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جمع‌آوری و تعیین ویژگی جدایه‌های ایرانی ویروس چندوجهی هسته‌ای کرم غوزه پنبه Helicoverpa armigera Single Nucleopolyhedro virus (HearSNPV) بر مبنای ژن پلی‌هدرین (polh)

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه گیاه‌پزشکی، دانشکده علوم و مهندسی کشاورزی، پردیس کشاورزی و منابع طبیعی دانشگاه تهران، کرج.

2 عضو هیات علمی دانشگاه تهران

3 استادیار پژوهش، موسسه تحقیقات ثبت و گواهی بذر و نهال، سازمان تحقیقات، آموزش و ترویج کشاورزی، کرج، ایران

4 دانشگاه شهید مدنی آذربایجان

5 عضو هیات علمی

6 بخش تحقیقات آنفلونزا و سایر بیماریهای تنفسی انستیتو پاستور ایران، تهران

چکیده

استفاده از روش‌های مبتنی بر اسیدنوکلئیک در شناسایی ویروس‌ها به دلیل ساختار کوچک ژنومی آن‌ها و تفاوت‌های جزئی در سطح نوکلئوتید بین جدایه‌ها یا استرین‌ها، از اهمیت زیادی برخوردار است. در پژوهش حاضر، لاروهای مرده و/یا بیمار از مزارع گوجه‌فرنگی مناطق مختلف جغرافیایی ایران جمع‌آوری و برای آلودگی باکولوویروسیHelicoverpa armigera Single nucleopolyhedrovirus (HearSNPV) با روش بهینه‌سازی شده بر اساس سدیم دودسیل سولفات برای استخراج OBs، بررسی شدند. واکنش زنجیره‌ای پلی‌مراز (PCR) با استفاده از دو جفت آغازگر اختصاصی (Polh-a, Polh-b) طراحی شده بر اساس ناحیه حفاظت شده ژن پلی‌هدرین(Polh) برای 26 جدایه ثبت شده از ویروس Hear/H. zea-SNPV انجام یافت. آلودگی به HearSNPVبا تکثیر باندهای مونومورفیک 370 و 790 نوکلئوتیدی در 28 لارو تایید شد. همسانه‌سازی و تعیین توالی نوکلئوتیدی باندهای تکثیر شده، تعلق آن‌ها به ژن Polhویروس چندوجهی‌هسته‌ای گونه‌هایarmigera H. و H. zea با شباهت بیش از 6/99 درصد را نشان داد. تجزیه و تحلیل تبارزایی جدایه‌های ایرانی HearSNPV همراه با سایر جدایه‌های ثبت شده از این ویروس در دنیا، جدایه‌های ایرانی را در گروه II آلفاباکولوویروس‌ها طبقه‌بندی و کارایی بیش‌تر روش توسعه داده شده را اثبات کرد. پژوهش حاضر اولین گزارش از معرفی و شناسایی مولکولی جدایه‌های ایرانی HearSNPV با به کارگیری تکنینک PCR و DNA ویروس از لاروهای با هویت نامشخص بیمارگر، می‌باشد. با توجه به حصول اطمینان از موثر و مفید بودن این روش در غربال‌گری سریع لاروهای حشرات از نظر آلودگی به این ویروس و بررسی تبارزایش، بهره‌مندی از آن در پژوهش‌های آتی توصیه می‌گردد.

کلیدواژه‌ها


عنوان مقاله [English]

Collecting and partial characterization of Iranian Helicoverpa armigera Single NucleopolyhedroVirus isolates based on polh gene

نویسندگان [English]

  • Raheleh Shahbazi 1
  • masoud Naderpour 3
  • Ali Mehrvar 4
  • Akbar Dizadji 5
  • FATEMEH FOTOUHI 6
1 Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
3 Research Assistant Professor, Seed and Plant Improvement Institute, Education and Extension Organization, Karaj, Iran
4 Department of Plant Protection, Faculty of Agriculture, Azarbaijan Shahid Madani University, Tabriz, Iran
5 پردیس کشاورزی و منابع طبیعی دانشگاه تهران- گروه گیاهپزشکی
6 Pasteur Institute, Tehran, Iran
چکیده [English]

