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Article: Generation of Stable Cell Lines Expressing Akabane Virus N Protein and Insight into Its Function in Viral Replication.

Wang, Jingjing / Chen, Dongjie / Wei, Fang / Deng, Junhua / Su, Jia / Lin, Xiangmei / Wu, Shaoqiang

2023 Volume 12, Issue 8

Abstract: Akabane virus (AKAV) is a world wide epidemic arbovirus belonging to ... ...

Abstract	Akabane virus (AKAV) is a world wide epidemic arbovirus belonging to the
Language	English
Publishing date	2023-08-18
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2695572-6
ISSN	2076-0817
ISSN	2076-0817
DOI	10.3390/pathogens12081058
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Article ; Online: Characterization and Double-Antibody Sandwich ELISA Application of a Monoclonal Antibody Against Akabane Virus Nucleocapsid Protein.

Chen, Dongjie / Wang, Jingjing / Wei, Fang / Jing, Hongli / Wang, Di / Zhang, Zhou / Lin, Xiangmei / Wu, Shaoqiang

Journal of AOAC International

2023 Volume 106, Issue 4, Page(s) 931–938

Abstract: Background: Akabane virus (AKAV) is a Culicoides-borne Orthobunyavirus that is teratogenic to the fetus of cattle and small ruminant species.: Objective: This study aimed to develop an effective diagnostic assay for the detection of AKAV using ... ...

Abstract	Background: Akabane virus (AKAV) is a Culicoides-borne Orthobunyavirus that is teratogenic to the fetus of cattle and small ruminant species. Objective: This study aimed to develop an effective diagnostic assay for the detection of AKAV using produced monoclonal antibody (mAb). Method: First, the mAb against N protein of AKAV was produced and characterized by Western blot (WB) and indirect immunofluorescence (IFA) assays. Then, the linear epitope of AKAV N protein against the mAb was identified and the mAb was applied to establish a double-antibody sandwich ELISA (DAS-ELISA). Results: One AKAV N-reactive monoclonal mAb was generated and designated as 2D3. WB and IFA assays indicated that 2D3 could react with both recombinant N protein and AKAV isolate TJ2016. The linear epitope recognized by mAb 2D3 was located at amino acids 168-182 of AKAV N protein. The DAS-ELISA established on based mAb 2D3 was able to detect both the purified AKAV N protein (with a detection limit of 6.25 ng/mL) and AKAV-infected cell culture supernatant (with a detection limit of 250 TCID50/mL). Conclusions: Taken together, we successfully prepared a mAb 2D3 against AKAV N protein and identified its corresponding linear epitope, and then established a DAS-ELISA for the detection of AKAV antigen. Highlights: A produced mAb against AKAV N protein was used to define a linear epitope of AKAV and establish a DAS-ELISA for AKAV antigen detection.
MeSH term(s)	Cattle ; Animals ; Bunyaviridae Infections/veterinary ; Antibodies, Monoclonal ; Orthobunyavirus ; Enzyme-Linked Immunosorbent Assay ; Nucleocapsid Proteins
Chemical Substances	Antibodies, Monoclonal ; Nucleocapsid Proteins
Language	English
Publishing date	2023-02-13
Publishing country	England
Document type	Journal Article
ZDB-ID	1103149-9
ISSN	1944-7922 ; 1060-3271
ISSN (online)	1944-7922
ISSN	1060-3271
DOI	10.1093/jaoacint/qsad025
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Article: Characterization and reverse genetic establishment of cattle derived Akabane virus in China

Chen, Dongjie / Wang, Di / Wei, Fang / Kong, Yufang / Deng, Junhua / Lin, Xiangmei / Wu, Shaoqiang

BMC veterinary research. 2021 Dec., v. 17, no. 1

2021

Abstract: BACKGROUND: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. RESULTS: An AKAV strain defined as TJ2016 was firstly isolated ...

