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En-Min Zhou
Northwest Agriculture and Forestry University

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Preprint content
Published: 08 June 2021
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Background: Nanobodies are the third generation of genetically-engineered antibodies, possessing advantages of small molecular weight, simple genetic engineering, and low production cost for promising diagnostic application. In this study, a competitive ELISA (cELISA) using nanobody-horseradish peroxidase (HRP) fusion protein was utilized as an ultrasensitive probe for the first time to detect antibodies against genotype 2 porcine reproductive and respiratory syndrome virus (PRRSV). In addition, a platform for easily producing the nanobody-HRP fusion protein against PRRSV was designed and developed. Results: In the present study, three nanobodies against the PRRSV-N protein were screened by Camel immunization, library construction, and phage display. Subsequently, a recombinant HEK293S cell line stably secreting nanobody-HRP fusion protein against PRRSV-N protein was successfully designed and constructed using the lentivirus transduction assay. Using the cell lines, the fusion protein was easily produced. Then, a novel cELISA was developed using the nanobody-HRP fusion protein for detecting the antibodies against genotype 2 PRRSV in pig sera, exhibiting a cut‑off value of 23.19% and good sensitivity (100%), specificity, and reproducibility. The compliance rate of cELISA with a commercial IDEXX ELISA kit was 96.4%. By testing the sequential sera from the challenged pigs, the results showed that the sensitivity of cELISA was higher than the commercial IDEXX ELISA kit. In addition, the commercial IDEXX ELISA kit can be combined with the developed cELISA for the differential detection of antibodies against genotype 1 and 2 PRRSV in pig sera.Conclusions: By screening the three nanobodies against the genotype 2 PRRSV-N protein, a recombinant HEK293S cell line stably secreting nanobody-HRP fusion protein against PRRSV-N protein was developed (Scheme 1a). Subsequently, a cELISA was developed for detecting the antibodies against genotype 2 PRRSV using the fusion protein and demonstrated high sensitivity, specificity, and reproducibility (Scheme 1b). More importantly, the production of the fusion protein using the developed platform is simple and low cost.

ACS Style

Hong Duan; Xu Chen; Jiakai Zhao; Jiahong Zhu; Guixi Zhang; Mengnan Fan; Beibei Zhang; Xueting Wang; Yani Sun; BaoYuan Liu; En-Min Zhou; Qin Zhao. Easily Prepared Nanobody-horseradish Peroxidase Fusion Protein-based Immunoassay to Detect Antibodies Against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses. 2021, 1 .

AMA Style

Hong Duan, Xu Chen, Jiakai Zhao, Jiahong Zhu, Guixi Zhang, Mengnan Fan, Beibei Zhang, Xueting Wang, Yani Sun, BaoYuan Liu, En-Min Zhou, Qin Zhao. Easily Prepared Nanobody-horseradish Peroxidase Fusion Protein-based Immunoassay to Detect Antibodies Against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses. . 2021; ():1.

Chicago/Turabian Style

Hong Duan; Xu Chen; Jiakai Zhao; Jiahong Zhu; Guixi Zhang; Mengnan Fan; Beibei Zhang; Xueting Wang; Yani Sun; BaoYuan Liu; En-Min Zhou; Qin Zhao. 2021. "Easily Prepared Nanobody-horseradish Peroxidase Fusion Protein-based Immunoassay to Detect Antibodies Against Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses." , no. : 1.

Journal article
Published: 16 March 2021 in Veterinary Research
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Neutralizing antibodies (NAbs) have attracted attention as tools for achieving PRRSV control and prevention, but viral antigenic variation undermines the abilities of NAbs elicited by attenuated PRRSV vaccines to confer full protection against heterogeneous PRRSV field isolates. As demonstrated in this study, the monoclonal antibody (mAb) mAb-PN9cx3 exhibited broad-spectrum recognition and neutralizing activities against PRRSV-1 and PRRSV-2 strains in vitro. Furthermore, in vivo experiments revealed that the administration of two 10-mg doses of mAb-PN9cx3 before and after the inoculation of piglets with heterologous PRRSV isolates (HP-PRRSV-JXA1 or PRRSV NADC30-like strain HNhx) resulted in significant reduction of the PRRSV-induced pulmonary pathological changes and virus loads in porcine alveolar macrophages (PAMs) compared with the results obtained with mAb-treated isotype controls. Moreover, minimal hilar lymph node PRRSV antigen levels were observed in mAb-PN9cx3-treated piglets. A transcriptome profile analysis of PAMs extracted from lung tissues of piglets belonging to different groups (except for antibody-isotype controls) indicated that mAb-PN9cx3 treatment reversed the PRRSV infection-induced alterations in expression profiles. A gene ontology (GO) enrichment analysis of these genes traced their functions to pathways that included the immune response, inflammatory response, and response to steroid hormone, and their functions in oogenesis and positive regulation of angiogenesis have been implicated in PRRSV pathogenesis. Overall, NADC30-like HNhx infection affected more gene pathways than HP-PRRSV infection. In conclusion, our research describes a novel immunologic approach involving the use of mAbs that confer cross-protection against serious illness resulting from infection with heterogeneous PRRSV-2 isolates, which is a feat that has not yet been achieved through vaccination. Ultimately, mAb-PN9cx3 will be a powerful addition to our current arsenal for achieving PRRSV prevention and eradication.

ACS Style

Zhigang Zhang; Tianshu Zhai; Mingshuo Li; Kun Zhang; Jingrui Li; Xu Zheng; Chaonan Tian; Rui Chen; Jianhui Dong; En-Min Zhou; Yuchen Nan; Chunyan Wu. A broadly neutralizing monoclonal antibody induces broad protection against heterogeneous PRRSV strains in piglets. Veterinary Research 2021, 52, 1 -23.

AMA Style

Zhigang Zhang, Tianshu Zhai, Mingshuo Li, Kun Zhang, Jingrui Li, Xu Zheng, Chaonan Tian, Rui Chen, Jianhui Dong, En-Min Zhou, Yuchen Nan, Chunyan Wu. A broadly neutralizing monoclonal antibody induces broad protection against heterogeneous PRRSV strains in piglets. Veterinary Research. 2021; 52 (1):1-23.

