Phylogenetic analysis of two Iranian grapevine virus A isolates using coat protein gene sequence

Document Type : Research paper


1 Department of Plant Protection, Faculty of Agriculture, University of Zanjan, P. O. Box: 45371-38111, Zanjan, Iran.

2 Department of Plant Protection, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran.


Symptomatic grapevine samples were collected from vineyards in Zanjan province to detect Grapevine virus A. Total RNA was extracted from symptomatic leaf samples and subjected to cDNA synthesis using random hexamer primers. Then, a DNA fragment around 800 bp including the complete coat protein (CP) gene was amplified from nine out of 57 samples by polymerase chain reaction (PCR) using specific primers. The infection rate of GAV in vineyards was around 4%, 6%, 2%, and 6% in Zanjan, Abhar, Tarom, and Khoramdareh, respectively. Two DNA fragments corresponding to samples Abhar (p25) and Zanjan (p26), were purified and sequenced. The CP-nucleotide sequence identity between two Iranian isolates was 97.3%. However, sequence identity with previously reported isolates were 76 to 95% and 82 to 98% at the nt and amino acid levels, respectively. CP-based phylogenetic trees showed three main groups (I, II, III) in which p25 (MG977013) and p26 (MG977013) isolates were placed in the group I together with isolates from different geographical regions including Palestine (Israel), Italy, Czech Republic, Jordan, USA and South Africa. To our knowledge, this is the first report of detection and phylogenetic analysis of GVA isolates from Iranian vineyards based on the complete CP gene. Positive selection value was observed on codon 25 indicating the role of this position probably in virus survival and flexibility against evolutionary forces.


