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IBDV isolated in UK Penguins |
Sequence Analysis Of An Infectious
Bursal Disease Virus Isolated From Penguins In The United Kingdom.
D. J. Jackwood1, S. E. Sommer1, R. E. Gough2,
S. E. Drury3, D. de B. Welchman4, J. R. Chitty5 and G. E. S. Summerhays6
INFECTIOUS bursal disease virus (IBDV) is an avian
Birnavirus. Serotype 1 strains cause an immunosuppressive disease in
chickens. Serotype 2 viruses were originally isolated from turkeys and
studies demonstrated these isolates to be avirulent for chickens and turkeys
(Ismail, Saif, and Moorehead 1988; Jackwood, Saif, and Hughes 1982).
During a disease outbreak in 1999, Gough and others
isolated an avian Birnavirus from African black-footed penguins (Spheniscus
demersus) and Macaroni penguins (Eudyptes chrysolophus) that died at a
zoological park in the UK (Gough and others 2002). Birnavirus-like particles
were observed by electron microscopy in samples of the virus propagated in
cell culture. In addition, one-way virus neutralization assays were
conducted using both serotype 1 and 2 reference antiserum. The results
suggested the Birnavirus isolated was related to IBDV serotype 2. Although
antibodies to IBDV have been reported in Antarctic penguins (Gardner,
Knowles, and Riddle 1997), this was the first report of a Birnavirus
isolated from penguins. To confirm the identity of the penguin Birnavirus,
an isolate was obtained for molecular sequence analysis.
MATERIALS AND
METHODS
Prior to shipment to the United States for nucleotide
sequence analysis, a sample of the Birnavirus isolated from penguins (Gough
and others 2002) was treated for 7 days in an equal volume of
phenol:chloroform:isoamyl alcohol (25:24:1) according to U.S. Import Permit
#44226.
A modification of the procedures for isolation of
Birnaviral RNA was used (Jackwood and Jackwood 1994). The aqueous layer was
collected and a 1/10 volume of 10X TNE (100mM Tris-HCL [pH 8.0], 1.0 M NaCl,
and 10 mM ethylenediaminetetraacetic acid) was added. An equal volume of
chloroform was used to extract the sample and the aqueous layer was
collected. A volume of sodium dodecyl sulfate (Sigma Chemical Co., St.
Louis, MO) was added to achieve a final concentration of 0.5% (w/v) and
proteinase K (Sigma) was added to a final concentration of 1.0mg/ml.
Incubation was for 1 hr at 37 C. The sample was then extracted with an equal
volume of acid phenol (AMRESCO, Solon, OH), followed by extraction with an
equal volume of chloroform:isoamyl alcohol (24:1) (AMRESCO). Viral RNA was
precipitated using ethanol.
Prior to nucleotide sequence analysis, the Birnavirus
sample was amplified using RT-PCR. The primers and procedures used have been
described (Jackwood and Sommer 1997). The primers are located in conserved
sequence regions and amplify a 743 bp fragment of the VP2 gene of IBDV.
Their sequences are: Primer #1, 5-GCCCAGAGTCTACACCAT-3' and Primer #2,
5'-CCCGGATTATGTCTTTGA-3'. The GeneAmp RNA PCR kit (Perkin Elmer, Roche
Molecular Systems, Inc., Branchburg, NJ) was used for RT and PCR according
to the manufacturer’s instructions. The RT reaction was at 42 C for 1 hr. It
was followed by 30 cycles of PCR at 95 C for 2 min, 53 C for 1 min and 72 C
for 2 min. The samples were incubated for 7 min at 72 C after the last PCR
cycle. A positive internal control and negative control were used to help
insure the integrity of the RT-PCR (Smiley, Sommer, and Jackwood 1999).
