Journal of Virological Methods 220 (2015) 43–48
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
A rapid immunochromatographic test to detect the lily mottle virus Yubao Zhang a , Yajun Wang a , Wanrong Yang b , Zhongkui Xie a,∗ , Ruoyu Wang a , Hadley Randal Kutcher c , Zhihong Guo a a b c
Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, China Ningxia Agricultural Comprehensive Development Office, Yinchuan 750002, China College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon SK, S7N 5A8, Canada
a b s t r a c t Article history: Received 13 January 2015 Received in revised form 2 April 2015 Accepted 2 April 2015 Available online 17 April 2015 Keywords: Immunochromatographic strip LMoV PCR Rapid detection
We developed a rapid immunochromatographic strip (ICS) test for lily mottle virus (LMoV). The test is based on a double-antibody sandwich format and employs two distinct anti-LMoV polyclonal antibodies (IgG3 and IgG4 ). The first antibody, IgG3 was conjugated with colloidal gold, and the second antibody, IgG4 was used as the capture antibody at the test line. The performance of the ICS test was evaluated and the results obtained were compared with a quadruplex RT-PCR assay. When serial dilutions of purified LMoV were tested, the LMoV detection limit of the ICS test was 8.0 × 10−9 mg/mL, which was in complete agreement with the results of quadruplex RT-PCR. Compared with quadruplex RT-PCR, the specificity and sensitivity of ICS were 98.7 and 100%, respectively. There was therefore significant agreement between the results obtained from the two tests ( = 0.982). The ICS test therefore appears to be broadly applicable, and will be especially useful in the field, as well as in areas without laboratory facilities, to support efforts to detect and control LMoV. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lily (Lilium spp.) is an important economic crop in the floricultural industry, and additional value is obtained from the bulbs, which are edible and have medicinal properties. Almost all lilies are propagated vegetatively. Therefore, viruses may accumulate in bulbs from one generation to the next. Viruses cause quantitative and qualitative yield reductions of lilies around the world (Wang et al., 2010). More than 10 different viruses have been reported to infect lilies worldwide, and the lily mottle virus (LMoV) is one of the most common (Ryu et al., 2002; Zhang et al., 2014). LMoV is closely related to the tulip breaking virus (TBV; Alper et al., 1982), and is a member of the Potyvirus genus within the Potyviridae family. LMoV is flexuous, non-enveloped, and rod-shaped; it is 680–900 nm long and 11–15 nm wide (King et al., 2011). LMoV is composed of a single-stranded 9.7 kb RNA with a positive polarity, and is surrounded by about 2000 copies of the coat protein (CP). The virus encodes a polyprotein of 3095 aa with a molecular weight of
∗ Corresponding author at: Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, No 320 Dong Gang West Road, Lanzhou 730000, China. Tel.: +86 931 496 7206; fax: +86 931 827 3894. E-mail addresses: [email protected] (Y. Zhang), [email protected] (Y. Wang), [email protected] (W. Yang), [email protected] (Z. Xie), [email protected] (R. Wang), [email protected] (H.R. Kutcher), [email protected] (Z. Guo). http://dx.doi.org/10.1016/j.jviromet.2015.04.010 0166-0934/© 2015 Elsevier B.V. All rights reserved.
351.0 kDa (King et al., 2011). The polyprotein self-cleaves and form proteins of different sizes and functions, including CP and a cytoplasmic inclusion protein (CI). The CP and CI contain 274 and 635 amino acids of 30 and 70 kDa in size, respectively (Urcuqui-Inchima et al., 2001). Symptoms of LMoV may vary from vein clearing, leaf mottle, leaf mosaic, chlorotic and yellow streaking, leaf curling, and narrowing and reddish-brownish-necrotic spots, to milder forms of leaf symptoms, or plants may even be symptomless at some stages of growth in the field (Fig. 1). Diseased plants are often shorter than healthy plants and the symptoms may be more severe if plants are also infected by Lily symptomless virus (LSV) (Asjes, 2000). The most commonly used LMoV detection methods for lily samples are electron microscopy (Wang et al., 2007), enzyme-linked immunosorbent assay (ELISA; Sharma et al., 2005), reversetranscriptase polymerase chain reaction (RT-PCR; Zhang et al., 2010), and real-time PCR (Zhang et al., 2014). However, these methods are time consuming, require technical expertise and specialized laboratory equipment. Therefore, these methods are not suitable for rapid detection during the cultivation of lily, it is necessary to develop a rapid, specific, and easily performed assay to detect LMoV. The immunochromatographic assay is one possibility. This technique offers several advantages over traditional immunoassays, such as its low cost, procedural simplicity, limited requirement for special skills or expensive equipment, and rapid results (Alvarez et al., 2010). The immunochromatographic assay has been widely applied to detect hormones, bioactive molecules,
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Fig. 1. (A) Electron microscopy image of LMoV particles and (B) leaves showing mottle symptoms.
contagious human diseases, animal and plant viruses, bacteria and parasite antigens as well as specific antibodies (Weiss, 1999; Alvarez et al., 2010; Sun et al., 2013). The aim of the present study was to develop an immunochromatographic test strip (ICS) that could be used to rapidly detect LMoV. The sensitivity and specificity of the ICS were evaluated using a quadruplex RT-PCR assay as a reference test with 118 lily samples from the field. The results showed strong agreement between the two assays ( = 0.982). 2. Materials and methods
electron microscopy (TEM) measurements using a JEM-1230 transmission electron microscope (TEM; JEOL, Tokyo, Japan). To prepare colloidal gold-IgG3 , the IgG3 (180 L at 1.0 mg/mL) was added drop-wise to 10 mL of the pH-adjusted colloidal gold solution. The mixture was stirred vigorously for 30 min, and then 2.5 mL of 10% bovine serum albumin (BSA) was added to stabilize the conjugate solution. The mixture was then stirred for an additional 30 min, then was centrifuged at 10,000 × g at 4 ◦ C for 30 min, after which the gold pellets were suspended in 1 mL of dilution buffer: 20 mM Tris/HCl buffer (pH 8.2) containing 1% (w/v) BSA, 3% (w/v) sucrose, and 0.02% (w/v) sodium azide. The colloidal gold conjugate was stored at 4 ◦ C until use.
