Publications by authors named "Xiujie Shi"

17 Publications

  • Page 1 of 1

Hirudin ameliorates diabetic nephropathy by inhibiting Gsdmd-mediated pyroptosis.

Cell Biol Toxicol 2021 Jul 2. Epub 2021 Jul 2.

Henan University of Chinese Medicine, No. 156 Jinshui Dong Road, Zhengdong New District, Zhengzhou, 450046, Henan, China.

Our group previously reported that hirudin ameliorated diabetic nephropathy (DN) in streptozotocin (STZ)-injected rats, but the mechanism remained largely unknown. Therefore, we further explored its possible mechanism. We subcutaneously injected 5 U hirudin into STZ-induced WT mice or Gasdermin D (Gsdmd) (KO) mice daily for 12 weeks, respectively, and evaluated their kidney injury. Next, glomerular endothelial cells (GECs), renal tubular epithelial cells (RTECs), and bone-marrow-derived macrophages (BMDMs) were isolated from WT mice and treated with hirudin in the presence of high glucose/lipopolysaccharides and ATP to measure the release of interleukin-18 and interleukin-1β. Kidney injury induced by STZ injection was significantly ameliorated by hirudin through inhibiting Gsdmd-mediated pyroptosis in the mice, not Caspase 1-mediated apoptosis. Meanwhile, hirudin also suppressed pyroptosis in primary GECs, RTECs, and BMDMs in vitro. Moreover, the deletion of Gsdmd reduced pyroptosis and kidney injury both in vivo and in vitro. We also found that hirudin regulated the expression of Gsdmd by inhibiting interferon regulatory factor 2 (Irf2). Hirudin ameliorated Gsdmd-mediated pyroptosis by inhibiting irf2, leading to the improvement of kidney injury. Therefore, hirudin might serve as a potential therapeutic strategy to treat DN.
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http://dx.doi.org/10.1007/s10565-021-09622-zDOI Listing
July 2021

Ameliorative role of SIRT1 in peritoneal fibrosis: an in vivo and in vitro study.

Cell Biosci 2021 Apr 27;11(1):79. Epub 2021 Apr 27.

Department of Nephropathy, The First Affiliated Hospital of Zhengzhou University, No. 1 East Jianshe Road, Zhengzhou, 450052, Henan, China.

Background: Peritoneal fibrosis is one of the major complications induced by peritoneal dialysis (PD). Damaged integrity and function of peritoneum caused by peritoneal fibrosis not only limits the curative efficacy of PD and but affects the prognosis of patients. However, the detailed mechanisms underlying the process remain unclear and therapeutic strategy targeting TGF-β is deficient. Transforming growth factor-β (TGF-β) signaling participates in the progression of peritoneal fibrosis through enhancing mesothelial-mesenchymal transition of mesothelial cells.

Methods: The study aims to demonstrate the regulatory role of Sirtuin1 (SIRT1) to the TGF-β signaling mediated peritoneal fibrosis. SIRT1 mice were used to establish animal model. Masson's staining and peritoneal equilibration assay were performed to evaluate the degree of peritoneal fibrosis. QRT-PCR assays were used to estimate the RNA levels of Sirt1 and matrix genes related to peritoneal fibrosis, and their protein levels were examined by Western blot assays.

Results: SIRT1 significantly decreased in vivo post PD treatment. SIRT1 knockout exacerbated peritoneal fibrosis both in vivo and vitro. Overexpression of SIRT1 efficiently inhibited peritoneal fibrosis by inhibiting the peritoneal inflammation and the activation of TGF-β signaling.

Conclusion: SIRT1 ameliorated peritoneal fibrosis both in vivo and in vitro through inhibiting the expression of protein matrix induced by TGF-β signaling.
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http://dx.doi.org/10.1186/s13578-021-00591-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8077771PMC
April 2021

Ginkgo Biloba Extract EGB761 Ameliorates the Extracellular Matrix Accumulation and Mesenchymal Transformation of Renal Tubules in Diabetic Kidney Disease by Inhibiting Endoplasmic Reticulum Stress.

Biomed Res Int 2021 23;2021:6657206. Epub 2021 Mar 23.

Henan University of Chinese Medicine, Zhengzhou, 450046 Henan, China.