The RT-PCR products were prepared for sequencing using
the Geneclean Spin Kit (BIO 101, Vista, CA) as previously described (Jackwood,
Sommer, and Knoblich 2001). Sequencing was done at the Molecular and
Cellular Imaging Center at The Ohio State University O.A.R.D.C., Wooster,
Ohio. They used the BigDye Terminator Sequencing Kit (Perkin-Elmer
Biosystems, Foster, City, CA) and a 64 lane Perkin-Elmer ABI 377 instrument
(Perkin-Elmer Biosystems). Data were viewed using Chromas (Technelysium Pty
Ltd. Queensland, Australia) and analyzed with Omiga software (Oxford
Molecular, Campbell, CA).
RESULTS AND
DISCUSSION
The RT-PCR primers amplified a 743 bp fragment of
the VP2 gene from the penguin IBDV isolate. The nucleotide sequence of this
RT-PCR fragment was determined and submitted to GenBank (Accession #
AY539855). The predicted amino acid sequence of the penguin IBDV was
determined to be similar but not identical to a European serotype 2 IBDV
strain 23/82 isolated from turkeys (Chettle, Eddy, and Wyeth 1985). An amino
acid sequence comparison is shown in figure 1. There were 5 amino acid
differences between these two serotype 2 viruses within this hypervariable
sequence region of VP2. When compared to the amino acid sequence of the OH
serotype 2 virus from the U.S. (Kibenge, McKenna, and Dybing 1991), 13 amino
acid differences were observed over the same region. A phylogenetic analysis
of the penguin nucleotide sequence with serotype 1 and 2 IBDV strains was
prepared using the Mega 3.0 program and the UPGMA method (Nei and Kumar,
2000) (Figure 2).
Four IBDV isolates were obtained from penguins at the
U.K. zoological park (Gough and others 2002). These isolates were tested
with mono-specific antiserum to both serotype 1 and 2 IBDV in a virus
neutralization assay. The results suggested the isolates were serotype 2
because the serotype 1 antiserum did not neutralize the viruses while the
serotype 2 antiserum completely neutralized these viruses. Our sequence
results demonstrate and confirm the penguin IBDV is a serotype 2 strain and
that it is most closely related to the 23/82 European serotype 2 virus from
turkeys (Figure 2).
The role of the serotype 2 penguin IBDV isolates in the
clinical signs and mortality of the Macaroni and African black-footed
penguins is not known (Gough and others 2002). In addition to finding IBDV,
these penguins were infected with reovirus and had evidence of a Plasmodium
infection. Earlier studies in chickens and turkeys demonstrated that the
serotype 2 IBDV strains tested were apathogenic for these species (Ismail,
Saif, and Moorhead 1988; Jackwood and others 1984; McFerran and others
1980). However, the pathogenicity of serotype 2 IBDV in penguins has not
been determined. Further studies are needed to determine if serotype 2 IBDV
can cause an immunosuppressive disease in penguins that is similar to the
one observed in chickens, which leads to secondary and opportunistic
infections.
This is the first report of the nucleotide and
predicted amino acid sequence of an IBDV isolate from penguins. Previous
studies reported on the presence of antibodies to IBDV in Antarctic penguins
(Gardner, Knowles, and Riddle 1997), but viruses were not isolated from
those birds.
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1Food Animal Health Research Program Ohio Agricultural Research and
Development Center The Ohio State University 1680 Madison Ave., Wooster,
Ohio 44691 USA 2FIMLS, CBiol, MIBiol, Veterinary Laboratories Agency –
Weybridge New Haw, Addlestone, Surrey KT15 3NB, United Kingdom. 3Avian
Virology, Veterinary Laboratories Agency – Weybridge New Haw, Addlestone,
Surrey KT15 3NB, United Kingdom 4Veterinary Laboratories Agency –
Winchester, Itchen Abbas, Winchester Hampshire SO21 1BX, United Kingdom
5Strathmore Veterinary Clinic, 6 London Road, Andover, Hampshire SP10 2PH,
United Kingdom 6Liphook Equine Hospital, Forest Mere, Liphook, Hampshire
GU30 7JG, United Kingdom 5Strathmore Veterinary Clinic, 6 London Road,
Andover, Hampshire SP10 2PH, United Kingdom 6Liphook Equine Hospital, Forest
Mere, Liphook, Hampshire GU30 7JG, United Kingdom
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