2.1. Reagents and materials 2.3. Preparation of the immunochromatographic strip (ICS) Leaves infected by LMoV, LSV or CMV served as sources of these viruses and were stored at −70 ◦ C at the Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Science (Lanzhou, China). The first antibody, IgG3 -rabbit IgG antiLMoV CP-CI200 (CPI200) and the second antibody IgG4 -rabbit IgG anti-LMoV CP were obtained following the procedure of Tong et al. (2010). Both of the antibodies were kept in aliquots of 1 mg at −20 ◦ C for one year. We purchased chloroauric acid (HAuCl4 ) and goat anti-rabbit IgG from the Sigma Company (St. Louis, MO, USA), nitrocellulose membranes (HiFlow-120) and cellulose filter from Millipore (Billerica, MA, USA), and special cellulose and absorbent papers from Jieyi Company (Shanghai, China). 2.2. Preparation of colloidal gold and a colloidal gold-IgG3 conjugate Colloidal gold particles with a mean diameter of 30 nm were prepared using the method of Hermanson (2008) in 1.4 mL of 1% trisodium citrate (w/v) which was rapidly added to 100 mL of 0.01% aqueous chloroauric acid solution (w/v) at 100 ◦ C and boiled for 5 min. As the resulting colloidal gold cooled gradually to room temperature with continuous stirring, the pH was maintained at 7.4 by adding 0.1 M potassium carbonate. Sodium azide was added to a final concentration of 0.01% (w/v) before storage at 4 ◦ C in a glass bottle covered with foil. The absorption maxima (max ) of the solutions were analyzed by means of ultraviolet/visible spectroscopy (UV/VIS) using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) to determine the approximate particle sizes, which were confirmed by transmission
The ICS included four components: a sample pad (special cellulose paper), a conjugate release pad (cellulose filter), a nitrocellulose membrane, and an absorbent pad (absorbent paper). The sample pad was treated with 20 mM phosphate buffer containing 1% (w/v) BSA, 0.5% (v/v) Tween-20, and 0.05% (w/v) sodium azide (pH 7.4), and was then dried for 2 h at 37 ◦ C. For the conjugated pad, we first impregnated the cellulose filter with 15 L colloidal gold-IgG3 and then dried the pad for 2 h at 37 ◦ C. Subsequently, we formed a control line by carefully dragging a pipette tip containing 100 L of the goat anti-rabbit IgG (1.5 mg/mL) along the long axis of the nitrocellulose membrane. This was done by leaning the tip against a ruler placed 6 mm from one end of the membrane. Immediately afterwards, we repeated this process to generate a test line using the IgG4 (1.0 mg/mL). We used the rule to maintain a 5-mm gap between the two lines. The remaining active sites on the membrane were blocked by incubation with 2% (w/v) BSA in PBS for 30 min at room temperature. The membrane was washed once with PBS, a second time with distilled water, and was then dried for at least 2 h at room temperature. Next, the sample pad, the prepared conjugated pad, the nitrocellulose membrane, and the absorbent pad were adhered to a backing plate (300 mm × 70 mm) in the proper order, as described previously (Peng et al., 2008). The assembled plate was then cut into 4-mm-wide pieces with a pair of small scissors. The resulting strips were mounted in plastic cassettes with windows over the sample pad and the nitrocellulose membrane (Jieyi Company, Ltd., Shanghai China). The cassettes were then packaged in plastic bags containing desiccant, and stored at 4 ◦ C until use.
Y. Zhang et al. / Journal of Virological Methods 220 (2015) 43–48
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Fig. 2. (A) UV/Vis spectrum of the solution containing the colloidal gold particles and (B) TEM image of the particles.
2.4. The immunochromatographic assay If the sample tested contained LMoV, the LMoV reacted with the IgG3 conjugated to colloidal gold. The colloidal gold-labeled IgG3 –LMoV complex then migrated into the nitrocellulose membrane by means of capillary action and subsequently reacted with the immobilized IgG4 in the test line. Unbound colloidal goldlabeled IgG3 passed through the test line and reacted with the goat anti-rabbit IgG in the control line to form a second visible purple-red band. About 100 L of the test samples were applied to the plastic cassette sample window. The result was read between 5 and 10 min after addition of the sample. The sample was considered positive if two distinct purple-red lines appeared, one in the test region and the other in the control region; negative when no line appeared in the test region, and invalid if the control line failed to appear.
2.5. Specificity and sensitivity of the ICS test To evaluate the cross-reactivity of the ICS test strip, LMoV and two other common lily viruses (LSV, the lily symptomless virus; CMV, the cucumber mosaic virus) from strongly positive field samples, were tested with the test strip. LMoV-infected leaves were used as a positive control, and virus extraction buffer (0.2 M Tris/HCl buffer (at pH 7.5) containing 10 mM EDTA–Na2 , 0.1% (w/v) polyvinyl polypyrrolidone and 0.1% (v/v) 2-mercaptoethanol) was used as a negative control. The sensitivity of this test strip was evaluated by testing a series of 10-fold dilutions of the purified LMoV solution (at 8.0 × 10−2 mg/mL), which was analyzed as the virus antigen by means of western blotting using the IgG3 protein at the concentration of 1.2 × 10−3 mg/mL. Each dilution was then added to the ICS test strip, and the sensitivity was determined by finding the endpoint dilution. In addition, we compared the specificity and sensitivity of the ICS test strip with the results of quadruplex RT-PCR test developed by Zhang (2010). Briefly, we used four pairs of primers (LMoV forward, 5 -TGGCACCTCACCAAATGTA3 and LMoV reverse, 5 -CATCATCTGCTGTATGCCTCT-3 ; LSV forward, 5 -TATGGGCTTCCAATACAAC-3 and LSV reverse, 5 TATTCGGTTTCCAGGTT-3 ; CMV forward, 5 -CTTTGTAGGGAGTGAACGCTGTA-3 and CMV reverse, 5 -AGATGGCGGCAACGGATA-3 ; and 18S rRNA forward, 5 -ATACCGTCCTAGTCTCAACC-3 and 18S rRNA reverse, 5 -ACAAATCGCTCCACCAAC-3 ) to simultaneously detect LMoV, LSV, CMV, and the lily 18S rRNA housekeeping gene (as an internal control). Takara Ex taq (Takara, Japan) DNA polymerase was used in amplification (94 ◦ C for 30 s, 52.5 ◦ C for 45 s, and 72 ◦ C 1 min, 30 cycles). The quadruplex RT-PCR products
