凤果花叶病毒高灵敏度血清学检测技术

Highly sensitive serological approaches for Pepino mosaic virus detection 凤果花叶病毒高灵敏度血清学检测技术 1 first-author 2 2 1 3 4 1 3 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:ffli@ippcaas.cnwujx@zju.edu.cn †E-mail:ffli@ippcaas.cnwujx@zju.edu.cn ffli@ippcaas.cn 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:ffli@ippcaas.cnwujx@zju.edu.cn †E-mail:ffli@ippcaas.cnwujx@zju.edu.cn ffli@ippcaas.cn State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China Department of Applied Biological Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China Department of Applied Biological Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China Hunan Plant Protection Institute, Chinese Academy of Agricultural Sciences, Changsha 410125, China Hunan Plant Protection Institute, Chinese Academy of Agricultural Sciences, Changsha 410125, China Pepino mosaic virus (PepMV) causes severe disease in tomato and other Solanaceous crops around globe. To effectively study and manage this viral disease, researchers need new, sensitive, and high-throughput approaches for viral detection. In this study, we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies (mAbs). A panel of highly specific and sensitive murine mAbs (15B2, 8H6, 23D11, 20D9, 3A6, and 8E3) could be produced through cell fusion, antibody selection, and cell cloning. Using the mAbs as the detection antibodies, we established double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Dot-ELISA, and Tissue print-ELISA for detecting PepMV infection in tomato plants. Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1 310 720 and 1:20 480 (weight/volume ratio (w/v), g/mL), respectively. Among the three methods developed, the Tissue print-ELISA was found to be the most practical detection technique. Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing, demonstrating all three serological methods are reliable and effective for monitoring PepMV. Anti-PepMV mAbs and the newly developed DAS-ELISA, Dot-ELISA, and Tissue print-ELISA can benefit PepMV detection and field epidemiological study, and management of this viral disease, which is already widespread in tomato plants in Yunnan Province of China. 目的 2 建立基于单克隆抗体的检测番茄等植物中凤果花叶病毒(PepMV)的血清学方法,为PepMV的田间调查和诊断及其科学防控提供快速实用的检测技术. 创新点 2 首次制备了抗PepMV的高度特异和灵敏的单克隆抗体,并利用制备的单抗建立了3种能特异且灵敏地检测PepMV的血清学方法. 方法 2 以差速离心方法提纯的PepMV粒子作为免疫原免疫BALB/c小鼠,通过杂交瘤技术获得了能稳定传代并分泌PepMV单克隆抗体的杂交瘤细胞株;杂交瘤细胞注射到小鼠腹腔获得单克隆抗体腹水,并以制备单抗为核心,根据血清学原理建立检测植物中PepMV的双抗夹心酶联免疫吸附试验(DAS-ELISA)、斑点酶联免疫吸附试验(Dot-ELISA)和组织印迹酶联免疫吸附试验(Tissue print-ELISA)三种血清学检测方法;利用田间番茄样品分析建立的血清学方法检测PepMV的有效性. 结论 2 利用杂交瘤技术获得了6株能分泌高度特异灵敏PepMV单克隆抗体的杂交瘤细胞株,以分泌的单抗为核心建立了检测植株中PepMV的DAS-ELISA、Dot-ELISA和Tissue print-ELISA三种高度灵敏的血清学新技术.三种建立的血清学技术检测感染PepMV的番茄植株均呈强阳性反应,而检测健康番茄及感染其他5种植物病毒的植株呈阴性反应,且DAS-ELISA和Dot-ELISA血清学技术检测番茄病叶粗提液的灵敏度分别达到1:1 310 720和1:20 480倍稀释(质量体积比,g/mL).田间样品检测结果发现,建立的血清学技术的检测结果与反转录聚合酶链反应(RT-PCR)的检测结果一致,表明建立的血清学方法可有效地用于植物中PepMV的检测.同时,本研究首次发现PepMV已在我国云南番茄作物上发生流行.PepMV单克隆抗体的制备及其灵敏血清学检测方法的建立有益于PepMV的田间调查和诊断及其科学防控. Pepino mosaic virus Monoclonal antibody Serological method Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) Dot-ELISA Tissue print-ELISA 凤果花叶病毒 单克隆抗体 血清学方法 双抗夹心酶联免疫吸附试验(DAS-ELISA) 斑点酶联免疫吸附试验(Dot-ELISA) 组织印迹酶联免疫吸附试验(Tissue print-ELISA) Introduction 1 Tomato ( Solanum lycopersicum ) is an important vegetable crop grown worldwide, and its yield and planting area are very huge, particularly in China and India. These plants suffer enormously from many viral diseases including Pepino mosaic virus (PepMV), which can devastate tomato production. PepMV was originally isolated from pepino in Peru in 1974, and has now become a widespread virus of greenhouse tomato plants worldwide, especially in Europe and North America (Jones et al., 1980 1980 ; van der Vlugt et al., 2000 2000 ; French et al., 2001 2001 ; Pagán et al., 2006 2006 ; Ling et al., 2008 2008 ; Hanssen and Thomma, 2010 