The Experts below are selected from a list of 717 Experts worldwide ranked by ideXlab platform
Sakol Panyim - One of the best experts on this subject based on the ideXlab platform.
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A multi-target dsRNA for simultaneous inhibition of Yellow Head Virus and white spot syndrome Virus in shrimp
Journal of biotechnology, 2020Co-Authors: Dam Chaimongkon, Sakol Panyim, Wanchai Assavalapsakul, Pongsopee AttasartAbstract:Abstract Outbreaks of diseases caused by Yellow Head Virus (YHV) and white spot syndrome Virus (WSSV) infection in shrimp have resulted in economic losses worldwide. DsRNA-mediated RNAi has been used to control these Viruses, and the best target genes for efficient inhibition of YHV and WSSV are the protease and ribonuleotide reductase small subunit (rr2), respectively. However, one dsRNA can suppress only one Virus, and therefore the production of multi-target dsRNA to effectively inhibit both YHV and WSSV is needed. In this study, plasmids pETpro-rr2_one stem and pETpro-rr2_two stems were constructed to produce two different forms of multi-target dsRNA in E. coli, which were designed specifically to both YHV protease and WSSV rr2 genes. The potency of each dsRNA in inhibiting YHV and WSSV and reducing shrimp death were investigated in L. vannamei. Shrimp were injected with the dsRNAs into the hemolymph before challenge with YHV or WSSV. The results showed that both dsRNAs could inhibit the Viruses, however the one stem construct was more effective than the two stems construct when shrimp were infected with WSSV. This study establishes a potential strategy for dual inhibition of YHV and WSSV for further application in shrimp aquaculture.
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Administration of co-expressed Penaeus stylirostris densoVirus-like particles and dsRNA-YHV-Pro provide protection against Yellow Head Virus in shrimp.
Journal of biotechnology, 2018Co-Authors: Rapee Sinnuengnong, Sakol Panyim, Duncan R. Smith, Pongsopee Attasart, Wanchai AssavalapsakulAbstract:Abstract The activation of the innate RNA interference pathway through double-stranded RNAs (dsRNAs) is one of the approaches to protecting shrimp from Viruses. Previous studies have shown that injection of specific dsRNAs can successfully inhibit viral infection in shrimp. However, inhibition requires high levels of dsRNA and dsRNA stability in shrimp is limited. Virus-like particles (VLPs) have been applied to deliver nucleic acids into host cells because of the protection of dsRNAs from host endonucleases as well as the target specificity provided by VLPs. Therefore, this study aimed to develop Penaeus stylirostris densoVirus (PstDNV) VLPs for dsRNA deliver to shrimp. The PstDNV capsid protein was expressed and can be self-assembled to form PstDNV VLPs. Co-expression of dsRNA-YHV-Pro and PstDNV capsid protein was achieved in the same bacterial cells, whose structure was displayed as the aggregation of VLPs by TEM. Tested for their inhibiting Yellow Head Virus (YHV) from infecting shrimp, the dsRNA-YHV-Pro-PstDNV VLPs gave higher levels of YHV suppression and a greater reduction in shrimp mortality than the delivery of naked dsRNA-YHV-Pro. Therefore, PstDNV-VLPs are a promising vehicle for dsRNA delivery that maintains the anti-Virus activity of dsRNA in shrimp over a longer period of time as compared to native dsRNAs.
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In vitro neutralization of Yellow Head Virus infection in shrimp using recombinant PmYRP65 protein
Aquaculture, 2018Co-Authors: Sitthichai Kanokudom, Sakol Panyim, Duncan R. Smith, Pongsopee Attasart, Tantikhon Prateeprat, Sittiruk Roytrakul, Wanchai AssavalapsakulAbstract:Abstract Yellow Head Virus (YHV) is a major pathogen in the Southeast Asia shrimp aquaculture industry especially in Thailand, and YHV associated mortality results in significant economic loss. Although appropriate farm management strategies can decrease the YHV infection rate in shrimp, an epidemic of the Virus remains, with no specific anti-YHV therapy. Previous reports have identified PmYRP65 as a protein that mediates YHV entry into susceptible cells, and studies using RNA interference have shown that systemic administration of double-stranded RNAs (dsRNAs) directed to PmYRP65 are able to provide protection against infection. This study sought to determine whether recombinant PmYRP65 protein (rPmYRP65) would be able to act as a competitive binding protein to neutralize YHV infection. To undertake this a cDNA encoding PmYRP65 was cloned, and rPmYRP65 was expressed, purified and characterized for its ability to neutralize YHV infection before injection into shrimp. The results showed that rPmYRP65 protein could significantly inhibit YHV infection by its receptor characteristic, through acting as a competitor to bind YHV. This study showed that rPmYRP65 has potential applications for neutralizing YHV infection of shrimp.
