Genetic variability is a key problem in the prevention and therapy of RNA-based virus infections. Infectious Salmon Anemia virus (ISAv) is an RNA virus which aggressively attacks salmon producing farms worldwide and in particular in Chile. Just as with most of the Orthomyxovirus, ISAv displays high variability in its genome which is reflected by a wider infection potential, thus hampering management and prevention of the disease. Although a number of widely validated detection procedures exist, in this case there is a need of a more complex approach to the characterization of virus variability. We have adapted a procedure of High Resolution Melting (HRM) as a fine-tuning technique to fully differentiate viral variants detected in Chile and projected to other infective variants reported elsewhere. Out of the eight viral coding segments, the technique was adapted using natural Chilean variants for two of them, namely segments 5 and 6, recognized as virulence-associated factors. Our work demonstrates the versatility of the technique as well as its superior resolution capacity compared with standard techniques currently in use as key diagnostic tools.
References
[1]
Thorud K, Djupvik HO (1988) lnfectious salmon anaemia in Atlantic salmon (Salmo salar L.). B Euro Asso Fish Pat. 8: 109–111.
[2]
Mullins JE, Groman D, Wadowska D (1998) Infectious salmon anaemia in salt water Atlantic salmon (Salmo salar L.) in New Brunswick, Canada. B Euro Asso Fish Pat(18): 110–114.
[3]
Bouchard D, Keleher W, Opitz HM, Blake S, Edwards KC, et al. (1999) Isolation of infectious salmon anemia virus (ISAV) from Atlantic salmon in New Brunswick, Canada. Dis Aquat Organ. 35: 131–137.
[4]
Lovely JE, Dannevig BH, Falk K, Hutchin L, MacKinnon AM, et al. (1999) First identification of infectious salmon anaemia virus in North America with haemorrhagic kidney syndrome. Dis Aquat Organ. 35: 145–148.
[5]
Bouchard DA, Brockway K, Giray C, Keleher W, Merrill PL (2001) First report of infectious salmon anemia (ISA) in the United States. B Euro Asso Fish Pat. 21: 86–88.
[6]
Rodger HD, Turnbull T, Muir F, Millar S, Richards RH (1998) Infectious salmon anaemia (ISA) in the United Kingdom. B Euro Asso Fish Pat(18): 115–116.
[7]
Lyngoy C (2003) Infectious salmon anaemia in Norway and the Faroe Islands: An industrial appproach. 2003: 97–109.
[8]
Kibenge FS, Garate ON, Johnson G, Arriagada R, Kibenge MJ, et al. (2001) Isolation and identification of infectious salmon anaemia virus (ISAV) from Coho salmon in Chile. Dis Aquat Organ. 45: 9–18.
[9]
Godoy M, Aedo A, Kibenge M, Groman D, Yason C, et al. (2008) First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile. BMC Vet Res. 4: 28.
[10]
Krossoy B, Hordvik I, Nilsen F, Nylund A, Endresen C (1999) The putative polymerase sequence of infectious salmon anemia virus suggests a new genus within the Orthomyxoviridae. J Virol. 73: 2136–2142.
[11]
Kawaoka Y, Cox NJ, Haller O, Hongo S, Kaverin N, et al. (2005) Infectious salmon anaemia virus. Virus Taxonomy–Eight Report of the International Committee on Taxonomy Viruses. pp. 681–693.
[12]
Falk K, Namork E, Rimstad E, Mjaaland S, Dannevig BH (1997) Characterization of infectious salmon anemia virus, an orthomyxo-like virus isolated from Atlantic salmon (Salmo salar L.). J Virol 71: 9016–9023.
[13]
Mjaaland S, Rimstad E, Falk K, Dannevig BH (1997) Genomic characterization of the virus causing infectious salmon anemia in Atlantic salmon (Salmo salar L.): an orthomyxo-like virus in a teleost. J Virol 71: 7681–7686.
[14]
Brown EG (2000) Influenza virus genetics. Biomed Pharmacothe. 54: 196–209.