2010 ). The hosts of PepMV were initially thought to be mainly Solanaceous species such as tomato, eggplant, potato, and species from the genera Nicotiana , Datura , Capsicum , and Physalis (Jones et al., 1980 1980 ; Salomone and Roggero, 2002 2002 ; Verhoeven et al., 2003 2003 ). However, researchers have discovered that the virus can also be found in many other hosts including Amaranthus sp., Calendula arvensis , Chrysanthemum segetum , Convolvulus arvensis , and Malva parviflora (Jordá et al., 2001 2001 ; Córdoba et al., 2004 2004 ). The PepMV-infected sweet pepino plants exhibit mosaic, dark green enations on the abaxial surfaces (Jones et al., 1980 1980 ). Virus-affected tomato leaves show mosaic symptoms (Roggero et al., 2001 2001 ). Other typical symptoms of PepMV-infected plants include yellow angular spots on the leaves, the so-called nettleheads, leaf scorching, leaf blistering or bubbling, and fruit marbling (Spence et al., 2006 2006 ; Hanssen et al., 2008 2008 ). PepMV is sap-transmissible and also readily spread mechanically by contaminated pruning and cultivating equipment and is, therefore, very contagious via regular crop handling procedures (Jones et al., 1980 1980 ). In addition, bumblebees are suspected of spreading PepMV (Shipp et al., 2008 2008 ) and seed transmission of PepMV has been reported in several studies albeit at relatively low levels (Córdoba-Sellés et al., 2007 2007 ; Ling, 2008 2008 ; Hanssen et al., 2010 2010 ). PepMV is a member in the genus Potexvirus , family Flexiviridae. PepMV virions are non-enveloped flexuous thread-shaped particles, 508 nm long and 13 nm wide (Jones et al., 1980 1980 ). The viral positive single-stranded RNA genome is 6.4 kb in length and consists of five open reading frames (ORFs), 5' and 3' untranslated regions (UTRs), with a 3' poly-A tail. The viral genome encodes a 164-kDa RNA-dependent RNA polymerase (RdRp), three triple gene block (TGB) proteins of 26, 14, and 9 kDa (assigned TGBp1, TGBp2, and TGBp3, respectively), and a 25-kDa coat protein (CP) (Aguilar et al., 2002 2002 ; Cotillon et al., 2002 2002 ). Currently, virus detection and roguing of infected plants are used to limit spread of PepMV infection, and constitute to the major approach for the prevention and control of the disease in the tomato industry (Hanssen and Thomma, 2010 2010 ). Creating new, fast, simple, sensitive, inexpensive, and high-throughput detection approaches will greatly improve the ability of the industry to cost-effectively maintain healthy crops. Today, the methods described for detecting PepMV in plants mainly include reverse transcription polymerase chain reaction (RT-PCR) (Maroon-Lango et al., 2005 2005 ; Pagán et al., 2006 2006 ; Hasiów-Jaroszewska et al., 2009 2009 ), immunocapture RT-PCR (IC-RT-PCR) (Mansilla et al., 2003 2003 ), the RT-loop-mediated isothermal amplification (LAMP) assay (Hasiów-Jaroszewska and Borodynko, 2013 2013 ), and polyclonal antibody (pAb)-based enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic strip (Salomone and Roggero, 2002 2002 ). Although molecular techniques have been acknowledged to be specific and sensitive to detect PepMV, these techniques are not easily scalable. By contrast, serological assays are known to be quick, simple, low-cost, and high-throughput (Zhang MH et al., 2018 2018 ; Zhang Y et al., 2020 2020 ), although the effectiveness of serological methods depends primarily on the quality and availability of specific antibodies (Zhang et al., 2020 2020 ). To develop sensitive serological techniques for PepMV detection, we prepared a panel of PepMV-specific monoclonal antibodies (mAbs) and further developed double antibody sandwich ELISA (DAS-ELISA), Dot-ELISA, and Tissue print-ELISA for highly sensitive and specific monitoring PepMV in plants. Since PepMV is already widespread in Yunnan Province, China, the mAbs and