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Nonstructural protein NS4 of Rice Stripe Virus plays a critical role in viral spread in the body of vector insects.

Wu W, Zheng L, Chen H, Jia D, Li F, Wei T - PLoS ONE (2014)

Bottom Line: We demonstrated that the ribonucleoprotein particles of RSV were closely associated with NS4-specific inclusions in the body of viruliferous SBPH through a direct interaction between NS4 and nucleoprotein of RSV.Moreover, the knockdown of NS4 expression due to RNA interference induced by dsRNA from NS4 gene significantly prevented the spread of RSV in the bodies of SBPHs without a significant effect on viral replication in continuous cell culture derived from SBPH.All these results suggest that the nonstructural protein NS4 of RSV plays a critical role in viral spread by the vector insects.

View Article: PubMed Central - PubMed

Affiliation: Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian, PR China ; Key Laboratory of Agricultural Biodiversity for Pest Management of China's Ministry of Education, Yunnan Agricultural University, Kunming, Yunnan, PR China.

ABSTRACT
Rice stripe virus (RSV), a tenuivirus, is transmitted by small brown planthopper (SBPH) in a persistent-propagative manner. In this study, sequential infection of RSV in the internal organs of SBPH after ingestion of virus indicated that RSV initially infected the midgut epithelium, and then progressed to the visceral muscle tissues, through which RSV spread to the entire alimentary canal. Finally, RSV spread into the salivary glands and reproductive system. During viral infection, the nonstructural protein NS4 of RSV formed cytoplasmic inclusions in various tissues of viruliferous SBPH. We demonstrated that the ribonucleoprotein particles of RSV were closely associated with NS4-specific inclusions in the body of viruliferous SBPH through a direct interaction between NS4 and nucleoprotein of RSV. Moreover, the knockdown of NS4 expression due to RNA interference induced by dsRNA from NS4 gene significantly prevented the spread of RSV in the bodies of SBPHs without a significant effect on viral replication in continuous cell culture derived from SBPH. All these results suggest that the nonstructural protein NS4 of RSV plays a critical role in viral spread by the vector insects.

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Accumulation of RNPs of RSV in reproductive system of viruliferous SBPHs.At 14 days padp, reproductive organs were labelled with RSV dye RNP-rhodamine and then examined with confocal microscopy. RNP antigens were detected in the testis, seminal vesicles and ejaculatory duct of males (panel A) and in the ovarioles and oviduct of females (panel B). Inset in panel B: enlargement of boxed area to show RNPs of RSV present in the follicular cells of ovariole. Images are shown with red fluorescence (RNP antigens) under background visualized by transmitted lights. ed, ejaculatory duct; sv, seminal vesicles; fc, follicular cells of ovariole; ov, ovariole; od, oviduct. Bars, 100 µm.
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pone-0088636-g003: Accumulation of RNPs of RSV in reproductive system of viruliferous SBPHs.At 14 days padp, reproductive organs were labelled with RSV dye RNP-rhodamine and then examined with confocal microscopy. RNP antigens were detected in the testis, seminal vesicles and ejaculatory duct of males (panel A) and in the ovarioles and oviduct of females (panel B). Inset in panel B: enlargement of boxed area to show RNPs of RSV present in the follicular cells of ovariole. Images are shown with red fluorescence (RNP antigens) under background visualized by transmitted lights. ed, ejaculatory duct; sv, seminal vesicles; fc, follicular cells of ovariole; ov, ovariole; od, oviduct. Bars, 100 µm.

Mentions: The infection route of RSV in its vector insect was examined by immunofluorescence microscopy. About 30% of SBPHs become viruliferous after a latent period of about 14 days (data not shown). Here, at 1, 3, 5, 8 and 14 days post-first access to diseased plants (padp), the internal organs from 50 SBPHs were examined with immunofluorescence microscopy. RSV was labelled with RNP-rhodamine; actin was labelled with actin dye phalloidin-FITC to show the morphology of the internal organs of SBPHs. As described previously [21], [30], the alimentary canal of SBPHs consists of the esophagus, anterior diverticulum, midgut and hindgut (Fig. 1A).The midgut epithelium of SBPHs was surrounded by visceral muscle tissues (Fig. 1B). At 1 day padp, RSV was accumulated in the midgut lumen in about 56% of SBPHs (Table 1, Fig. 2A). Three days padp, RSV was firstly observed in single midgut epithelial cell in about 14% of SBPHs (Table 1, Fig. 2B), suggesting that the midgut epithelium was the initial infection site for RSV. Five days padp, RSV had spread from the initial infection site, forming the small infection foci including 10–12 midgut epithelial cells in about 20% of SBPHs (Table 1, Fig. 2C). At this time, RSV had spread to the visceral muscle tissues surrounding the infected midgut epithelial cells (Fig. 2C). Eight days padp, RSV had spread to the entire alimentary canal in about 28% of SBPHs (Table 1, Fig. 2D, E). At this time, RSV had spread into the principal salivary glands in about 6% of SBPHs (Table 1, Fig. 2F). Fourteen days padp, RSV had extensively spread to the entire digestive system including the alimentary canal and salivary glands in about 24% of SBPHs (Table 1, Fig. 2G, H). At this time, in about 6% of female or 4% of male SBPHs, RSV was observed in the reproductive system: in the ovarioles and oviduct of females or in the testis, seminal vesicles and ejaculatory duct of males (Table 1, Fig. 3). Taken together, the sequential infection studies indicated that RSV initially infected the epithelial cells of the midgut, moved to the visceral muscles bordering the infected epithelium regions, then spread to the entire alimentary canal, and finally into the salivary glands and reproductive system.