Nucleic acid-based diagnostic approaches are significant methods in detection and identification of viruses due to their small genome structures and minimal nucleotide differences among virus isolates or strains. In this study, dead or diseased larvae of Helicoverpa sp. were collected from tomato fields in distinct geographic areas in Iran and baculovirus infection was assayed by occlusion body extraction using an optimized Sodium Dodecyl Sulphate method. Two sets of specific polymerase chain reaction (PCR) primer pairs (Pol-a and Pol-b) were designed based on the conserved polyhedrin gene region of 26 fully sequenced HearSNPV/HzNPV isolates. Accordingly, infection by HearSNPV was confirmed in 28 out of 34 tested larvae by PCR amplification of monomorphic fragments of about 370 and 790 nucleotides, respectively. Cloning and sequencing of fragments showed that they belong to the corresponding polyhedron gene from nucleopolyhedriviruses of H. arimegra and H. zea species with 99.6% sequence identity. The phylogeny trees developed for Iranian isolates based on their polyhedron gene sequences showed that they all belong to the Group II Alphabaculovirus and formed one clade with other HearSNPV isolates. This, further confirmed the efficiency of the developed method for baculovirus detection and characterization. To our knowledge, this is the first report of introduction and molecular characterization of some HearSNPV isolates, circulating in these pests in Iran, using a robust DNA-based approach. The excellent efficacy of this method in virus detection makes it a valuable tool for rapid screening of insect cadavers for HearNPV infection, phylogenic studies and virus monitoring in bioinsecticides application.

کلیدواژه‌ها [English]