protocols developed in this study can be extremely useful and effective for PepMV detection, screening for PepMV-free seeds, and the management of this viral disease. Materials and methods 1 Virus sources and plant samples 2 We generated an infectious clone of PepMV (Yunnan strain) in our laboratory (Li et al., 2018 2018 ) and used it to inoculate Nicotiana benthamiana plants. Viral particles were then extracted from the infected plants following the methods described by Zhou et al. ( 1994 1994 ). The prepared PepMV particles were used as the immunogen to generate murine hybridomas secreting anti-PepMV mAbs. Twenty-three field tomato plant samples showing virus-like symptoms were gathered from tomato fields in Yunnan Province in China during 2019–2020. Known Tomato yellow leaf curl virus (TYLCV)-, Tomato mottle mosaic virus (ToMMV)-, Tomato mosaic virus (ToMV)-, Tomato spotted wilt virus (TSWV)-, or Potato virus X (PVX)-infected plants were stored in a −86 °C lab freezer. Generation of murine mAbs 2 The prepared PepMV particles were used to immunize five eight-week-old BALB/c female mice through intraperitoneal injection (Song et al., 2017 2017 ; Guo et al., 2020 2020 ). Preparation of hybridomas and ascitic fluids containing mAbs was described previously (Wu et al., 2011 2011 ; Huang et al., 2019 2019 ). Titers of the mAbs were determined with an indirect-ELISA using the PepMV particles as the antigen. Isotypes of the mAbs were determined with a mouse mAb isotyping kit according to manufacturer’s instructions (Sigma-Aldrich, MO, USA). Specificity and sensitivity of the produced mAbs were analyzed by antigen-coated-plate (ACP)-ELISA and western blot assays (Shang et al., 2011 2011 ; Guo et al., 2020 2020 ). ACP-ELISA 2 We performed serial dilution tests to determine the optimal working dilutions of anti-PepMV mAbs and the alkaline phosphatase (AP)-conjugated goat anti-mouse IgG second antibody (Sigma-Aldrich) for ACP-ELISA as described previously (Shang et al., 2011 2011 ; Song et al., 2017 2017 ). Briefly, the plant tissues were ground and homogenized in a 0.05 mol/L sodium bicarbonate buffer. After centrifugation, the supernatants were coated to 96-well ELISA microplates (100 µL supernatant/well) and the plate was incubated overnight at 4 °C. After three rinses with 0.01 mol/L phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST, pH 7.4), all plate wells were blocked with 0.01 mol/L PBS containing 3% (0.03 g/mL) skimmed milk powder for 30 min. Each serially two-fold diluted anti-PepMV mAb solution (primary antibody) was added into each well of a vertical column of the plate and the plate was incubated at 37 °C for 1 h. After three rinses with PBST, each serially two-fold diluted AP-conjugated goat anti-mouse IgG second antibody was added into each well of a transverse row of the plate followed by 1 h incubation at 37 °C. The optical density at 405 nm (OD 405 ) of each well was measured with the microplate reader at 30 min post adding substrate. The supernatants from a PepMV-infected and a healthy tomato plant were used as the positive and negative controls, respectively. ACP-ELISA was performed as described by Zhang et al. ( 2020 2020 ). DAS-ELISA 2 Individual purified mAb IgG against PepMV was conjugated with AP (Sigma-Aldrich) as previously described (Chen et al., 2017 2017 ). To develop DAS-ELISA for PepMV detection, the effectiveness of different combinations of capture mAb and AP-conjugated mAb IgG was analyzed. Serial dilution tests were also used to determine the working dilutions of the capture antibody and the AP-conjugated detection antibody for DAS-ELISA. DAS-ELISA procedure was carried out as described by Chen et al. ( 2017 2017 ). Dot-ELISA and Tissue print-ELISA 2 Dot-ELISA was performed as described previously with slight modifications (Wu et al., 2014 2014 ; Zhang et al., 2020 2020 ). Briefly, in a