were checked by electrophoresis on a 2.0% agarose gel and the sizes of the gene fragments were 395, 198, 248, and 303 bp, respectively. 2.6. Detection of the virus in field samples We collected 3–4 leaf samples per plant from 118 plants, for a total of 118 samples of oriental lily from fields at the Gaolan Ecological and Agricultural Integrated Experiment Station (Gaolan County, Lanzhou, Gansu Province, 36◦ 13 N, 103◦ 47 E), Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences. We placed 500-mg leaf samples from each plant in a sterile grinder and homogenized the tissue with 2.5 mL of virus extraction buffer to permit detection of LMoV. The homogenate was then centrifuged at 1500 × g for 5 min. The liquid supernatants were collected for use as the detection samples, and were tested by adding 100 L of the supernatant to the plastic cassette sample window of ICS. In addition, each leaf sample was tested using quadruplex RT-PCR, as described above. We then compared the ICS results with the results of the quadruplex RT-PCR assay. 3. Results 3.1. Identification of the colloidal gold particles The colloidal gold solution was deep red and had high light transmission. Fig. 2A shows the UV/Vis spectrum of the colloidal gold between 400 and 600 nm. A peak at about 529 nm resulted from surface resonance of the colloidal gold particles. A TEM image shows that the colloidal gold particles were almost of the same diameter, which provided the best basis for preparation of the ICS (Fig. 2B). 3.2. Specificity and sensitivity of the ICS To determine the specificity of the ICS, we tested it with LMoV and two other common lily viruses (LSV, CMV). The samples from healthy leaves and leaves infected with LSV or CMV produced one strong band in the control line, whereas LMoV produced an additional band in the test line in the ICS (Fig. 3A). All of the samples were also tested by means of quadruplex RT-PCR, which confirmed the ICS results (Fig. 3B). Thus, the ICS has high specificity for LMoV. We determined the sensitivity of the ICS by assaying serial dilutions of purified products of LMoV, ranging from 8.0 × 10−2 mg/mL to 8.0 × 10−10 mg/mL. Western blotting revealed that the purified native CP of LMoV (30 kDa) bound to the IgG3 protein (Fig. 4). All
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Fig. 4. Western blot analysis of the purified LMoV using IgG3 antibodies: M, protein markers; blank lane, negative control. The 30 kDa fragment is the LMoV target protein.
Fig. 3. (A) Specificity of LMoV detection by the ICS and (B) amplification by means of quadruplex RT-PCR for healthy leaves and for leaf samples infected with LSV, CMV, or LMoV. Lane M, DL600 marker; lane 1, negative control; lane 2, healthy control; lane 3, LSV; lane 4, CMV; lane 5, LMoV; lane 6, positive plasmids control (LSV + CMV + 18S rRNA + LMoV).
dilutions were tested by means of ICS and quadruplex RT-PCR. The diluted samples revealed a detection limit of approximately 8.0 × 10−9 mg/mL for ICS and quadruplex RT-PCR (Fig. 5A and B). The results of the quantification test showed that the ICS was
capable of detecting very small concentrations of the virus, at the same detection limit as in the quadruplex assay. 3.3. Experimental detection of LMoV in field samples The 118 field samples were analyzed using the quadruplex RTPCR assay and the ICS. Of the 118 samples, 41 positives and 77 negatives were obtained with the ICS, versus 40 positives and 78 negatives by means of quadruplex RT-PCR (Table 1). Thus, compared with the quadruplex RT-PCR assay, the ICS test had a specificity of 98.7%, and a sensitivity of 100%. There was excellent
Fig. 5. Comparison of the sensitivities of the ICS test and the quadruplex RT-PCR assay. (A) 8.0 × 10−2 mg/mL LMoV was serially diluted to 8.0 × 10−10 mg/mL; LMoV could be detected to 8.0 × 10−9 mg/mL by the ICS test. (B) The quadruplex RT-PCR assay could also detect LMoV at 8.0 × 10−9 mg/mL: lanes 2 to 10 represent serial dilutions from 8.0 × 10−2 mg/mL to 8.0 × 10−10 mg/mL LMoV; M, DL600 marker; lane 1, negative control; lane 11, healthy control; lane 12, positive plasmids control (LSV + CMV + 18S rRNA + LMoV).
Y. Zhang et al. / Journal of Virological Methods 220 (2015) 43–48 Table 1 Results of LMoV detection in field samples using the ICS and quadruplex RT-PCR. RT-PCR
ICS Positive Negative Total
Positive
Negative
Total
40 0 40
1 77 78
41 77 118
Mixed infections have been reported in lily plants (Wang et al., 2010; Zhang et al., 2014). For the field samples in Table 1, 71 are healthy, 42 are infected with single LMoV, LSV or CMV, and 5 are mixed infections with LMoV + CMV or CMV + LSV. Therefore, the samples including healthy, single LSV or CMV infections, or mixed infections (CMV + LSV) were included in the LMoV negative group. Similarly, the samples infected by single LMoV or mixed (LMoV + CMV) were included in the LMoV positive group. Compared with the quadruplex RT-PCR: Specificity of the ICS test: 77/(1 + 77) × 100% = 98.7%. Sensitivity of the ICS test: 40/(0 + 40) × 100% = 100%. Po = (40 + 77)/118 = 0.992. Pe = (40/118) × (41/118) + (78/118) × (77/118) = 0.549. = (Po – Pe )/(1 – Pe ) = 0.982.