Chicago/Turabian Style

Zhigang Zhang; Tianshu Zhai; Mingshuo Li; Kun Zhang; Jingrui Li; Xu Zheng; Chaonan Tian; Rui Chen; Jianhui Dong; En-Min Zhou; Yuchen Nan; Chunyan Wu. 2021. "A broadly neutralizing monoclonal antibody induces broad protection against heterogeneous PRRSV strains in piglets." Veterinary Research 52, no. 1: 1-23.

Correction
Published: 03 March 2021 in Journal of Nanobiotechnology
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An amendment to this paper has been published and can be accessed via the original article.

ACS Style

Yang Mu; Cunyu Jia; Xu Zheng; Haipeng Zhu; Xin Zhang; Haoran Xu; BaoYuan Liu; Qin Zhao; En-Min Zhou. Correction to: A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2. Journal of Nanobiotechnology 2021, 19, 1 -1.

AMA Style

Yang Mu, Cunyu Jia, Xu Zheng, Haipeng Zhu, Xin Zhang, Haoran Xu, BaoYuan Liu, Qin Zhao, En-Min Zhou. Correction to: A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2. Journal of Nanobiotechnology. 2021; 19 (1):1-1.

Chicago/Turabian Style

Yang Mu; Cunyu Jia; Xu Zheng; Haipeng Zhu; Xin Zhang; Haoran Xu; BaoYuan Liu; Qin Zhao; En-Min Zhou. 2021. "Correction to: A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2." Journal of Nanobiotechnology 19, no. 1: 1-1.

Journal article
Published: 17 February 2021 in Veterinary Research
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Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious virus that has led to enormous economic loss worldwide because of ineffective prevention and treatment. In view of their minimized size, high target specificity and affinity, nanobodies have been extensively investigated as diagnostic tools and treatments of many diseases. Previously, a PRRSV Nsp9-specific nanobody (Nb6) was identified as a PRRSV replication inhibitor. When it was fused with cell-penetrating peptide (CPP) TAT, Nb6-TAT could enter the cells for PRRSV suppression. However, delivery of molecules by CPP lack cell specificity and have a short duration of action. PRRSV has a tropism for monocyte/macrophage lineage, which expresses high levels of Fcγ receptors. Herein, we designed a nanobody containing porcine IgG Fc (Fcγ) to inhibit PRRSV replication in PRRSV permissive cells. Fcγ fused Nb6 chimeric antibody (Nb6-pFc) was assembled into a dimer with interchain disulfide bonds and expressed in a Pichia pastoris system. The results show that Nb6-pFc exhibits a well-binding ability to recombinant Nsp9 or PRRSV-encoded Nsp9 and that FcγR-mediated endocytosis of Nb6-pFc into porcine alveolar macrophages (PAM) was in a dose-dependent manner. Nb6-pFc can inhibit PRRSV infection efficiently not only by binding with Nsp9 but also by upregulating proinflammatory cytokine production in PAM. Together, this study proposes the design of a porcine IgG Fc-fused nanobody that can enter PRRSV susceptible PAM via FcγR-mediated endocytosis and inhibit PRRSV replication. This research reveals that nanobody-Fcγ chimeric antibodies might be effective for the control and prevention of monocyte/macrophage lineage susceptible pathogeneses.

ACS Style

Lu Zhang; Lizhen Wang; Shuaishuai Cao; Huanhuan Lv; Jingjing Huang; Guixi Zhang; Kaissar Tabynov; Qin Zhao; En-Min Zhou. Nanobody Nb6 fused with porcine IgG Fc as the delivering tag to inhibit porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages. Veterinary Research 2021, 52, 1 -14.

AMA Style

Lu Zhang, Lizhen Wang, Shuaishuai Cao, Huanhuan Lv, Jingjing Huang, Guixi Zhang, Kaissar Tabynov, Qin Zhao, En-Min Zhou. Nanobody Nb6 fused with porcine IgG Fc as the delivering tag to inhibit porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages. Veterinary Research. 2021; 52 (1):1-14.

Chicago/Turabian Style

Lu Zhang; Lizhen Wang; Shuaishuai Cao; Huanhuan Lv; Jingjing Huang; Guixi Zhang; Kaissar Tabynov; Qin Zhao; En-Min Zhou. 2021. "Nanobody Nb6 fused with porcine IgG Fc as the delivering tag to inhibit porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages." Veterinary Research 52, no. 1: 1-14.

Journal article
Published: 01 February 2021 in Journal of Nanobiotechnology
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Background The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)‑horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum. Results Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15‑HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti‑PCV2 antibodies. The cut‑off value of the cELISA is 20.72 %. Three hundreds and sixty porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68 % and 95.92 %, respectively. The coincidence rate of the two methods was 99.17 %. When detecting 620 clinical porcine serum samples, a good consistent (kappa value = 0.954) was found between the results of the cELISA and those of commercial kits. Conclusions In brief, the newly developed cELISA based PCV2-Nb15‑HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccine efficacy and the indirect diagnosis of PCV2 infection.

ACS Style

Yang Mu; Cunyu Jia; Xu Zheng; Haipeng Zhu; Xin Zhang; Haoran Xu; BaoYuan Liu; Qin Zhao; En-Min Zhou. A nanobody‐horseradish peroxidase fusion protein‐based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2. Journal of Nanobiotechnology 2021, 19, 1 -13.

AMA Style

Yang Mu, Cunyu Jia, Xu Zheng, Haipeng Zhu, Xin Zhang, Haoran Xu, BaoYuan Liu, Qin Zhao, En-Min Zhou. A nanobody‐horseradish peroxidase fusion protein‐based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2. Journal of Nanobiotechnology. 2021; 19 (1):1-13.

Chicago/Turabian Style

Yang Mu; Cunyu Jia; Xu Zheng; Haipeng Zhu; Xin Zhang; Haoran Xu; BaoYuan Liu; Qin Zhao; En-Min Zhou. 2021. "A nanobody‐horseradish peroxidase fusion protein‐based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2." Journal of Nanobiotechnology 19, no. 1: 1-13.