mortar, the collected plant tissues were ground and homogenized in 0.01 mol/L PBS at a ratio of 0.1 g tissue per 2 mL buffer. After being centrifuged at 5000 g for 3 min, the supernatant was plant tissue crude extract. Approximately 2.0 μL of plant crude extract from each sample was spotted onto a nitrocellulose membrane. After being air-dried at room temperature (RT), the membrane was soaked for 30 min in blocking buffer followed by 1 h incubation in anti-PepMV mAb solution. After washing with PBST, the membrane was incubated for another 1 h in AP-conjugated goat anti-mouse IgG second antibody solution. After four washes with PBST, the membrane was immersed in nitro-blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3'-indolylphosphate (BCIP) substrate for color development. The samples that developed purple color within 20 min were considered PepMV-positive. Tissue print-ELISA was carried out as the protocol described by Liu et al. ( 2017 2017 ). Briefly, stems were obtained from PepMV-, TYLCV-, ToMMV-, ToMV-, TSWV-, or PVX-infected plants and healthy tomato plants. After being cut with a blade, the cut surface was quickly printed on a nitrocellulose membrane for 3 s. The following procedure was the same as that of Dot-ELISA described above. RT-PCR and sequence analysis 2 Based on CP gene sequence of PepMV in GenBank, the forward primer (5'-ATGGCTGACA ATACCCCAGT-3', nucleotide position 8122–8141) and the reverse primer (5'-AAGTTCTGGGGGTGCA TCAAT-3', nucleotide position 8812–8832) (PepMV-YN: MN939787) were designed to amplify the segment of PepMV CP gene in RT-PCR. RT-PCR detection of PepMV was carried out as described previously (Hasiów-Jaroszewska et al., 2009 2009 ). The PCR-amplified gene segments were individually sequenced and aligned with PepMV CP genes in GenBank. Results 1 Virus purification 2 PepMV particles were extracted from PepMV-infected N . benthamiana leaves by an ultracentrifugation method. The PepMV virion preparation was negatively stained with 1% phosphotungstic acid (pH 7.5), prior to electron microscopic observation. Under an electron microscope, we observed a large number of flexuous and thread-shaped virions with 508-nm length and 13-nm width in the virion preparation. Morphology of the virion is similar to the viruses in the genus Potexvirus (Fig. 1 1 ). PepMV: Pepino mosaic virus . Bar=100 nm Generation and characterization of anti-PepMV mAbs 2 On Day 3 after the third immunization, splenocytes were isolated from immunized mice and then fused with Sp2/0 myeloma cells to produce hybridomas. Via cell selection by hypoxanthine-aminopterin-thymidine (HAT) medium and anti-PepMV antibody detection by indirect-ELISA, we discovered 869 hybridoma wells containing anti-PepMV antibodies. Then, we selected 21 hybridomas secreting highly sensitive anti-PepMV antibodies for cell clone. Finally, six hybridoma lines (i.e., 15B2, 8H6, 23D11, 20D9, 3A6, and 8E3) secreting mAbs against PepMV were obtained by the limited dilution cloning. These hybridomas were then injected intraperitoneally into pristine-primed BALB/c mice for producing ascitic fluids containing mAbs. All selected mAbs were of the IgG1 isotypes with κ light chain (Table 1 1 ). Titers of all six mAbs in ascites were up to 1×10 −7 through indirect-ELISA (Table 1 1 ). The IgG yields of mAbs from ascitic fluids ranged from 6.62 to 9.48 mg/mL (Table 1 1 ). The ascites titer was ascertained by indirect-enzyme-linked immunosorbent assay (ELISA) mAb Isotype and subclass Ascites titer 1 1 Yield of IgG in ascites (mg/mL)  15B2 IgG1, κ chain 1×10 −7 8.49  8H6 IgG1, κ chain 1×10 −7 7.76  23D11 IgG1, κ chain 1×10 −7 9.03  20D9 IgG1, κ chain 1×10 −7 7.54  3A6 IgG1, κ chain 1×10 −7 6.62  8E3 IgG1, κ chain 1×10 −7 9.48 Western blot assay was further used to ascertain the specificity of the anti-PepMV mAbs. The resulting data showed that all six prepared