agreement ( = 0.982) between the ICS results and the quadruplex RT-PCR results. 4. Discussion LMoV occurs frequently in many Lilium species, but most virus researchers have focused on laboratory diagnostic techniques such as RT-PCR, real-time PCR, and ELISA, which are very useful in larger laboratories with appropriate equipment but are not suitable for field use outside the laboratory because of the requirement for skilled technicians or special equipment. Therefore, these methods would not be practical for detecting and rapidly manage an outbreak of a viral disease in the field. It is necessary to develop a convenient and rapid detection method for common lily viruses. The present study describes a new approach to detect LMoV, based on a technique that has been used to detect many other antigens and virus-specific antibodies. The ICS test could detect the virus at 8.0 × 10−9 mg/mL, which was as sensitive as the quadruplex RT-PCR assay; compared with the latter, which was used as a reference test, the ICS test had a very high specificity (98.7%) and sensitivity (100%). There was significant agreement between the results obtained by quadruplex RT-PCR and the ICS tests ( = 0.982) with the field samples. The sensitivity and specificity of the ICS depend strongly on the antibodies used in the test strip. We used IgG3 conjugated with colloidal gold as the detector antibody and IgG4 as capture antibody in the test line. Both are core reagents, and they determine the specificity and sensitivity of the ICS strip. In previous research, we used the CP and CI of LMoV to construct a recombinant protein (CP-CI200), and produced the first antibody (Tong et al., 2010). We chose these proteins because the CP and CI of a potyvirus are involved in intercellular movement of virions (Rodríguez-Cerezo et al., 1997), and their abundance in infected cytoplasm is relatively high. Therefore, these are effective antigens to produce antibodies. Furthermore, western blotting analysis showed that IgG3 specifically reacted with the viral CP and CI of LMoV (Tong et al., 2010). CI plays a complementary role in the reaction between viral antigens and polyclonal antibodies, and this may explain the 100% sensitivity of the ICS test compared with quadruplex RT-PCR. Additionally, to avoid the competing reaction with the first antibody, we used the second antibody (IgG4 ) with high specificity as the capture antibody, which may bind to different epitopes of LMoV antigen. This may contribute to the higher specificity of ICS test for LMoV. In addition, the colloidal gold conjugate, the sample and
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the conjugated pads should be pretreated appropriately because these components are also crucial for the development of an ICS to detect LMoV. Furthermore, lily pigments in the solution from the leaf samples may potentially interfere with release of the colloidal gold-antibody conjugates and protein-binding capacity of the membrane if they migrated into the conjugate release pad and the nitrocellulose membrane. Reduced protein-binding capacity will lead to a reduction in sensitivity, so the selected sample pad must be able to effectively filter out these pigments. A special cellulosebased pad will be suitable for this purpose. In conclusion, the ICS assay is a useful method for detecting LMoV in both field and laboratory samples. The ICS was highly specific and sensitive, and was able to detect LMoV at low concentrations. This assay has the advantage over other techniques that it can be completed within 10 min without the need for special instruments or skilled personnel, and can be used virtually anywhere. These benefits would contribute to detection and control of this viral disease in China. Based on these promising results, the ICS has been adapted for monitoring LMoV in field samples from lily production areas in China’s Gansu Province and Ningxia Hui Autonomous Region. To the best of our knowledge, this is the first description of a practical ICS test to detect LMoV infection. Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant No. 31201651), by the “West Light” project of the Chinese Academy of Sciences in 2014, by the Gansu Agricultural Biotechnology Research and Development Projects (Grants Nos. GNSW-2011-05 and GNSW-2010-20), and by the Ningxia Agricultural Comprehensive Development Office (NTKJ-2014-09-1). References Alper, M.K., Lesemann, D.E., Loebenstein, G., 1982. Mechanical transmission of a strain of tulip breaking virus from Lilium longiflorum to Chenopodium spp. Phytoparasitica 10, 193–199. Alvarez, I., Gutierrez, G., Barrandeguy, M., Trono, K., 2010. Immunochromatographic lateral flow test for detection of antibodies to equine infections anemia virus. J. Virol. Methods 167, 152–157. Asjes, C.J., 2000. Control of aphid-borne Lily symptomless virus and Lily mottle virus in Lilium in the Netherlands. Virus Res. 71, 23–32. Hermanson, G.T., 2008. Bioconjugate Techniques, 2nd ed. Academic Press, San Diego, pp. 928–930. King, A.M.Q., Lefkowitz, E., Adams, M.J., Carstens, E.B., 2011. Virus Taxonomy: Classification and Nomenclature of Viruses. Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego, pp. p1075. Peng, F., Wang, Z., Zhang, S., Wu, R., Hu, S., Li, Z., Wang, X., Bi, D., 2008. Development of an immunochromatographic strip for rapid detection of H9 subtype avian influenza viruses. Clin. Vaccine Immunol. 15, 569–574. Rodríguez-Cerezo, E., Findlay, K., Shaw, J.G., Lomonossoff, G.P., Qiu, S.G., Linstead, P., Shanks, M., Risco, C., 1997. The coat and cylindrical inclusion proteins of a potyvirus are associated with connections between plant cells. Virology 236, 296–306. Ryu, K.H., Park, H.W., Choi, J.K., 2002. Characterization and sequence analysis of a lily isolate of cucumber mosaic virus from Lilium tsingtauense. Plant Pathol J. 18 (2), 85–92. Sharma, A., Mahinghara, B.K., Singh, A.K., Kulshrestha, S., Raikhy, G., Singh, L., Verma, N., Hallan, V., Ram, R., Zaidi, A.A., 2005. Identification, detection and frequency of lily viruses in northern India. Sci. Hortic. 106, 213–227. Sun, C., Zhao, K., Chen, K.Y., He, W.Q., Su, G.L., Sun, X.P., Wang, L., Pan, W., Zhang, W., Gao, F., Song, D.G., 2013. Development of a convenient immunochromatographic strip for the diagnosis of vesicular stomatitis virus serotype Indiana infections. J. Virol. Methods 188, 57–63. Tong, X.Z., Wang, Y.J., Xie, Z.K., Zhang, Y.B., An, L.P., Guo, Z.H., Guo, F., 2010. Production and application of antisera to expression product of CP and CI fusion gene of Lily mottle virus. Acta Phytopathol. Sinica 40 (5), 475–481 (in Chinese with English summary). Urcuqui-Inchima, S., Haenni, A., Bernardi, F., 2001. Potyvirus proteins: a wealth of functions. Virus Res. 74, 157–175. Wang, R., Wang, G., Zhao, Q., Zhu, Y., Zhan, J., Xie, Z., An, L., Wang, Y., 2010. Molecular and cytopathologic evidences for a mixed infection of multiple viruses on
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Lanzhou lily (Lilium davidii var. unicolor) in northwestern China. J. Plant Dis. Protect. 117 (4), 145–149. Wang, R.Y., Wang, J.H., Wang, Y., Xie, Z.K., An, L.Z., 2007. Comparison of two gel filtration chromatographic methods for the purification of lily symptomless virus. J. Virol. Methods 139, 125–131. Weiss, A., 1999. Concurrent engineering for lateral-flow diagnostics. IVD Technol. Arch. 11, 48–58.
Zhang, Y.B., Xie, Z.K., Kutcher, H.R., Wang, Y.J., Wang, R.Y., Guo, Z.H., 2014. Effects of lily mottle virus on photosynthetic pigment content, phenol and flavonoid concentration, and defense enzyme activity of oriental lily (Lilium auratum cv. Sorbonne). Philipp. Agric. Sci. 97 (1), 60–66. Zhang, Y.B., Xie, Z.K., Wang, Y.J., Guo, Z.H., Tong, X.Z., 2010. Simultaneous detection of two main Lanzhou lily (Lilium davidii var. unicolor) viruses by RT-PCR. J. Wuhan Bot. Res. 28 (6), 744–749 (in Chinese with English abstract).