Journal article
Published: 23 November 2020 in Journal of Virology
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Porcine reproductive and respiratory syndrome (PRRS), which is caused by PRRS virus (PRRSV), is of great economic significance to the swine industry. Due to the complicated immune escape mechanisms of PRRSV, there are no effective vaccines or therapeutic drugs currently available against PRRS. Identification of cellular factors and underlying mechanisms that establish an effective antiviral state against PRRSV can provide unique strategies for developing antiviral vaccines or drugs. As an interferon (IFN)-stimulated gene, the role of IFN-induced transmembrane 3 (IFITM3) in PRRSV infection has not been reported as of yet. In the present study, it was shown that IFITM3 can exert a potent anti-PRRSV effect, and PRRS virions are trafficked to IFITM3-containing cell vesicles, where viral membrane fusion is impaired by cholesterol accumulation that is induced by IFITM3. Additionally, both endogenous and exogenous IFITM3 are incorporated into newly assembled progeny virions, and this decreased their intrinsic infectivity.

ACS Style

Angke Zhang; Hong Duan; Huijun Zhao; Huancheng Liao; Yongkun Du; Liangliang Li; Dawei Jiang; Bo Wan; Yanan Wu; Pengchao Ji; En-Min Zhou; Gaiping Zhang. Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion. Journal of Virology 2020, 94, 1 .

AMA Style

Angke Zhang, Hong Duan, Huijun Zhao, Huancheng Liao, Yongkun Du, Liangliang Li, Dawei Jiang, Bo Wan, Yanan Wu, Pengchao Ji, En-Min Zhou, Gaiping Zhang. Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion. Journal of Virology. 2020; 94 (24):1.

Chicago/Turabian Style

Angke Zhang; Hong Duan; Huijun Zhao; Huancheng Liao; Yongkun Du; Liangliang Li; Dawei Jiang; Bo Wan; Yanan Wu; Pengchao Ji; En-Min Zhou; Gaiping Zhang. 2020. "Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion." Journal of Virology 94, no. 24: 1.

Methods and protocols
Published: 07 November 2020 in Applied Microbiology and Biotechnology
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Canine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)–conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID50 per 100 μL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores. Key points • Three CDV mAbs that recognized different epitopes and bound to virion were generated. • The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed. • The sandwich ELISA is an ideal method for detecting CDV infections in the field.

ACS Style

Yuan Zhang; Gang Xu; Lu Zhang; Jiakai Zhao; Pinpin Ji; Yaning Li; BaoYuan Liu; Jingfei Zhang; Qin Zhao; Yani Sun; En-Min Zhou. Development of a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus. Applied Microbiology and Biotechnology 2020, 104, 10725 -10735.

AMA Style

Yuan Zhang, Gang Xu, Lu Zhang, Jiakai Zhao, Pinpin Ji, Yaning Li, BaoYuan Liu, Jingfei Zhang, Qin Zhao, Yani Sun, En-Min Zhou. Development of a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus. Applied Microbiology and Biotechnology. 2020; 104 (24):10725-10735.

Chicago/Turabian Style

Yuan Zhang; Gang Xu; Lu Zhang; Jiakai Zhao; Pinpin Ji; Yaning Li; BaoYuan Liu; Jingfei Zhang; Qin Zhao; Yani Sun; En-Min Zhou. 2020. "Development of a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus." Applied Microbiology and Biotechnology 104, no. 24: 10725-10735.

Journal article
Published: 14 October 2020 in Journal of Virology
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PRRSV has haunted the swine industry for over 30 years since its emergence. Besides the limited efficacy of PRRSV modified live vaccines (MLVs) against heterogeneous PRRSV isolates, rapid induction of nonneutralizing antibodies (non-NAbs) against PRRSV NSPs after MLV immunization or wild-strain infection is one of the reasons why development of an effective vaccine has been hampered. By using in vitro -generated BMDCs as models to understand the antigen presentation process of PRRSV, we obtained data indicating that PRRSV infection of BMDCs promotes functional SLA-DR upregulation to present PRRSV NSP-derived immunopeptides for evoking a non-NAb response in vivo . Our work not only uncovered a novel mechanism for interference in host antigen presentation by PRRSV but also revealed a novel insight for understanding the rapid production of nonneutralizing antibodies against PRRSV NSPs, which may have benefit for developing an effective vaccine against PRRSV in the future.

ACS Style

Chunyan Wu; Bingjun Shi; Di Yang; Kun Zhang; Jie Li; Jie Wang; Hongliang Liu; Qin Zhao; En-Min Zhou; Yuchen Nan. Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo. Journal of Virology 2020, 94, 1 .

AMA Style

Chunyan Wu, Bingjun Shi, Di Yang, Kun Zhang, Jie Li, Jie Wang, Hongliang Liu, Qin Zhao, En-Min Zhou, Yuchen Nan. Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo. Journal of Virology. 2020; 94 (21):1.

Chicago/Turabian Style

Chunyan Wu; Bingjun Shi; Di Yang; Kun Zhang; Jie Li; Jie Wang; Hongliang Liu; Qin Zhao; En-Min Zhou; Yuchen Nan. 2020. "Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo." Journal of Virology 94, no. 21: 1.

Journal article
Published: 14 October 2020 in Viruses
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

ACS Style

Shona C. Moore; Rebekah Penrice-Randall; Muhannad Alruwaili; Nadine Randle; Stuart Armstrong; Catherine Hartley; Sam Haldenby; Xiaofeng Dong; Abdulrahman Alrezaihi; Mai Almsaud; Eleanor Bentley; Jordan Clark; Isabel García-Dorival; Paul Gilmore; Ximeng Han; Benjamin Jones; Lisa Luu; Parul Sharma; Ghada Shawli; Yani Sun; Qin Zhao; Steven T. Pullan; Daniel P. Carter; Kevin Bewley; Jake Dunning; En-Min Zhou; Tom Solomon; Michael Beadsworth; James Cruise; Derrick W. Crook; David A. Matthews; Andrew D. Davidson; Zana Mahmood; Waleed Aljabr; Julian Druce; Richard Vipond; Lisa Ng; Laurent Renia; Peter J. M. Openshaw; J. Kenneth Baillie; Miles W. Carroll; James Stewart; Alistair Darby; Malcolm Semple; Lance Turtle; Julian A. Hiscox. Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism. Viruses 2020, 12, 1164 .