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Cholesterol-based cationic liposome increases dsRNA protection of Yellow Head Virus infection in Penaeus vannamei.
Journal of biotechnology, 2016Co-Authors: Poohrawind Sanitt, Sakol Panyim, Wanchai Assavalapsakul, Nuttapon Apiratikul, Nattisa Niyomtham, Boon-ek Yingyongnarongkul, Apinunt UdomkitAbstract:Abstract Protection of shrimp from Yellow Head Virus (YHV) infection has been demonstrated by injection and oral delivery of dsRNA-YHV protease gene (dsYHV) or shrimp endogenous gene (dsRab7). However, to achieve complete viral suppression and to prolong dsRNA activity, the development of an effective dsRNA delivery system is required. In this study, four cationic liposomes were synthesized and tested for their ability to increase dsRNA efficiency. The results demonstrated that entrapping dsYHV in a cholesterol-based cationic liposome gave the best protection against YHV infection when compared with other cationic lipids. The cholesterol-based cationic liposome-dsYHV (Chol-dsYHV) complex conferred YHV protection in a dose-dependent manner. Injection with Chol-dsYHV at 0.05 μg dsYHV/g shrimp could give comparable level of YHV protection to the injection with 1.25 μg naked dsYHV/g shrimp. The shrimp injected with Chol- dsYHV at 1.25 μg dsRNA/g shrimp showed only 50% mortality at 60 days post injection whereas the naked dsYHV at the same concentration gave 90% mortality. Thus, the liposome-entrapped dsYHV could lower an effective dsRNA concentration in viral protection and prolong dsRNA activity. In addition, encapsulating dsRab7 in the cholesterol-based cationic liposome could protect the dsRab7 from enzymatic digestion, and continuous feeding the shrimp with the diet formulated with the liposome-entrapped dsRab7 for 4 days in the total of 960 μg dsRab7/g shrimp could enhance YHV protection efficiency compared with the naked dsRab7. Our studies reveal that cholesterol-based cationic liposome is a promising dsRNA carrier to enhance dsRNA efficiency in both injection and oral delivery systems.
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Rab5, an early endosomal protein required for Yellow Head Virus infection of Penaeus monodon
Aquaculture, 2016Co-Authors: Pratsaneeyaporn Posiri, Sakol Panyim, Chalermporn OngvarrasoponeAbstract:Abstract Yellow Head Virus (YHV) is a virulent pathogen in black tiger shrimp. It causes high mortality within a few days after infection. In this study, colocalization between YHV and P. monodon Rab5 (PmRab5) which is a small GTPase protein was visualized at 10 min to 3 h post-YHV challenge under a confocal microscope. The result indicated that PmRab5 plays a key role in the early stage of endocytosis, and is involved in YHV trafficking process. Molecular cloning showed that the open reading frame of PmRab5 is 633 bp encoding a putative protein of 210 amino acids with an estimated molecular weight of 23 kDa. PmRab5 contained all Rab-specific signature motifs: Rab family motif (RabF), Rab subfamily motif (RabSF), G-boxes and cysteine prenylation signal. Silencing of PmRab5 by specific dsRNA reduced YHV replication inside the cells. Furthermore, lack of PmRab5 expression exhibited a delay of shrimp mortality after YHV infection. The results demonstrated that PmRab5 is involved in YHV early endosomal trafficking, suggesting its role in the early step of infection. Statement of relevance Silencing of PmRab5 may be applicable for inhibition of YHV.
Donald V. Lightner - One of the best experts on this subject based on the ideXlab platform.
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Protection from Yellow Head Virus (YHV) infection in Penaeus vannamei pre-infected with Taura syndrome Virus (TSV).