The study is aimed at investigating the effects of Ginkgo biloba extract EGB761 on renal tubular damage and endoplasmic reticulum stress (ERS) in diabetic kidney disease (DKD). A total of 50 C57BL/6 N mice were randomly divided into the normal group, DKD group, DKD+EGB761 group (36 mg/kg), and DKD+4-phenylbutyrate (4-PBA) group (1 g/kg). The DKD model was replicated by high-fat diet combined with intraperitoneal injection of streptozotocin (STZ). Renal tubular epithelial cells (HK-2) were divided into the control group, high-glucose group (30 mmol/L), EGB761 group (40 mg/L, 20 mg/L, 10 mg/L), TM group, and TM+4-PBA group. After 8 weeks of administration, expressions of serum creatinine (Scr), blood urea nitrogen (BUN), 24 h urinary protein (24 h Pro), fasting blood glucose (FBG), -microglobulin ( -MG), and retinol binding protein 4 (RBP4) of mice were tested. The pathological changes of renal tissue were observed. The expressions of extracellular matrix (ECM) accumulation and epithelial-mesenchymal transition (EMT) markers -smooth muscle actin (-SMA), E-cadherin, fibronectin, and collagen IV, as well as the ERS markers GRP78 and ATF6, were tested by Western blot, qPCR, immunohistochemistry, or immunofluorescence. EGB761 could decrease the Scr, BUN, 24 h Pro, and FBG levels in the DKD group, alleviate renal pathological injury, decrease urine -MG, RBP4 levels, and decrease the expression of -SMA, collagen IV, fibronectin, and GRP78, as well as ATF6, while increase the expression of E-cadherin. These findings demonstrate that EGB761 can improve renal function, reduce tubular injury, and ameliorate ECM accumulation and EMT in DKD kidney tubules, and the mechanism may be related to the inhibition of ERS.
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http://dx.doi.org/10.1155/2021/6657206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009711PMC
May 2021

Tongluo Digui decoction treats renal injury in diabetic rats by promoting autophagy of podocytes.

J Tradit Chin Med 2021 02;41(1):125-132

Department of Nephropathy, The Second Hospital Affiliated to Henan University of Chinese Medicine, Zhengzhou 450002, China.

Objective: To investigate the effects of Tongluo Digui decoction on renal injury and streptozotocin-induced podocyte autophagy in diabetic rats.

Methods: Male Sprague-Dawley rats were randomly divided into six groups: normal, model, Tongluo Digui decoction (high, medium, and low dose) and valsartan. Streptozotocin was injected intraperitoneally to replicate the diabetic animal model. After 8 weeks, proteinuria was evaluated to establish the diabetic nephropathy model. Treatments were administered daily via the intragastric route. At 16 weeks after gavage, we determined 24 h urine protein concentration, and blood glucose, serum creatinine, and urea nitrogen concentrations. Then, rats were sacrificed, and kidneys were harvested and stained with periodic acid-Schiff to evaluate the pathological changes in glomeruli, including glomerular podocytes by transmission electron microscopy. Western blot analysis was used to determine the expression of nephrin, podocin, p62, beclin-1, LC3Ⅱ/Ⅰ, and p-mTOR/mTOR protein in kidney tissues.

Results: Compared with the model group, Tongluo Digui decoction was associated with decreases in 24 h urine protein concentration, and blood glucose, hemoglobin A1c, serum creatinine, urea nitrogen concentrations, total serum protein and albumin. Concurrently, mesangial mesenteric broadening and fusion of foot processes were reduced, the glomerular basement membrane was not significantly thickened, and the number of podocytes and the number of autophagosomes in the podocytes was increased. Further, expression of nephrin, podocin, LC3Ⅱ, and beclin-1 protein in kidney tissue was up-regulated, while expression of p62 protein was down-regulated and mTOR phosphorylation was inhibited.

Conclusion: Tongluo Digui decoction may inhibit the progression of diabetic nephropathy by inhibiting mTOR phosphorylation, thereby increasing autophagy to protect podocytes and reducing proteinuria.
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http://dx.doi.org/10.19852/j.cnki.jtcm.2021.01.014DOI Listing
February 2021

Hirudin Protects Against Kidney Damage in Streptozotocin-Induced Diabetic Nephropathy Rats by Inhibiting Inflammation via P38 MAPK/NF-κB Pathway.

Drug Des Devel Ther 2020 7;14:3223-3234. Epub 2020 Aug 7.

Department of Nephropathy, Henan Provincial Hospital of Traditional Chinese Medicine, Zhengzhou, Henan Province, People's Republic of China.