Abstract	BACKGROUND: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. RESULTS: An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. CONCLUSION: We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.
Keywords	Akabane orthobunyavirus ; cattle ; genome ; growth models ; herbivores ; pathogenesis ; plasmids ; reverse genetics ; sequence analysis ; vaccines ; veterinary medicine ; viruses ; China ; Europe
Language	English
Dates of publication	2021-12
Size	p. 349.
Publishing place	BioMed Central
Document type	Article
ZDB-ID	2191675-5
ISSN	1746-6148
ISSN	1746-6148
DOI	10.1186/s12917-021-03054-x
Database	NAL-Catalogue (AGRICOLA)

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Article: Application of an AlphaLISA method for rapid sensitive detection of African swine fever virus in porcine serum

Chen, Dongjie / Wang, Di / Wang, Caixia / Wei, Fang / Zhao, Hongyuan / Lin, Xiangmei / Wu, Shaoqiang

Applied microbiology and biotechnology. 2021 June, v. 105, no. 11

2021

Abstract: Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich- ... ...

Abstract	Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and “sandwiched” by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78–100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. KEY POINTS: • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.
Keywords	African swine fever virus ; DNA ; antigen detection ; biotechnology ; blood serum ; diagnostic techniques ; epitopes ; immunoassays ; luminescence ; streptavidin ; swine ; China
Language	English
Dates of publication	2021-06
Size	p. 4751-4759.
Publishing place	Springer Berlin Heidelberg
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	392453-1
ISSN	1432-0614 ; 0171-1741 ; 0175-7598
ISSN (online)	1432-0614
ISSN	0171-1741 ; 0175-7598
DOI	10.1007/s00253-021-11339-2
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Article ; Online: Development of a novel droplet digital PCR assay for the sensitive detection of carp edema virus

Wang, Na / Zhang, Zhou / Jing, Hongli / Zhang, Min / Wu, Shaoqiang / Lin, Xiangmei

Aquaculture. 2021 Dec., v. 545 p.737162-

2021

Abstract: Carp edema virus (CEV) is a poxvirus associated with outbreaks of clinical disease that may lead to high mortality in both koi and common carp populations, potentially causing severe economic losses for carp aquaculture and the global koi trade. ... ...

Abstract	Carp edema virus (CEV) is a poxvirus associated with outbreaks of clinical disease that may lead to high mortality in both koi and common carp populations, potentially causing severe economic losses for carp aquaculture and the global koi trade. Therefore, the establishment of a sensitive and specific assay for detection of CEV is an essential requirement for the prevention of further spread of this emerging viral disease. In this study, a novel droplet-digital PCR (ddPCR) assay, targeting the p4a gene of CEV was developed. The assay exhibits good linearity, repeatability and reproducibility. Linearity was maintained at extremely low concentrations of CEV nucleic acid templates. The detection limit of ddPCR was 2.2 ± 0.26 copies of CEV DNA per reaction (n = 8), which represents a greater sensitivity than the quantitative PCR (qPCR) assay. Specificity analysis showed that the ddPCR assay for CEV had no cross-reactivity with other important aquatic pathogens. In clinical diagnosis of 151 koi and common-carp samples, 58 and 45 tested positively by ddPCR and qPCR assays, respectively. The overall agreement between the two assays was 91.39% (138/151). Linear regression analysis demonstrated a significant correlation between ddPCR and qPCR results with an R² value of 0.9627. Our findings indicate that the ddPCR assay could provide a robust diagnostic tool for the sensitive detection of CEV, even in samples with a low viral load.
Keywords	Carp edema virus ; Cyprinus carpio ; DNA ; aquaculture ; cross reaction ; detection limit ; diagnostic techniques ; droplets ; genes ; koi ; mortality ; quantitative polymerase chain reaction ; regression analysis ; trade ; viral load ; Carp edema virus (CEV) ; Droplet digital PCR (ddPCR) ; Common carp ; Detection
Language	English
Dates of publication	2021-12
Publishing place	Elsevier B.V.
Document type	Article ; Online
ZDB-ID	185380-6
ISSN	0044-8486 ; 0044-8516
ISSN	0044-8486 ; 0044-8516
DOI	10.1016/j.aquaculture.2021.737162
Database	NAL-Catalogue (AGRICOLA)

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Z 3275: Show issues

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Article ; Online: Genetically modified adipose-derived stem cells with matrix metalloproteinase 3 promote scarless cutaneous repair.

