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Inactivation of White Spot Syndrome Virus (WSSV) by normal rabbit serum: Implications for the role of the envelope protein VP28 in WSSV infection of shrimp

Wageningen University, Wageningen, Gelderland, Netherlands
Virus Research (Impact Factor: 2.83). 07/2006; 118(1-2):55-61. DOI: 10.1016/j.virusres.2005.11.011
Source: PubMed

ABSTRACT White Spot Syndrome Virus (WSSV) is a highly pathogenic and prevalent virus affecting crustacea. A number of WSSV envelope proteins, including vp28, have been proposed to be involved in viral infectivity based on the ability of specific antibodies to attenuate WSSV-induced mortality in vivo. In the present study, a series of monoclonal and polyclonal antibodies targeting vp28 were tested for their ability to neutralize WSSV infectivity, with the purpose of identifying epitopes potentially involved in vp28-mediated infection of shrimp. Surprisingly, when used as protein A-purified immunoglobulin, none of the antibodies tested were capable of inhibiting WSSV infectivity. This included one polyclonal preparation that has been previously shown to inactivate WSSV, when used as whole rabbit serum. Moreover, strong inactivation of WSSV by some rabbit sera was observed, in a manner independent of anti-vp28 antibodies. These results underscore the problems associated with using heterogeneous reagents (e.g. whole rabbit antiserum) in viral neutralization experiments aimed at defining proteins involved in infection by WSSV. In light of this, the potential of anti-vp28 antibodies to specifically neutralize WSSV should be reconsidered.

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    • "Protection conferred from WSSV neutralization assays in shrimp have led to passive immunization strategies [24e28]. Neutralization assays employing monoclonal antibodies (MAbs) has also led to identification of key epitopes involved in WSSV attachment to host cells [9] [12] [13]. Antiviral activity of neutralizing antibodies could be exploited in the development of therapeutic strategies to control white spot disease (WSD). "
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    ABSTRACT: A panel of six monoclonal antibodies (MAbs) against the major envelope proteins VP18, VP26 and VP28 of white spot syndrome virus (WSSV) was evaluated for neutralization of the virus in vivo in Penaeus monodon. WSSV stock diluted to 1×10−6 resulting in 100% mortality on 12 day post injection (dpi) was used as optimum infectious dose of virus for challenge. Constant quantity (100μg/ml) of MAbs C-5, C-14, C-33, C-38, C-56 and C-72 was incubated separately with WSSV (1×10−6 dilution) at 27°C for 90min and injected to shrimp. WSSV infection was neutralized by the MAbs C-5, C-14 and C-33 with a relative percent survival (RPS) of 60, 80 and 60 on 12dpi, respectively compared to 100% mortality in positive control injected with WSSV alone. MAbs C-38, C-56 and C-72 could neutralize WSSV infection with RPS on 12dpi of 40, 30 and 30, respectively. Shrimp injected with WSSV (1×10−6 dilution) incubated with panel of the MAbs at 100μg/ml separately were subjected to nested PCR analysis at 0, 8, 12, 24, 36, 48 and 72hour post injection (hpi) to provide further evidence for neutralization. MAbs C-5, C-14 and C-33 showed delay in WSSV positivity by 24 and 48hpi by 2nd and 1st step PCR, respectively. MAbs C-38, C-56 and C-72 showed WSSV positivity by 12 and 24hpi by 2nd and 1st step PCR, respectively. Shrimp injected with WSSV alone showed WSSV positivity by 8 and 12hpi by 2nd and 1st step PCR, respectively. The study clearly shows that infectivity of WSSV could be delayed by MAbs C-14, C-5 and C-33.
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    • "Protection conferred from WSSV neutralization assays in shrimp have led to passive immunization strategies [24e28]. Neutralization assays employing monoclonal antibodies (MAbs) has also led to identification of key epitopes involved in WSSV attachment to host cells [9] [12] [13]. Antiviral activity of neutralizing antibodies could be exploited in the development of therapeutic strategies to control white spot disease (WSD). "
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    ABSTRACT: A panel of six monoclonal antibodies (MAbs) against the major envelope proteins VP18, VP26 and VP28 of white spot syndrome virus (WSSV) was evaluated for neutralization of the virus in vivo in Penaeus monodon. WSSV stock diluted to 1 x 10⁻⁶ resulting in 100% mortality on 12 day post injection (dpi) was used as optimum infectious dose of virus for challenge. Constant quantity (100 μg/ml) of MAbs C-5, C-14, C-33, C-38, C-56 and C-72 was incubated separately with WSSV (1 x 10⁻⁶ dilution) at 27 °C for 90 min and injected to shrimp. WSSV infection was neutralized by the MAbs C-5, C-14 and C-33 with a relative percent survival (RPS) of 60, 80 and 60 on 12 dpi, respectively compared to 100% mortality in positive control injected with WSSV alone. MAbs C-38, C-56 and C-72 could neutralize WSSV infection with RPS on 12 dpi of 40, 30 and 30, respectively. Shrimp injected with WSSV (1 x 10⁻⁶ dilution) incubated with panel of the MAbs at 100 μg/ml separately were subjected to nested PCR analysis at 0, 8, 12, 24, 36, 48 and 72 hour post injection (hpi) to provide further evidence for neutralization. MAbs C-5, C-14 and C-33 showed delay in WSSV positivity by 24 and 48 hpi by 2nd and 1st step PCR, respectively. MAbs C-38, C-56 and C-72 showed WSSV positivity by 12 and 24 hpi by 2nd and 1st step PCR, respectively. Shrimp injected with WSSV alone showed WSSV positivity by 8 and 12 hpi by 2nd and 1st step PCR, respectively. The study clearly shows that infectivity of WSSV could be delayed by MAbs C-14, C-5 and C-33.
    Fish &amp Shellfish Immunology 02/2011; 30(4-5):1007-13. DOI:10.1016/j.fsi.2011.01.023 · 3.03 Impact Factor
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    • "Yi et al. [21] have reported that prokaryotic expressed VP28 could attach to host cell directly, but so sorry to tell that the result could not be repeated in our lab. Moreover, it has been reported that when immunoglobulin was purified by protein A from antiserum against VP28, neutralization activity was vanished [22]. Thus more work is necessary for further study a real role of VP28 in the viral infection. "
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    ABSTRACT: White spot syndrome virus (WSSV) is a major pathogen in shrimp aquaculture. VP28 is one of the most important envelope proteins of WSSV. In this study, a recombinant antibody library, as single-chain fragment variable (scFv) format, displayed on phage was constructed using mRNA from spleen cells of mice immunized with full-length VP28 expressed in Escherichia coli. After several rounds of panning, six scFv antibodies specifically binding to the epitopes in the N-terminal, middle, and C-terminal regions of VP28, respectively, were isolated from the library. Using these scFv antibodies as tools, the epitopes in VP28 were located on the envelope of the virion by immuno-electron microscopy. Neutralization assay with these antibodies in vitro suggested that these epitopes may not be the attachment site of WSSV to host cell receptor. This study provides a new way to investigate the structure and function of the envelope proteins of WSSV.
    Biochemical and Biophysical Research Communications 09/2008; 372(4):902-6. DOI:10.1016/j.bbrc.2008.05.152 · 2.28 Impact Factor
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