Diseases of Aquatic Organisms, 2012Co-Authors: Luis Fernando Aranguren, Kathy F.j. Tang, Donald V. LightnerAbstract:Pacific white shrimp Penaeus vannamei that were pre-exposed to Taura syndrome Virus (TSV) and then challenged with Yellow Head Virus (YHV) acquired partial protection from Yellow Head disease (YHD). Experimental infections were carried out using specific-pathogen-free (SPF) shrimp which were first exposed per os to TSV; at 27, 37 and 47 d post infection they were then challenged by injection with 1 × 104 copies of YHV per shrimp (designated the TSV-YHV group). Shrimp not infected with TSV were injected with YHV as a positive control. Survival analyses comparing the TSV-YHV and YHV (positive control) groups were conducted, and significant survival rates were found for all the time groups (p < 0.001). A higher final survival was found in the TSV-YHV group (mean 55%) than in the positive control (0%) (p < 0.05). Duplex reverse transcription quantitative PCR was used to quantify both TSV and YHV. Lower YHV copy numbers were found in the TSV-YHV group than in the positive control in pleopods (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.001) and lymphoid organ (LO) samples (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.01). In situ hybridization assays were conducted, and differences in the distribution of the 2 Viruses in the target tissues were found. The foci of LO were infected with TSV but were not infected with YHV. This study suggests that a viral interference effect exists between TSV and YHV, which could, in part, explain the absence of YHD in the Americas, where P. vannamei are often raised in farms where TSV is present.
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An improved Taura syndrome Virus (TSV) RT-PCR using newly designed primers.
Aquaculture, 2009Co-Authors: Solangel A. Navarro, Kathy F.j. Tang, Donald V. LightnerAbstract:Taura syndrome Virus (TSV)-specific primers, designated as 7171F/7511R, were designed to improve the sensitivity of RT-PCR detection. This pair of primers was shown to detect TSV isolates representative of four phylogenetic lineages: Belize, Americas, SE Asia, and Venezuela. Its detection limit was determined to be 20 copies of the TSV genome, 100 times more sensitive than the TSV primers 9992/9195 currently being used by many laboratories and recommended for surveillance and diagnostic applications by the Office International des Epizooties. Primers 7171F/7511R were found to be specific to TSV and did not to react to either Infectious hypodermal and hematopoietic necrosis Virus (IHHNV), White spot syndrome Virus (WSSV), Yellow Head Virus (YHV), or Infectious myonecrosis Virus (IMNV). This new RT-PCR method was shown to detect TSV in chronically infected Penaeus vannamei that had survived up to 236 days after exposure to TSV.
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In situ detection of Australian gill-associated Virus with a Yellow Head Virus gene probe
Aquaculture (Amsterdam Netherlands), 2002Co-Authors: Kathy F.j. Tang, Kirsten Spann, Leigh Owens, Donald V. LightnerAbstract:A digoxigenin-labeled gene probe for Yellow Head Virus (YHV) was used to detect gill-associated Virus (GAV) in Penaeus monodon from Australia via in situ hybridization. In GAV-infected shrimp, positively reacting tissues included: lymphoid organ, gills, antennal gland, and cuticular epithelium of the stomach. This demonstrates that a YHV probe can be used as a diagnostic tool for GAV and supports previous suggestions that these two Viruses are closely related.
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Frozen Commodity Shrimp: Potential Avenue for Introduction of White Spot Syndrome Virus and Yellow Head Virus
Journal of Aquatic Animal Health, 2000Co-Authors: S. V. Durand, Kathy F.j. Tang, Donald V. LightnerAbstract:Abstract Since 1992, white spot syndrome Virus (WSSV) and Yellow Head Virus (YHV) have caused mortalities in cultured shrimp throughout Asia. By 1995, WSSV was detected in Texas and South Carolina, and the Virus has also been recently reported in Central and South America (Nicaragua, Honduras, Guatemala, Panama, Colombia, Peru, and Ecuador). The importation of live infected shrimp is the principal mechanism by which exotic Viruses may be introduced to new geographic regions. However, another probable mechanism is via the importation of infected commodity shrimp from regions where the pathogens are enzootic. Ten different lots of imported frozen tails of Penaeus monodon were screened for WSSV and YHV by polymerase chain reaction (PCR) and reverse transcriptase (RT) PCR analysis. In 8 of 10 samples tested, WSSV was detected, and YHV was found in 3 out of the 10 samples. Six of the 10 sample sets of frozen shrimp gave strong positive tests for WSSV or YHV by PCR, and these were selected for bioassay with spe...
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A Yellow Head Virus gene probe: nucleotide sequence and application for in situ hybridization.