AMA Style

Shona C. Moore, Rebekah Penrice-Randall, Muhannad Alruwaili, Nadine Randle, Stuart Armstrong, Catherine Hartley, Sam Haldenby, Xiaofeng Dong, Abdulrahman Alrezaihi, Mai Almsaud, Eleanor Bentley, Jordan Clark, Isabel García-Dorival, Paul Gilmore, Ximeng Han, Benjamin Jones, Lisa Luu, Parul Sharma, Ghada Shawli, Yani Sun, Qin Zhao, Steven T. Pullan, Daniel P. Carter, Kevin Bewley, Jake Dunning, En-Min Zhou, Tom Solomon, Michael Beadsworth, James Cruise, Derrick W. Crook, David A. Matthews, Andrew D. Davidson, Zana Mahmood, Waleed Aljabr, Julian Druce, Richard Vipond, Lisa Ng, Laurent Renia, Peter J. M. Openshaw, J. Kenneth Baillie, Miles W. Carroll, James Stewart, Alistair Darby, Malcolm Semple, Lance Turtle, Julian A. Hiscox. Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism. Viruses. 2020; 12 (10):1164.

Chicago/Turabian Style

Shona C. Moore; Rebekah Penrice-Randall; Muhannad Alruwaili; Nadine Randle; Stuart Armstrong; Catherine Hartley; Sam Haldenby; Xiaofeng Dong; Abdulrahman Alrezaihi; Mai Almsaud; Eleanor Bentley; Jordan Clark; Isabel García-Dorival; Paul Gilmore; Ximeng Han; Benjamin Jones; Lisa Luu; Parul Sharma; Ghada Shawli; Yani Sun; Qin Zhao; Steven T. Pullan; Daniel P. Carter; Kevin Bewley; Jake Dunning; En-Min Zhou; Tom Solomon; Michael Beadsworth; James Cruise; Derrick W. Crook; David A. Matthews; Andrew D. Davidson; Zana Mahmood; Waleed Aljabr; Julian Druce; Richard Vipond; Lisa Ng; Laurent Renia; Peter J. M. Openshaw; J. Kenneth Baillie; Miles W. Carroll; James Stewart; Alistair Darby; Malcolm Semple; Lance Turtle; Julian A. Hiscox. 2020. "Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism." Viruses 12, no. 10: 1164.

Preprint content
Published: 01 October 2020
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Background: The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)‑horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum.Results: Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15‑HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti‑PCV2 antibodies. The cut‑off value of the cELISA was 20.72%, 360 porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68% and 95.92%, respectively. The coincidence rate of the two methods was 99.17%. When detecting 620 clinical porcine serum samples, a good consistent (kappa value=0.954) was found between the result of the cELISA and that of commercial kits.Conclusions: In brief, the newly developed cELISA based PCV2-Nb15‑HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccines efficacy and indirect diagnosis of PCV2 infection.

ACS Style

Yang Mu; Cunyu Jia; Xu Zheng; Haipeng Zhu; Xin Zhang; Haoran Xu; BaoYuan Liu; Qin Zhao; En-Min Zhou. A Nanobody-horseradish Peroxidase Fusion Protein-based Competitive ELISA for Rapid Detection of Antibodies Against Porcine Circovirus Type 2. 2020, 1 .

AMA Style

Yang Mu, Cunyu Jia, Xu Zheng, Haipeng Zhu, Xin Zhang, Haoran Xu, BaoYuan Liu, Qin Zhao, En-Min Zhou. A Nanobody-horseradish Peroxidase Fusion Protein-based Competitive ELISA for Rapid Detection of Antibodies Against Porcine Circovirus Type 2. . 2020; ():1.

Chicago/Turabian Style

Yang Mu; Cunyu Jia; Xu Zheng; Haipeng Zhu; Xin Zhang; Haoran Xu; BaoYuan Liu; Qin Zhao; En-Min Zhou. 2020. "A Nanobody-horseradish Peroxidase Fusion Protein-based Competitive ELISA for Rapid Detection of Antibodies Against Porcine Circovirus Type 2." , no. : 1.

Journal article
Published: 31 August 2020 in Vaccines
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Although widespread administration of attenuated porcine reproductive and respiratory syndrome virus (PRRSV) vaccines has been implemented since they first became commercially available two decades ago, PRRSV infection prevalence in swine herds remains high. The limited success of PRRSV vaccines is partly due to the well-established fact that a given vaccine strain confers only partial or no protection against heterologous strains. In our past work, A2MC2-P90, a novel PRRSV vaccine candidate that induced a type I IFNs response in vitro, conferred complete protection against challenge with genetically heterologous PRRSV strains. Here we assessed the ability of the PRRSV vaccine candidate A2MC2-P90 to protect piglets against the HP-PRRSV challenge and compared its efficacy to that of a licensed HP-PRRSV-specific vaccine (TJM-F92) assessed in parallel. A2MC2-P90 provided vaccinated piglets with 100% protection from a lethal challenge with extremely virulent HP-PRRSV-XJA1, while 100% mortality was observed for unvaccinated piglets by day 21 post-challenge. Notably, comparison of partial sequence (GP5) of XJA1 to A2MC2-P90 suggested there was only 88.7% homology. When comparing post-HP-PRRSV challenge responses between piglets administered A2AMC2-P90 versus those immunized with licensed vaccine TJM-F92, A2MC2-P90-vaccinated piglets rapidly developed a stronger protective humoral immune response, as evidenced by much higher titers of neutralizing antibodies, more rapid clearance of viremia and less nasal virus shedding. In conclusion, our data suggest that this novel vaccine candidate A2MC2-P90 has improved protection spectrum against heterologous HP-PRRSV strains.

ACS Style

Yafei Li; Junhui Li; Sun He; Wei Zhang; Jian Cao; Xiaomei Pan; Huifen Tang; En-Min Zhou; Chunyan Wu; Yuchen Nan. Interferon Inducing Porcine Reproductive and Respiratory Syndrome Virus Vaccine Candidate Protected Piglets from HP-PRRSV Challenge and Evoke a Higher Level of Neutralizing Antibodies Response. Vaccines 2020, 8, 490 .

AMA Style

Yafei Li, Junhui Li, Sun He, Wei Zhang, Jian Cao, Xiaomei Pan, Huifen Tang, En-Min Zhou, Chunyan Wu, Yuchen Nan. Interferon Inducing Porcine Reproductive and Respiratory Syndrome Virus Vaccine Candidate Protected Piglets from HP-PRRSV Challenge and Evoke a Higher Level of Neutralizing Antibodies Response. Vaccines. 2020; 8 (3):490.

