Virologica Sinica

, Volume 33, Issue 2, pp 173–180 | Cite as

Monoclonal Antibody-Based Serological Detection Methods for Wheat Dwarf Virus

  • Minghao Zhang
  • Rui Chen
  • Xueping Zhou
  • Jianxiang Wu
Research Article


Wheat dwarf disease caused by wheat dwarf virus (WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein (CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines (i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies (MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840 (w/v, g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120 (w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.


Wheat dwarf virus (WDV) Monoclonal antibody ACP-ELISA Dot-ELISA 



We are grateful to Dr. Xinshun Ding (Samuel Roberts Noble Foundation, Ardmore, USA) for his valuable comments and manuscript edits. This work was supported by Public Science and Technology Research Funds Projects of Agriculture (201303021), and the National Basic Research Program (973) of China (No. 2014CB138400).

Author Contributions

MHZ prepared the MAbs and carried out the immunoassays. RC performed the PCR detection of wheat samples. XPZ and JXW conceived of the study, participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

The animal experiments were performed in accordance with the Principles of the Helsinki accords and approved by the Animal Experimentation Ethics Committee of Zhejiang University, Hangzhou, China.


  1. Achon MA, Serrano L, Ratti C, Rubies-Autonell C (2006) First detection of wheat dwarf virus in barley in Spain associated with an outbreak of barley yellow dwarf. Plant Dis 90:970CrossRefGoogle Scholar
  2. Bendahmane M (1995) Identification and characterization of wheat dwarf virus from france using a rapid method for geminivirus DNA preparation. Phytopathology 85:1449–1455CrossRefGoogle Scholar
  3. Chen Z, Zhang MH, Zhou XP, Wu JX (2017) Development and detection application of monoclonal antibodies against Zucchini yellow mosaic virus. J Integr Agr 16:115–124CrossRefGoogle Scholar
  4. Ekzayez AM, Kumari SG, Ismail I (2010) First report of wheat dwarf virus and its vector (Psammotettix provincialis) affecting wheat and barley crops in Syria. Plant Dis 95:76CrossRefGoogle Scholar
  5. Gadiou S, Ripl J, Janourova B, Jarosova J, Kundu JK (2012) Real-time PCR assay for the discrimination and quantification of wheat and barley strains of wheat dwarf virus. Virus Genes 44:349–355CrossRefPubMedGoogle Scholar
  6. Gawel NJ, Jarret RL (1991) A modified CTAB DNA extraction procedure for Musa and Ipomoea. Plant Mol Biol Rep 9:262–266CrossRefGoogle Scholar
  7. Jin W, Zhang J, Liu Y, Wang X (2015) Development and application of nucleic acid spot hybridization (NASH) assay for rapid detection of Wheat dwarf virus. Plant Protect 41:100–103 (in Chinese) Google Scholar
  8. Kapooria RG, Ndunguru J (2004) Occurrence of viruses in irrigated wheat in Zambia. EPPO Bull 34:413–419CrossRefGoogle Scholar
  9. Koklu G, Ramsell JN, Kvarnheden A (2007) The complete genome sequence for a Turkish isolate of wheat dwarf virus (WDV) from barley confirms the presence of two distinct WDV strains. Virus Genes 34:359–366CrossRefPubMedGoogle Scholar
  10. Lemmetty A, Huusela-Veistola E (2005) First report of wheat dwarf virus in winter wheat in Finland. Plant Dis 89:912–912CrossRefGoogle Scholar
  11. Li N, Chen Z, Liu Y, Liu Y, Zhou XP, Wu JX (2015) Development of monoclonal antibodies and serological assays specific for Barley yellow dwarf virus GAV strain. Virol J 12:136CrossRefPubMedPubMedCentralGoogle Scholar