Diseases of aquatic organisms, 1999Co-Authors: Kathy F.j. Tang, Donald V. LightnerAbstract:A portion of the genome of Yellow Head Virus (YHV) of penaeid shrimp was cloned and the cDNA fragment (1161 bp) was designated clone 3-27. The fragment was labeled with digoxigenin and hybridized in situ to tissue sections of YHV-infected Penaeus vannamei. Positively reacting tissues included those of the lymphoid organ, cuticular epithelium, and gills. In addition, connective tissue of hepatopancreas, heart, antennal gland, hematopoietic organ, nerve tract, midgut cecum and muscle reacted to the probe. The probe was highly specific since it hybridized only to tissues from YHV-infected shrimp. It did not react to those of uninfected shrimp or shrimp infected with WSSV (white spot syndrome Virus), IHHNV (infectious hypodermal and hematopoietic necrosis Virus), or TSV (Taura syndrome Virus). The clone was sequenced, and primers were synthesized for rapid detection of YHV in hemolymph using RT-PCR (reverse transcription-polymerase chain reaction). The strand that constituted the viral sequence in the cDNA was also determined via RT-PCR and in situ hybridization with a single-stranded RNA (ssRNA) probe.
Timothy W. Flegel - One of the best experts on this subject based on the ideXlab platform.
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Effective suppression of Yellow Head Virus replication in Penaeus monodon hemocytes using constitutive expression vector for long-hairpin RNA (lhRNA)
Journal of invertebrate pathology, 2020Co-Authors: Phathrawarin Thedcharoen, Hoa Khanh Tran Kiem, Timothy W. Flegel, Suparat Taengchaiyaphum, Kallaya Sritunyalucksana, Yongyut Pewkliang, Lalana Nuntakarn, Vanvimon Saksmerprome, Suparerk BorwornpinyoAbstract:Abstract Double-stranded RNA (dsRNA) is employed to down-regulate the expression of specific genes of shrimp viral pathogens through the RNA interference (RNAi) pathway. The administration of dsRNA into shrimp has been shown to be an effective strategy to block Yellow Head Virus (YHV) progression. In this study, a vector (pLVX-AcGFP1-N1) was developed to introduce a long-hairpin RNA (lhRNA) silencing cassette under a CMV promoter, so-called “pLVX-lhRdRp”, against the RNA-dependent RNA polymerase (RdRp) gene of YHV. A primary culture of hemocytes isolated from Penaeus monodon was transfected with the pLVX-lhRdRp vector, generating transcripts of lhRNAs as early as 12 h post transfection. Twelve hours prior to YHV challenge, the primary hemocyte cell culture was transfected with pLVX-lhRdRp, whereas control groups were transfected with pLVX-AcGFP1-N1 or no transfection. The group treated with pLVX-lhRdRp significantly suppressed YHV replication at 24–72 h after YHV challenge. The results from RT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of YHV were effectively inhibited by the pLVX-lhRdRp vector. Thus, our hemocyte culture and dsRNA expression plasmid with constitutive promoter have potential as a platform to test DNA constructs expressing long-hairpin RNA against pathogenic viral infection and as a RNAi-based DNA vaccine in shrimp.
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A novel lectin domain-containing protein (LvCTLD) associated with response of the whiteleg shrimp Penaeus (Litopenaeus) vannamei to Yellow Head Virus (YHV)
Developmental and Comparative Immunology, 2011Co-Authors: Kingkamon Junkunlo, Timothy W. Flegel, Saengchan Senapin, Suparerk Borwornpinyo, Anuphap Prachumwat, Amornrat Tangprasittipap, Kallaya SritunyalucksanaAbstract:When using mRNA from gills of normal whiteleg shrimp Penaeus (Litopenaeus) vannamei as the tester and mRNA from Yellow Head Virus (YHV)-infected shrimp as the driver, subtractive suppression hybridization (SSH) revealed that a novel EST clone of 198 bp with a putative C-type lectin-like domain (CTLD) was downregulated in YHV-infected shrimp. The clone nucleotide sequence had 99% identity with one contig MGID1052359 (1,380 bp) reported in an EST database of P. vannamei, and the presence of this target in normal shrimp was confirmed by RT-PCR using primers designed from the MGID1052359 sequence. Analysis of the primary structure of the deduced amino acid (a.a.) sequence of the contig revealed a short portion (40 a.a. residues) at its N-terminus with high similarity to a low density lipoprotein receptor (LDLR) class A domain and another 152 a.a. residues at its C-terminus with high similarity to a C-type lectin domain. Thus, the clone was named LvCTLD and three recombinant proteins (LvCTLD, the LDLR domain and the CTLD domain) were synthesized in a bacterial system based on its sequence. An in vitro encapsulation assay revealed that Sepharose 4B beads coated with rLvCTLD were encapsulated by shrimp hemocytes and that melanization followed by 24 h post-encapsulation. The encapsulation activity of rLvCTLD was inhibited by 100 mM galactose, but not mannose or EDTA. In vivo injection of rLvCTLD or rLvCTLD plus YHV resulted in a significant elevation of PO activity in the hemolymph of the challenged shrimp when compared to shrimp injected with buffer, suggesting that rLvCTLD could activate the proPO system. An ELISA test revealed that rLvCTLD could bind to YHV particles in the presence of shrimp hemolymph. Phylogenetic analysis suggested that the LvCTLD sequence was more closely related to an antiviral gene found in Penaeus monodon (PmAV) than to other reported shrimp lectins. Taken together, we conclude that a novel shrimp LvCTLD is a host recognition molecule involved in the shrimp defense mechanism against YHV via recruitment of hemocytes, probably at the site of viral infection, and via activation of the proPO system.