Nonstructural protein NS4 of Rice Stripe Virus plays a critical role in viral spread in the body of vector insects.

Wu W, Zheng L, Chen H, Jia D, Li F, Wei T - PLoS ONE (2014)

Accumulation of RNPs of RSV in reproductive system of viruliferous SBPHs.At 14 days padp, reproductive organs were labelled with RSV dye RNP-rhodamine and then examined with confocal microscopy. RNP antigens were detected in the testis, seminal vesicles and ejaculatory duct of males (panel A) and in the ovarioles and oviduct of females (panel B). Inset in panel B: enlargement of boxed area to show RNPs of RSV present in the follicular cells of ovariole. Images are shown with red fluorescence (RNP antigens) under background visualized by transmitted lights. ed, ejaculatory duct; sv, seminal vesicles; fc, follicular cells of ovariole; ov, ovariole; od, oviduct. Bars, 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3921211&req=5

pone-0088636-g003: Accumulation of RNPs of RSV in reproductive system of viruliferous SBPHs.At 14 days padp, reproductive organs were labelled with RSV dye RNP-rhodamine and then examined with confocal microscopy. RNP antigens were detected in the testis, seminal vesicles and ejaculatory duct of males (panel A) and in the ovarioles and oviduct of females (panel B). Inset in panel B: enlargement of boxed area to show RNPs of RSV present in the follicular cells of ovariole. Images are shown with red fluorescence (RNP antigens) under background visualized by transmitted lights. ed, ejaculatory duct; sv, seminal vesicles; fc, follicular cells of ovariole; ov, ovariole; od, oviduct. Bars, 100 µm.
Mentions: The infection route of RSV in its vector insect was examined by immunofluorescence microscopy. About 30% of SBPHs become viruliferous after a latent period of about 14 days (data not shown). Here, at 1, 3, 5, 8 and 14 days post-first access to diseased plants (padp), the internal organs from 50 SBPHs were examined with immunofluorescence microscopy. RSV was labelled with RNP-rhodamine; actin was labelled with actin dye phalloidin-FITC to show the morphology of the internal organs of SBPHs. As described previously [21], [30], the alimentary canal of SBPHs consists of the esophagus, anterior diverticulum, midgut and hindgut (Fig. 1A).The midgut epithelium of SBPHs was surrounded by visceral muscle tissues (Fig. 1B). At 1 day padp, RSV was accumulated in the midgut lumen in about 56% of SBPHs (Table 1, Fig. 2A). Three days padp, RSV was firstly observed in single midgut epithelial cell in about 14% of SBPHs (Table 1, Fig. 2B), suggesting that the midgut epithelium was the initial infection site for RSV. Five days padp, RSV had spread from the initial infection site, forming the small infection foci including 10–12 midgut epithelial cells in about 20% of SBPHs (Table 1, Fig. 2C). At this time, RSV had spread to the visceral muscle tissues surrounding the infected midgut epithelial cells (Fig. 2C). Eight days padp, RSV had spread to the entire alimentary canal in about 28% of SBPHs (Table 1, Fig. 2D, E). At this time, RSV had spread into the principal salivary glands in about 6% of SBPHs (Table 1, Fig. 2F). Fourteen days padp, RSV had extensively spread to the entire digestive system including the alimentary canal and salivary glands in about 24% of SBPHs (Table 1, Fig. 2G, H). At this time, in about 6% of female or 4% of male SBPHs, RSV was observed in the reproductive system: in the ovarioles and oviduct of females or in the testis, seminal vesicles and ejaculatory duct of males (Table 1, Fig. 3). Taken together, the sequential infection studies indicated that RSV initially infected the epithelial cells of the midgut, moved to the visceral muscles bordering the infected epithelium regions, then spread to the entire alimentary canal, and finally into the salivary glands and reproductive system.

Bottom Line: We demonstrated that the ribonucleoprotein particles of RSV were closely associated with NS4-specific inclusions in the body of viruliferous SBPH through a direct interaction between NS4 and nucleoprotein of RSV.Moreover, the knockdown of NS4 expression due to RNA interference induced by dsRNA from NS4 gene significantly prevented the spread of RSV in the bodies of SBPHs without a significant effect on viral replication in continuous cell culture derived from SBPH.All these results suggest that the nonstructural protein NS4 of RSV plays a critical role in viral spread by the vector insects.

View Article: PubMed Central - PubMed

Affiliation: Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian, PR China ; Key Laboratory of Agricultural Biodiversity for Pest Management of China's Ministry of Education, Yunnan Agricultural University, Kunming, Yunnan, PR China.

ABSTRACT
Rice stripe virus (RSV), a tenuivirus, is transmitted by small brown planthopper (SBPH) in a persistent-propagative manner. In this study, sequential infection of RSV in the internal organs of SBPH after ingestion of virus indicated that RSV initially infected the midgut epithelium, and then progressed to the visceral muscle tissues, through which RSV spread to the entire alimentary canal. Finally, RSV spread into the salivary glands and reproductive system. During viral infection, the nonstructural protein NS4 of RSV formed cytoplasmic inclusions in various tissues of viruliferous SBPH. We demonstrated that the ribonucleoprotein particles of RSV were closely associated with NS4-specific inclusions in the body of viruliferous SBPH through a direct interaction between NS4 and nucleoprotein of RSV. Moreover, the knockdown of NS4 expression due to RNA interference induced by dsRNA from NS4 gene significantly prevented the spread of RSV in the bodies of SBPHs without a significant effect on viral replication in continuous cell culture derived from SBPH. All these results suggest that the nonstructural protein NS4 of RSV plays a critical role in viral spread by the vector insects.

Show MeSH
Related in: MedlinePlus