Chicago/Turabian Style

Yafei Li; Junhui Li; Sun He; Wei Zhang; Jian Cao; Xiaomei Pan; Huifen Tang; En-Min Zhou; Chunyan Wu; Yuchen Nan. 2020. "Interferon Inducing Porcine Reproductive and Respiratory Syndrome Virus Vaccine Candidate Protected Piglets from HP-PRRSV Challenge and Evoke a Higher Level of Neutralizing Antibodies Response." Vaccines 8, no. 3: 490.

Research article
Published: 14 August 2020 in Nano Research
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Protein degradation technology, which is one of the most direct and effective ways to regulate the life activities of cells, is expected to be applied to the treatment of various diseases. However, current protein degradation technologies such as some small-molecule degraders which are unable to achieve spatiotemporal regulation, making them difficult to transform into clinical applications. In this article, an upconversion optogenetic nanosystem was designed to attain accurate regulation of protein degradation. This system worked via two interconnected parts: 1) the host cell expressed light-sensitive protein that could trigger the ubiquitinproteasome pathway upon blue-light exposure; 2) the light regulated light-sensitive protein by changing light conditions to achieve regulation of protein degradation. Experimental results based on model protein (Green Fluorescent Protein, GFP) validated that this system could fulfill protein degradation both in vitro (both Hela and 293T cells) and in vivo (by upconversion optogenetic nanosystem), and further demonstrated that we could reach spatiotemporal regulation by changing the illumination time (0–25 h) and the illumination frequency (the illuminating frequency of 0–30 s every 1 min). We further took another functional protein (The Nonstructural Protein 9, NSP9) into experiment. Results confirmed that the proliferation of porcine reproductive and respiratory syndrome virus (PRRSV) was inhibited by degrading the NSP9 in this light-induced system, and PRRSV proliferation was affected by different light conditions (illumination time varies from 0–24 h). We expected this system could provide new perspectives into spatiotemporal regulation of protein degradation and help realize the clinical application transformation for treating diseases of protein degradation technology.

ACS Style

Yafeng Hao; Taofeng Du; Gaoju Pang; Jiahua Li; Huizhuo Pan; Yingying Zhang; Lizhen Wang; Jin Chang; En-Min Zhou; Hanjie Wang. Spatiotemporal regulation of ubiquitin-mediated protein degradation via upconversion optogenetic nanosystem. Nano Research 2020, 1 -8.

AMA Style

Yafeng Hao, Taofeng Du, Gaoju Pang, Jiahua Li, Huizhuo Pan, Yingying Zhang, Lizhen Wang, Jin Chang, En-Min Zhou, Hanjie Wang. Spatiotemporal regulation of ubiquitin-mediated protein degradation via upconversion optogenetic nanosystem. Nano Research. 2020; ():1-8.

Chicago/Turabian Style

Yafeng Hao; Taofeng Du; Gaoju Pang; Jiahua Li; Huizhuo Pan; Yingying Zhang; Lizhen Wang; Jin Chang; En-Min Zhou; Hanjie Wang. 2020. "Spatiotemporal regulation of ubiquitin-mediated protein degradation via upconversion optogenetic nanosystem." Nano Research , no. : 1-8.

Journal article
Published: 30 July 2020 in Vaccine
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Pseudorabies Virus (PRV) is the causative agent of Pseudorabies (PR), also known as Aujeszky’s Disease, one of the most important infectious diseases in swine, resulting in huge economic losses to the swine industry globally. The emergence of mutant PRV strains after 2011 resulted in a sharp decrease in the efficacy of available commercial vaccines. To develop a more effective vaccine that can prevent the spread of PRV, glycoprotein B (gB), glycoprotein C (gC) and glycoprotein D (gD) from recent PRV isolates were expressed in a baculovirus system and their protective efficacy was tested in mice and piglets. Neutralizing antibody titers (NAs) in mice vaccinated with gB, gC and gD peaked at 28 days after immunization and then slowly declined. NAs in the mice immunized with gD were remarkably higher than other groups. After a lethal challenge of 5 LD50 with mutant PRV-HNLH strain, the survival rates of gB and gD were 100% and 87.5% respectively, which was significantly higher than gC group (50%). Piglets vaccinated with the gD and gB + D vaccines developed the highest NAs 7 days post immunization. No piglets in these two groups exhibited clinical symptoms, high body temperature or virus shedding following challenge with 106.6 TCID50 with the mutant PRV-HNLH strain. Histopathology and immunohistochemistry showed remarkably reduced pathological damage and viral loads in gD and gB + D groups. Furthermore, the duration of the NAs induced by gD vaccine could maintain as long as four months after a single dose. The current study indicates that a gD-based vaccine could be developed for the efficient control of PRV.

ACS Style

Teng Zhang; Yunchao Liu; Yumei Chen; Aiping Wang; Hua Feng; Qiang Wei; Enmin Zhou; Gaiping Zhang. A single dose glycoprotein D-based subunit vaccine against pseudorabies virus infection. Vaccine 2020, 38, 6153 -6161.

AMA Style

Teng Zhang, Yunchao Liu, Yumei Chen, Aiping Wang, Hua Feng, Qiang Wei, Enmin Zhou, Gaiping Zhang. A single dose glycoprotein D-based subunit vaccine against pseudorabies virus infection. Vaccine. 2020; 38 (39):6153-6161.

Chicago/Turabian Style

Teng Zhang; Yunchao Liu; Yumei Chen; Aiping Wang; Hua Feng; Qiang Wei; Enmin Zhou; Gaiping Zhang. 2020. "A single dose glycoprotein D-based subunit vaccine against pseudorabies virus infection." Vaccine 38, no. 39: 6153-6161.