mAbs could react consumingly and specifically with approximately 27-kDa protein in PepMV-infected tomato plant tissues or in the PepMV particle preparation (Fig. 2 2 ). We speculate that the detected protein band is PepMV CP based on the molecular weight of the reactive protein. No protein signals were observed in the lane loaded with the total protein extract from healthy tomato plant tissues (Fig. 2 2 ). Total plant protein was probed with prepared anti- Pepino mosaic virus (PepMV) monoclonal antibodies (mAbs). The identity of mAb is indicated at the bottom of the figure Serial dilution tests indicate that in ACP-ELISA, PepMV can be optimally monitored in the infected tomato plant tissues using 1:7000 (v/v) diluted mAbs (i.e., primary antibody) and 1:8000 (v/v) diluted AP-conjugated goat anti-mouse IgG second antibody. Specificity analysis experiment demonstrated that all six mAbs could specifically react with PepMV-infected tomato plant extracts, not with TYLCV-, ToMMV-, ToMV-, TSWV-, or PVX-infected or healthy plant tissue extracts (Fig. 3a 3a ), indicating that these six mAbs are specific for PepMV detection. (a) Crude extract from Pepino mosaic virus (PepMV)-infected tomato plants was diluted at 1:30 (weight/volume ratio (w/v), g/mL). (b) Two crude extracts from a PepMV-infected and a healthy tomato plant tissue were serially two-fold diluted from 1:10 to 1:2 621 440 (w/v) in the coating buffer, prior to use. The values of optical density at 405 nm (OD 405 ) were read at 30 min post adding the substrate at room temperature (RT) and expressed as mean±standard deviation (SD) of three biological replications. ** P ≤0.01. mAb: monoclonal antibody; CK−: a healthy tomato plant tissue used as a negative control Sensitivity analysis using serially two-fold diluted crude extracts from a PepMV-infected or a healthy tomato plant tissue revealed that the mAbs (15B2, 8H6, 23D11, 20D9, 3A6, and 8E3) can monitor the virus in PepMV-infected tomato plant tissue crude extracts diluted at 1:163 840, 1:163 840, 1:81 920, 1:163 840, 1:81 920, and 1:81 920 (weight/volume ratio (w/v), g/mL), respectively (Fig. 3b 3b ), indicating that all six mAbs are highly sensitive for PepMV detection. DAS-ELISA for PepMV detection 2 Our experiments showed that DAS-ELISA using mAb 8H6 as the capture antibody and AP-conjugated mAb 20D9 as the detection antibody gave the best detection effectiveness of PepMV. Serial dilution tests of DAS-ELISA showed that the optimal working dilutions for mAb 8H6 and AP-conjugated mAb 20D9 were 1:5000 and 1:8000, respectively. Serial two-fold dilutions of PepMV-infected or healthy tomato plant tissue crude extracts were used to determine the detection sensitivity of DAS-ELISA. Resulting data demonstrated that the detection endpoint of DAS-ELISA was 1:1 310 720 dilution for infected plant crude extracts (Fig. 4a 4a ). The specificity analysis result indicated that the DAS-ELISA could specifically detect PepMV infection in plants but had negative reactions for other viruses tested in this study (Fig. 4b 4b ). These data indicate that the developed DAS-ELISA is specific and sensitive to monitor PepMV infection in plants. (a) Six different viruses-infected extracts and a healthy tomato plant extract were diluted at 1:30 (weight/volume ratio (w/v), g/mL) in 0.01 mol/L phosphate-buffered saline (PBS) prior to use. The values of optical density at 405 nm (OD 405 ) were expressed as mean±standard deviation (SD) of three biological replications. ** P ≤0.01 vs. Pepino mosaic virus (PepMV)-infected extract. (b) PepMV-infected extract and a healthy tomato crude extract were serially two-fold diluted in 0.01 mol/L PBS from 1:10 to 1:5 242 880 (w/v, g/mL). These diluted extracts were used to determine the sensitivity of the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The OD 405 values were read at 30 min post addition of the