Citrus yellow vein clearing virus (CYVCV) is considered as the causal agent of Citrus yellow vein clearing disease and belongs to the genus Mandarivirus in the family Alphaflexiviridae.  Capsid protein (CP) of CYVCV Chongqing isolate (CYVCV-CQ) was produced using a prokaryotic expression system and used as the immunogen for monoclonal antibody (MAb) production.  Four highly specific and sensitive murine MAbs and one polyclonal antibody were prepared in this study.  Titers of the four MAbs in ascites fluids ranged from 10^–6 to 10^–7 as determined by indirect enzyme-linked immunosorbent assay (ELISA).  Three serological assays, including dot enzyme-linked immunosorbent assay (dot-ELISA), tissue blot-ELISA, and double-antibody sandwich (DAS)-ELISA, were developed for quick and reliable detections of CYVCV in citrus samples.  The developed dot-ELISA and DAS-ELISA methods could detect CYVCV in the infected citrus leaf crude extracts diluted at 1:2 560 and 1:10 240 (w/v, g mL^–1), respectively.  The detection result of 125 citrus leaf samples collected from citrus groves in Yunnan Province and Chongqing Municipality of China showed that approximately 36% samples were positive for CYVCV.  This virus was, however, not detected in any sample collected from Zhejiang or Jiangxi Province, China.

Aphid-borne Zucchini yellow mosaic virus (ZYMV) is one of the most economically important viruses of cucurbitaceous plants.  To survey and control this virus, it is necessary to develop an efficient detection technique.  Using purified ZYMV virion and the conventional hybridoma technology, three hybridoma cell lines (16A11, 5A7 and 3B8) secreting monoclonal antibodies (MAbs) against ZYMV Zhejiang isolate were obtained.  The working titers of the ascitic fluids secreted by the three hybridoma cell lines were up to 10^–7 by indirect enzyme-linked immunosorbent assay (ELISA).  All MAbs were isotyped as IgG1, kappa light chain.  Western blot analysis indicated that the MAb 3B8 could specifically react with the coat protein of ZYMV while MAbs 5A7 and 16A11 reacted strongly with a protein of approximately 51 kDa from the ZYMV-infected leaf tissues.  According to this molecular weight, we consider this reactive protein is likely to be the HC-Pro protein.  Using these three MAbs, we have now developed five detection assays, i.e., antigen-coated-plate ELISA (ACP-ELISA), dot-ELISA, tissue blot-ELISA, double-antibody sandwich ELISA (DAS-ELISA), and immunocapture-RT-PCR (IC-RT-PCR), for the sensitive, specific, and easy detection of ZYMV.  The sensitivity test revealed that ZYMV could be readily detected respectively by ACP-ELISA, dot-ELISA, DAS-ELISA and IC-RT-PCR in 1:163 840, 1:2 560, 1:327 680 and 1:1 310 720 (w/v, g mL^–1) diluted crude extracts from the ZYMV-infected plants.  We demonstrated in this study that the dot-ELISA could also be used to detect ZYMV in individual viruliferous aphids.  A total of 275 cucurbitaceous plant samples collected from the Zhejiang, Jiangsu, Shandong and Hainan provinces, China, were screened for the presence of ZYMV with the described assays.  Our results showed that 163 of the 275 samples (59%) were infected with ZYMV.  This finding indicates that ZYMV is now widely present in cucurbitaceous crops in China.  RT-PCR followed by DNA sequencing and sequence analyses confirmed the accuracy of the five assays.  We consider that these detection assays can significantly benefit the control of ZYMV in China.  

Rice ragged stunt virus (RRSV) is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein (CP) gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21 (DE3) using the pMAL-C2X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody (MAb) against RRSV was obtained by fusing mouse myeloma cells (Sp 2/0) with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), a dot enzyme-linked immunosorbent assay (dot-ELISA), and immunocapture-RT-PCR (IC-RT-PCR) assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1:40 960, 1:1280 and 1:655360 (w/v, g mL-1), respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1:12800 and 1:1600 (an individual planthopper μL-1), respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.

Tomato yellow leaf curl virus (TYLCV) is a species of the family Geminiviridae, causing serious yield losses in tomato production. The coat protein (CP) gene of TYLCV isolate SH2 was expressed in Escherichia coli BL21 (DE3) using pET- 32a as the expression vector. The recombinant protein was purified through Ni+-NTA affinity column and used to immunize BALB/c mice. Three hybridoma cell lines (2B2, 2E3 and 3E10) secreting monoclonal antibodies (MAbs) against TYLCV CP were obtained by fusing mouse myeloma cells (Sp 2/0) with spleen cells from the immunized BALB/c mouse. The titers of ascitic fluids of three MAbs ranged from 10-6 to 10-7 in indirect-ELISA. Isotypes and subclasses of all the MAbs belonged to IgG1, κ light chain. Triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) showed that the MAb 3E10 could react with five begomoviruses infecting tomato, while the other two (2B2 and 2E3) mainly reacted with TYLCV. TAS-ELISA was set up using the MAb 3E10, and the established method could successfully detect virus in plant sap at 1:2 560 (w/v, g mL-1). Detection of field samples showed that begomoviruses were common in tomato crops in Zhejiang Province, China.