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
A rapid immunochromatographic test to detect the lily mottle virus Yubao Zhang a , Yajun Wang a , Wanrong Yang b , Zhongkui Xie a,∗ , Ruoyu Wang a , Hadley Randal Kutcher c , Zhihong Guo a a b c
Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 730000, China Ningxia Agricultural Comprehensive Development Office, Yinchuan 750002, China College of Agriculture and Bioresources, University of Saskatchewan, Saskatoon SK, S7N 5A8, Canada
a b s t r a c t Article history: Received 13 January 2015 Received in revised form 2 April 2015 Accepted 2 April 2015 Available online 17 April 2015 Keywords: Immunochromatographic strip LMoV PCR Rapid detection
We developed a rapid immunochromatographic strip (ICS) test for lily mottle virus (LMoV). The test is based on a double-antibody sandwich format and employs two distinct anti-LMoV polyclonal antibodies (IgG3 and IgG4 ). The first antibody, IgG3 was conjugated with colloidal gold, and the second antibody, IgG4 was used as the capture antibody at the test line. The performance of the ICS test was evaluated and the results obtained were compared with a quadruplex RT-PCR assay. When serial dilutions of purified LMoV were tested, the LMoV detection limit of the ICS test was 8.0 × 10−9 mg/mL, which was in complete agreement with the results of quadruplex RT-PCR. Compared with quadruplex RT-PCR, the specificity and sensitivity of ICS were 98.7 and 100%, respectively. There was therefore significant agreement between the results obtained from the two tests ( = 0.982). The ICS test therefore appears to be broadly applicable, and will be especially useful in the field, as well as in areas without laboratory facilities, to support efforts to detect and control LMoV. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Lily (Lilium spp.) is an important economic crop in the floricultural industry, and additional value is obtained from the bulbs, which are edible and have medicinal properties. Almost all lilies are propagated vegetatively. Therefore, viruses may accumulate in bulbs from one generation to the next. Viruses cause quantitative and qualitative yield reductions of lilies around the world (Wang et al., 2010). More than 10 different viruses have been reported to infect lilies worldwide, and the lily mottle virus (LMoV) is one of the most common (Ryu et al., 2002; Zhang et al., 2014). LMoV is closely related to the tulip breaking virus (TBV; Alper et al., 1982), and is a member of the Potyvirus genus within the Potyviridae family. LMoV is flexuous, non-enveloped, and rod-shaped; it is 680–900 nm long and 11–15 nm wide (King et al., 2011). LMoV is composed of a single-stranded 9.7 kb RNA with a positive polarity, and is surrounded by about 2000 copies of the coat protein (CP). The virus encodes a polyprotein of 3095 aa with a molecular weight of
∗ Corresponding author at: Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, No 320 Dong Gang West Road, Lanzhou 730000, China. Tel.: +86 931 496 7206; fax: +86 931 827 3894. E-mail addresses: [email protected] (Y. Zhang), [email protected] (Y. Wang), [email protected] (W. Yang), [email protected] (Z. Xie), [email protected] (R. Wang), [email protected] (H.R. Kutcher), [email protected] (Z. Guo). http://dx.doi.org/10.1016/j.jviromet.2015.04.010 0166-0934/© 2015 Elsevier B.V. All rights reserved.
351.0 kDa (King et al., 2011). The polyprotein self-cleaves and form proteins of different sizes and functions, including CP and a cytoplasmic inclusion protein (CI). The CP and CI contain 274 and 635 amino acids of 30 and 70 kDa in size, respectively (Urcuqui-Inchima et al., 2001). Symptoms of LMoV may vary from vein clearing, leaf mottle, leaf mosaic, chlorotic and yellow streaking, leaf curling, and narrowing and reddish-brownish-necrotic spots, to milder forms of leaf symptoms, or plants may even be symptomless at some stages of growth in the field (Fig. 1). Diseased plants are often shorter than healthy plants and the symptoms may be more severe if plants are also infected by Lily symptomless virus (LSV) (Asjes, 2000). The most commonly used LMoV detection methods for lily samples are electron microscopy (Wang et al., 2007), enzyme-linked immunosorbent assay (ELISA; Sharma et al., 2005), reversetranscriptase polymerase chain reaction (RT-PCR; Zhang et al., 2010), and real-time PCR (Zhang et al., 2014). However, these methods are time consuming, require technical expertise and specialized laboratory equipment. Therefore, these methods are not suitable for rapid detection during the cultivation of lily, it is necessary to develop a rapid, specific, and easily performed assay to detect LMoV. The immunochromatographic assay is one possibility. This technique offers several advantages over traditional immunoassays, such as its low cost, procedural simplicity, limited requirement for special skills or expensive equipment, and rapid results (Alvarez et al., 2010). The immunochromatographic assay has been widely applied to detect hormones, bioactive molecules,
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Fig. 1. (A) Electron microscopy image of LMoV particles and (B) leaves showing mottle symptoms.
contagious human diseases, animal and plant viruses, bacteria and parasite antigens as well as specific antibodies (Weiss, 1999; Alvarez et al., 2010; Sun et al., 2013). The aim of the present study was to develop an immunochromatographic test strip (ICS) that could be used to rapidly detect LMoV. The sensitivity and specificity of the ICS were evaluated using a quadruplex RT-PCR assay as a reference test with 118 lily samples from the field. The results showed strong agreement between the two assays ( = 0.982). 2. Materials and methods
electron microscopy (TEM) measurements using a JEM-1230 transmission electron microscope (TEM; JEOL, Tokyo, Japan). To prepare colloidal gold-IgG3 , the IgG3 (180 L at 1.0 mg/mL) was added drop-wise to 10 mL of the pH-adjusted colloidal gold solution. The mixture was stirred vigorously for 30 min, and then 2.5 mL of 10% bovine serum albumin (BSA) was added to stabilize the conjugate solution. The mixture was then stirred for an additional 30 min, then was centrifuged at 10,000 × g at 4 ◦ C for 30 min, after which the gold pellets were suspended in 1 mL of dilution buffer: 20 mM Tris/HCl buffer (pH 8.2) containing 1% (w/v) BSA, 3% (w/v) sucrose, and 0.02% (w/v) sodium azide. The colloidal gold conjugate was stored at 4 ◦ C until use.