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Successful propagation of shrimp Yellow Head Virus in immortal mosquito cells.
Diseases of aquatic organisms, 2010Co-Authors: Warachin Gangnonngiw, Nipaporn Kanthong, Timothy W. FlegelAbstract:Research on crustacean Viruses is hampered by the lack of continuous cell lines susceptible to them. To overcome this problem, we previously challenged immortal mosquito and lepidopteran cell lines with shrimp Yellow Head Virus (YHV), followed by serial, split-passage of whole cells, and showed that this produced cells that persistently expressed YHV antigens. To determine whether such insect cultures positive for YHV antigens could be used to infect shrimp Penaeus monodon with YHV, culture supernatants and whole-cell homogenates were used to challenge shrimp by injection. Shrimp injected with culture supernatants could not be infected. However, shrimp injection-challenged with whole-cell homogenates from Passage 5 (early-passage) of such cultures died with histological and clinical signs typical for Yellow Head disease (YHD), while homogenates of mock-passaged, YHV-challenged cells did not. By contrast, shrimp challenged with cell homogenates of late-passage cultures became infected with YHV, but survived, suggesting that YHV attenuation had occurred during its long-term serial passage in insect cells. Thus, YHV could be propagated successfully in C6/36 mosquito cells and used at low passage numbers as a source of inoculum to initiate lethal infections in shrimp. This partially solves the problem of lack of continuous shrimp cell lines for cultivation of YHV.
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Impact of Yellow Head Virus outbreaks in the whiteleg shrimp, Penaeus vannamei (Boone), in Thailand.
Journal of fish diseases, 2010Co-Authors: Saengchan Senapin, Warachin Gangnonngiw, Siriporn Sriurairatana, Y Thaowbut, Niti Chuchird, Timothy W. FlegelAbstract:Yellow Head Virus (YHV) is known as a major pathogen in the black tiger shrimp, Penaeus (Penaeus) monodon. It can also cause serious mortality in farmed whiteleg shrimp, Penaeus (Litopenaeus) vannamei. However, there is no published information on the economic and/or production impact of the disease in P. vannamei. Shrimp with gross signs of YHV disease (faded body colour and 60-70% mortality) were observed in 20 study farms rearing P. vannamei in the central part of Thailand from the end of 2007 through early 2008. The estimated economic loss for these farms according to the Thai Animal Aquaculture Association was approximately US$3 million. Detailed sequence analysis of RT-PCR amplicons from shrimp in all the study ponds revealed the presence of YHV Type 1b (YHV-1b) alone (characterized by a 162-bp deletion in the ORF3 region encoding the structural gene for gp116) and the absence of YHV Type 1a (YHV-1a), the original YHV type reported from Thailand. Despite the large 162-bp deletion (= 54 deduced amino acids) in the gp116 structural gene, histopathology of YHV-1b infections was identical to that of YHV-1a infections, and electron microscopy revealed that YHV-1b virions were morphologically indistinguishable from those previously reported for YHV-1a. In addition, an existing commercial RT-PCR detection kit and an immunochromatographic test strip for the detection of YHV were proven to have been valid tests for both YHV-1b and YHV-1a. The source of the Virus for these outbreaks was unlikely to have been the post-larvae used to stock the ponds, as they were derived from domesticated specific pathogen-free stocks free of YHV. Thus, it is possible that they originated from an unknown, natural reservoir.