Background: Inflammation-induced podocyte apoptosis plays an important role in kidney injury during diabetic nephropathy (DN). Hirudin (HIR), a natural compound extracted from leeches, can inhibit inflammation. However, whether HIR can protect the kidneys against inflammation during DN is unknown. In the present study, we aimed to study the effects of HIR on kidney damage in a DN rat model and explore its anti-inflammatory properties.

Methods: A streptozotocin-induced DN rat model was generated, and HIR was administered subcutaneously. Immortal podocytes and primary peritoneal macrophages were used for vitro studies. Hematoxylin and eosin staining was used to evaluate renal pathological changes; quantitative polymerase chain reaction and immunoblotting were used to detect gene expression; and TUNEL staining was used to detect apoptotic cells.

Results: Our results showed that HIR protected against renal injury, as indicated by kidney weight/body weight, serum creatinine, renal pathological changes, blood urea nitrogen, and detection of urine proteins. Notably, HIR treatment reduced macrophage infiltration, pro-inflammatory cytokine expression, and podocyte apoptosis in the kidney tissues of DN rats. In vitro, high glucose (HG) induced the activation of M1 macrophages, which was accompanied by increased podocyte apoptosis. HIR could decrease HG-induced podocyte apoptosis and suppress pro-inflammatory cytokine expression in podocytes in vitro. This was achieved via inhibition of p38 MAPK/NF-κB activation in renal tissues and podocytes.

Conclusion: HIR could inhibit inflammation via the p38 MAPK/NF-κB pathway, prevent podocyte apoptosis, and protect against kidney damage in a DN rat model.
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http://dx.doi.org/10.2147/DDDT.S257613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425656PMC
July 2021

SIRT1-modified human umbilical cord mesenchymal stem cells ameliorate experimental peritoneal fibrosis by inhibiting the TGF-β/Smad3 pathway.

Stem Cell Res Ther 2020 08 18;11(1):362. Epub 2020 Aug 18.

Department of Nephropathy, The First Affiliated Hospital of Zhengzhou University, No. 1 East Jianshe Road, Zhengzhou, 450052, Henan, China.

Introduction: Peritoneal fibrosis is a serious complication of long-term peritoneal dialysis (PD). Combination therapies are emerging as a promising treatment for tissue damage. Here, we investigated the therapeutic potential of SIRT1-modified human umbilical cord mesenchymal stem cells (hUCMSCs) for peritoneal fibrosis.

Methods: SIRT1 was overexpressed in hUCMSCs to establish SIRT1-modified hUCMSCs. Co-culture and transplantation experiments were performed in TGF-β-stimulated Met-5A cells and peritoneal damage rodent model to assess the therapeutic potential of SIRT1-modified hUCMSCs for peritoneal fibrosis through qPCR, Western blot, and peritoneal function analyses.

Results: SIRT1-modified hUCMSC administration had more potent anti-fibrosis ability than hUCMSCs, which significantly inhibited the expression of fibrotic genes and suppressed EMT process, increased ultrafiltration volume, and restored homeostasis of bioincompatible factors in dialysis solution. Mechanistically, SIRT1-modified hUCMSCs attenuated peritoneal fibrosis through reducing peritoneal inflammation and inhibiting the TGF-β/Smad3 pathway in peritoneal omentum tissues.

Conclusion: SIRT1-modified hUCMSCs might work as a promising therapeutic strategy for the treatment of peritoneal dialysis-induced peritoneal damage and fibrosis.
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http://dx.doi.org/10.1186/s13287-020-01878-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7436980PMC
August 2020

Hirudin Reduces the Expression of Markers of the Extracellular Matrix in Renal Tubular Epithelial Cells in a Rat Model of Diabetic Kidney Disease Through the Hypoxia-Inducible Factor-1α (HIF-1α)/Vascular Endothelial Growth Factor (VEGF) Signaling Pathway.

Med Sci Monit 2020 May 30;26:e921894. Epub 2020 May 30.

Henan University of Chinese Medicine, Second Clinical Medical College, Zhengzhou, Henan, China (mainland).