2.1. Reagents and materials 2.3. Preparation of the immunochromatographic strip (ICS) Leaves infected by LMoV, LSV or CMV served as sources of these viruses and were stored at −70 ◦ C at the Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Science (Lanzhou, China). The first antibody, IgG3 -rabbit IgG antiLMoV CP-CI200 (CPI200) and the second antibody IgG4 -rabbit IgG anti-LMoV CP were obtained following the procedure of Tong et al. (2010). Both of the antibodies were kept in aliquots of 1 mg at −20 ◦ C for one year. We purchased chloroauric acid (HAuCl4 ) and goat anti-rabbit IgG from the Sigma Company (St. Louis, MO, USA), nitrocellulose membranes (HiFlow-120) and cellulose filter from Millipore (Billerica, MA, USA), and special cellulose and absorbent papers from Jieyi Company (Shanghai, China). 2.2. Preparation of colloidal gold and a colloidal gold-IgG3 conjugate Colloidal gold particles with a mean diameter of 30 nm were prepared using the method of Hermanson (2008) in 1.4 mL of 1% trisodium citrate (w/v) which was rapidly added to 100 mL of 0.01% aqueous chloroauric acid solution (w/v) at 100 ◦ C and boiled for 5 min. As the resulting colloidal gold cooled gradually to room temperature with continuous stirring, the pH was maintained at 7.4 by adding 0.1 M potassium carbonate. Sodium azide was added to a final concentration of 0.01% (w/v) before storage at 4 ◦ C in a glass bottle covered with foil. The absorption maxima (max ) of the solutions were analyzed by means of ultraviolet/visible spectroscopy (UV/VIS) using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) to determine the approximate particle sizes, which were confirmed by transmission
The ICS included four components: a sample pad (special cellulose paper), a conjugate release pad (cellulose filter), a nitrocellulose membrane, and an absorbent pad (absorbent paper). The sample pad was treated with 20 mM phosphate buffer containing 1% (w/v) BSA, 0.5% (v/v) Tween-20, and 0.05% (w/v) sodium azide (pH 7.4), and was then dried for 2 h at 37 ◦ C. For the conjugated pad, we first impregnated the cellulose filter with 15 L colloidal gold-IgG3 and then dried the pad for 2 h at 37 ◦ C. Subsequently, we formed a control line by carefully dragging a pipette tip containing 100 L of the goat anti-rabbit IgG (1.5 mg/mL) along the long axis of the nitrocellulose membrane. This was done by leaning the tip against a ruler placed 6 mm from one end of the membrane. Immediately afterwards, we repeated this process to generate a test line using the IgG4 (1.0 mg/mL). We used the rule to maintain a 5-mm gap between the two lines. The remaining active sites on the membrane were blocked by incubation with 2% (w/v) BSA in PBS for 30 min at room temperature. The membrane was washed once with PBS, a second time with distilled water, and was then dried for at least 2 h at room temperature. Next, the sample pad, the prepared conjugated pad, the nitrocellulose membrane, and the absorbent pad were adhered to a backing plate (300 mm × 70 mm) in the proper order, as described previously (Peng et al., 2008). The assembled plate was then cut into 4-mm-wide pieces with a pair of small scissors. The resulting strips were mounted in plastic cassettes with windows over the sample pad and the nitrocellulose membrane (Jieyi Company, Ltd., Shanghai China). The cassettes were then packaged in plastic bags containing desiccant, and stored at 4 ◦ C until use.
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Fig. 2. (A) UV/Vis spectrum of the solution containing the colloidal gold particles and (B) TEM image of the particles.
2.4. The immunochromatographic assay If the sample tested contained LMoV, the LMoV reacted with the IgG3 conjugated to colloidal gold. The colloidal gold-labeled IgG3 –LMoV complex then migrated into the nitrocellulose membrane by means of capillary action and subsequently reacted with the immobilized IgG4 in the test line. Unbound colloidal goldlabeled IgG3 passed through the test line and reacted with the goat anti-rabbit IgG in the control line to form a second visible purple-red band. About 100 L of the test samples were applied to the plastic cassette sample window. The result was read between 5 and 10 min after addition of the sample. The sample was considered positive if two distinct purple-red lines appeared, one in the test region and the other in the control region; negative when no line appeared in the test region, and invalid if the control line failed to appear.
2.5. Specificity and sensitivity of the ICS test To evaluate the cross-reactivity of the ICS test strip, LMoV and two other common lily viruses (LSV, the lily symptomless virus; CMV, the cucumber mosaic virus) from strongly positive field samples, were tested with the test strip. LMoV-infected leaves were used as a positive control, and virus extraction buffer (0.2 M Tris/HCl buffer (at pH 7.5) containing 10 mM EDTA–Na2 , 0.1% (w/v) polyvinyl polypyrrolidone and 0.1% (v/v) 2-mercaptoethanol) was used as a negative control. The sensitivity of this test strip was evaluated by testing a series of 10-fold dilutions of the purified LMoV solution (at 8.0 × 10−2 mg/mL), which was analyzed as the virus antigen by means of western blotting using the IgG3 protein at the concentration of 1.2 × 10−3 mg/mL. Each dilution was then added to the ICS test strip, and the sensitivity was determined by finding the endpoint dilution. In addition, we compared the specificity and sensitivity of the ICS test strip with the results of quadruplex RT-PCR test developed by Zhang (2010). Briefly, we used four pairs of primers (LMoV forward, 5 -TGGCACCTCACCAAATGTA3 and LMoV reverse, 5 -CATCATCTGCTGTATGCCTCT-3 ; LSV forward, 5 -TATGGGCTTCCAATACAAC-3 and LSV reverse, 5 TATTCGGTTTCCAGGTT-3 ; CMV forward, 5 -CTTTGTAGGGAGTGAACGCTGTA-3 and CMV reverse, 5 -AGATGGCGGCAACGGATA-3 ; and 18S rRNA forward, 5 -ATACCGTCCTAGTCTCAACC-3 and 18S rRNA reverse, 5 -ACAAATCGCTCCACCAAC-3 ) to simultaneously detect LMoV, LSV, CMV, and the lily 18S rRNA housekeeping gene (as an internal control). Takara Ex taq (Takara, Japan) DNA polymerase was used in amplification (94 ◦ C for 30 s, 52.5 ◦ C for 45 s, and 72 ◦ C 1 min, 30 cycles). The quadruplex RT-PCR products