Rong, Shouxiang / Li, Chunlan / Li, Shuting / Wu, Shaoqiang / Sun, Fei

Dermatologic therapy

2020 Volume 33, Issue 6, Page(s) e14112

Abstract: Adipose-derived stem cells (ASCs) possess strong regenerative potencies and have been used to improve wound healing in animal models and clinical studies. However, the use of ASCs on scarless wound healing is not satisfactory. Matrix metalloproteinase 3 ( ...

Abstract	Adipose-derived stem cells (ASCs) possess strong regenerative potencies and have been used to improve wound healing in animal models and clinical studies. However, the use of ASCs on scarless wound healing is not satisfactory. Matrix metalloproteinase 3 (MMP-3) is involved in extracellular matrix (ECM) remolding and scar formation. We aimed to investigate the effect of ASCs stable expressing MMP-3 (ASCs-MMP-3) on wound healing and scarring. A cutaneous wound healing animal model was used to assess the effect of ASCs and ASCs-MMP-3 on wound healing and scar formation. The target protein levels in the wound tissues were determined by western blot assay. Our results demonstrated that ASCs alone promoted wound healing but had a negligible effect on reducing scarring. ASCs-MMP-3 not only possessed the ability of ASCs to speed up wound healing, but also incorporated the capability of MMP-3 to reduce scaring. Overexpressing of MMP-3 decreased the collagen I, transforming growth factor (TGF)-β1, and α-smooth muscle actin (α-SMA) levels and enhanced collagen III and TGF-β3 levels which contributed to reducing scar formation. Our studies suggested that ASCs-MMP-3 is a potential candidate for developing effective therapeutic strategies for scarless wound healing.
MeSH term(s)	Adipocytes ; Adipose Tissue ; Animals ; Cicatrix/pathology ; Cicatrix/prevention & control ; Matrix Metalloproteinase 3/genetics ; Stem Cells/pathology ; Wound Healing
Chemical Substances	Matrix Metalloproteinase 3 (EC 3.4.24.17)
Language	English
Publishing date	2020-08-27
Publishing country	United States
Document type	Journal Article
ZDB-ID	1354801-3
ISSN	1529-8019 ; 1396-0296
ISSN (online)	1529-8019
ISSN	1396-0296
DOI	10.1111/dth.14112
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Zs.A 5181: Show issues

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bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
Jg. 1995 - 2021: Lesesall (2.OG)
ab Jg. 2022: Lesesaal (EG)

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Article ; Online: Characterization and reverse genetic establishment of cattle derived Akabane virus in China.

Chen, Dongjie / Wang, Di / Wei, Fang / Kong, Yufang / Deng, Junhua / Lin, Xiangmei / Wu, Shaoqiang

BMC veterinary research

2021 Volume 17, Issue 1, Page(s) 349

Abstract: Background: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health.: Results: An AKAV strain defined as TJ2016 was firstly ... ...

Abstract	Background: Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. Results: An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. Conclusion: We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.
MeSH term(s)	Animals ; Bunyaviridae Infections/veterinary ; Bunyaviridae Infections/virology ; Cattle ; Cattle Diseases/virology ; Cell Line ; Cricetinae ; Genotype ; Orthobunyavirus/genetics ; Orthobunyavirus/isolation & purification ; Orthobunyavirus/pathogenicity ; Phylogeny ; Reverse Genetics/veterinary
Language	English
Publishing date	2021-11-15
Publishing country	England
Document type	Journal Article
ISSN	1746-6148
ISSN (online)	1746-6148
DOI	10.1186/s12917-021-03054-x
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Application of an AlphaLISA method for rapid sensitive detection of African swine fever virus in porcine serum.

