Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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 123 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, 123 576 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 123 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)