were checked by electrophoresis on a 2.0% agarose gel and the sizes of the gene fragments were 395, 198, 248, and 303 bp, respectively. 2.6. Detection of the virus in field samples We collected 3–4 leaf samples per plant from 118 plants, for a total of 118 samples of oriental lily from fields at the Gaolan Ecological and Agricultural Integrated Experiment Station (Gaolan County, Lanzhou, Gansu Province, 36◦ 13 N, 103◦ 47 E), Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences. We placed 500-mg leaf samples from each plant in a sterile grinder and homogenized the tissue with 2.5 mL of virus extraction buffer to permit detection of LMoV. The homogenate was then centrifuged at 1500 × g for 5 min. The liquid supernatants were collected for use as the detection samples, and were tested by adding 100 L of the supernatant to the plastic cassette sample window of ICS. In addition, each leaf sample was tested using quadruplex RT-PCR, as described above. We then compared the ICS results with the results of the quadruplex RT-PCR assay. 3. Results 3.1. Identification of the colloidal gold particles The colloidal gold solution was deep red and had high light transmission. Fig. 2A shows the UV/Vis spectrum of the colloidal gold between 400 and 600 nm. A peak at about 529 nm resulted from surface resonance of the colloidal gold particles. A TEM image shows that the colloidal gold particles were almost of the same diameter, which provided the best basis for preparation of the ICS (Fig. 2B). 3.2. Specificity and sensitivity of the ICS To determine the specificity of the ICS, we tested it with LMoV and two other common lily viruses (LSV, CMV). The samples from healthy leaves and leaves infected with LSV or CMV produced one strong band in the control line, whereas LMoV produced an additional band in the test line in the ICS (Fig. 3A). All of the samples were also tested by means of quadruplex RT-PCR, which confirmed the ICS results (Fig. 3B). Thus, the ICS has high specificity for LMoV. We determined the sensitivity of the ICS by assaying serial dilutions of purified products of LMoV, ranging from 8.0 × 10−2 mg/mL to 8.0 × 10−10 mg/mL. Western blotting revealed that the purified native CP of LMoV (30 kDa) bound to the IgG3 protein (Fig. 4). All
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Fig. 4. Western blot analysis of the purified LMoV using IgG3 antibodies: M, protein markers; blank lane, negative control. The 30 kDa fragment is the LMoV target protein.
Fig. 3. (A) Specificity of LMoV detection by the ICS and (B) amplification by means of quadruplex RT-PCR for healthy leaves and for leaf samples infected with LSV, CMV, or LMoV. Lane M, DL600 marker; lane 1, negative control; lane 2, healthy control; lane 3, LSV; lane 4, CMV; lane 5, LMoV; lane 6, positive plasmids control (LSV + CMV + 18S rRNA + LMoV).
dilutions were tested by means of ICS and quadruplex RT-PCR. The diluted samples revealed a detection limit of approximately 8.0 × 10−9 mg/mL for ICS and quadruplex RT-PCR (Fig. 5A and B). The results of the quantification test showed that the ICS was
capable of detecting very small concentrations of the virus, at the same detection limit as in the quadruplex assay. 3.3. Experimental detection of LMoV in field samples The 118 field samples were analyzed using the quadruplex RTPCR assay and the ICS. Of the 118 samples, 41 positives and 77 negatives were obtained with the ICS, versus 40 positives and 78 negatives by means of quadruplex RT-PCR (Table 1). Thus, compared with the quadruplex RT-PCR assay, the ICS test had a specificity of 98.7%, and a sensitivity of 100%. There was excellent
Fig. 5. Comparison of the sensitivities of the ICS test and the quadruplex RT-PCR assay. (A) 8.0 × 10−2 mg/mL LMoV was serially diluted to 8.0 × 10−10 mg/mL; LMoV could be detected to 8.0 × 10−9 mg/mL by the ICS test. (B) The quadruplex RT-PCR assay could also detect LMoV at 8.0 × 10−9 mg/mL: lanes 2 to 10 represent serial dilutions from 8.0 × 10−2 mg/mL to 8.0 × 10−10 mg/mL LMoV; M, DL600 marker; lane 1, negative control; lane 11, healthy control; lane 12, positive plasmids control (LSV + CMV + 18S rRNA + LMoV).
Y. Zhang et al. / Journal of Virological Methods 220 (2015) 43–48 Table 1 Results of LMoV detection in field samples using the ICS and quadruplex RT-PCR. RT-PCR
ICS Positive Negative Total
Positive
Negative
Total
40 0 40
1 77 78
41 77 118
Mixed infections have been reported in lily plants (Wang et al., 2010; Zhang et al., 2014). For the field samples in Table 1, 71 are healthy, 42 are infected with single LMoV, LSV or CMV, and 5 are mixed infections with LMoV + CMV or CMV + LSV. Therefore, the samples including healthy, single LSV or CMV infections, or mixed infections (CMV + LSV) were included in the LMoV negative group. Similarly, the samples infected by single LMoV or mixed (LMoV + CMV) were included in the LMoV positive group. Compared with the quadruplex RT-PCR: Specificity of the ICS test: 77/(1 + 77) × 100% = 98.7%. Sensitivity of the ICS test: 40/(0 + 40) × 100% = 100%. Po = (40 + 77)/118 = 0.992. Pe = (40/118) × (41/118) + (78/118) × (77/118) = 0.549. = (Po – Pe )/(1 – Pe ) = 0.982.