[15]
Rumschlag-Booms E, Guo Y, Wang J, Caffrey M, Rong L (2009) Comparative analysis between a low pathogenic and a high pathogenic influenza H5 hemagglutinin in cell entry. Virol J. 6: 76.
[16]
Jackson D, Hossain M (2008) A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity. PNAS USA. 105: 4381–4386.
[17]
Coleman JR (2007) The PB1-F2 protein of Influenza A virus: increasing pathogenicity by disrupting alveolar macrophages. Virol J. 4: 9.
[18]
Goto H, Wells K, Takada A, Kawaoka Y (2001) Plasminogen-binding activity of neuraminidase determines the pathogenicity of influenza A virus. J Virol. 75: 9297–92301.
[19]
Rimstad E, Mjaaland S, Snow M, Mikalsen AB, Cunningham CO (2001) Characterization of the infectious salmon anemia virus genomic segment that encodes the putative hemagglutinin. J Virol. 75: 5352–5356.
[20]
Falk K, Aspehaug V, Vlasak R, Endresen C (2004) Identification and characterization of viral structural proteins of infectious salmon anemia virus. J Virol. 78: 3063–3071.
[21]
Devold M, Falk K, Dale B, Krossoy B, Biering E, et al. (2001) Strain variation, based on the hemagglutinin gene, in Norwegian ISA virus isolates collected from 1987 to 2001: indications of recombination. Dis Aquat Organ. 47: 119–128.
[22]
Kibenge FS, Kibenge MJ, McKenna PK, Stothard P, Marshall R, et al. (2001) Antigenic variation among isolates of infectious salmon anaemia virus correlates with genetic variation of the viral haemagglutinin gene. J Gen Virol. 82: 2869–2879.
[23]
Krossoy B, Devold M, Sanders L, Knappskog PM, Aspehaug V, et al. (2001) Cloning and identification of the infectious salmon anaemia virus haemagglutinin. J Gen Virol. 82: 1757–1765.
[24]
Mjaaland S, Hungnes O, Teig A, Dannevig BH, Thorud K, et al. (2002) Polymorphism in the infectious salmon anemia virus hemagglutinin gene: importance and possible implications for evolution and ecology of infectious salmon anemia disease. Virology. 304: 379–391.
[25]
Kibenge FSB, Godoy MG, Wang Y, Kibenge MJT, Gherardelli V, et al. (2009) Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences. Virol J. 6: 88.
[26]
Cunningham CO, Snow M (2000) Genetic analysis of infectious salmon anaemia virus (ISAV) from Scotland. Dis Aquat Organ. 41: 1–8.
[27]
Christiansen DH, ？stergaard PS, Snow M, Dale OB, Falk K (2011) A low-pathogenic variant of infectious salmon anemia virus (ISAV-HPR0) is highly prevalent and causes a non-clinical transient infection in farmed Atlantic salmon (Salmo salar L.) in the Faroe Islands. J Gen Virol. 92: 909–918.
[28]
McBeath AJA, Bain N, Snow M (2009) Surveillance for infectious salmon anaemia virus HPR0 in marine Atlantic salmon farms across Scotland. Dis Aquat Organ. 87: 161–169.
[29]
Markussen T, Jonassen CM, Numanovic S, Braaen S, Hjortaas M, et al. (2008) Evolutionary mechanisms involved in the virulence of infectious salmon anaemia virus (ISAV), a piscine orthomyxovirus. Virology. 374: 515–527.
[30]
Kibenge FS, Kibenge MJ, Wang Y, Qian B, Hariharan S, et al. (2007) Mapping of putative virulence motifs on infectious salmon anemia virus surface glycoprotein genes. J Gen Virol. 88: 3100–3111.
[31]
Aspehaug V, Mikalsen AB, Snow M, Biering E, Villoing S (2005) Characterization of the infectious salmon anemia virus fusion protein. J Virol. 79: 12544–12553.
[32]
Devold M, Karlsen M, Nylund A (2006) Sequence analysis of the fusion protein gene from infectious salmon anemia virus isolates: evidence of recombination and reassortment. J Gen Virol. 87: 2031–2040.