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Yellow Head Virus (YHV) transmission risk from commodity shrimp is reduced to negligible levels by normal processing
Aquaculture, 2010Co-Authors: Kallaya Sritunyalucksana, Jiraporn Srisala, Watcharakorn Wangnai, Timothy W. FlegelAbstract:Abstract The aim of this study was to examine whether shrimp Yellow Head Virus (YHV) from processed shrimp tissue infected at the pre-patent disease level could be transmitted to naive shrimp in a laboratory setting. In a preliminary test, 120 YHV-free shrimp were injected intramuscularly with a virulent YHV stock to yield 5 × 10 5 (pre-patent disease level) and 2500 (carrier level) viral copies/g shrimp tissue (60 shrimp each dose). These are possible infection levels for grossly normal shrimp from normal harvest ponds (i.e., not shrimp from disease outbreak ponds). These yielded 1-step and 2-step positive (nested) RT-PCR reactions, respectively, in pleopods at 6 h post-injection of the viral stock. After being subjected to standard industrial processing conditions, only fresh frozen whole or peeled shrimp injected with pre-patent dose gave positive RT-PCR test results for YHV. None of the naive shrimp exposed to the chopped processed products for 24 h and then reared on a standard diet for 14 days showed any significant mortality or gave any positive test results for YHV using nested RT-PCR assays. Based on these preliminary test results, a large-scale test was carried out using only the high, pre-patent injection dose with 1000 fresh frozen whole shrimp. The negative control consisted of 1000 buffer-injected shrimp. A random sample of 60 shrimp from the YHV-injected group after processing, revealed 57 positive for YHV by 1-step RT-PCR assay. Of the 3 remaining, 2 were positive and 1 negative by nested RT-PCR assay. All 60 shrimp from the buffer-injected, control group were negative for YHV by nested RT-PCR assay. Exposure of these whole shrimp to naive shrimp resulted in no significant mortality and no positive RT-PCR test results for YHV by nested RT-PCR assay in the exposed naive shrimp. Our results showed that grossly normal whole shrimp processed by chilling and freezing would present negligible YHV disease transmission risks, even if they were 1-step RT-PCR positive for YHV. Thus, shrimp subjected to any additional processing steps such as peeling or cooking should present even lower transmission risks.
Witoon Tirasophon - One of the best experts on this subject based on the ideXlab platform.
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Therapeutic inhibition of Yellow Head Virus multiplication in infected shrimps by YHV-protease dsRNA
Antiviral research, 2006Co-Authors: Witoon Tirasophon, Supansa Yodmuang, Wanlop Chinnirunvong, Nongluk Plongthongkum, Sakol PanyimAbstract:Abstract Yellow Head Virus (YHV) is an invertebrate nidoVirus which causes a severe mortality in cultured Penaeus monodon. The mortality may be prevented by prior treatment of shrimps with YHV-protease dsRNA. Whether the YHV infected shrimp might be cured by the dsRNA remains to be investigated. P. monodon injected with 10−6 YHV showed a high Virus replication and mortality within 2 days. Injection of 25 μg YHV-protease dsRNA at 3, 6, 12 or 24 h post YHV infection showed a strong inhibition of YHV replication up to 12 h. Unrelated dsRNA-GFP showed no inhibition, indicating that the inhibition was nucleic acid sequence specific through RNAi pathway. Shrimp mortality could be prevented at 3 h post YHV infection by the dsRNA, but not at 24 h. These results demonstrate that YHV-protease dsRNA gives therapeutic effect and pave the way to develop a cure for YHV-infected shrimps.