agreement ( = 0.982) between the ICS results and the quadruplex RT-PCR results. 4. Discussion LMoV occurs frequently in many Lilium species, but most virus researchers have focused on laboratory diagnostic techniques such as RT-PCR, real-time PCR, and ELISA, which are very useful in larger laboratories with appropriate equipment but are not suitable for field use outside the laboratory because of the requirement for skilled technicians or special equipment. Therefore, these methods would not be practical for detecting and rapidly manage an outbreak of a viral disease in the field. It is necessary to develop a convenient and rapid detection method for common lily viruses. The present study describes a new approach to detect LMoV, based on a technique that has been used to detect many other antigens and virus-specific antibodies. The ICS test could detect the virus at 8.0 × 10−9 mg/mL, which was as sensitive as the quadruplex RT-PCR assay; compared with the latter, which was used as a reference test, the ICS test had a very high specificity (98.7%) and sensitivity (100%). There was significant agreement between the results obtained by quadruplex RT-PCR and the ICS tests ( = 0.982) with the field samples. The sensitivity and specificity of the ICS depend strongly on the antibodies used in the test strip. We used IgG3 conjugated with colloidal gold as the detector antibody and IgG4 as capture antibody in the test line. Both are core reagents, and they determine the specificity and sensitivity of the ICS strip. In previous research, we used the CP and CI of LMoV to construct a recombinant protein (CP-CI200), and produced the first antibody (Tong et al., 2010). We chose these proteins because the CP and CI of a potyvirus are involved in intercellular movement of virions (Rodríguez-Cerezo et al., 1997), and their abundance in infected cytoplasm is relatively high. Therefore, these are effective antigens to produce antibodies. Furthermore, western blotting analysis showed that IgG3 specifically reacted with the viral CP and CI of LMoV (Tong et al., 2010). CI plays a complementary role in the reaction between viral antigens and polyclonal antibodies, and this may explain the 100% sensitivity of the ICS test compared with quadruplex RT-PCR. Additionally, to avoid the competing reaction with the first antibody, we used the second antibody (IgG4 ) with high specificity as the capture antibody, which may bind to different epitopes of LMoV antigen. This may contribute to the higher specificity of ICS test for LMoV. In addition, the colloidal gold conjugate, the sample and
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the conjugated pads should be pretreated appropriately because these components are also crucial for the development of an ICS to detect LMoV. Furthermore, lily pigments in the solution from the leaf samples may potentially interfere with release of the colloidal gold-antibody conjugates and protein-binding capacity of the membrane if they migrated into the conjugate release pad and the nitrocellulose membrane. Reduced protein-binding capacity will lead to a reduction in sensitivity, so the selected sample pad must be able to effectively filter out these pigments. A special cellulosebased pad will be suitable for this purpose. In conclusion, the ICS assay is a useful method for detecting LMoV in both field and laboratory samples. The ICS was highly specific and sensitive, and was able to detect LMoV at low concentrations. This assay has the advantage over other techniques that it can be completed within 10 min without the need for special instruments or skilled personnel, and can be used virtually anywhere. These benefits would contribute to detection and control of this viral disease in China. Based on these promising results, the ICS has been adapted for monitoring LMoV in field samples from lily production areas in China’s Gansu Province and Ningxia Hui Autonomous Region. To the best of our knowledge, this is the first description of a practical ICS test to detect LMoV infection. Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant No. 31201651), by the “West Light” project of the Chinese Academy of Sciences in 2014, by the Gansu Agricultural Biotechnology Research and Development Projects (Grants Nos. GNSW-2011-05 and GNSW-2010-20), and by the Ningxia Agricultural Comprehensive Development Office (NTKJ-2014-09-1). References Alper, M.K., Lesemann, D.E., Loebenstein, G., 1982. Mechanical transmission of a strain of tulip breaking virus from Lilium longiflorum to Chenopodium spp. Phytoparasitica 10, 193–199. Alvarez, I., Gutierrez, G., Barrandeguy, M., Trono, K., 2010. Immunochromatographic lateral flow test for detection of antibodies to equine infections anemia virus. J. Virol. Methods 167, 152–157. Asjes, C.J., 2000. Control of aphid-borne Lily symptomless virus and Lily mottle virus in Lilium in the Netherlands. Virus Res. 71, 23–32. Hermanson, G.T., 2008. Bioconjugate Techniques, 2nd ed. Academic Press, San Diego, pp. 928–930. King, A.M.Q., Lefkowitz, E., Adams, M.J., Carstens, E.B., 2011. Virus Taxonomy: Classification and Nomenclature of Viruses. Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego, pp. p1075. Peng, F., Wang, Z., Zhang, S., Wu, R., Hu, S., Li, Z., Wang, X., Bi, D., 2008. Development of an immunochromatographic strip for rapid detection of H9 subtype avian influenza viruses. Clin. Vaccine Immunol. 15, 569–574. Rodríguez-Cerezo, E., Findlay, K., Shaw, J.G., Lomonossoff, G.P., Qiu, S.G., Linstead, P., Shanks, M., Risco, C., 1997. The coat and cylindrical inclusion proteins of a potyvirus are associated with connections between plant cells. Virology 236, 296–306. Ryu, K.H., Park, H.W., Choi, J.K., 2002. Characterization and sequence analysis of a lily isolate of cucumber mosaic virus from Lilium tsingtauense. Plant Pathol J. 18 (2), 85–92. Sharma, A., Mahinghara, B.K., Singh, A.K., Kulshrestha, S., Raikhy, G., Singh, L., Verma, N., Hallan, V., Ram, R., Zaidi, A.A., 2005. Identification, detection and frequency of lily viruses in northern India. Sci. Hortic. 106, 213–227. Sun, C., Zhao, K., Chen, K.Y., He, W.Q., Su, G.L., Sun, X.P., Wang, L., Pan, W., Zhang, W., Gao, F., Song, D.G., 2013. Development of a convenient immunochromatographic strip for the diagnosis of vesicular stomatitis virus serotype Indiana infections. J. Virol. Methods 188, 57–63. Tong, X.Z., Wang, Y.J., Xie, Z.K., Zhang, Y.B., An, L.P., Guo, Z.H., Guo, F., 2010. Production and application of antisera to expression product of CP and CI fusion gene of Lily mottle virus. Acta Phytopathol. Sinica 40 (5), 475–481 (in Chinese with English summary). Urcuqui-Inchima, S., Haenni, A., Bernardi, F., 2001. Potyvirus proteins: a wealth of functions. Virus Res. 74, 157–175. Wang, R., Wang, G., Zhao, Q., Zhu, Y., Zhan, J., Xie, Z., An, L., Wang, Y., 2010. Molecular and cytopathologic evidences for a mixed infection of multiple viruses on
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Zhang, Y.B., Xie, Z.K., Kutcher, H.R., Wang, Y.J., Wang, R.Y., Guo, Z.H., 2014. Effects of lily mottle virus on photosynthetic pigment content, phenol and flavonoid concentration, and defense enzyme activity of oriental lily (Lilium auratum cv. Sorbonne). Philipp. Agric. Sci. 97 (1), 60–66. Zhang, Y.B., Xie, Z.K., Wang, Y.J., Guo, Z.H., Tong, X.Z., 2010. Simultaneous detection of two main Lanzhou lily (Lilium davidii var. unicolor) viruses by RT-PCR. J. Wuhan Bot. Res. 28 (6), 744–749 (in Chinese with English abstract).