BACKGROUND This study aimed to investigate the effects of hirudin on the production of extracellular matrix (ECM) factors by renal tubular epithelial cells in a rat model of diabetic kidney disease (DKD) and HK-2 human renal tubule epithelial cells. MATERIAL AND METHODS Sprague-Dawley rats were divided into the normal control group (n=10), the normal control+hirudin group (n=10), the DKD model group (n=12) and the DKD+hirudin group (n=12). At the end of the study, renal histopathology was undertaken, and the expression of type IV collagen, fibronectin, hypoxia-inducible factor-1alpha (HIF-1alpha), and vascular endothelial growth factor (VEGF) were evaluated using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). HK-2 cells were cultured in glucose and treated with hirudin. Protein and mRNA expression of fibronectin, type IV collagen, HIF-1alpha, and VEGF were evaluated following knockdown or overexpression of HIF-1alpha. RESULTS Hirudin significantly improved renal function in the rat model of DKD (P<0.01), and significantly down-regulated the expression of fibronectin, type IV collagen, HIF-1alpha, and VEGF proteins (P<0.05). The expression of ECM associated proteins was increased in HK-2 cells treated with high glucose and reduced in the high glucose+shRNA HIF-1alpha group (P<0.05). Compared with the control group, the expression of ECM associated proteins was increased in the HIF-1alpha over-expressed group, and decreased following treatment with hirudin (P<0.05). CONCLUSIONS Hirudin reduced the expression of markers of ECM by inhibiting the HIF-1alpha/VEGF signaling pathway in DKD renal tubular epithelial cells.
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http://dx.doi.org/10.12659/MSM.921894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282348PMC
May 2020

Hirudin reduces nephropathy microangiopathy in STZ-induced diabetes rats by inhibiting endothelial cell migration and angiogenesis.

Life Sci 2020 Aug 15;255:117779. Epub 2020 May 15.

Department of Nephropathy, Henan Provincial Hospital of Traditional Chinese Medicine, China; Department of Nephropathy, The Second Hospital Affiliated to Henan University of Chinese Medicine, China.

Objective: Kidney is the most common location of microangiopathy in diabetic patients, and we designed this study to investigate the effects of hirudin on renal microangiopathy in STZ-induced diabetes rats and in vitro.

Methods: We established a diabetes model by intraperitoneal injection of STZ and administered hirudin daily by subcutaneous injection. HE staining was used to assess kidney pathological changes. Western blot and immunochemistry was used to detect the protein expression. Glomerular endothelial cells (GEC) in normal rats were assessed by cell scratch test for migration ability and tubule formation experiment for angiogenesis ability.

Results: Compared with DN rats without any treatment, the serum creatinine, serum Cys C, 24-hour urine protein of DN rats with hirudin treatment were significantly decrease, the kidney/body weight and glomerular area of DN rats with hirudin treatment were all significantly decrease, and also significant improvement in renal pathology revealed by HE staining in DN rats after treating with hirudin. Moreover, we also found that hirudin coun not only significantly increase the prothrombin time and aivated partial thromboplastin time in DN rats, but also significantly decrease the expression of VEGF and TM-1 protein in kidney tissues of DN rats. In vitro, we found that high glucose could promote the migration and angiogensis of GEC, and significantly increased the expression of VEGF and Ang protein, but significantly decreased the expression of THBS1 and Arg1 protein. More importantly was that hirudin could inhibit the migration and angiogensis of GEC, and reversed HG-induced the expression of VEGF, Ang, THBS1 and Arg1 protein in GEC. In addition, we also found that hirudin could not only decrease HG-enhanced the activity of RhoA in GEC, but also decrease HG-enhanced the expression of p-MYPT1/MYPT1, p-p38/p38 protein in GEC.

Conclusion: Hirudin reduces nephropathy microangiopathy in STZ-induced diabetes, and might be related to hirudin inhibiting glomerular endothelial cell migration and angiogenesis through Rho-kinase and subsequent p38MAPK/NF-kB signaling pathway.
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http://dx.doi.org/10.1016/j.lfs.2020.117779DOI Listing
August 2020

ERp44 depletion exacerbates ER stress and aggravates diabetic nephropathy in db/db mice.

Biochem Biophys Res Commun 2018 10 14;504(4):921-926. Epub 2018 Sep 14.