  12. Lindblad M, Sigvald R (2004) Temporal spread of wheat dwarf virus and mature plant resistance in winter wheat. Crop Protect 23:229–234CrossRefGoogle Scholar
  13. Liu H, Song XJ, Ni YQ, Lu LN, Zhou XP, Wu JX (2014) Highly sensitive and specific monoclonal antibody-based serological methods for rice ragged stunt virus detection in rice plants and rice brown planthopper vectors. J Integr Agr 13:1943–1951CrossRefGoogle Scholar
  14. Liu Z, Chen Z, Hong J, Wang XF, Zhou CY, Zhou XP, Wu JX (2016) Monoclonal antibody-based serological methods for detecting Citrus tristeza virus in citrus groves. Virol Sin 31:324–330CrossRefPubMedGoogle Scholar
  15. Liu Z, SunZhu YJ, Zhou XP, Hong J, Wu JX (2017) Monoclonal antibody-based serological detection of Citrus yellow vein clearing virus in citrus groves. J Integr Agr 16:884–891CrossRefGoogle Scholar
  16. Nygren J, Shad N, Kvarnheden A, Westerbergh A (2015) Variation in susceptibility to wheat dwarf virus among wild and domesticated wheat. PLoS ONE 10:e0121580CrossRefPubMedPubMedCentralGoogle Scholar
  17. Ramsell JNE, Lemmetty A, Jonasson J, Andersson A, Sigvald R, Kvarnheden A (2008) Sequence analyses of Wheat dwarf virus isolates from different hosts reveal low genetic diversity within the wheat strain. Plant Pathol 57:834–841CrossRefGoogle Scholar
  18. Schalk HJ, Matzeit V, Schiller B, Schell J, Gronenborn B (1989) Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication. EMBO J 8:359–364PubMedPubMedCentralGoogle Scholar
  19. Shang HL, Xie Y, Zhou XP, Qian YJ, Wu JX (2011) Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus. Virol J 8:228CrossRefPubMedPubMedCentralGoogle Scholar
  20. Vacke J (1961) Wheat dwarf virus disease. Biol Plantarum 3:228–233Google Scholar
  21. Wang Y, Mao Q, Liu W, Mar T, Wei T, Liu Y, Wang X (2014) Localization and distribution of Wheat dwarf virus in its vector leafhopper, Psammotettix alienus. Phytopathol 104:897–904CrossRefGoogle Scholar
  22. Wang L, Liu Y, Wang XF (2016) Detection of Wheat dwarf virus by TaqMan LNA probe real-time PCR. Acta Phytopathologica Sin 46:313–319 (in Chinese) Google Scholar
  23. Wu J, Ni Y, Liu H, Rao L, Zhou Y, Zhou X (2013) Development and use of three monoclonal antibodies for the detection of rice black-streaked dwarf virus in field plants and planthopper vectors. Virol J 10:114CrossRefPubMedPubMedCentralGoogle Scholar
  24. Wu J, Ni Y, Liu H, Ding M, Zhou X (2014) Monoclonal antibody-based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors. J Virol Methods 195:134–140CrossRefPubMedGoogle Scholar
  25. Wu B, Shang X, Schubert J, Habekuß A, Elena SF, Wang X (2015) Global-scale computational analysis of genomic sequences reveals the recombination pattern and coevolution dynamics of cereal-infecting geminiviruses. Sci Rep 5:8153CrossRefPubMedPubMedCentralGoogle Scholar
  26. Xie J, Wang X, Liu Y, Peng Y, Zhou G (2007) First report of the occurrence of wheat dwarf virus in wheat in China. Plant Dis 91:111CrossRefGoogle Scholar
  27. Zhang X, Zhou G, Wang X (2010) Detection of wheat dwarf virus (WDV) in wheat and vector leafhopper (Psammotettix alienus Dahlb.) by real-time PCR. J Virol Methods 169:416–419CrossRefPubMedGoogle Scholar
  28. Zhou X, Xie Y, Peng Y, Zhang Z (2003) Malvastrum yellow vein virus, a newBegomovirus species associated with satellite DNA molecule. Chin Sci Bull 48:2206–2210CrossRefGoogle Scholar

Copyright information

© Wuhan Institute of Virology, CAS and Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Rice Biology, Institute of BiotechnologyZhejiang UniversityHangzhouChina
  2. 2.State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijingChina

Personalised recommendations