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RSMV virions were purified from RSMV-infected rice plant tissues through differential centrifugation. Numerous bacilliform virions of 300–375 nm long and 45–55 nm wide, similar to viruses in the genus Cytorhabdovirus, were found in the purified preparation under a transmission electron microscope (Fig. 1, with black arrows). Virions shorter than 300–375 nm long were also observed in the preparation (Fig. 1), indicating that many virions were broken in the purified process.
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Purified RSMV virions were used to immunize BALB/c mice through intraperitoneal injection. After four immunizations, spleen cells were isolated from immunized mice and used for hybridoma preparations. Four hybridoma cell lines secreting RSMV MAb (1D4, 4A8, 8E4 and 11F11) were generated through cell fusion, cell culture, antibody detection and then cell cloning. These four hybridoma cell lines were individually injected intraperitoneally into pristine-primed mice to produce ascites fluids. Yields of IgG in the four ascites fluids were found to range from 6.93 to 8.16 mg mL-1 (Table 1). Immunoglobulin class and subclass isoform of the four MAbs were identified as IgG1, kappa light chain (Table 1). Titers of the four MAbs in ascites were determined to be 10-7 by an indirect ELISA (Table 1).
MAb Isotype titer IgG yield (mg mL-1) 1D4 IgG1, κ chain 10-7 7.34 8E4 IgG1, κ chain 10-7 6.93 4A8 IgG1, κ chain 10-7 8.16 11F11 IgG1, κ chain 10-7 7.82 Table 1. Properties of the four anti-RSMV monoclonal antibodies.
Specificities of the four anti-RSMV MAbs were then determined by ACP-ELISA and Western blot, respectively. ACP-ELISA results indicated that all the four MAbs reacted strongly with RSMV in crude extracts from RSMV-infected rice tissues or in homogenates from RSMV viruliferous leafhoppers (Fig. 2A, 2B). No positive reaction was obtained when crude extracts from a healthy rice plant or from the RGDV-, RSV-, RBSDV- or SRBSDV-infected rice plants, or from non-viruliferous leafhoppers were used for ACP-ELISA (Fig. 2A, 2B). Western blot assays were also performed to determine the specificities of the four anti-RSMV MAbs. Results showed that all the four MAbs detected a protein band of ~60 kDa in crude extracts from RSMV-infected rice plants or RSMV viruliferous leafhoppers, but not in the crude extracts from healthy rice plants or from non-viruliferous leafhoppers (Fig. 2C, 2D). Based on the estimated molecular weight, we decided that the detected protein is the nucleocapsid protein of RSMV (Fig. 2C, 2D).
Figure 2. Specificities of the four RSMV MAbs in ACP-ELISA and Western blot. A Specificity analyses of the MAbs through ACP-ELISA. Crude extracts were prepared by grinding tissues from a healthy rice plant, or from the RSMV-, RGDV-, RSV-, RBSDV- or SRBSDV-infected rice plants in coating buffer at a 1:30 ratio (w/v, g/mL). These crude extracts were tested for RSMV infection using the four anti-RSMV MAbs diluted 1:7000 (v/v) in PBST? M. The results are shown as the mean ± standard deviation (SD) from three biological replicates. B RSMV viruliferous and non-viruliferous leafhoppers were homogenized individually in coating buffer, and tested for RSMV infection through ACP-ELISA using three biological replicates per treatment. C Specificity analyses of the four MAbs were done using crude extracts from a RSMV-infected or a healthy rice plant through Western blot. D Specificity analyses of the four MAbs were done using RSMV viruliferous and non-viruliferous leafhopper homogenates through Western blot. Lanes loaded with a protein marker are indicated. Dilutions of the four MAbs were the same as described for ACP-ELISA.
