White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacter...White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect L itopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus(WSSV+Vp), or we fi rst infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection(Vp+WSSV). The effect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of V ibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction(PCR) method. L v My D88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the different mortality rates. Our results show that(1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection;(2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed signifi cant increases in the numbers of Vibrio( P <0.05);(3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10 5 to 10 7 /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.展开更多
The peritrophic membrane plays an important role in the defense system of the arthropod gut. The digestive tract is considered one of the major tissues targeted by white spot syndrome virus （WSSV） in shrimp. In this...The peritrophic membrane plays an important role in the defense system of the arthropod gut. The digestive tract is considered one of the major tissues targeted by white spot syndrome virus （WSSV） in shrimp. In this study, the nucleotide sequence encoding peritrophin-like protein of Litopenaeus vannamei （LvPT） was amplified from a yeast two-hybrid library of L. vannamei. The epitope peptide of LvPT was predicted with the GenScript OptimumAntigenTM design tool. An anti-LvPT polyclonal antibody was produced and shown to specifically bind a band at -27 kDa, identified as LvPT. The LvPT protein was expressed and its concentration determined. LvPT dsRNA （4 pg per shrimp） was used to inhibit LvPT expression in shrimp, and a WSSV challenge experiment was then performed with reverse gavage. The pleopods, stomachs, and guts were collected from the shrimp at 0, 24, 48, and 72 h post-infection （hpi）. Viral load quantification showed that the levels of WSSV were significantly lower in the pleopods, stomachs, and guts of shrimp after LvPT dsRNA interference than in those of the controls at 48 and 72 hpi. Our results imply that LvPT plays an important role during WSSV infection of the digestive tract.展开更多
Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31(WSV340/WSSV396), an envelope protein of white spot syndrome virus(WSSV), contains...Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31(WSV340/WSSV396), an envelope protein of white spot syndrome virus(WSSV), contains an Arg-Gly-Asp(RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into p ET30a(+), expressed in Escherichia coli strain BL21 and purifi ed with immobilized metal ion affi nity chromatography. Four gill cellular proteins of shrimp( Fenneropenaeus c hinensis) were pulled down by an affi nity column coupled with recombinant VP31(r VP31), and the amino acid sequences were identifi ed with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase(AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's(r AK) binding activity with r VP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the fi rst evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.展开更多
Anti-lipopolysaccharide factors （ALFs） are basic components of the crustacean immune system that defend against a range of pathogens. The cDNA sequence of a new ALF, designated nLvALF2, with an open reading flame en...Anti-lipopolysaccharide factors （ALFs） are basic components of the crustacean immune system that defend against a range of pathogens. The cDNA sequence of a new ALF, designated nLvALF2, with an open reading flame encoding 132 amino acids was cloned. Its deduced amino acid sequence contained the conserved functional domain of ALFs, the LPS binding domain （LBD）. its genotnic sequence consisted of three exons and four introns, nLvALF2 was mainly expressed in the Oka organ and gills of shrimps. The transcriptional level of nLw4LF2 increased significantly after white spot syndrome virus （WSSV） infection, suggesting its important roles in protecting shrimps from WSSV. Single nucleotide polymorphisms （SNPs） were found in the genomic sequence of nLvALF2, of which 38 were analyzed for associations with the susceptibility/resistance of shrimps to WSSV. The loci g.2422 A〉G, g.2466 T〉C, and g.2529 G〉A were significantly associated with the resistance to WSSV （P〈0.05）. These SNP loci could be developed as markers for selection of WSSV-resistant varieties ofLitopenaeus vannamei.展开更多
White spot syndrome virus (WSSV),Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming ind...White spot syndrome virus (WSSV),Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province,China,in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations,8 farms were positive for WSSV,8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV,while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples:Bingjiang (93.3%),liuao (66.7%),Jianshan (46.7%) and Xianxiang (46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.展开更多
