doi: 10.15389/agrobiology.2016.3.385eng

UDC 634.2:632.3:578.1:57.083

Supported by Russian Science Foundation, grant № 14-24-00007



A.A. Sheveleva, N.A. Nikitin, E.A. Trifonova, A.V. Zakubanskiy,
S.N. Chirkov

M.V. Lomonosov Moscow State University, Department of Virology, Biological Faculty, 1/12, Leninskie gory, Moscow, 119234 Russia,

Received November 19, 2015


Molecular weight heterogeneity of the coat protein (CP) is commonly observed in the purified potyvirus preparations due to partial proteolysis of the N-terminal domain exposed on the surface of viral particles. The cellular proteases released from disrupted plant tissues are believed to attribute to the partial CP proteolysis during purification and storage of the virus. The N-terminal domain is the most variable part of the CP. It displays virus- and strain-specific epitopes and is highly immunogenic. Degradation of the N-terminal domain creates a potential problem in the production of virus-specific antisera and strain-specific monoclonal antibodies and complicates the serological analysis of virus isolates. Plum pox virus (PPV) is considered the most important viral pathogen of stone fruit crops. Serological methods are of great importance for the diagnosis of PPV, strain identification, elaboration of PPV-resistant cultivars, epidemiological surveys and prevention of the uncontrolled spread of the virus in stone fruit plantings. The CP of PPV was studied by polyacrylamide gel electrophoresis, Western blotting and immunoelectron microscopy using monoclonal antibodies 5B and 4DG5 specific to the PPV universal and strain D epitopes, respectively. The purified virus particles have been shown to contain three types of subunits: full-size CP as well as the 28 and 31 kDa products of its partial proteolysis. Three bands of the similar molecular weight were also detected in the analysis of fresh extracts from PPV- infected Nicotiana benthamiana tobacco leaves and a number of stone fruits (peach, plum and cherry plum), homogenized directly in the sample  buffer, by Western blotting with the antibody 5B suggesting that the CP can be proteolytically processed already in infected plant tissues. The improved procedure of PPV purification, based on the combination of two original methods (H.J. van Oosten, 1972; S. Lain et al., 1988) has been developed. The procedure included accumulation of the virus in N. benthamiana plants, its extraction from the infected leaves using a neutral HEPES buffer, the incubation of the clarified extract with 5 % Triton X-100, ultracentrifugation on a 20 % sucrose cushion and purification of the virus using the ultracentrifugation in sucrose concentration gradient (10-40 %) in a 0.1 М sodium borate buffer, рН 8.2. The method provided the yield up to 10 mg of the purified virus from 100 g of infected leaves. The virus particles contained mainly the full-size CP. The high yield of the virus seems to be due to an effective extraction of the virus using HEPES buffer as well as its negligible losses during clarification of the extract, treatment with Triton X-100 and ultracentrifugation on a sucrose cushion. The use of Triton X-100 in the specified concentration leads to a more complete separation of the virus from membrane complexes and solubilization of aggregates of viral particles, that increases the yield of the virus. Application of such the virus preparations for animal immunization can facilitate the high specific antisera and monoclonal antibodies production for reliable detection and serological analysis of PPV.

Keywords: plum pox virus, purification, coat protein, Western blot, immunoelectron microscopy, monoclonal antibody, epitope.


Full article (Rus)

Full text (Eng)