substrate at room temperature Dot-ELISA and Tissue print-ELISA for PepMV detection 2 The resulting data of serial dilution tests indicated that 1:6000 (v/v) diluted anti-PepMV mAbs (primary antibody) and 1:8000 (v/v) diluted AP-conjugated goat anti-mouse IgG secondary antibody were optimal working dilution for detecting PepMV in Dot-ELISA and Tissue print-ELISA. Specificity of the Dot-ELISA was confirmed using PepMV-infected tomato plant tissues and TYLCV-, ToMMV-, ToMV-, TSWV-, or PVX-infected or healthy plant tissues (Fig. 5a 5a ). Two crude extracts respectively from a PepMV-infected and a healthy tomato plant were serially two-fold diluted from 1:20 to 1:20 480 (w/v, g/mL) and used to ascertain the detection endpoints of the developed Dot-ELISA. Sensitivity analysis experiment indicated that PepMV could be effectively monitored in 1:20 480 (w/v, g/mL) diluted crude extracts from PepMV-infected tomato plant tissues (Fig. 5b 5b ). (a) Specificity analysis results of Dot-enzyme-linked immunosorbent assay (ELISA) for monitoring Pepino mosaic virus (PepMV) in tomato plants. On each membrane, two dots (upper and lower) were made using two crude extracts from PepMV-, Tomato yellow leaf curl virus (TYLCV)-, Tomato mottle mosaic virus (ToMMV)-, Tomato mosaic virus (ToMV)-, Tomato spotted wilt virus (TSWV)-, or Potato virus X (PVX)-infected plant tissues, or healthy tomato plant tissues. (b) Sensitivity analysis results of Dot-ELISA for monitoring PepMV in tomato plants. Two crude extracts from PepMV-infected and healthy tomato plant tissues were serially two-fold diluted from 1:20 to 1:20 480 (weight/volume ratio (w/v), g/mL) in phosphate-buffered saline (PBS) (pH 7.4). Purple color represents a positive reaction. The mAb names were indicated on the right side of the figure To explore whether Tissue print-ELISA can be used to monitor PepMV infection in tomato plants, stems were transversely cut from known TYLCV-, ToMMV-, ToMV-, TSWV-, or PVX-infected, and healthy plants and the cut surfaces were printed individually on a nitrocellulose membrane. The printed membrane was probed by anti-PepMV mAb followed by the AP-conjugated goat anti-mouse IgG second antibody. After adding NBT/BCIP substrate, dark purple color indicating PepMV-positive was observed on the print made with the PepMV-infected tomato plant. No color development was found on the prints made with TYLCV-, ToMMV-, ToMV-, TSWV-, or PVX-infected, or healthy plant stems (Fig. 6 6 ), indicating the established Tissue print-ELISA was highly specific for PepMV and had no cross-reaction with the tested five other plant viruses. Specificity analyses of the established Tissue print enzyme-linked immunosorbent assay (ELISA). Columns from 1 to 6 were the prints from Pepino mosaic virus (PepMV)-, Tomato yellow leaf curl virus (TYLCV)-, Tomato mottle mosaic virus (ToMMV)-, Tomato mosaic virus (ToMV)-, Tomato spotted wilt virus (TSWV)-, and Potato virus X (PVX)-infected plant stems. A healthy tomato stem was used as the negative control. The prints were probed with anti-PepMV monoclonal antibodies (mAbs) followed by the alkaline phosphatase (AP)-conjugated goat anti-mouse IgG second antibody. Purple color prints indicated PepMV-positive, while light green color prints represented PepMV-negative Detection of PepMV infection in the field-collected tomato samples 2 The presence of PepMV in 23 field-collected tomato plants was screened simultaneously through the developed DAS-ELISA, Dot-ELISA, Tissue print-ELISA, and RT-PCR. Six of the twenty-three field samples tested positive for PepMV through these three serological approaches (Figs. 7a–7c 7a–7c ). The results of the serological detection methods agreed with those obtained by RT-PCR method (Fig. 7d 7d ). Results of sequencing and sequence alignment of the resulting PCR products indicated that the PepMV isolates were detected in