[33]
Snow M, McKay P, McBeath AJ, Black J, Doig F, et al. (2006) Development, application and validation of a taqman？ real-time RT-PCR assay for the detection of infectious salmon anaemia virus(ISAV) in atlantic salmon (Salmo salar). Dev Biol (Basel). 126: 133–145.
Wittwer CT (2009) High resolution DNA melting analysis: advancements and limitations. Hum Mutat. 30: 857–859.
[36]
Vossen RHAM, Aten E, Roos A, Den Dunnen JT (2009) High Resolution Melting Analysis (HRMA)–More than just sequence variant screening. Hum Mutat. 30: 860–866.
[37]
Taylor CF (2009) Mutation scanning using high-resolution melting. Biochem Soc Trans 37: 433–437.
[38]
Ugo V, Tondeur S, Menot ML, Bonnin N, Le Gac G, et al. (2010) Interlaboratory development and validation of a HRM method applied to the detection of JAK2 exon 12 mutations in polycythemia vera patients. PLoS One 5: e8893.
[39]
Naze F, Jouen E, Randriamahazo RT, Simac C, Laurent P, et al. (2010) Pseudomonas aeruginosa outbreak linked to mineral water bottles in a neonatal intensive care unit: fast typing by use of high-resolution melting analysis of a variable-number tandem-repeat locus. J Clin Microbiol. 48: 3146–3152.
[40]
Cheng JC, Huang CL, Lin CC, Chen CC, Chang YC, et al. (2006) Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR. Clin Chem. 52: 1997–2004.
[41]
Fortini D, Ciammaruconi A, De Santis R, Fasanella A, Battisti A, et al. (2007) Optimization of high-resolution melting analysis for low-cost and rapid screening of allelic variants of Bacillus anthracis by multiple-locus variable-number tandem repeat analysis. Clin Chem. 53: 1377–1380.
[42]
Liew M, Pryor R, Palais R, Meadows C, Erali M, et al. (2004) Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem(50): 1156–1164.
[43]
Tajiri-Utagawa E, Hara M, Takahashi K, Watanabe M, Wakita T (2009) Development of a rapid high-throughput method for high-resolution melting analysis for routine detection and genotyping of noroviruses. J Clin Microbiol. 47: 435–440.
[44]
Ghorashi SA, O’Rourke D, Ignjatovic J, Noormohammadi AH (2011) Differentiation of infectious bursal disease virus strains using real-time RT-PCR and high resolution melt curve analysis. J Virol Methods. 171: 264–271.
[45]
Lin JH, Tseng CP, Chen YJ, Lin CY, Chang SS, et al. (2008) Rapid differentiation of influenza A virus subtypes and genetic screening for virus variants by high-resolution melting analysis. J Clin Microbiol. 46: 1090–1097.
[46]
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948.
[47]
Waterhouse AM, Procter JB, Martin D, Clamp M, Barton GJ (2009) Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189–1191.
[48]
Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL (2008) GenBank. Nucleic Acids Res 36: D25–30.
[49]
Erali M, Wittwer CT High resolution melting analysis for gene scanning. Methods 50: 250–261.
[50]
Erali M, Voelkerding KV, Wittwer CT (2008) High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol. 85: 50–58.
[51]
Rice P, Longden I, Bleasby A (2000) EMBOSS: the European molecular biology open software suite. Trends Genet 16: 276–277.
[52]
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
[53]
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386.
[54]
Kibbe WA (2007) OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res 35: 43–46.
[55]
Néron B, Ménager H, Maufrais C, Joly N, Maupetit J, et al. (2009) Mobyle: a new full web bioinformatics framework. Bioinformatics 25: 3005–3011.
[56]
Breslauer KJ, Frank R, Bl？cker H, Marky LA (1986) Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci 83: 3746–3750.
[57]
Baldino Jr F, Chesselet MF, Lewis ME (1989) High-resolution in situ hybridization histochemistry. Methods Enzymol 168: 761–777.