Journal article
Published: 25 April 2020 in Veterinary Microbiology
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Hepatitis E virus (HEV) is a public health concern because of its zoonotic potential; however, the host species spectrum and the genetic diversity of HEV in many birds are unknown. In the present study, a novel genotype avian HEV was isolated from a bird, silkie fowl, and designated CHN-GS-aHEV (GenBank No. MN562265). The genome of CHN-GS-aHEV was analyzed in comparison with other avian HEVs’ and the pathogenicity in silkie fowl was characterized. The results show that the CHN-GS-aHEV shares about 81 % identity with known avian HEV in chickens, ORF3 shares the highest identity (85.1 %–88.0 %) at the nucleotide level, while ORF2 shares the highest identity (96.5 %–98.0 %) at the amino acid level, indicating that the CHN-GS-aHEV belongs to a new genotype avian HEV. The pathogenicity study showed that silkie fowl experimentally infected with the CHN-GS-aHEV demonstrated seroconversion, viremia, fecal virus shedding, liver lesions, and increased ALT level. Furthermore, ultrastructural changes in hepatocyte cells by transmission electron microscopy were characterized by the loss of mitochondrial cristae and swollen mitochondria and endoplasmic reticulum in the infected birds, suggesting that these two organelles may play a significant role in HEV replication. Overall, this study reports the complete genome characterization of a novel avian HEV and successful experimental infection in silkie fowl, and may be serving as a prominent indicator for additional avian HEV detection in other species.

ACS Style

BaoYuan Liu; Yiyang Chen; Liang Zhao; Meimei Zhang; Xiaolei Ren; Yuan Zhang; Beibei Zhang; Mengnan Fan; Qin Zhao; En-Min Zhou. Identification and pathogenicity of a novel genotype avian hepatitis E virus from silkie fowl (gallus gallus). Veterinary Microbiology 2020, 245, 108688 .

AMA Style

BaoYuan Liu, Yiyang Chen, Liang Zhao, Meimei Zhang, Xiaolei Ren, Yuan Zhang, Beibei Zhang, Mengnan Fan, Qin Zhao, En-Min Zhou. Identification and pathogenicity of a novel genotype avian hepatitis E virus from silkie fowl (gallus gallus). Veterinary Microbiology. 2020; 245 ():108688.

Chicago/Turabian Style

BaoYuan Liu; Yiyang Chen; Liang Zhao; Meimei Zhang; Xiaolei Ren; Yuan Zhang; Beibei Zhang; Mengnan Fan; Qin Zhao; En-Min Zhou. 2020. "Identification and pathogenicity of a novel genotype avian hepatitis E virus from silkie fowl (gallus gallus)." Veterinary Microbiology 245, no. : 108688.

Journal article
Published: 13 April 2020 in Vaccines
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Genotype 4 hepatitis E virus (HEV) is a zoonotic pathogen transmitted to humans through food and water. Previously, three genotype 4 swine HEV ORF2 peptides (407EPTV410, 410VKLYTS415, and 458PSRPF462) were identified as epitopes of virus-neutralizing monoclonal antibodies that partially blocked rabbit infection with swine HEV. Here, individual and tandem fused peptides were synthesized, conjugated to keyhole limpet hemocyanin (KLH), then evaluated for immunoprotection of rabbits against swine HEV infection. Forty New Zealand White rabbits were randomly assigned to eight groups; groups 1 thru 5 received three immunizations with EPTV-KLH, VKLYTS-KLH, PSRPF-KLH, EPTVKLYTS-KLH, or EPTVKLYTSPSRPF-KLH, respectively; group 6 received truncated swine HEV ORF2 protein (sp239), and group 7 received phosphate-buffered saline. After an intravenous swine HEV challenge, all group 7 rabbits exhibited viremia and fecal virus shedding by 2–4 weeks post challenge (wpc), seroconversion by 4–9 wpc, elevated alanine aminotransferase (ALT) at 2 wpc, and severe liver lymphocytic venous periphlebitis. Only 1–2 rabbits/group in groups 1–4 exhibited delayed viremia, fecal shedding, seroconversion, increased ALT levels, and slight liver lymphocytic venous periphlebitis; groups 5–6 showed no pathogenic effects. Collectively, these results demonstrate that immunization with a polypeptide containing three genotype 4 HEV ORF2 neutralizing epitopes completely protected rabbits against swine HEV infection.

ACS Style

Yiyang Chen; Tianxiang Chen; Yuhang Luo; Jie Fan; Meimei Zhang; Qin Zhao; Yuchen Nan; BaoYuan Liu; En-Min Zhou. Synthetic Peptides Containing Three Neutralizing Epitopes of Genotype 4 Swine Hepatitis E Virus ORF2 induced Protection against Swine HEV Infection in Rabbit. Vaccines 2020, 8, 178 .

AMA Style

Yiyang Chen, Tianxiang Chen, Yuhang Luo, Jie Fan, Meimei Zhang, Qin Zhao, Yuchen Nan, BaoYuan Liu, En-Min Zhou. Synthetic Peptides Containing Three Neutralizing Epitopes of Genotype 4 Swine Hepatitis E Virus ORF2 induced Protection against Swine HEV Infection in Rabbit. Vaccines. 2020; 8 (2):178.

Chicago/Turabian Style

Yiyang Chen; Tianxiang Chen; Yuhang Luo; Jie Fan; Meimei Zhang; Qin Zhao; Yuchen Nan; BaoYuan Liu; En-Min Zhou. 2020. "Synthetic Peptides Containing Three Neutralizing Epitopes of Genotype 4 Swine Hepatitis E Virus ORF2 induced Protection against Swine HEV Infection in Rabbit." Vaccines 8, no. 2: 178.

Journal article
Published: 14 March 2020 in Journal of Nanobiotechnology
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Background Traditional sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies as reagents presents several drawbacks, including limited amounts, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies can be easily expressed with different systems and fused with several tags in their tertiary structure by recombinant technology, thus offering an effective detection method for diagnostic purposes. Recently, the fenobody (ferritin-fused nanobody) and RANbody (nanobody-fused reporter) have been designed and derived from the nanobody for developing the diagnostic immunoassays. However, there was no report about developing the sandwich ELISA using the fenobody and RANbody as pairing reagents. Results A platform for developing a sandwich ELISA utilizing fenobody as the capture antibody and RANbody as the detection antibody was firstly designed in the study. Newcastle disease virus (NDV) was selected as the antigen, from which 13 NDV-specific nanobodies were screened from an immunized Bactrian camel. Then, 5 nanobodies were selected to produce fenobodies and RANbodies. The best pairing of fenobodies (NDV-fenobody-4, 800 ng/well) and RANbodies (NDV-RANbody-49, 1:10) was determined to develop the sandwich ELISA for detecting NDV. The detection limits of the assay were determined to be 22 of hemagglutination (HA) titers and 10 ng of purified NDV particles. Compared with two commercial assays, the developed assay shows higher sensitivity and specificity. Meanwhile, it exhibits 98.7% agreement with the HA test and can detect the reference NDV strains belonging to Class II but not Class I. Conclusions In the presented study, the 13 anti-NDV nanobodies binding the NDV particles were first produced. Then, for the first time, the sandwich ELISA to detect the NDV in the different samples has been developed using the fenobody and RANbody as reagents derived from the nanobodies. Considering the rapidly increasing generation of nanobodies, the platform can reduce the cost of production for the sandwich ELISA and be universally used to develop assays for detecting other antigens.