Department of Nephropathy, The Second Hospital Affiliated to Henan University of Chinese Medicine, China; Department of Nephropathy, Henan Provincial Hospital of Traditional Chinese Medicine, China. Electronic address:

Diabetic nephropathy (DN) is a major complication of diabetes, and the dysfunction of endoplasmic reticulum (ER) plays an important role in its pathogenesis. ERp44, an ER resident chaperone protein, has been implicated in the modulation of ER stress, however, its role and mechanism in DN are not determined. Here, we show that ERp44 expression is upregulated in the glomeruli of db/db mice, a rodent model of type 2 diabetes. When ERp44 is depleted by in vivo shRNA-mediated knockdown, the features associated with DN including albuminuria level and glomerular basement membrane (GBM) thickness are aggravated, therefore suggesting a detrimental role of ERp44 depletion in DN progression. We further show that ERp44 depletion exacerbates ER stress in DN in db/db mice, and that attenuating ER stress with the chemical chaperone TUDCA remarkably diminishes the aggravated DN features caused by ERp44 depletion. These results suggest that the exacerbated ER stress is a critical factor for the detrimental effect of ERp44 depletion on DN progression in db/db mice. Thus, our study links the role of ERp44 in DN with ER stress regulation and may offer a potential therapeutic strategy to interfere DN progression.
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http://dx.doi.org/10.1016/j.bbrc.2018.09.037DOI Listing
October 2018

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus.

J Virol Methods 2017 07 24;245:73-80. Epub 2017 Mar 24.

Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen, 518045, People's Republic of China; Shenzhen Academy of Inspection and Quarantine Sciences, Shenzhen, 518045, People's Republic of China. Electronic address:

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4-100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.
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http://dx.doi.org/10.1016/j.jviromet.2017.03.010DOI Listing
July 2017

Identification of Infectious Salmon Anaemia Virus from Imported Iced Atlantic Salmons.

Bing Du Xue Bao 2017 Jan;33(1):96-102

Infectious Salmon anaemia virus (ISAV) has become a threat to the salmon industry worldwide and has caused considerable economic loss. In the present study, 9 suspect cases of ISAV infection were identified from iced Atlantic salmons imported from Norway in 2014 through Shenzhen port (Shenzhen, China) using methods recommended by the World Organization for Animal Health. However, the results of virus isolation were negative., Based on the sequence analysis of ISAV segment 6, the 9 ISAV isolates belonged to the HPRO type, had high homology (98.3%~100.0%) and closest relationship with Norway strains. We identified the 9 positive HPRO ISAVs from 491 iced Atlantic salmons (1. 8%). Therefore, we should strengthen the quarantine of iced Atlantic salmons from Norway in case of HPRO ISAV into China.
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January 2017

Toll-like receptors and interferon associated immune factors responses to spring viraemia of carp virus infection in common carp (Cyprinus carpio).

Fish Shellfish Immunol 2016 Aug 1;55:568-76. Epub 2016 Jun 1.

Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen 518045, China. Electronic address:

Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play a fundamental role in the innate immune response against viruses among all vertebrate classes. Common carp is a host for spring viraemia of carp virus (Rhabdovirus carpio, SVCV), which belong to Rhabdoviridae family. The present in-vivo experiment was conducted to investigate the expression of these innate immune factors in early phase as well as during recovery of SVCV infection by real-time quantitative reverse transcriptase polymerase chain reaction. A less lethal SVCV infection was generated in common carp (Cyprinus carpio) and was sampled at 3, 6, 12 h post infection (hpi), 1, 3, 5, 7 and 10 days post infection (dpi). At 3 hpi, the SVCV N gene was detected in all three fish and all three fish showed a relative fold increase of TLR2, TLR3 and TLR7, IFNa1, ISG15 and Vig1. Viral copies rapidly increased at 12 hpi then remained high until 5 dpi. When viral copy numbers were high, a higher expression of immune genes TLR2, TLR3, TLR7, IFNa1, IFNa2, IFNa1S, IFN regulatory factor 3 (IRF3), IRF7, interleukin 1β (IL1β), IL6, IL10, ADAR, ISG15, Mx1, PKR and Vig1 were observed. Viral copies were gradually reduced in 5 to 10 dpi fish, and also the immune response was considerably reduced but remained elevated. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against SVCV.
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http://dx.doi.org/10.1016/j.fsi.2016.05.043DOI Listing
August 2016

Selection and characterization of single-chain recombinant antibodies against infectious haematopoietic necrosis virus from mouse phage display library.

J Virol Methods 2014 09 18;205:61-7. Epub 2014 Apr 18.