Recent studies showed that white spot syndrome virus(WSSV) isolates from different geographic locations share a high genetic similarity except the variable regions in ORF23/24 and ORF14/15,and variable number of tande...Recent studies showed that white spot syndrome virus(WSSV) isolates from different geographic locations share a high genetic similarity except the variable regions in ORF23/24 and ORF14/15,and variable number of tandem repeats(VNTR) within ORF94.In this study,genotyping was performed according to these three variable regions among WSSV isolates collected during 1998/1999 from Southern China.These WSSV isolates contain a deletion of 1168,5657,5898,9316 and 11093 bp,respectively in the variable region ORF23/24 compared with WSSV-TW,and a deletion of 4749 or 5622 bp in the variable region ORF14/15 relative to TH-96-II.Four types of repeat units(RUs)(6,8,9 and 13 RUs) in ORF94 were detected in these isolates,with the shortest 6 RUs as the most prevalent type.Our results provide important information for a better understanding of the spatio-temporal transmission mode and the WSSV genetic evolution lineage.展开更多
To elucidate the molecular response of shrimp hepatopancreas to white spot syndrome virus(WSSV) infection, microarray was applied to investigate the differentially expressed genes in the hepatopancreas of ‘Huanghai N...To elucidate the molecular response of shrimp hepatopancreas to white spot syndrome virus(WSSV) infection, microarray was applied to investigate the differentially expressed genes in the hepatopancreas of ‘Huanghai No. 2'(Fenneropenaeus chinensis). A total of 59137 unigenes were designed onto a custom-made 60 K Agilent chip. After infection, the gene expression profiles in the hepatopancreas of the shrimp with a lower viral load at early(48–96 h), peak(168–192 h) and late(264–288 h) infection phases were analyzed. Of 18704 differentially expressed genes, 6412 were annotated. In total, 5453 differentially expressed genes(1916 annotated) expressed at all three phases, and most of the annotated were either up-or down-regulated continuously. These genes function diversely in, for example, immune response, cytoskeletal system, signal transduction, stress resistance, protein synthesis and processing, metabolism among others. Some of the immune-related genes, including antilipopolysaccharide factor, Kazal-type proteinase inhibitor, C-type lectin and serine protease encoding genes, were up-regulated after WSSV infection. These genes have been reported to be involved in the anti-WSSV responses. The expression of genes related to the cytoskeletal system, including β-actin and myosin but without tubulin genes, were down-regulated after WSSV infection. Astakine was found for the first time in the WSSV-infected F. chinensis. To further confirm the expression of differentially expressed genes, quantitative real-time PCR was performed to test the expression of eight randomly selected genes and verified the reliability and accuracy of the microarray expression analysis. The data will provide valuable information to understanding the immune mechanism of shrimp's response to WSSV.展开更多
It is necessary to take measures against infectious diseases in the Southeast Asian prawn farming industry. In giant tiger prawn （Penaeus monodon） aquaculture, diseases caused by viruses such as white spot syndrome ...It is necessary to take measures against infectious diseases in the Southeast Asian prawn farming industry. In giant tiger prawn （Penaeus monodon） aquaculture, diseases caused by viruses such as white spot syndrome virus （WSSV） and bacteria such as Vibrio have become a menace. Appropriate treatments of prawn culture pond＇s waters are advocated for preventing and controlling pathogens. The purpose of this study was to conduct an antimicrobial water treatment using a low-voltage pulsed electric field sterilization. Here we prepared a mechanical low-voltage pulsed electric field system with copper wire coiling around a titanium ring. The viability of WSSV in seawater was examined by prawn infectivity experiments. We inoculated healthy prawns （approximately 10-15 g） with the WSSV master sample that passed 0-3 times through the system. WSSV infection in prawns decreased according to the number of passes through the system. Healthy prawns survived for〉 10 days without feeding, where as prawns inoculated with the WSSV master sample showed symptoms of white spot and died in about 4 days. Two-thirds of the prawns inoculated with the WSSV master sample that was passed once through the system developed WSSV symptoms and were polymerase chain reaction （PCR） positive. However, no symptoms were observed and prawns were PCR negative when the WSSV master sample was passed three times through the system. Based on these results, we purpose that a low-voltage pulsed electric field system will serve as an efficient pond drainage sterilization system and will replace conventional treatments using chemicals such as sodium hypochlorite.展开更多
The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21.After induction,the recombinant VP28 (rVP...The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21.After induction,the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production.It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection.Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay.Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.展开更多