  1. Cambra M., Boscia D., Myrta A., Llacer G. Plum pox virus and estimated cost associated with Sharka disease. EPPO Bull., 2006, 36: 202-204 CrossRef
  2. Chirkov S.N., Prikhod'ko Yu.N. Geneticheskoe raznoobrazie i struktura populyatsii virusa ospy (sharki) slivy v Rossii [Genetic diversity and population structure of Plum pox virus in Russia]. Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2015, 50(5): 529-539 CrossRef CrossRef
  3. Garcia J.A., Glasa M., Cambra M., Candresse T. Plum pox virus and sharka: a model potyvirus and a major disease. Mol. PlantPathol., 2014, 15: 226-241 () CrossRef
  4. Llacer G., Cambra M. Host and symptoms of Plum pox virus: fruiting Prunus species. EPPO Bull., 2006, 36: 219-221 CrossRef
  5. James D., Thompson D. Host and symptoms of Plum pox virus: ornamental and wild Prunus species. EPPO Bull., 2006, 36: 222-224 CrossRef
  6. Polak J. Host and symptoms of Plum pox virus: woody species other than fruit and ornamental species of Prunus. EPPO Bull., 2006, 36: 225-226 CrossRef
  7. Llacer G. Host and symptoms of Plum pox virus: herbaceous hosts. EPPO Bull., 2006, 36: 227-228 CrossRef
  8. van Oosten H.J. Herbaceous host plants for the sharka (plum pox) virus. Neth. J. Pl. Path., 1970, 76: 253-260 CrossRef
  9. van Oosten H.J. Purification of plum pox (sharka) virus with the use of Triton X-100. Neth. J. Pl. Path., 1972, 78: 33-44 CrossRef
  10. Laín S., Riechmann J.L., Méndez E., García J.A. Nucleotide sequence of the 3' terminal region of plum pox potyvirus RNA. Virus Res., 1988, 10: 325-341 CrossRef
  11. Shukla D.D., Jilka J., Tosic M., Ford R.E. A novel approach to the serology of potyviruses involving affinity-purified polyclonal antibodies directed towards virus-specific N termini of coat protein. J. Gen. Virol., 1989, 70: 13-23 CrossRef
  12. Shukla D.D., Strike P.M., Tracy S.L., Gough K.H., Ward C.W. The N and C termini of the coat proteins of potyviruses are surface-located and the N terminus contains the major virus-specific epitopes. J. Gen. Virol., 1988, 69: 1497-1508 CrossRef
  13. Subr Z., Glasa M. Plum pox virus variability detected by the advanced analytical methods. Acta Virologica, 2008, 52: 75-90. 
  14. Kollerova E., Glasa M., Subr Z.W. Western blotting analysis of the Plum pox virus capsid protein. J. Plant Pathol., 2008, 90: S1.19-S1.22 CrossRef
  15. Sheveleva A., Ivanov P., Prihodko Y., James D., Chirkov S. Occurrence and genetic diversity of Winona-like Plum pox virus isolates in Russia. Plant Dis., 2012, 96: 1135-1142 CrossRef
  16. Lopez-Moya J.J., Canto T., Lopez-Abella D., Diaz-Ruiz J.R. Differentiation of Mediterranean plum pox virus isolates by coat protein analysis. Plant Pathol., 1994, 43: 164-171 CrossRef
  17. Lopez-Moya J.J., Sanz A., Cambra M., Gorris M.T., Anaya C., Miguet J.G., Cortes E., Lopez-Abella D. Production and characterization of monoclonal antibodies to plum pox virus and their use in differentiation of Mediterranean isolates. Arch. Virol., 1994, 135: 293-304 CrossRef
  18. Maiss E., Timpe U., Brisske A., Jelkman W., Casper R., Himmler G., Mattanovich D., Kattinger H.W. The complete nucleotide sequence of plum pox virus RNA. J. Gen. Virol., 1989, 70: 513-524 CrossRef
  19. Chirkov S., Ivanov P., Sheveleva A., Kudryavtseva A., Prikhodko Y., Mitrofanova I. Occurrence and characterization of Plum pox virus strain D isolates from European Russia and Crimea. Arch. Virol., 2016, 161: 425-430 CrossRef 
  20. Nikitin N., Trifonova E., Karpova O., Atabekov J. Examination of biologically active nanocomplexes by nanoparticle tracking analysis. Microsc. Microanal., 2013, 19: 808-813 CrossRef
  21. Cambra M., Asensio M., Gorris M.T., Perez E., Camarasa E., Garcia J.A., Moya J.J., Lopez-Abella D., Vela C., Sanz A. Detection of plum pox potyvirus using monoclonal antibodies to structural and non-structural proteins. EPPO Bull., 1994, 24: 569-577 CrossRef
  22. Candresse T., Saenz P., Garcia J.A., Boscia D., Navratil M., Gorris M.T., Cambra M. Analysis of the epitope structure of Plum pox virus coat protein. Phytopathology, 2011, 101: 611-619 CrossRef
  23. Candresse T., Cambra M., Dallot S., Lanneau M., Asensio M., Gorris M.T., Revers F., Macquaire G., Olmos A., Boscia D., Quiot J.B., Dunez J. Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the D and M serotypes of plum pox potyvirus. Phytopathology, 1998, 88(3): 198-204 CrossRef
  24. Sheveleva A., Chirkov S., Nemova E. Detection of a new Winona-like Plum pox virus isolate in naturally infected Canadian plum (Prunus nigra) in Russia. Acta Horticulturae, 2011, 899: 49-55 CrossRef 
  25. Garcia J.A., Martin M.T., Cervera M.T., Reichmann J.L. Proteolytic processing of the plum pox virus polyprotein by the NIa protease at a novel cleavage site. Virology, 1992, 188: 697-703 CrossRef
  26. Adams M.J., Antoniw J.F., Beaudoin F. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Mol. Plant Pathol., 2005, 6: 471-487 CrossRef