College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China. Electronic address:

Six single-chain fragment variable (scFv) antibodies against infectious haematopoietic necrosis virus (IHNV) were selected from an antibody phage display library by phage display technology. The soluble scFv antibodies showed a molecular weight 32kDa by Western blot. Dot blot analysis revealed that the six scFv antibodies could recognize IHNV. For enzyme linked immunosorbent assay (ELISA), four scFv antibodies (P1A4, P1A12, P1D5 and P3E2) showed cross-reactivity with spring viraemia of carp virus (SVCV). However, none of the six scFv antibodies had cross-reaction with Pike fry rhabdovirus (PFRV), Soft-shelled turtle iridovirus (STIV), viral haemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the IHNV-infected cells. These scFv antibodies will be useful in diagnostic test development and pathogenesis studies for IHNV.
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http://dx.doi.org/10.1016/j.jviromet.2014.04.008DOI Listing
September 2014

Development and evaluation of a loop-mediated isothermal amplification assay for diagnosis of Cyprinid herpesvirus 2.

J Virol Methods 2013 Dec 4;194(1-2):206-10. Epub 2013 Sep 4.

Shenzhen Exit and Entry Inspection and Quarantine Bureau, Shenzhen, Guangdong 518001, China.

Goldfish Haematopoietic Necrosis caused by Cyprinid herpesvirus 2 (CyHV-2) is a severe fish disease with high level of mortality. This is the first study on detection of this disease by loop-mediated isothermal amplification (LAMP). A set of six primers targeting terminase gene (accession no. EU349285.1) was determined after a serial of tests. Detection limit was 1.09 × 10(-4)μg/μL, which was superior to conventional PCR and real-time PCR. No cross reaction with 28 other viruses or bacteria commonly found in fish was observed. The application of commercial kit and instrument for the LAMP assay could reduce the risk of cross contamination, which is suitable for detection of infection under field conditions.
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http://dx.doi.org/10.1016/j.jviromet.2013.08.028DOI Listing
December 2013

Selection and characterization of single-chain recombinant antibodies against spring viraemia of carp virus from mouse phage display library.

J Virol Methods 2013 Dec 29;194(1-2):178-84. Epub 2013 Aug 29.

The Key Laboratory of Aquatic Animal Diseases, Shenzhen Exit & Entry Inspection and Quarantine Bureau, Shenzhen 518001, PR China; College of Fisheries, Huazhong Agricultural University, Wuhan 430070, PR China.

Antibody-displaying phage library was selected after three rounds of panning against spring viraemia of carp virus (SVCV) by phage display technology. Eight positive clones which could produce soluble single-chain fragment variable (scFv) antibody induced by isopropyl-beta-d-thiogalactopyranoside (IPTG) were obtained. Dot blot results showed that the eight scFv antibodies could recognize SVCV. The soluble scFv antibodies showed a molecular weight 29 kD by Western blot. All scFv antibodies could recognize SVCV proteins specifically without cross-reaction with other virus proteins by ELISA. Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the SVCV-infected cells. These scFv antibodies will be useful tools to establish immunological detection methods for SVCV.
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http://dx.doi.org/10.1016/j.jviromet.2013.08.017DOI Listing
December 2013

Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR.

J Virol Methods 2010 Nov 17;169(2):391-6. Epub 2010 Aug 17.

College of Animal Science and Veterinary Medicine, Jilin University, No. 5333, Xi'an Road, Changchun 130062, China.

The aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay using a TaqMan probe to detect and quantify hirame rhabdovirus (HRV). The results demonstrated that the assay had a detection limit of 100 copies of RNA per reaction and a log-linear range up to 10(8) copies of HRV RNA. Regression analysis demonstrated a significant correlation with an R(2) value of 0.9963 and a slope of -3.18 between the mean C(t) values and HRV cRNA. This assay was 100 times more sensitive than the conventional one-step RT-PCR assay. The qRT-PCR assay was found to be highly reproducible with intra- and inter-assay coefficients of variation of 0.37-1.72% and 1.37-3.79%, respectively. The primers and TaqMan probe were specific for HRV and did not react with either the spring viraemia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV), marine birnavirus (MABV), viral hemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). This assay was evaluated using 40 fish samples, indicating that such method offers considerable advantages over the classical virus isolation method currently used for HRV surveillance. In conclusion, the developed qRT-PCR assay was a reliable, specific and sensitive tool for the quantitative diagnosis of HRV in fish samples.
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http://dx.doi.org/10.1016/j.jviromet.2010.08.011DOI Listing
November 2010

Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay.

J Virol Methods 2008 Apr 4;149(1):103-9. Epub 2008 Mar 4.

College of Fisheries, Huazhong Agricultural University, Wuhan 430070, PR China; Show-how Bio-tech Institute, Beihai 536000, PR China.

Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.
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http://dx.doi.org/10.1016/j.jviromet.2007.12.017DOI Listing
April 2008
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