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yhv protease dsrna inhibits yhv replication in penaeus monodon and prevents mortality
Biochemical and Biophysical Research Communications, 2006Co-Authors: Supansa Yodmuang, Witoon Tirasophon, Wanlop Chinnirunvong, Yaowaluck Roshorm, Sakol PanyimAbstract:Yellow Head Virus infects cultured shrimps and causes severe mortality resulting in a great economic loss. Haemolymph injection of dsRNA(pro) corresponding to the protease motif of YHV genome resulted in a complete inhibition of YHV replication. The effect of dsRNA lasted for at least 5 days. Injecting sequence-unrelated dsRNA(gfp) or dsRNA(TSV-pol) also resulted in an inhibition of YHV replication but at a comparatively much less extent. Shrimp mortality was monitored for 10 days when more than 90% shrimps receiving no dsRNA died within 8 dpi. However, those receiving dsRNA(pro) showed no mortality. A partial mortality was observed among the shrimps receiving dsRNA(gfp) or dsRNA(TSV-pol). Thus, Penaeus monodon possesses the sequence-specific protection to YHV infection, most likely through the RNAi pathway, in addition to sequence-independent protection. It gives a new notion that dsRNA induction of antiviral immunity in shrimp goes through two pathways, sequence-independent and sequence-dependent.
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Silencing of Yellow Head Virus replication in penaeid shrimp cells by dsRNA.
Biochemical and biophysical research communications, 2005Co-Authors: Witoon Tirasophon, Yaowaluck Roshorm, Sakol PanyimAbstract:RNA interference (RNAi) has been shown to inhibit viral replication in some animals and plants. Whether the RNAi is functional in shrimp remains to be demonstrated. In vitro transcribed dsRNAs of YHV helicase, polymerase, protease, gp116, and gp64 were transfected into shrimp primary cell culture and found to inhibit YHV replication. dsRNA targeted to nonstructural genes (protease, polymerase, and helicase) effectively inhibited YHV replication. Those targeted structural genes (gp116 and gp64) were the least effective. These findings are the first evidence that RNAi-mediated gene silencing is operative in shrimp cells. This could be a powerful tool for studying gene function and to develop effective control of viral infection in shrimp.
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Antiserum to the gp116 glycoprotein of Yellow Head Virus neutralizes infectivity in primary lymphoid organ cells of Penaeus monodon.
Diseases of aquatic organisms, 2005Co-Authors: Wanchai Assavalapsakul, Witoon Tirasophon, Sakol PanyimAbstract:Yellow Head Virus (YHV) is an invertebrate nidoVirus that has caused mass mortality of cultured Penaeus monodon in Asia. In this study, we investigated whether mouse polyclonal anti- serum raised against the YHV gp116 or gp64 structural glycoproteins could neutralize YHV infectiv- ity as determined using an in vitro quantal assay in primary cultures of lymphoid organ cells. Anti- gp116 antiserum showed Virus-neutralizing activity whereas anti-gp64 antiserum failed to inhibit infection. The results suggest that gp116 antiserum blocks binding of virions to cellular receptors to facilitate YHV entry into lymphoid organ cells.
Chalermporn Ongvarrasopone - One of the best experts on this subject based on the ideXlab platform.
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Rab5, an early endosomal protein required for Yellow Head Virus infection of Penaeus monodon
Aquaculture, 2016Co-Authors: Pratsaneeyaporn Posiri, Sakol Panyim, Chalermporn OngvarrasoponeAbstract:Abstract Yellow Head Virus (YHV) is a virulent pathogen in black tiger shrimp. It causes high mortality within a few days after infection. In this study, colocalization between YHV and P. monodon Rab5 (PmRab5) which is a small GTPase protein was visualized at 10 min to 3 h post-YHV challenge under a confocal microscope. The result indicated that PmRab5 plays a key role in the early stage of endocytosis, and is involved in YHV trafficking process. Molecular cloning showed that the open reading frame of PmRab5 is 633 bp encoding a putative protein of 210 amino acids with an estimated molecular weight of 23 kDa. PmRab5 contained all Rab-specific signature motifs: Rab family motif (RabF), Rab subfamily motif (RabSF), G-boxes and cysteine prenylation signal. Silencing of PmRab5 by specific dsRNA reduced YHV replication inside the cells. Furthermore, lack of PmRab5 expression exhibited a delay of shrimp mortality after YHV infection. The results demonstrated that PmRab5 is involved in YHV early endosomal trafficking, suggesting its role in the early step of infection. Statement of relevance Silencing of PmRab5 may be applicable for inhibition of YHV.
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Successful Yellow Head Virus infection of Penaeus monodon requires clathrin heavy chain.