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The optimal working dilutions of the four MAbs and the AP-conjugated or the HRP-conjugated goat anti-mouse IgG secondary antibody were determined using the phalanx tests. Results from three independent phalanx tests indicated that RSMV could be reliably detected in the infected rice crude extracts and in viruliferous leafhopper homogenates with the four MAbs diluted at 1:7000 (v/v) and the AP-conjugated or the HRP-conjugated goat anti-mouse IgG diluted at 1:8000 (v/v). Cross reaction assay demonstrated that the ACP-ELISA developed in this study gave strong and specific reactions with the RSMV-infected rice plant crude extracts and RSMV viruliferous leafhopper homogenates (Fig. 2A, 2B). No positive reaction was observed when the crude extracts from the RBSDV-, SRBSDV-, RSV- or RGDV-infected or a healthy rice plant, or from a non-viruliferous leafhopper were tested. Serial two-fold dilution assays using crude extracts from a RSMV-infected or a healthy rice plant revealed that this ACP-ELISA was able to detect RSMV in infected crude extracts diluted at 1:2, 621, 440 (w/v, g/mL) using MAb 1D4, 1:5, 242, 880 using MAb 4A8, 1:10, 485, 760 using MAb 11F11 and 1:20, 971, 520 using MAb 8E4, respectively (Fig. 3A). When individual RSMV viruliferous leafhoppers were used, this assay could detect RSMV in the homogenates diluted at 1:307, 200 (i.e., one leafhopper in 307, 200 μL of PBS) using MAb 4A8, 8E4, 11F11, or 1D4 (Fig. 3B). These results indicate that these ACPELISAs are highly sensitive and specific for detections of RSMV infection in rice plant and leafhopper crude extracts.
Figure 3. Sensitivity analyses of MAbs through ACP-ELISA. A A crude extract from a RSMV-infected rice plant and a crude extract from a healthy rice plant were separately diluted in a coating buffer, starting from 1:10 to 1:83, 886, 080 (w/v). These samples were tested for RSMV infection through ACP-ELISA using the four MAbs followed by the AP-conjugated goat anti-mouse IgG second antibody. The OD405 values were read at 30 min after the addition of substrate at room temperature. The presented mean values were from three independent assays. B Homogenates from RSMV viruliferous or nonviruliferous leafhoppers were diluted from 1:600 to 1:614, 400 (one insect/μL of coating buffer) and then tested for RSMV infection as described in A.
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Based on the results of phalanx tests, we used 1:5000 diluted anti-RSMV MAbs and 1:8000 diluted AP-conjugated or HRP-conjugated goat anti-mouse IgG secondary antibody for the Dot-ELISA and Tissue print-ELISA. Crude extracts from RSMV-, RGDV-, RSV-, RBSDV- or SRBSDV-infected or healthy rice plants, or homogenates from RSMV viruliferous or non-viruliferous leafhoppers were blotted onto nitrocellulose membranes for Dot-ELISA. Results showed that strong purple color reactions were developed on the dots from the RSMV-infected rice plants (Fig. 4A). No purple color reaction was observed on the dots from the RGDV-, RSV-, RBSDV- or SRBSDV-infected rice plants or from the healthy rice plants (Fig. 4A). Same results were obtained for the RSMV viruliferous or non-viruliferous leafhopper samples (Fig. 4A). Results of sensitivity assays indicated that using Dot-ELISA, RSMV could be reliably detected in the dots made with 1:327, 680 (w/v, g/mL) diluted RSMV-infected crude extract (MAb 4A8 and 11F11), 1:163, 840 diluted crude extract (MAb 8E4) or 1:81, 920 diluted crude extract (MAb 1D4) (Fig. 4B). For RSMV viruliferous leafhoppers, the virus could be detected in the dots made with 1:163, 840 (individual leafhopper/μL) diluted homogenate (MAb 4A8), 1:81, 920 diluted homogenate (MAb 8E4) or 1:40, 960 diluted homogenate (MAb 1D4 and 11F11) (Fig. 4B, 4C).