Alkowni R., Digiaro M., and Savino V. (1998). Viruses and virus diseases of grapevine in Palestine. European and Mediterranean Plant Protection Organization Bulletin, 28: 189-195.
Alkowni R., Rowhani A., Daubert S., and Golino D. (2004). Partial characterization of a new ampelovirus associated with grapevine leafroll disease. Journal of Plant Pathology, 86: 123-133.
Andret-Link P., Laporte C., Valat L., Ritzenthaler C., Demangeat G., Vigne E., Laval V., Pfeiffer P., Stussi-Garaud C., and Fuchs M. (2004). Grapevine fanleaf virus: still a major threat to the grapevine industry. Journal of  Plant Pathology, 86: 183-195.
Anfoka G., Shahrour W., and Nakhla M. (2004). Detection and molecular characterization of Grapevine fanleaf virus and Grapevine leafroll-associated virus 3 in Jordan. Journal of Plant Pathology, 86: 203-207.
Basso M. F., Fajardo T. V., and Saldarelli P. (2017). Grapevine virus diseases: economic impact and current advances in viral prospection and management. Revista Brasileira de Fruticultura, 39: 1-22.
Bonavia M., Digiaro M., Boscia D., Boari A., Bottalico G., Savino V., and Martelli G. (1996). Studies on” corky rugose wood” of grapevine and on the diagnosis of grapevine virus B. Vitis, 35: 53-58.
Boscia D., Greif C., Gugerli P., Martelli G., Walter B., and Gonsalves D. (1995). Nomenclature of grapevine leafroll-associated putative closteroviruses. Vitis, 34: 171-175.
Delatte H., Holota H., Moury B., Reynaud B., Lett J. M., and Peterschmitt M. (2007). Evidence for a founder effect after introduction of Tomato Yellow Leaf Curl Virus–Mild in an insular environment. Journal of Molecular Evolution, 65: 112-118.
Digiaro M., Martelli G., and Savino V. (1999). Phloem-limited viruses of the grapevine in the Mediterranean and Near East: a synopsis. Options Méditerrranéennes, Ser. B Studies and Research, 29: 83-92.
Fattouh F., Ratti C., El-Ahwany A., Aleem E. A., Babini A., and Autonell C. R. (2014). Detection and molecular characterization of Egyptian isolates of grapevine viruses. Acta Virologica, 58: 137-145.
Fu Y. X., and Li W. H. (1993). Statistical tests of neutrality of mutations. Genetics, 133: 693-709.
Galiakparov N., Goszczynski D. E., Che X., Batuman O., Bar-Joseph M., and Mawassi M. (2003). Two classes of subgenomic RNA of grapevine virus A produced by internal controller elements. Virology, 312: 434-448.
Garau R., Prota V.A., Piredda R., Boscia D., and Prota U. (1994). On the possible relationship between Kober stem grooving and grapevine virus A. Vitis, 33: 161-163.
García-Arenal F., Fraile A., and Malpica J. M. (2001). Variability and genetic structure of plant virus populations. Annual Review of Phytopathology, 39: 157-186.
Goszczynski D., Du Preez J., and Burger J. (2008). Molecular divergence of Grapevine virus A (GVA) variants associated with Shiraz disease in South Africa. Virus Research, 138: 105-110.
Goszczynski D., and Habili N. (2012). Grapevine virus A variants of group II associated with Shiraz disease in South Africa are present in plants affected by Australian Shiraz disease, and have also been detected in the USA. Plant Pathology, 61: 205-214.
Goszczynski D., and Jooste A. (2003a). Identification of grapevines infected with divergent variants of Grapevine virus A using variant-specific RT-PCR. Journal of Virological Methods, 112: 157-164.
Goszczynski D. E., and Jooste A. (2003b). Identification of divergent variants of Grapevine virus A. European Journal of Plant Pathology, 109: 397-403.
Haidar M., Digiaro M., Khoury W., and Savino V. (1996). Viruses and virus diseases of grapevine in Lebanon. European and Mediterranean Plant Protection Organization Bulletin, 26: 147-153.
Hu G., Dong Y., Zhang Z., Fan X., Fang R., and Zhu H. (2014). Detection and sequence analysis of grapevine virus B isolates from China. Acta Virologica, 58: 180-184.
Hudson R., Boos D. D., and Kaplan N. (1992). A statistical test for detecting geographic subdivision. Molecular Biology and Evolution, 9: 138-151.
Hudson R. R. (2000). A new statistic for detecting genetic differentiation. Genetics, 155: 2011-2014.
Ioannou N., (1993). Occurrence and natural spread of grapevine leafroll-associated closteroviruses in Cyprus. Proceedings 11th Congress of ICVG, 5–10 September, Montreux, Switzland, 111-112.
King A. M., Lefkowitz E., Adams M. J., and Carstens E. B. (2011). Virus taxonomy: ninth report of the International Committee on Taxonomy of Viruses. New York, Elsevier.
Koklu G., Digiaro M., and Savino V. (1998). A survey of grapevine viruses in Turkish Thrace. Phytopathologia Mediterranea, 37: 140-142.