ACS Style

Pinpin Ji; Jiahong Zhu; Xiaoxuan Li; Wenqi Fan; Qianqian Liu; Kun Wang; Jiakai Zhao; Yani Sun; BaoYuan Liu; En-Min Zhou; Qin Zhao. Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus. Journal of Nanobiotechnology 2020, 18, 1 -19.

AMA Style

Pinpin Ji, Jiahong Zhu, Xiaoxuan Li, Wenqi Fan, Qianqian Liu, Kun Wang, Jiakai Zhao, Yani Sun, BaoYuan Liu, En-Min Zhou, Qin Zhao. Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus. Journal of Nanobiotechnology. 2020; 18 (1):1-19.

Chicago/Turabian Style

Pinpin Ji; Jiahong Zhu; Xiaoxuan Li; Wenqi Fan; Qianqian Liu; Kun Wang; Jiakai Zhao; Yani Sun; BaoYuan Liu; En-Min Zhou; Qin Zhao. 2020. "Fenobody and RANbody-based sandwich enzyme-linked immunosorbent assay to detect Newcastle disease virus." Journal of Nanobiotechnology 18, no. 1: 1-19.

Journal article
Published: 09 March 2020 in Vaccines
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Newcastle disease (ND) is a highly contagious avian disease, causing considerable economic losses to the poultry industry. To obtain a safe, inexpensive, and effective ND vaccine to meet the international trade requirements of differentiating infected from vaccinated animals (DIVA), here we report the production of Oryza sativa recombinant fusion (F) protein in stably transformed transgenic rice seeds via agroinfiltration. The F protein expression level was enhanced 3.6-fold with a genetic background in low glutelin. Inoculation of plant-produced F antigen into Specific Pathogen Free (SPF) chickens markedly elicited neutralizing antibody responses against homologous and heterologous ND virus strains. Two doses of 4.5 μg fully protected chickens from a lethal ND challenge without any clinical symptoms. The mean weight gain of F protein-immunized chickens within 15 days after challenge was significantly higher than that of traditional whole virus vaccine-immunized chickens, thereby obtaining higher economic benefits. Moreover, the sera from the chickens vaccinated with the plant-produced F vaccine did not show reactivity in an immunochromatographic strip targeting the haemagglutinin-neuraminidase protein (HN) protein, and DIVA could be achieved within 10 minutes. Our results demonstrate that the plant-derived F vaccine along with immunochromatographic strips could be useful in the implementation of an NDV eradication program.

ACS Style

Fanshu Ma; Erqin Zhang; Qingmei Li; Qianru Xu; Jiquan Ou; Heng Yin; Kunpeng Li; Li Wang; Xiangyue Zhao; Xiangxiang Niu; Xueyang Li; Shenli Zhang; Yanan Wang; Ruiguang Deng; Enmin Zhou; Gaiping Zhang. A Plant-Produced Recombinant Fusion Protein-Based Newcastle Disease Subunit Vaccine and Rapid Differential Diagnosis Platform. Vaccines 2020, 8, 122 .

AMA Style

Fanshu Ma, Erqin Zhang, Qingmei Li, Qianru Xu, Jiquan Ou, Heng Yin, Kunpeng Li, Li Wang, Xiangyue Zhao, Xiangxiang Niu, Xueyang Li, Shenli Zhang, Yanan Wang, Ruiguang Deng, Enmin Zhou, Gaiping Zhang. A Plant-Produced Recombinant Fusion Protein-Based Newcastle Disease Subunit Vaccine and Rapid Differential Diagnosis Platform. Vaccines. 2020; 8 (1):122.

Chicago/Turabian Style

Fanshu Ma; Erqin Zhang; Qingmei Li; Qianru Xu; Jiquan Ou; Heng Yin; Kunpeng Li; Li Wang; Xiangyue Zhao; Xiangxiang Niu; Xueyang Li; Shenli Zhang; Yanan Wang; Ruiguang Deng; Enmin Zhou; Gaiping Zhang. 2020. "A Plant-Produced Recombinant Fusion Protein-Based Newcastle Disease Subunit Vaccine and Rapid Differential Diagnosis Platform." Vaccines 8, no. 1: 122.

Journal article
Published: 23 January 2020 in Antiviral Research
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Porcine reproductive and respiratory syndrome (PRRS) is the most economically important infectious disease affecting the global swine industry, especially since vaccination has had limited impact on PRRSV prevention and control. In this study, the monoclonal antibody PR5nf1 (Mab-PR5nf1, IgM isotype) was shown to react with heterogeneous PRRSV isolates belonging to both PRRSV-1 and PRRSV-2 species. Pepsin digestion of Mab-PR5nf1 did not affect Mab binding to virions, as F(ab)2 fragments demonstrated the same reactivity as undigested Mab. Upon further investigation, Mab-PR5nf1 could neutralize all tested PRRSV isolates of both PRRSV-1 and PRRSV-2, suggesting it was a broadly neutralizing Mab against PRRSV. Interestingly, Mab-PR5nf1 appeared to recognize a specific virus epitope that required post-translational modification within the host cellular Golgi apparatus. Deglycosylation of PRRSV virions with PNGase F abolished Mab binding, suggesting that a novel Mab-binding epitope may exist that confers cross-protection against isolates of both PRRSV species. Additionally, immunization of mice with a cocktail of inactivated PRRSV virus and Mab-PR5nf1 enhanced cell-mediated immunity, as determined by IFN-γ ELIspot. In conclusion, this is the first report describing a novel Mab that recognizes a conserved epitope common to both PRRSV-1 and PRRSV-2 and provides valuable insights to guide future PRRSV vaccine development.