The present study was conducted to investigate the effect of gamma irradiation on white spot syndrome virus （WS SV）. White spot syndrome virus is a pathogen of major economic importance in cultured penaeid shrimp in...The present study was conducted to investigate the effect of gamma irradiation on white spot syndrome virus （WS SV）. White spot syndrome virus is a pathogen of major economic importance in cultured penaeid shrimp industries. White spot disease can cause mortalities reaching 100% within 3-10 days of gross signs appearing. During the period of culture, immunostimulant agents and vaccines may provide potential methods to protect shrimps from opportunistic and pathogenic microrganisms. In this study, firstly, WSSV was isolated from infected shrimp and then multiplied in crayfish. WSSV was purified from the infected crayfish haemolymph by sucrose gradient and confirmed by transmission electron microscopy. In vivo virus titration was performed in shrimp, Penaeus semisulcatus. The LD50 of live virus stock was calculated 1054/mL. Shrimp post-larvae （1-2 g） were treated with gamma-irradiated （different doses） WSSV （10^o to 10^-4 dilutions） for a period of 10 days. The dose/survival curve for irradiated and un-irradiated WSSV was drawn; the optimum dose range for inactivation of WSSV and unaltered antigenicity was obtained 14- 15 kGy. This preliminary information suggests that shrimp appear to benefit from treatment with gamma- irradiated WSSV especially at 14-15 KGy.展开更多
基金Supported by the National Basic Research Program of China(973 Program)(No.2012CB114405)the Special Foundation Under the Construction Program for the“Taishan Scholarship”of Shandong Province of Chinathe Program for Chinese Outstanding Talents in Agricultural Scientific Research
文摘White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect L itopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus(WSSV+Vp), or we fi rst infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection(Vp+WSSV). The effect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of V ibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction(PCR) method. L v My D88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the different mortality rates. Our results show that(1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection;(2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed signifi cant increases in the numbers of Vibrio( P <0.05);(3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10 5 to 10 7 /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.
基金Supported by the National Basic Research Program of China(973 Program)(No.2012CB114403)the China Agriculture Research System-47(CARS-47)
文摘The peritrophic membrane plays an important role in the defense system of the arthropod gut. The digestive tract is considered one of the major tissues targeted by white spot syndrome virus （WSSV） in shrimp. In this study, the nucleotide sequence encoding peritrophin-like protein of Litopenaeus vannamei （LvPT） was amplified from a yeast two-hybrid library of L. vannamei. The epitope peptide of LvPT was predicted with the GenScript OptimumAntigenTM design tool. An anti-LvPT polyclonal antibody was produced and shown to specifically bind a band at -27 kDa, identified as LvPT. The LvPT protein was expressed and its concentration determined. LvPT dsRNA （4 pg per shrimp） was used to inhibit LvPT expression in shrimp, and a WSSV challenge experiment was then performed with reverse gavage. The pleopods, stomachs, and guts were collected from the shrimp at 0, 24, 48, and 72 h post-infection （hpi）. Viral load quantification showed that the levels of WSSV were significantly lower in the pleopods, stomachs, and guts of shrimp after LvPT dsRNA interference than in those of the controls at 48 and 72 hpi. Our results imply that LvPT plays an important role during WSSV infection of the digestive tract.
基金Supported by the National Science Foundation for Post-Doctoral Scientists of China(No.2013M541965)the International Postdoctoral Academic Exchange Program+2 种基金the Qingdao Postdoctoral Science Foundation Funded Projectthe Construction Program for“Taishan Scholarship”of Shandong Province of Chinathe Program for Chinese Outstanding Talents in Agricultural Scientific Research
文摘Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31(WSV340/WSSV396), an envelope protein of white spot syndrome virus(WSSV), contains an Arg-Gly-Asp(RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into p ET30a(+), expressed in Escherichia coli strain BL21 and purifi ed with immobilized metal ion affi nity chromatography. Four gill cellular proteins of shrimp( Fenneropenaeus c hinensis) were pulled down by an affi nity column coupled with recombinant VP31(r VP31), and the amino acid sequences were identifi ed with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase(AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's(r AK) binding activity with r VP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the fi rst evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.