Figure 4. Specificity and sensitivity analyses of the developed dotELISAs. A Specificity analyses of the dot-ELISAs. RSMV, RGDV, RSV, RBSDV, SRBSDV indicate rice crude extracts from the RSMV-, RGDV-, RSV-, RBSDV- or SRBSDV-infected rice plants, respectively. Samples from RSMV viruliferous or non-viruliferous leafhoppers are also indicated above the blot. MAbs used in this study are indicated on the right side of the blots. Purple color and blue color indicate RSMV positive reactions, after addition of NBT/BCIP or TMB substrate. B Sensitivity analyses of the dot-ELISAs for rice samples. Crude extracts from a healthy rice plant or a RSMV-infected rice plant were diluted separately from 1:20 to 1:1, 340, 620 in PBS prior to use. Purple color indicated a positive reaction, after addition of NBT/BCIP substrate. C Sensitivity analyses of the dot-ELISAs for leafhopper samples. Homogenates from an RSMV viruliferous or a non-viruliferous leafhopper were diluted from 1:10 to 1:163, 840 prior to use. Blue color indicated a positive reaction, after addition of TMB substrate.
To determine if Tissue print-ELISA could also be used to detect RSMV infection in rice plants, stems of healthy rice plants and the RSMV-, RGDV-, RSV-, RBSDV- or SRBSDV-infected rice plants were cut and printed individually on nitrocellulose membranes. The membranes were probed with MAbs followed by the AP-conjugated goat anti-mouse IgG secondary antibody. After addition of substrate, strong purple reactions were observed on the prints made with the RSMV-infected rice stems. No purple reaction was observed on the prints made with the RGDV-, RSV-, RBSDV- or SRBSDV-infected rice stems, or with the healthy rice stem (Fig. 5).
Figure 5. Detection of RSMV infection through the Tissue print-ELISA. Column 1 to 5 had the prints from the RSMV-, RGDV-, RSV-, RBSDVor SRBSDV-infected rice stems. Each treatment had two stem prints (up and down). Prints made with healthy rice stems were used as negative controls. The blot was probed with anti-RSMV MAbs followed by an AP-conjugated goat anti-mouse IgG secondary antibody. Purple color reactions indicated the presence of RSMV in the prints.
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Because MAb 4A8 showed the highest sensitivity for RSMV detection in above assays, we used it to detect the virus in 33 rice plants and in 16 leafhoppers collected from rice fields in 2018 through ACP-ELISA, Dot-ELISAs and Tissue print-ELISA, respectively. Those three assays took 3–4 h to get the test results, and the results from the three assays indicated that 19 of the 33 rice samples were RSMV-positive (Fig. 6A–6C). Results of ACP-ELISA and Dot-ELISA also showed that 5 of the 16 leafhoppers were RSMV-viruliferous (Fig. 7A, 7B). To validate these serological assay results, we tested the same rice and leafhopper samples by RT-PCR using RSMV P3 gene specific primers. The RT-PCR result was the same as that obtained through the serological assays. All the RSMV positive samples gave a specific PCR product band at approximately 500 bp (Fig. 6D, 7C). DNA sequencing and blast searching showed that the 24 RT-PCR products were all from the RSMV P3 gene. Sequence alignment with the known RSMV P3 gene sequences retrieved from the GenBank showed that the PCR-products of RSMV-infected samples obtained in this study shared 99% sequence similarities with the known RSMV P3 gene sequences.
Figure 6. Detection of RSMV infection in the field-collected rice samples through ACP-ELISA (A), Tissue print-ELISA (B), Dot-ELISA (C) and RT-PCR (D). Samples labeled as a1 to a5, b1 to b5, c1 to c5, d1 to d5, e1 to e5, f1 to f4, g1 to g4 were 33 field-collected rice samples. Samples labeled as f5 and g5 were from a RSMV-infected and a healthy rice plant, respectively, and were used as a positive and a negative control. MAb 4A8 was used in these serological assays. Lane M in (D) was loaded with a 1-kb DNA marker. All the RSMV positive samples gave a specific PCR product band at approximately 500 bp in (D).
Figure 7. Detection of RSMV infection in the field-collected leafhoppers through ACP-ELISA (A), Dot-ELISA (B) and RT-PCR (C). Samples labeled as a1 to a3, b1 to b3, c1 to c3, d1 to d3, e1, e2, f1 and f2 were from 16 field-collected leafhoppers. Sample labeled as e3 and f3 were from a RSMV viruliferous and a non-viruliferous leafhopper, respectively, and were used as a positive and a negative control. Lane M in (C) was loaded with a 1-kb DNA marker. All the RSMV positive samples gave a specific PCR product band at approximately 500 bp in (C).