  • nucleopolyhedrovirus
  • polymerase chain reaction
  • Helicoverpa sp
  • polh gene
Adams, M. J., LeftKowitz, E. J., King, A. M. Q., Bamford, D. H., Breitbart, M., Davison, A. J., Ghabrial, S. A., Gobalenya, A. E., Knowles, N. J., Krell, P., Lavigne, R., Prangishvili, D., Sanfacon, H., Siddell, S. G., Simmonds, P., and Carstens, E. B. (2015). Ratification vote on taxonomic proposals to the International committee on toxonomy of viruses. Archives Virology, 160, 1837-1850.
Arrizubieta, M., Williams, T., Caballeroa, P., and Simon, O. (2013). Selection of a nucleopolyhedrovirus isolate from Helicoverpa armigera as the basis for a biological insecticide. Pest Management Science, 70, 967–976.
Arrizubieta, M., Simon, O., Williams, T., and Caballero, P. (2015). A novel binary mixture of Helicoverpa armigera single nucleopolyhedrovirus genotypic variants has improved insecticidal characteristics for control of cotton bollworms. Applied and Environmental Microbiology, 81, 3984 –3993.
Baillie, V. L., and Bouwer, G. (2011). Development of highly sensitive assays for detection of genetic variation in key Helicoverpa armigera nucleopolyhedrovirus genes. Journal of Virological Methods, 178, 179– 185.
Barrera, G., Simon, O., Villamizar, L., Williams, T., and Caballero, P. (2011). Spodoptera frugiperda multiple nucleopolyhedrovirus as a potential biological insecticide: genetic and phenotypic comparison of field isolates from Colombia. Biological Control, 58, 113-120.
Baillie, V. L., and Bouwer, G. (2012). High levels of genetic variation within Helicoverpa armigera nucleopolyhedrovirus populations in individual host insects. Archives of Virology, 157, 2281–2289.
Brown, D.A.; Allen, C.J. and Bignell, G.N. (1982). The use of a protein A conjugate in an indirect enzyme-linked immunosorbent assay (ELISA) of four closely related baculoviruses from Spodoptera species. Journal of General Virology, 62:375-378.
Chen, X. Zhang, W. J., Wong, J., Chun, G., Lu, A., McCutchen, B. F., Presnail, J. K., Herrmann, R., Dolan, M., Tingey, S., Hu, Z. H., and Vlak, J. M. (2002). Comparative analysis of the complete genome sequences of Helicoverpa zea and Helicoverpa armigera single-nucleocapsid nucleopolyhedroviruses. Journal of General Virology, 83, 673-684.
Christian, P. D., Gibb, N., Kasprzak, A. B., and Richards, A. (2001). A rapid method for the identification and differentiation of Helicoverpa nucleopolyhedroviruses (NPV Baculoviridae) isolated from the environment. Journal of Virological Methods, 96, 51–65.
10. Cory, J. S., Green, B. M., Paul, R. K., and Hunter-Fujita, F. (2005). Genotypic and phenotypic diversity of a baculovirus population within an individual insect host. Journal of Invertebrate Pathology, 89, 101–111.
11. Costa, V.H.D., Soares, M.A.  Dimate, F.A.R., de Sá, V.G.M., and Zanuncio, J.C. (2019). Genetic Identification and Biological Characterization of Baculovirus Isolated from Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil. Florida Entomological Society102(1), 59-64.
12. Ehrt, S., and Schnappinger, D. (2003). Methodes in Molecular Biology. E. Coli Plasmid Vectors Edited by: N. Casali and A. Preston © Humana Pree Inc., Totowa, Nj. Vol, 235
13. Figueiredo, E., Munoz, D., Escribano, A., Mexia, A., Vlak, J. M., and Cabllero, P. (1999). Biochemical identification and comparative insecticidal activity of nucleopolyhedrovirus pathogenic for Heliothis armigera (Lep.: Noctuidae) larvae. Journal of Applied Entomolgy, 123, 165-169.
14. Galal, F. (2009). Universal Primer for Early and Rapid Detection of Nucleopolyhedroviruses of Multiple Species Using Polymerase Chain Reaction.Egyption Academic Journal of Biological Sciences, 1(1), 57-64
15. Haase, S., Sciocco-Cap, A., and Romanowski, V. (2015). Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives. Viruses, 7, 2230-2267.
16. Ikeda, M., Hamajima, R., and Kobayashi, M. (2015). Baculoviruses: diversity, evolution and manipulation of insects.  Journal of Entomological Science, 18,1-20.
17. Jehle, J. A., Blissard, G. W., Bonning, B. C., Cory, J. S., Herniou, E. A., Rohrmann, G. F., Theilmann, D. A., Thiem, S. M., and Vlak, J. M. (2006). On the classification and nomenclature of baculoviruses: a proposal for revision. Archives of Virology, 151(7), 1257–66.
18. Knell, J.D.; Summers, M.D. and Smith, G.E. (1983). Serological analysis of 17 baculoviruses from subgroup A and B using protein blot immunoassay. Virology, 125, 381-392.
19. Kost, T. A., Condreay, J. P., and Jarvis, D. L. (2005). Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature Biotechnology, 23, 567-575.
20. Kreimer-Rabalska, M., Rabalska, L., Jukes, M., D., Souza, M., L., Moore, S., D., and Szewczyk, B. (2019). New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Real Time Polymerase Chain Reaction (Real-Time PCR). Viruses, 11, 115.
21. Lange M., Wang H., Zhihong H., and Jehle J. A. (2004). Towards a molecular identification and classification system of lepidopteran specific baculoviruses. Virology, 325, 36-47.
22. Mazzi, D., and Dorn, S. (2012). Movement of insect pests in agricultural landscapes. Annals of Applied Biology 160: 97–113.