基金Supported by the National High Technology Research and Development Program of China(863 Program)(Nos.2012AA10A404,2012AA092205)the General Program of National Natural Science Foundation of China(Nos.31272683,31072203)to Dr.LI Fuhua
文摘Anti-lipopolysaccharide factors （ALFs） are basic components of the crustacean immune system that defend against a range of pathogens. The cDNA sequence of a new ALF, designated nLvALF2, with an open reading flame encoding 132 amino acids was cloned. Its deduced amino acid sequence contained the conserved functional domain of ALFs, the LPS binding domain （LBD）. its genotnic sequence consisted of three exons and four introns, nLvALF2 was mainly expressed in the Oka organ and gills of shrimps. The transcriptional level of nLw4LF2 increased significantly after white spot syndrome virus （WSSV） infection, suggesting its important roles in protecting shrimps from WSSV. Single nucleotide polymorphisms （SNPs） were found in the genomic sequence of nLvALF2, of which 38 were analyzed for associations with the susceptibility/resistance of shrimps to WSSV. The loci g.2422 A〉G, g.2466 T〉C, and g.2529 G〉A were significantly associated with the resistance to WSSV （P〈0.05）. These SNP loci could be developed as markers for selection of WSSV-resistant varieties ofLitopenaeus vannamei.
基金State Key Program for Basic Research Grants (2006CB101801)
文摘White spot syndrome virus (WSSV),Taura syndrome virus (TSV) and Infectious hypodermal and haematopoietic necrosis virus (IHHNV) are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province,China,in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations,8 farms were positive for WSSV,8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV,while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples:Bingjiang (93.3%),liuao (66.7%),Jianshan (46.7%) and Xianxiang (46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.
基金supported by the State Key Program for Basic Research Grants (2006CB101801)
文摘Recent studies showed that white spot syndrome virus(WSSV) isolates from different geographic locations share a high genetic similarity except the variable regions in ORF23/24 and ORF14/15,and variable number of tandem repeats(VNTR) within ORF94.In this study,genotyping was performed according to these three variable regions among WSSV isolates collected during 1998/1999 from Southern China.These WSSV isolates contain a deletion of 1168,5657,5898,9316 and 11093 bp,respectively in the variable region ORF23/24 compared with WSSV-TW,and a deletion of 4749 or 5622 bp in the variable region ORF14/15 relative to TH-96-II.Four types of repeat units(RUs)(6,8,9 and 13 RUs) in ORF94 were detected in these isolates,with the shortest 6 RUs as the most prevalent type.Our results provide important information for a better understanding of the spatio-temporal transmission mode and the WSSV genetic evolution lineage.
基金the Central Public-interest Scientific Institution Basal Research Fund, CAFS (No. 2016HY-ZD04)the National Natural Science Foundation of China (No. 31372523)+2 种基金the Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02-03)the Taishan Scholar Program For Seed Industry, the Shandong Provincial Natural Science Foundation (No. ZR2014CQ001)the Special Fund for Postdoctoral Innovative Project of Shandong Province, and Central Public-Interest Scientific Institution Basal Research Fund of the Yellow Sea Fisheries Research Institute (Nos. 2060302013036 and 20603022015013)
文摘To elucidate the molecular response of shrimp hepatopancreas to white spot syndrome virus(WSSV) infection, microarray was applied to investigate the differentially expressed genes in the hepatopancreas of ‘Huanghai No. 2'(Fenneropenaeus chinensis). A total of 59137 unigenes were designed onto a custom-made 60 K Agilent chip. After infection, the gene expression profiles in the hepatopancreas of the shrimp with a lower viral load at early(48–96 h), peak(168–192 h) and late(264–288 h) infection phases were analyzed. Of 18704 differentially expressed genes, 6412 were annotated. In total, 5453 differentially expressed genes(1916 annotated) expressed at all three phases, and most of the annotated were either up-or down-regulated continuously. These genes function diversely in, for example, immune response, cytoskeletal system, signal transduction, stress resistance, protein synthesis and processing, metabolism among others. Some of the immune-related genes, including antilipopolysaccharide factor, Kazal-type proteinase inhibitor, C-type lectin and serine protease encoding genes, were up-regulated after WSSV infection. These genes have been reported to be involved in the anti-WSSV responses. The expression of genes related to the cytoskeletal system, including β-actin and myosin but without tubulin genes, were down-regulated after WSSV infection. Astakine was found for the first time in the WSSV-infected F. chinensis. To further confirm the expression of differentially expressed genes, quantitative real-time PCR was performed to test the expression of eight randomly selected genes and verified the reliability and accuracy of the microarray expression analysis. The data will provide valuable information to understanding the immune mechanism of shrimp's response to WSSV.
