Download Sequence analysis of infectious pancreatic necrosis virus isolated

Virus Genes (2013) 47:574–578
DOI 10.1007/s11262-013-0981-4
Sequence analysis of infectious pancreatic necrosis virus isolated
from Iranian reared rainbow trout (Oncorhynchus mykiss) in 2012
Maryam Dadar • Rahim Peyghan • Hamid Rajabi Memari
Masod Reza Seifi Abad Shapouri • Reza Hasanzadeh •
Laleh Moazzami Goudarzi • Vikram N. Vakharia
•
Received: 8 July 2013 / Accepted: 10 September 2013 / Published online: 19 September 2013
Ó Springer Science+Business Media New York 2013
Abstract Infectious pancreatic necrosis virus (IPNV) is
the causal agent of a highly contagious disease that affects
many species of fish and shellfish. This virus causes economically significant diseases of farmed rainbow trout,
Oncorhynchus mykiss (Walbaum), in Iran, which is often
associated with the transmission of pathogens from European resources. In this study, moribund rainbow trout fry
samples were collected during an outbreak of IPNV in
three different fish farms in north and west provinces of
Iran in 2012; and we investigated the full genome sequence
of Iranian IPNV and compared it with previously identified
IPNV sequences. The sequences of different structural and
nonstructural-protein genes were compared to those of
other aquatic birnaviruses sequenced to date. Our results
show that the Iranian isolate falls within genogroup 5,
serotype A2 strain SP, having 99 % identity with the strain
M. Dadar (&) R. Peyghan
Department of Aquatic Health, College of Veterinary Medicine,
Shahid Chamran University, Ahvaz, Iran
e-mail: [email protected]
H. R. Memari
Center of Biotechnology Research, College of Agriculture,
Shahid Chamran University, Ahvaz, Iran
M. R. S. A. Shapouri
Center of Virology Research, College of Veterinary Medicine,
University of Shahid Chamran University, Ahvaz, Iran
R. Hasanzadeh L. M. Goudarzi
Iranian Veterinary Organization, Central Veterinary Laboratory,
Tehran, Iran
V. N. Vakharia
Institute of Marine and Environmental Technology, University
of Maryland Baltimore County, Baltimore, MD 21202, USA
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1146 from Spain. These results suggest that the Iranian
isolate may have originated from Europe.
Keywords Molecular characterization IPNV Aquatic birnaviruses Rainbow trout
Introduction
Infectious pancreatic necrosis (IPN) is one of the most
important viral diseases of farmed salmonid fish caused by
infectious pancreatic necrosis virus (IPNV). IPNV is a
nonenveloped virus belonging to the family Birnaviridae,
genus Aquabirnavirus [1, 2]. Serologically, Aquabirnaviruses have been classified on the basis of cross-neutralization assays and divided into four serogroups, A–D [3, 4].
Most aquatic birnaviruses belong to serogroup A, which
include nine serotypes (A1–A9), while the minor serogroup
B consists of a single serotype, B1 [5–7]. Serotype A1
include USA isolates, serotypes A6–A9 are mainly detected in Canada, and serotypes A2–A5 and B1 are found in
Europe and Asia [4, 8–10]. These viruses show significant
antigenic variation [3, 11]. The genome of the virus has
two segments of double-stranded RNA that are surrounded
with a single shelled, icosahedral capsid with 60-nm in
diameter [1, 12, 13]. Segment A is 3,097-bp long and
encodes four viral proteins, namely structural proteins, VP2
and VP3, and nonstructural proteins, VP4 and VP5 [1].