the field tomato plants and shared 82.0%–99.7% sequence similarity with PepMV CP genes in GenBank. Taken together, these data confirm that the newly developed DAS-ELISA, Dot-ELISA, and Tissue print-ELISA approaches were highly sensitive and accurate for detecting PepMV infection in plants. Furthermore, to our knowledge, this is the first report on PepMV infection in plants in China. Labels a1–a5, b1–b5, c1–c5, d1–d5, and e1–e5 represented individual field samples. Labels a1 and b1 were a known uninfected and a PepMV-infected tomato plant, and used as a negative and a positive control, respectively. Lane M is a 1 kb DNA ladder. PepMV: Pepino mosaic virus ; DAS-ELISA: double antibody sandwich enzyme-linked immunosorbent assay; RT-PCR: reverse transcription polymerase chain reaction Discussion 1 Van der Vlugt et al. ( 2000 2000 ) first reported that PepMV could infect tomato crops in the Netherlands. Now, PepMV has become an important virus on tomato plants worldwide, and causes serious economic losses because of its high rates of infection (French et al., 2001 2001 ; Pagán et al., 2006 2006 ; Spence et al., 2006 2006 ; Ling et al., 2008 2008 ; Hanssen and Thomma, 2010 2010 ). Currently, strategies to prevent and control PepMV often fail because of limited understanding the disease (Hanssen and Thomma, 2010 2010 ). Development of a simple, fast, sensitive, cost-effective, and high-throughput technique for detecting PepMV is crucial for studying its epidemiology and managing this disease. Visual inspection is a common and simple method for diagnosing plant viral diseases in the fields. However, diagnosis based on visual inspection is often inaccurate as symptoms of infection are often overlapping and PepMV infection can be easily confused with other pathogens or horticultural problems. The current diagnostic approaches for PepMV are primarily dependent on RT-PCR (Maroon-Lango et al., 2005 2005 ; Pagán et al., 2006 2006 ; Hasiów-Jaroszewska et al., 2009 2009 ), IC-RT-PCR (Mansilla et al., 2003 2003 ), and the real-time RT-PCR (Ling et al., 2007 2007 ). RT-PCR is often used in laboratory for detecting plant RNA viruses because of its sensitivity and specificity. However, RT-PCR is costly, time-consuming, and not scalable. To overcome these constraints, the RT-LAMP assay was developed for PepMV detection (Hasiów-Jaroszewska and Borodynko, 2013 2013 ). However, it is well known that the accuracy of RT-LAMP is not good enough because of a high false positive rate (Li et al., 2017 2017 ). Compared with these PCR-based approaches above, serological methods are known to be rapid, simple, cost-effective, and suitable for high volume testing. Serological techniques have been widely applied in detection and field surveys of plant viral diseases (Shang et al., 2011 2011 ; Wu et al., 2011 2011 ; Zhang et al., 2020 2020 ). However, it is well known that the validity of a serological approach depends mainly on the sensitivity and specificity of the viral detection antibodies. Salomone and Roggero ( 2002 2002 ) developed ELISA and an immunochromatographic strip for PepMV detection using the prepared pAbs with detection endpoints of both DAS-ELISA and the test strip for crude extracts from the infected plant tissues set at 1:8192 and 1:2048 dilution, respectively. However, these pAbs have been shown to have some drawbacks such as limited yield, high cross-reactions with other viruses in the same genus or with host proteins, leading to high background noise (Song et al., 2017 2017 ). However, the serological approaches we developed in the current study are much more effective. mAb has these following merits: (1) researchers can use preliminary screening to select highly sensitive and specific mAbs; (2) once a hybridoma line that secretes mAb is available, unlimited number of mAb can be easily generated; (3) serological approaches based on the