文摘It is necessary to take measures against infectious diseases in the Southeast Asian prawn farming industry. In giant tiger prawn （Penaeus monodon） aquaculture, diseases caused by viruses such as white spot syndrome virus （WSSV） and bacteria such as Vibrio have become a menace. Appropriate treatments of prawn culture pond＇s waters are advocated for preventing and controlling pathogens. The purpose of this study was to conduct an antimicrobial water treatment using a low-voltage pulsed electric field sterilization. Here we prepared a mechanical low-voltage pulsed electric field system with copper wire coiling around a titanium ring. The viability of WSSV in seawater was examined by prawn infectivity experiments. We inoculated healthy prawns （approximately 10-15 g） with the WSSV master sample that passed 0-3 times through the system. WSSV infection in prawns decreased according to the number of passes through the system. Healthy prawns survived for〉 10 days without feeding, where as prawns inoculated with the WSSV master sample showed symptoms of white spot and died in about 4 days. Two-thirds of the prawns inoculated with the WSSV master sample that was passed once through the system developed WSSV symptoms and were polymerase chain reaction （PCR） positive. However, no symptoms were observed and prawns were PCR negative when the WSSV master sample was passed three times through the system. Based on these results, we purpose that a low-voltage pulsed electric field system will serve as an efficient pond drainage sterilization system and will replace conventional treatments using chemicals such as sodium hypochlorite.
基金NSFC (30901116)Zhejiang Provincial Natural Science Foundation of China (Y3080212)The Planned Science and Technology Project of Zhejiang Province,China (2008C32034)
文摘The gene encoding the VP28 envelope protein of White spot syndrome virus (WSSV) was cloned into expression vector pET-30a and transformed into the Escherichia coli strain BL21.After induction,the recombinant VP28 (rVP28) protein was purified and then used to immunize Balb/c mice for monoclonal antibody (MAb) production.It was observed by immuno-electron microscopy the MAbs specific to rVP28 could recognize native VP28 target epitopes of WSSV and dot-blot analysis was used to detect natural WSSV infection.Competitive PCR showed that the viral level was approximately 104 copies/mg tissue in the dilution of gill homogenate of WSSV-infected crayfish at the detection limit of dot-blot assay.Our results suggest that dot-blot analysis with anti-rVP28 MAb could rapidly and sensitively detect WSSV at the early stages of WSSV infection.
基金Supported by the Nuclear Science and Technology Research Institute,Karaj,Iran(No.A87A061,2009)
文摘The present study was conducted to investigate the effect of gamma irradiation on white spot syndrome virus （WS SV）. White spot syndrome virus is a pathogen of major economic importance in cultured penaeid shrimp industries. White spot disease can cause mortalities reaching 100% within 3-10 days of gross signs appearing. During the period of culture, immunostimulant agents and vaccines may provide potential methods to protect shrimps from opportunistic and pathogenic microrganisms. In this study, firstly, WSSV was isolated from infected shrimp and then multiplied in crayfish. WSSV was purified from the infected crayfish haemolymph by sucrose gradient and confirmed by transmission electron microscopy. In vivo virus titration was performed in shrimp, Penaeus semisulcatus. The LD50 of live virus stock was calculated 1054/mL. Shrimp post-larvae （1-2 g） were treated with gamma-irradiated （different doses） WSSV （10^o to 10^-4 dilutions） for a period of 10 days. The dose/survival curve for irradiated and un-irradiated WSSV was drawn; the optimum dose range for inactivation of WSSV and unaltered antigenicity was obtained 14- 15 kGy. This preliminary information suggests that shrimp appear to benefit from treatment with gamma- irradiated WSSV especially at 14-15 KGy.