Chen, Dongjie / Wang, Di / Wang, Caixia / Wei, Fang / Zhao, Hongyuan / Lin, Xiangmei / Wu, Shaoqiang

Applied microbiology and biotechnology

2021 Volume 105, Issue 11, Page(s) 4751–4759

Abstract	Infection with African swine fever virus (ASFV) causes an acute and highly lethal hemorrhagic disease that has been responsible for huge economic losses in China. To exactly detect the antigen of ASFV, we established a rapid, no-wash, one-step sandwich-type immunoassay based on the amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) using two monoclonal antibodies (mAbs) M-5 and M-6 against ASFV p72. ASFV p72 in samples was captured by biotinylated mAb M-5 connected to the donor bead surface via streptavidin and "sandwiched" by mAb M-6 which was coated onto the acceptor bead. Efficacy and sensitivity trials revealed that the AlphaLISA could detect ≥0.78 ng/ml of purified p72 and with a linear range of 0.78-100 ng/ml. The AlphaLISA was specific for ASFV and did not cross-react with other common pathogenic porcine viruses. Compared with RealPCR ASFV DNA test and ASFV antigen detection kit, the sensitivity of the AlphaLISA evaluated in 60 porcine serum samples was 93% and 100%, respectively. The specificity was 100% and 91.7%, respectively. This study presents a good laboratory diagnostic tool for sensitive and efficient detection of ASFV in porcine serum. KEY POINTS: • MAbs M-5 and M-6 recognized various epitopes of ASFV p72. • The established ASFV p72 AlphaLISA showed well specificity, high sensitivity, and satisfied correlation coefficient.
MeSH term(s)	African Swine Fever/diagnosis ; African Swine Fever Virus ; Animals ; Antibodies, Monoclonal ; China ; Serum ; Swine
Chemical Substances	Antibodies, Monoclonal
Language	English
Publishing date	2021-05-29
Publishing country	Germany
Document type	Journal Article
ZDB-ID	392453-1
ISSN	1432-0614 ; 0171-1741 ; 0175-7598
ISSN (online)	1432-0614
ISSN	0171-1741 ; 0175-7598
DOI	10.1007/s00253-021-11339-2
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Zs.A 2229: Show issues

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Article ; Online: Multiplex Real-Time PCR Method for Simultaneous Detection and Differentiation of Goat pox Virus, Sheeppox Virus, and Lumpy Skin Disease Virus.

Wang, Huiyu / Kong, Yufang / Mei, Lin / Lv, Jizhou / Wu, Shaoqiang / Lin, Xiangmei / Han, Xueqing

Journal of AOAC International

2021 Volume 104, Issue 5, Page(s) 1389–1393

Abstract: Background: The diseases caused by the Capripoxvirus species have very similar symptoms and are difficult to distinguish clinically. According to a recent report, Capripoxvirus are not strictly host specific.: Objective: This study aimed to identify ... ...

Abstract	Background: The diseases caused by the Capripoxvirus species have very similar symptoms and are difficult to distinguish clinically. According to a recent report, Capripoxvirus are not strictly host specific. Objective: This study aimed to identify the viruses from ovine (include sheep and goat) or bovine, which will assist in selecting the appropriate vaccine and correct measures to control diseases. Method: Universal primers for all Capripoxvirus and specific probes for lumpy skin disease virus, sheeppox virus, and goatpox virus were designed and analyzed to identify the viruses from ovine (including sheep and goats) or bovine species. The parameters of the system, such as the annealing temperatures and the quantities of primers and probes used, were optimized. The sensitivity, speciﬁcity, and reproducibility were tested. Results: Each probe showed a specific fluorescent signal, with no cross reaction with other pathogens that cause symptoms similar to those of the poxviruses. The LOD was 102 copies of the target genome DNA. The 557 local clinical samples and samples from Ethiopia were successfully detected and the results were consistent with a restriction fragment length polymorphism PCR analysis of the P32 and RPO30 genes and gene sequencing. Conclusions: This optimized real-time PCR detection system has good diagnostic sensitivity and specificity and can be used for the rapid and effective differential diagnosis of these diseases in goats, sheep, and cattle. Highlights: It is a rapid detection method to distinguish the viruses from ovine (include sheep and goat) or bovine.
MeSH term(s)	Animals ; Capripoxvirus/genetics ; Cattle ; Goat Diseases/diagnosis ; Goats ; Lumpy skin disease virus/genetics ; Poxviridae Infections/diagnosis ; Poxviridae Infections/veterinary ; Real-Time Polymerase Chain Reaction ; Reproducibility of Results ; Sheep ; Sheep Diseases/diagnosis
Language	English
Publishing date	2021-03-26
Publishing country	England
Document type	Journal Article
ZDB-ID	1103149-9
ISSN	1944-7922 ; 1060-3271
ISSN (online)	1944-7922
ISSN	1060-3271
DOI	10.1093/jaoacint/qsab040
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Zs.A 1229: Show issues

