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Localization studies of two white spot syndrome virus structural proteins VP51 and VP76.

Wu C, Yang F - Virol. J. (2006)

Bottom Line: To gather more evidence, we developed a method based on immunofluorescence flow cytometry.The results revealed that the mean fluorescence intensity of the viral capsids group was significantly higher than that of intact virions group after incubation with anti-VP51 or anti-VP76 serum and fluorescein isothiocyanate conjugated secondary antibody.All these results indicate that VP51 and VP76 are both capsid proteins of WSSV.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, 178 Daxue Road, Xiamen, PR China. ifyrsun@yahoo.com.cn

ABSTRACT
VP51 and VP76 are two structural proteins of white spot syndrome virus (WSSV). However, there is some controversy about their localization in the virion at present. In this study, we employ multiple approaches to reevaluate the location of VP51 and VP76. Firstly, we found VP51 and VP76 presence in viral nucleocapsids fraction by Western blotting. Secondly, after the high-salt treatment of nucleocapsids, VP51 and VP76 were still exclusively present in viral capsids by Western blotting and immunoelectron microscopy, suggesting two proteins are structural components of the viral capsid. To gather more evidence, we developed a method based on immunofluorescence flow cytometry. The results revealed that the mean fluorescence intensity of the viral capsids group was significantly higher than that of intact virions group after incubation with anti-VP51 or anti-VP76 serum and fluorescein isothiocyanate conjugated secondary antibody. All these results indicate that VP51 and VP76 are both capsid proteins of WSSV.

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Comparison of mean fluorescence intensity between WSSV capsid group (high-salt treated) and virion group stained with different antiserum (in order: non-immune mouse, anti-VP28, anti-VP51, anti-VP76 and anti-VP664 serum) by immunofluorescence flow cytometry. Data are expressed as the means ± standard deviation of three independent experiments.
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Figure 4: Comparison of mean fluorescence intensity between WSSV capsid group (high-salt treated) and virion group stained with different antiserum (in order: non-immune mouse, anti-VP28, anti-VP51, anti-VP76 and anti-VP664 serum) by immunofluorescence flow cytometry. Data are expressed as the means ± standard deviation of three independent experiments.

Mentions: It is well known that FCM could be used to analyze cell surface marker by means of a fluorescent labeled specific antibody. In order to gather more evidence to make a credible conclusion, we developed a practical method based on immunofluorescence flow cytometry to study the localization of VP51 and VP76 by comparing the mean fluorescence intensity of the population of intact virions or high-salt treated nucleocapsids (capsids). This notion derives from reports that virus particles could be counted directly by FCM, such as the quantification of marine viruses [31,32] and the detection of baculovirus [33-35]. Although WSSV is small relatively in size in comparison with cells, their size (virion: ~ 275 × 120 nm; nucleocapsid: ~ 300 × 70 nm, [9]) is enough to be detected by FCM. In this experiment, WSSV virion and capsid groups were incubated with anti-VP51 or anti-VP76 serum, followed by staining with FITC-conjugated goat anti-mouse IgG. The analysis results showed that the fluorescent signal of the viral capsid group was significantly higher than the virion group (Fig. 4). To ensure reliability of data, anti-VP664 or anti-VP28 serum was conducted as primary antibody in positive control experiments. As expected, a stronger fluorescent signal was observed in the capsid group of anti-VP664 serum treatment, whereas the fluorescence intensity from virion group was low, which only corresponded to the background signal caused by pre-immune serum. In contrast, the virion group displayed much strong fluorescence intensity by using anti-VP28 serum. This data further support the conclusion from Western blotting or IEM. Based on the experiment, we considered FCM is an effective alternative technique for the localization of the structural proteins of WSSV or other large viruses due to its facility, high efficiency and sensitivity.


Localization studies of two white spot syndrome virus structural proteins VP51 and VP76.

Wu C, Yang F - Virol. J. (2006)

Comparison of mean fluorescence intensity between WSSV capsid group (high-salt treated) and virion group stained with different antiserum (in order: non-immune mouse, anti-VP28, anti-VP51, anti-VP76 and anti-VP664 serum) by immunofluorescence flow cytometry. Data are expressed as the means ± standard deviation of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1586196&req=5

Figure 4: Comparison of mean fluorescence intensity between WSSV capsid group (high-salt treated) and virion group stained with different antiserum (in order: non-immune mouse, anti-VP28, anti-VP51, anti-VP76 and anti-VP664 serum) by immunofluorescence flow cytometry. Data are expressed as the means ± standard deviation of three independent experiments.
Mentions: It is well known that FCM could be used to analyze cell surface marker by means of a fluorescent labeled specific antibody. In order to gather more evidence to make a credible conclusion, we developed a practical method based on immunofluorescence flow cytometry to study the localization of VP51 and VP76 by comparing the mean fluorescence intensity of the population of intact virions or high-salt treated nucleocapsids (capsids). This notion derives from reports that virus particles could be counted directly by FCM, such as the quantification of marine viruses [31,32] and the detection of baculovirus [33-35]. Although WSSV is small relatively in size in comparison with cells, their size (virion: ~ 275 × 120 nm; nucleocapsid: ~ 300 × 70 nm, [9]) is enough to be detected by FCM. In this experiment, WSSV virion and capsid groups were incubated with anti-VP51 or anti-VP76 serum, followed by staining with FITC-conjugated goat anti-mouse IgG. The analysis results showed that the fluorescent signal of the viral capsid group was significantly higher than the virion group (Fig. 4). To ensure reliability of data, anti-VP664 or anti-VP28 serum was conducted as primary antibody in positive control experiments. As expected, a stronger fluorescent signal was observed in the capsid group of anti-VP664 serum treatment, whereas the fluorescence intensity from virion group was low, which only corresponded to the background signal caused by pre-immune serum. In contrast, the virion group displayed much strong fluorescence intensity by using anti-VP28 serum. This data further support the conclusion from Western blotting or IEM. Based on the experiment, we considered FCM is an effective alternative technique for the localization of the structural proteins of WSSV or other large viruses due to its facility, high efficiency and sensitivity.

Bottom Line: To gather more evidence, we developed a method based on immunofluorescence flow cytometry.The results revealed that the mean fluorescence intensity of the viral capsids group was significantly higher than that of intact virions group after incubation with anti-VP51 or anti-VP76 serum and fluorescein isothiocyanate conjugated secondary antibody.All these results indicate that VP51 and VP76 are both capsid proteins of WSSV.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, 178 Daxue Road, Xiamen, PR China. ifyrsun@yahoo.com.cn

ABSTRACT
VP51 and VP76 are two structural proteins of white spot syndrome virus (WSSV). However, there is some controversy about their localization in the virion at present. In this study, we employ multiple approaches to reevaluate the location of VP51 and VP76. Firstly, we found VP51 and VP76 presence in viral nucleocapsids fraction by Western blotting. Secondly, after the high-salt treatment of nucleocapsids, VP51 and VP76 were still exclusively present in viral capsids by Western blotting and immunoelectron microscopy, suggesting two proteins are structural components of the viral capsid. To gather more evidence, we developed a method based on immunofluorescence flow cytometry. The results revealed that the mean fluorescence intensity of the viral capsids group was significantly higher than that of intact virions group after incubation with anti-VP51 or anti-VP76 serum and fluorescein isothiocyanate conjugated secondary antibody. All these results indicate that VP51 and VP76 are both capsid proteins of WSSV.

Show MeSH
Related in: MedlinePlus