23. Mehrvar, A., Rabindra, R. J., Veenakumari, K. and Narabenchi, G. B. 2006. Comparative evaluation of yield productivity parameters in seven geographic isolates of nucleopolyhedrovirus of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). Insect Environment. 12(1): 14-15.
24. Mehrvar, A. 2011. Entomopathogenic viruses, mass production technology. pp. 281-305. In: Borgio, J.F., Sahayaraj, K. and Susurluk, A. (Eds.). Microbial insecticides, principles and applications. NOVA Science Publishers, USA. (ISBN: 978-1-61209-223-2).
25. Miller, L. K. (1988). Baculoviruses as gene expression vectors. Annual Review of Microbiology, 42, 177−199.
26. Motta, F. C., Rosado, A. S., & Couceiro, J. N. 2002. Standardization of denaturing gradient gel electrophoresis for mutant screening of influenza A (H3N2) virus samples. Journal of Virological Methods 101, 105–115.
27. Munoz, D., and Caballero, P. (2000). Persistence and effects of parasitic genotypes in a mixed population of the Spodoptera exigua nucleopolyhedrovirus. Biological Control 19, 259-264.
28. Naser, W.L. and Miltenburger, H.G. (1983). Rapid baculovirus detection, identification, and serological classification by western blotting-ELISA using a monoclonal antibody. Journal of General Virology, 64,639-647.
29. Noune, C., and Hauxwell, C. (2016). Complete genome sequences of seven Helicoverpa armigera SNPV-AC53-derived strains. Genome, Announc. 4(3), e00260-16
30. Ogembo, J. G., Kunjeku, E. C., and Sithanantham, S. (2005). A preliminary study on the pathogenicity of two isolates of nucleopolyhedroviruses infecting the African bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). International Journal of Tropical Insect Science, 25, 218-222.
31. Ogembo, J. G. Chaeychomsri, S. Kamiya, K., Ishikawa, H., Katou, Y., Ikeda, M., and Kobayashi, M. (2007). Cloning and Comparative Characterization of Nucleopolyhedroviruses Isolated from African Bollworm, Helicoverpa armigera, (Lepidoptera: Noctudiae) in Different Geographic Regions. Journal of Insect Biotechnology and Sericology, 76, 39-49.
32. Rowley, D., L., Farrar, J., R.,  Blackburn, M., B., and Harrison, R., L. (2010). Genetic and biological variation among nucleopolyhedrovirus isolates from the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). Virus Genes, 40(3), 458-68.
33. Rowley, D., Popham, H.J.R., and Harrison, R.L. (2011). Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens. Journal of Invertebrate Pathology, 107,112–126.
34. Rohrmann, G.F. (1992). Baculovirus structure proteins. Journal of General Virology, 73,749-761.
35. Rozen, S., and Skaletsky, H.J. (2000). Primer3 on the WWW for General Users and for Biologist Programmers. In: Krawetz, S. and Misener, S., Eds., Bioinformatics Methods and Protocols: Methods in Molecular Biology,365-386.
36. Smith, G.E. and Summers, M.D. (1981). Application of a novel radioimmunoassay to identify baculovirus structural proteins that share interspecies antigenic determinants. Journal of Virology, 39,125-137.
37. Smith, G. E., Summers, M. D., and Fraser, M. J. (1983). Produc-tion of human β-interferon in insect cells infected with a baculovirus expression vector. Molecular and Cellular Biology, 3, 2156−2165.
38. Sun, X. L., and Peng, H. (2007). Recent advances in biological pest insects by using viruses in China. Virologica Sinica, 22, 158-162.
39. Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6, Molecuar evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.
40. Traverner, M.P. and Connor, E.F. (1992). Optical enumeration technique for detection of baculoviruses in the environment. Environmental Entomology, 21, 307-313.
41. Wang, H., Deng, F., Pijlman, G. P., Chen, X., Sun, X., Vlak, J. M., and Hu, Z. (2003). Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome. Virus Research, 97, 57-63.
42. Webb, S.E. and Shelton, A.M. (1990). Effect of age structure on the outcome of viral epizootics in field populations of imported cabbageworm (Lepidoptera: Pieridae). Environmental Entomology, 19, 111-116.
43. Woo, S.D., Choi, J.Y., Je, Y.H., and Jin, B.R. (2006). Characterization of the Helicoverpa assulta nucleopolyhedrovirus genome and sequence analysis of the polyhedrin gene region. Journal of BioSciences, 31(3), 329-338.
44. Woo, S., D. (2001). Rapid Detection of Multiple Nucleopolyhedroviruses Using Polymerase Chain Reaction. Molecules and Cells, 11(3), 334-340.
45. Zanotto, P.M.; Kessing, B.D. and Maruniak, J.E. (1993). Phylogenetic interrelationships among baculoviruses: evolutionary rates and host associations. Journal of Invertebrate Pathology, 62,147-164.
46. Zhang, C. X., Ma, X. C., and Guo, Z. J. (2005). Comparison of the complete genome sequence between C1 and G4 isolates of the Helicoverpa armigera single nucleocapsidnucleopoly- hedrovirus. Virology, 333 (1),190-199.
47. Zhang, H., Yang, Q., Qin, Q., Zhu, W., Zhang, Z., Li, Y., Zhang, N., and Zhang, J. (2014). Genomic sequence analysis of Helicoverpa armigera nucleopolyhedrovirus isolated from Australia. Archives of Virology, 159, 595– 601.