high-quality mAbs can obtain more reliable and accurate detection results than those using pAbs (Liu et al., 2014 2014 ; Zhang et al., 2020 2020 ). The six produced mAbs in this study reacted strongly with PepMV in crude extracts from infected tomato plant tissues, but not with crude extracts from TYLCV-, ToMMV-, ToMV-, TSWV-, or PVX-infected plants, or from healthy tomato plants. To our knowledge, the six mAbs prepared in this study are the first ever reported about anti-PepMV mAbs use. Using the produced mAbs as the detection antibody, three serological approaches (i.e., DAS-ELISA, Dot-ELISA, and Tissue print-ELISA) were developed for specific and sensitive detection of PepMV infection in tomato plants. The dilution endpoints (i.e., sensitivities) of the developed DAS-ELISA and Dot-ELISA were 1:1 310 720 and 1:20 480 (w/v, g/mL) dilution, respectively, for crude extracts from infected tomato plants. Surprisingly, the DAS-ELISA approach developed in this study is 160 times more sensitive than DAS-ELISA conducted by Salomone and Roggero ( 2002 2002 ). We conclude that the Tissue print-ELISA is the fastest and most practical approach for PepMV test among these three newly established serological methods. The dependability of the three serological approaches for field plant sample detection was validated by RT-PCR followed by DNA sequencing. According to the field survey, we have demonstrated that PepMV is already widespread in Yunnan Province of China. In addition, tomato is one of the most important vegetable crops grown worldwide. Thus, we conclude that the three newly developed serological approaches are highly valuable for the diagnosis and management of this vial disease during tomato production all over the world. PepMV causes a contagious disease in crops because of its simple transmission by contaminated contact, grafting, and other significant vectors. Shipp et al. ( 2008 2008 ) proved that bumblebees contributed to the spread of PepMV since they are often used for pollination in commercial tomato production. In addition, and perhaps more seriously, several reports demonstrated that PepMV exists a low rate seed transmission, implying that there is a substantial risk associated with tomato seeds harvested from infected crops (Córdoba-Sellés et al., 2007 2007 ; Ling, 2008 2008 ; Hanssen et al., 2010 2010 ). In addition, PepMV in dry plant materials is thought to remain viable for as long as three months, and the virus in moist organic debris is considered capable of infection for a relatively long period. In our next study, the virus carrying rate of bumblebees will be further investigated using the serological approaches developed here, and the feasibility of the serological methods for detecting PepMV in tomato seeds will be analyzed. In summary, the obtained six anti-PepMV mAbs and the three newly established serological approaches are very useful for PepMV detection and epidemiological study, and establishment of prevention and control strategies for this viral disease. Furthermore, to our knowledge, this is the first report on PepMV infection in plants in China. References JM Aguilar , MD Hernández-Gallardo , JL Cenis , 等 . Complete sequence of the Pepino mosaic virus RNA genome . 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A method for high yield purification of potyviruse Compliance with ethics guidelines Wan-qin HE, Jia-yu WU, Yi-yi REN, Xue-ping ZHOU, Song-bai ZHANG, Ya-juan QIAN, Fang-fang LI, and Jian-xiang WU declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B2000255 Project supported by the National Key R&D Program of China (Nos. 2019YFD1001800 and 2017YFD0201604) and the National Natural Science Foundation of China (Nos. 31772125 and 31972234) 2020-05-17 2020-07-30 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2020 2020 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 21