| White spot syndrome virus (WSSV) is the most important viral pathogen that causes considerable economic damage to current aquaculture of shrimps, and it has been extensively studied since emerging. As WSSV infection is based on the interaction of the virus envelope and host cells, it became an important control strategy to figure out the mechanism of the process and then interdict it. In this paper, we produced monoclonal antibodies (MAb) specific to different WSSV envelope protein and obtained MAbs against linear epitope of VP28, VP26 and VP19 respectively. Further, we analyzed the neutralizing ability of the MAbs by in vivo and in vitro neutralization experiments and found that two MAbs had obvious neutralizing ability to WSSV. Then we detected and analyzed the correlation between VP28 and host cells with the MAbs, and confirmed the role of VP28 played in virus infection. Also, the paper analyzed the infection regulation of WSSV to host cells with WSSV MAbs using FCM, and provided clues to corresponding research. The followings are the details.(1) Production of WSSV envelope protein specific MAbs. WSSV was isolated from shrimp tissues with differential centrifugation and ultra centrifugation, then series WSSV proteins (VP31, VP28, VP26, VP19 and proteins around VP76 and VP 51) were obtained through SDS-PAGE and electro-elution. Following immunization with the purified proteins, and fusion, screening, and cloning, 3 types VP-specific MAbs were obtained, including 8 MAbs specific to VP28 (1D6,2C8,2G3,2H5,3A6,3E1,4B5,5D2), 3 MAbs specific to VP26 (2D10,3G7,3H9) and 2 MAbs specific to VP19 (2C7,2E9). Indirect immunofluoresence assay (IFA) showed that the MAbs react with native WSSV in gill slices of infected shrimp and western-blot experiments demonstrated that the 3 type Mabs bind with VP28, VP26 and VP19 of WSSV respectively. The results of isotyping analysis showed that MAb 1D6,2G3,2H5,3A6,3E1,4B5,5D2, 3G7,3H9,2C7 and 2E9 are IgG isotype, MAb 2C8 and 2D10 are IgM isotype.(2) In vivo neutralization assay of WSSV by the MAbs. The neutralizing ability of the MAbs was detected with crayfish (Cambarus proclarkii). WSSV stock prepared from naturally infected shrimp was diluted and incubated with different MAbs, then the mixture of virus and MAb were injected into crayfish and the mortality was recorded. The results showed that the death and the 100% mortality of crayfish tests injected with MAb 1D6 treated (4d, 13d) and 2G3 (3d, 12d) treated WSSV lagged behind that of positive control (injected with myeloma culture supernatant treated WSSV, 1d and 7d), tests with other MAbs showed a similar result to the control or ahead of that slightly. The experiment demonstrated that 1D6 and 2G3 could significantly delay the mortality of crayfish, partially neutralize the virus infection.(3) In vitro neutralization assay of WSSV by the MAbs. The neutralizing ability of the MAbs was further confirmed by blocking the binding of WSSV and shrimp haemocytes membrane with ELISA. Haemocyte membrane of Fenneropenaeus chinensis was purified and WSSV was labeled with DIG, the DIG-WSSV was incubated with MAb and the mixture was added to ELISA plate coated with membrane haemocyte, then the binding was detected with anti-DIG antibody. The result demonstrated that MAbs 1D6 (OD, 0.207) and 2G3 (OD, 0.235) significantly reduced the binding of WSSV with haemocytes membrane of F. chinensis (OD of control, 0.401), while OD values of the other MAbs were similar to the control. The result indicate that the MAbs inhibit WSSV infection by block the binding between virus and host cell, and Mab 1D6 and 2G3 could partilally neutralize WSSV.(4) Analyzation of correlation between VP28 and host haemocyte. F. chinensis haemocytes reacted with VP28 and detected by IFA using VP28 specific MAb, then specific green fluorescence dot were observed around the haemocytes. Haemocyte membrane of F. chinensis reacted with VP28 and detected by ELISA using VP28 specific MAb, which showed positive reaction. F. chinensis haemocytes was electrophoresed and transferred to NC membrane, and VP28 protein was lay on and detected by immunoblotting with the MAb, then the results showed that the eluted VP28 bind with 72kDa protein of haemocytes membrane. These experiments all approved that VP28 takes part in virus infection through attaching to receptor of host cells, and certain linear epitope of which might be the critical site.(5) Analyzation of the WSSV infection regulation in crayfish haemocyte. Crayfish were injected with WSSV then haemocytes were collected every 24h and detected with IFA and FCM, at the same time, accumulative mortality of the crayfish and the haemocyte density were recorded. Results showed that 1d-5d after virus injection: the mortality of crayfish was 3.3, 13.3, 16.7, 36.7, 70.0, (%); the infected ratio of haemocytes was 6.47, 6.93, 10.65, 26.08, 4.94, (%); the mean fluorescence intensity was 10.7, 10.89, 11.71, 13.77, 15.47; the haemocyte density (×106/ml) was 7.56±0.30, 5.60±0.24, 4.21±0.30, 1.45±0.26, 1.21±0.21 (uninfected was 5.34±0.22). The results indicated that in the WSSV infection process of the organism, the haemocyte density rose at first and then declined, the value was only 1/5 of that at normal; the percent of infected haemocyte rose and declined at the end as well, but the virus amount in the infected haemocytes kept rising; the organism was going to die when the infected ratio of haemocytes rose to the highest value, revealed that the WSSV infection degree of haemocyte has a close relationship to that of the organism.In conclusion, on the basis of VP-specific monoclonal antibodies production, we obtained two MAbs with partial neutralizing ability, which provide tools for the further research on blocking WSSV infection using MAbs. Moreover, the paper confirmed that the virus envelope protein VP28 was involved in WSSV infection by attaching to host cells, which offered clues for figuring out the function of WSSV envelope proteins in virus infection. The results of the paper were important to the research of WSSV infection mechanism and prevent approach.
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