Segment A contains a large open reading frame (ORF)
encoding a 106-kDa polyprotein which is cotranslationally
cleaved by VP4 to produce pre-VP2 (pVP2) and VP3 [14,
15]. There is a small ORF which overlaps with the amino
terminal end of the large ORF and generates a 15 kDa
(VP5) nonstructural-polypeptide [1, 16]. VP5 contains
Bcl-2 homology domains and is capable of enhancing cell
Virus Genes (2013) 47:574–578
575
viability with a notable strategy via VP5 to regulate the
host antiapoptosis pathway [17]. VP2 is an outer capsid
protein, which contains major neutralizing epitopes which
elicit a protective antibody response. It also contains the
markers for virulence and has a particular taxonomic
importance for genotyping [18–20]. VP3 is an internal
protein with several roles in organizing the IPNV replication cycle [21–23]. Segment B is 2,784 bp long and
encodes VP1 protein, a minor internal polypeptide
(94 kDa), which acts as the virion-associated RNAdependent RNA polymerase (RdRP) of IPNV [1, 24]. IPN
disease can induce high mortality, which can result in huge
economic losses in both fry and juveniles of rainbow trout,
brook trout and Atlantic salmon [25]. This virus is widespread in salmonid hatcheries from the America to Europe,
Asia, Australia and South Africa [26, 27]. Fish that survive
an IPNV infection may become carriers of the virus for
long period and serve to sequentially transmit the virus to
other susceptible species of fish and shellfish [28]. For the
first time, IPNV was detected using RT-PCR method in
several provinces of Iran in 2007, followed by other reports
[29–32]. Therefore, the aim of the present study was to
determine IPNV genotype(s) in Iran and compare them
with known genotypes of European and American IPNV
isolates. The approach taken was to sequence the coding
regions of the genomic segments A and B.
Results and Discussion
Fig. 1 Phylogenetic relationship of Iranian IPNV isolate and other
reference strains using the UPGMA method as implemented in mega
5 software. a Phylogenetic tree based on VP1 protein encoded by
genomic segment B. A scale bar shows 0.1 substitutions per sequence
position. b Phylogenetic tree based on the polyprotein encoded by
genomic segment A. In this tree, the Iranian isolate has the highest
identity with isolate 1146, which is of Sp serotype. A scale bar
indicates 0.01 substitutions per sequence position
The Iranian IPNV isolate, obtained in 2012, was propagated in CHSE cells, and the virus was collected after the
second passage in CHSE-214 cells. Specific primer pairs
were used for RT-PCR amplifications which produced
fragments of 1,347, 852, 2,535, 402, and 720 bp encoding
VP2, VP2-VP4, VP1, VP5, and VP3 genes, respectively, as
described [8]. The RT-PCR products were cloned into a
cloning vector, and the plasmid DNA (from three independent clones of each amplicon) was sequenced by
dideoxy chain termination method. The sequences were
deposited in the NCBI database with accession numbers
VP1: KC900161, VP2: KC489465, VP3: KC489466, VP4:
KC710379, VP5: KC900222 and polyprotein: KF279643.
Blast p alignment comparison of VP1 and polyprotein
amino acid sequences showed that the Iranian isolate was
closely related to the Sp strain (Fig. 1) and had the highest
similarity to isolate 1146 (Q8JK08). We included the
phylogenetic tree of VP1 to demonstrate that the Iranian
isolate is of Sp serotype, and it is not a natural reassortant
virus, as shown for one isolate found in wild fish from
Flemish Cap fishery in Newfoundland, Canada [9]. Since
there was adequate information about the Iranian IPNV
genome sequence, the existing IPNV sequences were
extracted from NCBI and aligned using Mega5 software [4,
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Virus Genes (2013) 47:574–578
Fig. 2 High similarity hits for blast p on UNIPROTKB of deduced
VP2 protein sequence sorted by descending score. The hypervariable
region in VP2 is between residues 245 and 257 (box). VP2 sequence
of Iranian IPNV isolate used in this study (black arrow). The residues
at positions 217, 221, and 247 in VP2, which are involved in virulence
motif (red arrows) (Color figure online)
33, 34]. Earlier studies have shown that VP2 is responsible
for the production of type-specific monoclonal antibodies
[6, 35, 36]. The residues of VP2 domains can alter the
properties of this protein. These alterations can influence
the antigenic characteristics of VP2 and also the mortality
rates in fish [37]. In sum, VP2 carries the determinant
factors for IPNV virulence [2]. In virulent strains of IPNV,
there are threonine and alanine at positions 217 and 221 of
VP2, respectively, whereas, moderate to low virulence
strains have a proline and alanine at these positions. Strains
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Virus Genes (2013) 47:574–578
with threonine at position 221 have been shown to be
almost avirulent [3, 38, 39]. However, some of the IPNV
strains with proline and alanine at positions 217 and 221 do
show high virulence in the field and experimental conditions, supporting the notion that viral, host, and environmental factors, as well as specific amino acid residues
influence pathogenicity [40]. The residue at position 247 of
VP2 is also highly variable and may be linked to virulence.