Meng B., Martelli G. P., Golino D. A., and Fuchs M., (2017). Grapevine Viruses: Molecular Biology, Diagnostics and Management. Switzerland, Springer, pp. 698.
Minafra A., and Hadidi A. (1994). Sensitive detection of grapevine virus A, B, or leafroll-associated III from viruliferous mealybugs and infected tissue by cDNA amplification. Journal of Virological Methods, 47: 175-187.
Minafra A., Saldarelli P., and Martelli G. (1997). Grapevine virus A: nucleotide sequence, genome organization, and relationship in the Trichovirus genus. Archives of Virology, 142: 417-423.
Mslmanieh T., Digiaro M., Elbeaino T., Boscia D., and Martelli G. (2006). Viruses of grapevine in Syria. European and Mediterranean Plant Protection Organization Bulletin, 36: 523-528.
Mullins M. G., Bouquet A., and Williams L.E. (1992). Biology of the grapevine. University of California, Davis, USA, pp. 252.
Murolo S., Romanazzi G., Rowhani A., Minafra A., La Notte P., Branzanti M. B., and Savino V. (2008). Genetic variability and population structure of Grapevine virus A coat protein gene from naturally infected Italian vines. European Journal of Plant Pathology, 120: 137-145.
Murphy F. A., Fauquet C. M., Bishop D. H., Ghabrial S. A., Jarvis A.W., Martelli G. P., Mayo M. A., and Summers M. D. (2012). Virus taxonomy: classification and nomenclature of viruses. New York, Elsevier Springer Science & Business.
Naidu R., Rowhani A., Fuchs M., Golino D., and Martelli G. P. (2014). Grapevine leafroll: A complex viral disease affecting a high-value fruit crop. Plant Disease, 98: 1172-1185.
Nickel O., Fajardo T. V., Aragão F. J., Chagas C. M., and Kuhn G. B. (2002). Detection and coat protein gene characterization of an isolate of Grapevine virus B from corky bark-affected grapevines in Southern Brazil. Fitopatologia Brasileira, 27: 279-284.
Osman F., Hodzic E., Omanska-Klusek A., Olineka T., and Rowhani A. (2013). Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D. Journal of Virological Methods, 194: 138-145.
Osman F., and Rowhani A. (2008). Real-time RT-PCR (TaqMan®) assays for the detection of viruses associated with Rugose wood complex of grapevine. Journal of Virological Methods, 154: 69-75.
Rakhshandehroo F., Pourrahim R., Zamani Zadeh H., Rezaee S., and Mohammadi M. (2005). Incidence and distribution of viruses infecting Iranian vineyards. Journal of Phytopathology, 153: 480-484.
Roomi V., Afsharifar A., and Izadpanah K. (2006). Identification, distribution and prevalence of grapevine leafroll associated viruses and grapevine virus A in Iran and their rate of incidence in grapevine cultivars. Iranian Journal of Plant Pathology, 42: 223-240.
Rowhani A., Chay C., Golino D., and Falk B. (1993). Development of a polymerase chain reaction technique for the detection of grapevine fanleaf virus in grapevine tissue. Phytopathology, 83: 749-758.
Rozas J., Ferrer-Mata A., Sánchez-DelBarrio J. C., Guirao-Rico S., Librado P., Ramos-Onsins S. E., and Sánchez-Gracia A. (2017). DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Molecular Biology and Evolution, 34: 3299-3302.
Rozas J., Sánchez-DelBarrio J. C., Messeguer X., and Rozas R. (2003). DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics, 19: 2496-2497.
Saldarelli P., Minafra A., and Martelli G. (1996). The nucleotide sequence and genomic organization of grapevine virus B. Journal of General Virology, 77: 2645-2652.
Tajima F. (1989). Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics, 123: 585-595.
Tsompana M., Abad J., Purugganan M., and Moyer J. (2005). The molecular population genetics of the Tomato spotted wilt virus (TSWV) genome. Molecular Ecology, 14: 53-66.
Wang Q., Mawassi M., Li P., Gafny R., Sela I., and Tanne E. (2003). Elimination of grapevine virus A (GVA) by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L. Plant Science, 165: 321-327.
Wetzel T., Jardak R., Meunier L., Ghorbel A., Reustle G., and Krczal G. (2002). Simultaneous RT/PCR detection and differentiation of arabis mosaic and grapevine fanleaf nepoviruses in grapevines with a single pair of primers. Journal of Virological Methods, 101: 63-69.
Zabalgogeazcoa I., De Blas C., Cabaleiro C., Segura A., and Ponz F. (1997). First report of grapevine virus A in Spain. Plant Disease, 81: 830-830.
Zeinali R., Rahmani F., Abaspour N., and Baneh H. (2012). Molecular and morphological diversity among grapevine (Vitis vinifera L.) cultivars in Iran. Intrnational Journal of  Agricculture: Research & Review, 2: 735-743.