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Article ; Online: A Novel, Reverse Transcription, Droplet Digital PCR Assay for the Combined, Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus 2 with Swine Acute Diarrhea Syndrome Coronavirus.

Zhang, Zhou / Wang, Na / Liu, Xiaofei / Lv, Jizhou / Jing, Hongli / Yuan, Xiangfen / Chen, Dongjie / Lin, Xiangmei / Wu, Shaoqiang

Journal of AOAC International

2022 Volume 105, Issue 5, Page(s) 1437–1446

Abstract: Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread over the world since its emergence. Although the dominant route of SARS-CoV-2 infection is respiratory, a number of studies revealed infection risk from ... ...

Abstract	Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread over the world since its emergence. Although the dominant route of SARS-CoV-2 infection is respiratory, a number of studies revealed infection risk from contaminated surfaces and products, including porcine-derived food and other products. The SARS-CoV-2 outbreak has been severely threatening public health, and disrupting porcine products trade and the pig industry. Swine acute diarrhea syndrome coronavirus (SADS-CoV), which was responsible for large-scale, fatal disease in piglets, emerged in 2017 and has caused enormous economic losses in the pig industry. Currently, reverse transcription real-time PCR (RT-rPCR) is the gold standard method for SARS-CoV-2 diagnosis and is most commonly used for SADS-CoV detection. However, inaccurate detection of the SARS-CoV-2 infection obtained by RT-rPCR is increasingly reported, especially in specimens with low viral load. Objective: This study aimed to develop an accurate reverse transcription droplet digital PCR (RT-ddPCR) assay for the detection of SARS-CoV-2 and SADS-CoV simultaneously. Methods: Two pairs of primers and one double-quenched probe targeting the RNA-dependent RNA polymerase (RDRP) region of the open reading frame 1ab (ORF1ab) gene of SARS-CoV-2 and the corresponding ORF1ab region of SADS-CoV were designed to develop the RT-ddPCR assay. The sensitivity, specificity, repeatability, and reproducibility were tested using complementary RNAs (cRNAs) and clinical specimens. Results: The detection limits of RT-ddPCR were 1.48 ± 0.18 and 1.38 ± 0.17 copies in a 20 μL reaction for SARS-CoV-2 and SADS-CoV cRNAs, respectively (n = 8), showing approximately 4- and 10-fold greater sensitivity than the RT-rPCR assay. This assay also exhibited good specificity, repeatability, and reproducibility. Conclusion: The established RT-ddPCR assay was shown to be a highly effective, accurate, and reliable method for the sensitive detection of SARS-CoV-2 and SADS-CoV. Highlights: This RT-ddPCR assay could be used to detect both SARS-CoV-2 and SADS-CoV in a sample with one double-quenched probe, and is also the first reported RT-ddPCR assay for SADS-CoV detection.
MeSH term(s)	Alphacoronavirus ; Animals ; COVID-19/diagnosis ; COVID-19 Testing ; Humans ; RNA, Viral/analysis ; Real-Time Polymerase Chain Reaction/methods ; Reproducibility of Results ; Reverse Transcription ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; Swine
Chemical Substances	RNA, Viral
Language	English
Publishing date	2022-04-04
Publishing country	England
Document type	Journal Article
ZDB-ID	1103149-9
ISSN	1944-7922 ; 1060-3271
ISSN (online)	1944-7922
ISSN	1060-3271
DOI	10.1093/jaoacint/qsac039
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