Santi and colleagues showed that the motifs Thr217,
Ala221, Thr247 were associated with high virulence, and
the motifs Pro217, Ala221, Ala247 are present in viruses
with low and moderate virulence. So far, all of detected
Iranian IPNV had proline and threonine at positions 217
and 221, respectively. Despite the presence of threonine at
position 221, which is indicative of a nonvirulent nature,
the mortality of Iranian rainbow trout fry corresponds to a
moderate virulence [30–32]. The moderate virulence of
Iranian IPNV isolates may be related to alanine residue
present in position 247. The VP2 has also been shown to
contain the central variable domain that encodes two
hypervariable regions. These hypervariable regions determine the virus-specific serotypes. High similarity hits for
blast p on UNIPROTKB of VP2 show changes in 11 residues at positions 52, 94, 96, 219, 245, 248, 252, 255, 257,
286, and 321, most of which are present in the second
hypervariable region between residues 245 and 257 of the
Iranian isolate (Fig. 2). The position of the start codon of
the VP5 protein may vary [27]. Earlier studies have shown
that the start codon of VP5 is located at position 68 [41],
although Heppell et al. [11] reported that it could be initiated from position 68 or 112. Also Weber et al. [42] and
Shivappa et al. [37] have demonstrated that the second in
frame methionine codon is responsible for the initiation of
VP5 in VR299 and SP strains, respectively. Since virulence
of IPNV isolates has been connected to segment A [43], no
specific sequences or motifs have been identified in segment B which are linked to virulence. It demonstrated that
all studied pathogenic isolates encoded a truncated VP5
protein [17, 38]. Blake et al. [8] and Nishizawa et al. [10]
demonstrated that IPNV strains could be grouped into six
genogroups based on deduced amino acid sequences of
genome segment A and VP2, which correlated with geographical and serological similarity. The results of our
phylogenetic analyses results indicate that Iranian isolate
fall within genogroup 5, serotype A2 with moderate to low
virulence, and appear to be closely related to the Spanish
strains. In conclusion, the IPNV isolate from Iran is of Sp
serotype which may have originated from Europe.
Acknowledgments The authors are grateful to veterinary faculty of
Shahid Chamran University of Ahvaz for funding this work and
would like to thank Iranian veterinary organization, especially Central
Veterinary Laboratory for their assistance in this project.
577
References
1. P. Dobos, Annu. Rev. Fish Dis. 5, 25–54 (1995)
2. H. Song, N. Santi, Ø. Evensen, V.N. Vakharia, J. Virol. 79,
10289–10299 (2005)
3. N. Ruane, L. McCarthy, D. Swords, K. Henshilwood, J. Fish Dis.
32, 979–987 (2009)
4. S. Mutoloki, Ø. Evensen, J. Gen. Virol. 92, 1721–1726 (2011)
5. R.M. Bowers, S.E. Lapatra, A.K. Dhar, J. Virol. Methods 147,
226–234 (2008)
6. P. Caswell-Reno, V. Lipipun, P. Reno, B. Nicholson, J. Clin.
Microbiol. 27, 1924–1929 (1989)
7. B. Hill, K. Way, Annu. Rev. Fish Dis. 5, 55–77 (1995)
8. S. Blake, J. Ma, D. Caporale, S. Jairath, B. Nicholson, Dis. Aquat.
Org. 45, 89 (2001)
9. I. Romero-Brey, I. Bandin, J. Cutrı́n, V. Vakharia, C. Dopazo,
J. Fish Dis. 32, 585–595 (2009)
10. T. Nishizawa, S. Kinoshita, M. Yoshimizu, J. Gen. Virol. 86,
1973–1978 (2005)