Aquaculture (Amsterdam Netherlands), 2014Co-Authors: Pratsaneeyaporn Posiri, Ikuo Hirono, Sakol Panyim, Hidehiro Kondo, Chalermporn OngvarrasoponeAbstract:Viral disease caused by the Yellow Head Virus (YHV) had great impact on economic loss in the aquaculture industry. Prevention or curing YHV disease is still not possible due to the lack of understanding of the basic mechanisms of YHV infection. In this report, the endocytosis inhibitors (chlorpromazine (CPZ), amiloride and methyl-β-cyclodextrin (MβCD)) were used to identify the cellular entry pathway of YHV. Pretreating shrimp with CPZ but not amiloride or MβCD followed by YHV challenge resulted in a significant reduction of YHV levels, suggesting that YHV entered the shrimp cells via clathrin-mediated endocytosis. Next, the major component of the clathrin-coated vesicle, Penaeus monodon clathrin heavy chain (PmCHC) was cloned and characterized. The complete coding sequence of PmCHC is 5055 bp encoding a putative protein of 1684 amino acids. Specific silencing of PmCHC mRNA by dsRNA-PmCHC showed an inhibition of YHV replication for 48 h post YHV injection as well as exhibiting a delay in shrimp mortality. These results indicated that PmCHC was an essential component for YHV infection of shrimp cells.
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Inhibition of Taura syndrome Virus replication in Litopenaeus vannamei through silencing the LvRab7 gene using double-stranded RNA
Archives of Virology, 2011Co-Authors: Chalermporn Ongvarrasopone, Pipop Saejia, Mayuree Chanasakulniyom, Sakol PanyimAbstract:Taura syndrome Virus (TSV) is a major cause of high mortality in Pacific white shrimp ( Litopenaeus vannamei, Lv) . Previously, silencing of Penaeus monodon Rab7 (PmRab7) by injecting double-stranded RNA corresponding to PmRab7 (dsRNA-PmRab7) prevented white spot syndrome Virus or Yellow Head Virus infection. Rab7 is proposed to be involved in intracellular trafficking of the Viruses. This study aimed to investigate whether knockdown of Rab7 in L. vannamei by dsRNA-PmRab7 could inhibit replication of TSV. RNA interference (RNAi) technology using dsRNA targeting the LvRab7 gene was used to silence the mRNA expression of LvRab7. The silencing of the LvRab7 gene inhibited TSV replication dramatically when compared to groups receiving dsRNA-GFP or NaCl. This is the first demonstration that dsRNA targeting the endogenous shrimp gene LvRab7 strongly reduces TSV replication. It provides further evidence that LvRab7 is involved in the endosomal trafficking pathway of Viruses infecting penaeid shrimp.
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Suppression of PmRab7 by dsRNA Inhibits WSSV or YHV Infection in Shrimp
Marine Biotechnology, 2008Co-Authors: Chalermporn Ongvarrasopone, Mayuree Chanasakulniyom, Kallaya Sritunyalucksana, Sakol PanyimAbstract:Viral entry into host cells requires endocytosis machineries of the host for viral replication. PmRab7, a Penaeus monodon small GTPase protein, was investigated for its function in vesicular transport during viral infection. The double-stranded RNA of Rab7 was injected into a juvenile shrimp before challenging with white spot syndrome Virus (WSSV) or Yellow Head Virus (YHV). PmRab7 mRNA was specifically decreased at 48 h after dsRNA-Rab7 injection. Silencing of PmRab7 dramatically inhibited WSSV-VP28 mRNA and protein expression. Unexpectedly, the silencing of PmRab7 also inhibited YHV replication in the YHV-infected shrimp. These results suggested that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp. Because PmRab7 should function in the endosomal trafficking pathway, its silencing prevents successful viral trafficking necessary for replication. Silencing of PmRab7 could be a novel approach to prevent both DNA Virus (WSSV) and RNA Virus (YHV) infection of shrimp.
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A Simple and Cost Effective Method to Generate dsRNA for RNAi Studies in Invertebrates
2007Co-Authors: Chalermporn Ongvarrasopone, Sakol PanyimAbstract:Using an E. coli strain deficient in ribonuclease III activity, we have developed a simple and cost effective in vivo bacterial expression system to generate large amounts of double-stranded RNA (dsRNA). This method, which involves E. coli culture, RNase A treatment of lysed cells and total RNA extraction, is easier and less expensive than traditional in vitro transcription techniques. The system was validated by knocking down Yellow Head Virus replication in shrimp OKA cell cultures. Results showed that the dsRNAs prepared in vitro and in vivo possessed similar potency in inhibiting viral replication. This methodology gives an alternative means to prepare large amounts of dsRNA at low cost.