ACS Style

Chunyan Wu; Guoqian Gu; Tianshu Zhai; Yajing Wang; Yongling Yang; Yafei Li; Xu Zheng; Qin Zhao; En-Min Zhou; Yuchen Nan. Broad neutralization activity against both PRRSV-1 and PRRSV-2 and enhancement of cell mediated immunity against PRRSV by a novel IgM monoclonal antibody. Antiviral Research 2020, 175, 104716 .

AMA Style

Chunyan Wu, Guoqian Gu, Tianshu Zhai, Yajing Wang, Yongling Yang, Yafei Li, Xu Zheng, Qin Zhao, En-Min Zhou, Yuchen Nan. Broad neutralization activity against both PRRSV-1 and PRRSV-2 and enhancement of cell mediated immunity against PRRSV by a novel IgM monoclonal antibody. Antiviral Research. 2020; 175 ():104716.

Chicago/Turabian Style

Chunyan Wu; Guoqian Gu; Tianshu Zhai; Yajing Wang; Yongling Yang; Yafei Li; Xu Zheng; Qin Zhao; En-Min Zhou; Yuchen Nan. 2020. "Broad neutralization activity against both PRRSV-1 and PRRSV-2 and enhancement of cell mediated immunity against PRRSV by a novel IgM monoclonal antibody." Antiviral Research 175, no. : 104716.

Journal article
Published: 07 January 2020 in Journal of Nanobiotechnology
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Background Antibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV. Results In the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni–NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests. Conclusions For the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed immunoassays eliminate the use of commercial secondary antibodies and shorten detection time. Meanwhile, both assays display great developmental prospect for further commercial production and application.

ACS Style

Qizhong Lu; Xiaoxuan Li; Jiakai Zhao; Jiahong Zhu; Yuhang Luo; Hong Duan; Pinpin Ji; Kun Wang; BaoYuan Liu; Xueting Wang; Wenqi Fan; Yani Sun; En-Min Zhou; Qin Zhao. Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays. Journal of Nanobiotechnology 2020, 18, 1 -17.

AMA Style

Qizhong Lu, Xiaoxuan Li, Jiakai Zhao, Jiahong Zhu, Yuhang Luo, Hong Duan, Pinpin Ji, Kun Wang, BaoYuan Liu, Xueting Wang, Wenqi Fan, Yani Sun, En-Min Zhou, Qin Zhao. Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays. Journal of Nanobiotechnology. 2020; 18 (1):1-17.

Chicago/Turabian Style

Qizhong Lu; Xiaoxuan Li; Jiakai Zhao; Jiahong Zhu; Yuhang Luo; Hong Duan; Pinpin Ji; Kun Wang; BaoYuan Liu; Xueting Wang; Wenqi Fan; Yani Sun; En-Min Zhou; Qin Zhao. 2020. "Nanobody‑horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays." Journal of Nanobiotechnology 18, no. 1: 1-17.

Journal article
Published: 29 December 2019 in Viruses
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MYH9 has been identified as an indispensable cellular protein for porcine reproductive and respiratory syndrome virus (PRRSV) entry into permissive cells using the monoclonal anti-idiotypic antibody (Mab2-5G2) recognizing an antibody that specifically interacts with PRRSV glycoprotein 5 (GP5). More recently, we found that Mab2-5G2 interacted with the MYH9 C-terminal domain, designated PRA, which is required for PRRSV internalization. In this study, we demonstrate that blocking of MYH9 with Mab2-5G2 significantly diminished PRRSV internalization by porcine alveolar macrophage (PAM) via interruption of direct interaction between GP5 and MYH9, and thus remarkably inhibited subsequent infection of PAMs by PRRSV-2 isolates. Moreover, the three-dimensional structure of the Mab2-5G2 Fab-PRA complex determined via homology modeling predicted potential docking sites required for PRRSV internalization. Further analysis of Mab2-5G2-binding sites within PRA highlighted that the amino acids E1670, K1673, E1679, and I1683 in PRA are the key Mab2-5G2-binding residues. Notably, recombinant PRA protein blocked the interaction between PRRSV GP5 and cellular MYH9 by preventing translocation of MYH9 from the cytoplasm to the cell membrane, an essential step for PRRSV virion internalization. Meanwhile, porcine cell line permissive for PRRSV bearing point mutation of E1670A in MYH9 demonstrated reduced susceptibility for PRRSV infection. In conclusion, this work increases understanding of both PRRSV pathogenesis and the mechanistic role played by MYH9 in PRRSV infection.

ACS Style

Liangliang Li; Lu Zhang; Qifan Hu; Liang Zhao; Yuchen Nan; Gaopeng Hou; Yiyang Chen; Ximeng Han; Xiaolei Ren; Qin Zhao; Hu Tao; Zhenzhao Sun; Gaiping Zhang; Chunyan Wu; Jingfei Wang; En-Min Zhou. MYH9 Key Amino Acid Residues Identified by the Anti-Idiotypic Antibody to Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 5 Involve in the Virus Internalization by Porcine Alveolar Macrophages. Viruses 2019, 12, 40 .

AMA Style

Liangliang Li, Lu Zhang, Qifan Hu, Liang Zhao, Yuchen Nan, Gaopeng Hou, Yiyang Chen, Ximeng Han, Xiaolei Ren, Qin Zhao, Hu Tao, Zhenzhao Sun, Gaiping Zhang, Chunyan Wu, Jingfei Wang, En-Min Zhou. MYH9 Key Amino Acid Residues Identified by the Anti-Idiotypic Antibody to Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 5 Involve in the Virus Internalization by Porcine Alveolar Macrophages. Viruses. 2019; 12 (1):40.

Chicago/Turabian Style

Liangliang Li; Lu Zhang; Qifan Hu; Liang Zhao; Yuchen Nan; Gaopeng Hou; Yiyang Chen; Ximeng Han; Xiaolei Ren; Qin Zhao; Hu Tao; Zhenzhao Sun; Gaiping Zhang; Chunyan Wu; Jingfei Wang; En-Min Zhou. 2019. "MYH9 Key Amino Acid Residues Identified by the Anti-Idiotypic Antibody to Porcine Reproductive and Respiratory Syndrome Virus Glycoprotein 5 Involve in the Virus Internalization by Porcine Alveolar Macrophages." Viruses 12, no. 1: 40.