11. J. Heppell, E. Tarrab, L. Berthiaume, J. Lecomte, M. Arella,
J. Gen. Virol. 76, 2091–2096 (1995)
12. P. Dobos, D. Rowe, J. Virol. 24, 805–820 (1977)
13. R.D. Macdonald, P. Dobos, Virology 114, 414–422 (1981)
14. P. Dobos, T. Roberts, Can. J. Microbiol. 29, 377–384 (1983)
15. M. Galloux, C. Chevalier, C. Henry, J.-C. Huet, B. Da Costa,
B. Delmas, J. Gen. Virol. 85, 2231–2236 (2004)
16. S.R. Saint-Jean, J.J. Borrego, S.I. Perez-Prieto, Adv. Virus Res.
62, 113–165 (2003)
17. J.-R. Hong, H.-Y. Gong, J.-L. Wu, Virology 295, 217–229 (2002)
18. M.B. Labus, S. Breeman, A.E. Ellis, D.A. Smail, M. Kervick,
W.T. Melvin, Fish Shellfish Immunol. 11, 203–216 (2001)
19. C. Moon, J. Do, S. Cha, J.-D. Bang, M. Park, D. Yoo, J. Lee,
H. Kim, D. Chung, J. Park, Arch. Virol. 149, 2059–2068 (2004)
20. B. Das, B. Collet, M. Snow, A. Ellis, Fish Shellfish Immunol. 23,
825–830 (2007)
21. M.W. Bahar, L.P. Sarin, S.C. Graham, J. Pang, D.H. Bamford,
D.I. Stuart, J.M. Grimes, J. Virol. 87, 3229–3236 (2013)
22. C.-L. Chiu, J.-L. Wu, G.-M. Her, Y.-L. Chou, J.-R. Hong,
Apoptosis 15, 653–668 (2010)
23. T. Pedersen, A. Skjesol, J.B. Jørgensen, J. Virol. 81, 6652–6663
(2007)
24. R. Duncan, E. Nagy, P.J. Krell, P. Dobos, J. Virol. 61, 3655–3664
(1987)
25. K. Wolf, M. Quimby, C. Carlson, G. Bullock, J. Fish. Board Can.
25, 383–391 (1968)
26. M. Crane, P. Hardy-Smith, L.M. Williams, A.D. Hyatt, L.M.
Eaton, A. Gould, J. Handlinger, J. Kattenbelt, N. Gudkovs, Dis.
Aquat. Org. 43, 1–14 (2000)
27. K.R. Davies, K.A. McColl, L.-F. Wang, M. Yu, L.M. Williams,
M.S.J. Crane, Dis. Aquat. Org. 93, 1–15 (2010)
28. E.S. Munro, P.J. Midtlyng, Fish diseases and disorders: viral.
Bact. Fungal Infect. 3, 1 (2011)
29. M. Akhlaghi, A. Hosseini, Bull. Eur. Assoc. Fish Pathol. 27, 205
(2007)
30. N. Ahmadi, A. Oryan, M. Akhlaghi, A. Hosseini, J. Fish Dis. 36,
629–637 (2013)
31. M. Raissy, H. Momtaz, M. Ansari, M. Moumeni, M. Hosseinifard, J. Food Agric. Environ. 8, 614–615 (2010)
32. A. Oryan, N. Ahmadi, M. Akhlaghi, A. Hosseini, Aquacu. Int. 20,
725–734 (2012)
33. K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, S.
Kumar, Mol. Biol. Evol. 28, 2731–2739 (2011)
34. S. Kumar, M. Nei, J. Dudley, K. Tamura, Brief. Bioinform. 9,
299–306 (2008)
35. B.L. Nicholson, Annu. Rev. Fish Dis. 3, 241–257 (1993)
123
578
36. H. Melby, K. Christie, J. Fish Dis. 17, 409–415 (1994)
37. R. Shivappa, H. Song, K. Yao, A. Aas-Eng, O. Evensen, V.
Vakharia, Dis. Aquat. Org. 61, 23–32 (2004)
38. N. Santi, H. Song, V.N. Vakharia, Ø. Evensen, J. Virol. 79,
9206–9216 (2005)
39. A. Skjesol, I. Skjæveland, M. Elnæs, G. Timmerhaus, B.N. Fredriksen, S.M. Jørgensen, A. Krasnov, J.B. Jørgensen, Virol. J. 8,
396 (2011)
123
Virus Genes (2013) 47:574–578
40. N. Bain, A. Gregory, R. Raynard, J. Fish Dis. 31, 37–47 (2008)
41. G. Magyar, P. Dobos, Virology 198, 437–445 (1994)
42. S. Weber, D. Fichtner, T.C. Mettenleiter, E. Mundt, J. Gen. Virol.
82, 805–812 (2001)
43. T. Sano, N. Okamoto, T. Nishimura, J. Fish Dis. 4, 127–139
(1981)