisionally assigned to serotype
D2.

Alternative Identifier Host Species Country of Serogroup

Origin
M16/8/93 Marble goby (Oxyeleotris marmorta) Cambodia C
V1
T2 M5/3/93 Marble goby Thailand C
C5 M18/8/93 Marble goby Cambodia C
M5/3/93 Marble goby Thailand C
77 T1
C4 M8/3/93 Marble goby Cambodia C
M5/3/93 Marble goby Cambodia C
100
C3
C1 M4/1/93a Marble goby Cambodia C
S1 M23/6/91 or SHV Giant snakehead (Channa micropeltes) Singapore C
M1 M15/8/93a Marble goby Malaysia C
BSNV - Blotched snakehead (Channa lucius) - C
C2 M4/1/93a Marble goby Cambodia C
100 M2 A08/12/95 Angelfish (Pterophyllum scalare) Malaysia D1
M3 A16/09/97 Dwarf gourami (Colisa lalia) Malaysia D1
83 M5 A16/02/98 Ram cichlid (Microgeophagus ramirezi) Malaysia D2
100 M4 Ram cichlid Malaysia D2
A03/04/97
A1 West Buxton Rainbow trout (Oncorhynchus mykiss) USA A1
100 A2 Sp Rainbow trout Denmark A2
81 A3 Ab Rainbow trout Denmark A3

0.05

Fig. 2 An unrooted neighbor-joining distance tree based on a 226-
nucleotide partial RNA-dependent RNA polymerase gene (VP1)
sequence (nt 1176-1401) for representatives of Serogroup A and C
aquabirnaviruses and the sequences obtained for isolates M2, M3, M4
and M5. The phylogenetic analysis was performed using MEGA
version 3.1 [9]. Analysis was done on 1000 bootstrapped data sets,
and values of [70% are shown on the tree. The scale bar represents
substitutions per nucleotide site. The host species and the country
of origin are also shown. The accession numbers for the sequences
used in the analysis are AM889013-AM889023 and AM889216-
AM889222

123
Proposal for a fourth aquabirnavirus serogroup
Acknowledgment This work was partly funded by Defra contract
F1136.
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