Identification and epitope mapping of anti-p72 single-chain antibody against African swine fever virus based on phage display antibody library

African swine fever virus(ASFV)is a lethal pathogen that causes severe threats to the global swine industry and it has already had catastrophic socio-economic effects.To date,no licensed prophylactic vaccine exists.Limited knowledge exists about the major immunogens of ASFV and the epitope mapping of the key antigens.As such,there is a considerable requirement to understand the functional monoclonal antibodies(mAbs)and the epitope mapping may be of utmost importance in our understanding of immune responses and designing improved vaccines,therapeutics,and diagnostics.In this study,we generated an ASFV antibody phage-display library from ASFV convalescent swine PBMCs,further screened a specific ASFV major capsid protein(p72)single-chain antibody and fused with an IgG Fc fragment(scFv-83-Fc),which is a specific recognition antibody against ASFV Pig/HLJ/2018 strain.Using the scFv-83-Fc mAb,we selected a conserved epitope peptide(221MTGYKH226)of p72 retrieved from a phage-displayed random peptide library.Moreover,flow cytometry and cell uptake experiments demonstrated that the epitope peptide can significantly promote BMDCs maturation in vitro and could be effectively uptaken by DCs,which indicated its potential application in vaccine and diagnostic reagent development.Overall,this study provided a valuable platform for identifying targets for ASFV vaccine development,as well as to facilitate the optimization design of subunit vaccine and diagnostic reagents.

Establishment of Indirect ELISA Diagnosis Technique based on the VP1 Protein of Foot and Mouth Disease Virus Serotype A

The VP1 protein of foot-and-mouth disease virus serotype A was prokaryotically expressed and purified to replace the traditional virus antigen for estab- lishing a fast, safe, effective indirect ELISA method, so as to detecting antibody of foot-and-mouth disease virus serotype A. Western-Blot test showed that the VP1 recombinant protein could be used as detective antigen as it can be specifically recognized by bovine positive serum of FMDV serotype A. By employing matrix titra- tion method, the optimal parameters were obtained as follows： 1 mg/L VP1 protein as coating antigen, Vserum：Vblocking solution = 1：50 dilution for serum and Vsecondary enzyme-linked antibedies：Vblocking solution ---1：2 000 for enzyme combined antibodies. The results showod that the sensitivity and specificity of this method were 94.32% and 99.09% respectively, the coefficients of variations in intra-assay and inter-assay reproducibility tests was lower than 8%. Compared with liquid phase blocking ELISA kits, the agreement of 201 serum samples reached 92.54%. The VP1-ELISA method established here is specific, sensitive, stable and simple, which can be used to monitor the antibody level of FMD serotype A.

Screening for Monoclonal Antibodies Against the Membrane Proteins of Chicken Embryo Fibroblast Blocking the Infection of IBDV

This article aims to establish an efficient assay for screening monoclonal antibodies （McAbs） against the membrane proteins of chicken embryo fibroblast （CEF） for further studies of the cellular receptors of infectious bursal disease virus （IBDV）. McAbs against the membrane proteins of CEF were prepared by cell fusion. The monolayer CEF pre-incubated with the CEF-specific McAbs for 2 h were infected with IBDV and incubated with F22-EA6-biotin postinfection. Then, the cells were reacted with streptavidin-horseradish peroxidase （HRP） and finally stained by 3-amino-9-ethylcarbazole （AEC）. The inhibitive percentage of IBDV infection was calculated by counting the IBDV-infected cells to determine the inhibition efficiency of the CEF-specific McAbs. Compared with the control cells, the IBDV-infected cells pretreated with CEF-specific antibody significantly decreased; supernatant fluids of a total of 768 hybridomas were analyzed. The results of immunohistochemistry assays showed that six of them （1A5, 1H11, 2B 12, 3G1, 4D10, and 4B8） have the abilities to block the infection of IBDV to CEF, among which 4B8 can perfectly block the infection. This novel method is a sensitive and specific assay for the screening of CEF membrane protein-specific McAbs, which can block the infection of IBDV to CEF, and these McAbs can be used for the further investigations of the cellular receptors of IBDV.

Crystal Structure of a CD163 Scavenger Receptor Cysteine-rich Domain Produced in Pichia Pastoris

CD163 is an endocytic receptor specifically expressed in monocytes/macrophages and belongs to class B of the scavenger receptor cysteine-rich（SRCR） superfamily（SRCR-SF）. It plays critical roles in various physiological and pathological pathways. In this study, the fifth SRCR domain（SRCR5） of porcine CD163（pCD163） was expressed in Pichia pastoris system. The recombinant protein was homogeneous after purification by metal-affinity and size-exclusion chromatography. The purified protein was then assayed for its biological function. Single crystals of pCD163 SRCR5 were obtained under the condition of 25% PEG 4000, 0.1 M citric acid pH3.5 and its structure was finally determined at 1.80 A. All these results provide functional pCD163 SRCR5 in large quantity for future research, and enrich the structural details for CD163 and the SRCR-SF.

A Pair of Novel Primers for Universal Detection of the NS1 Gene from Various Bluetongue Virus Serotypes

Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 (VP7) and the non-structural protein (NS1) gene from different serotypes of BTV by DNAstar showed that the 5' end of the NS1 gene is the most conserved region. The primer pairs (P1 and P2) were designed based on the highly conserved region of NS1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus (EHDV) serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes.

Expression,Purification and Activity Detection of Structural Protein VP1 of Foot-and-Mouth Disease Virus Serotype A

The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.

Research Progress of Using Phage Display Technology to Screen Virus Affinity Peptides

Development and application of phage display technology and research progress of virus affinity peptide were summarized in the paper,and a preliminary outlook for future development was put forward. The paper laid a foundation for development of polypeptide drugs and polypeptide vaccine.

Critical roles of non-coding RNAs in lifecycle and biology of Marek's disease herpesvirus

Over the past two decades, numerous non-coding RNAs(ncRNAs) have been identified in different biological systems including virology, especially in large DNA viruses such as herpesviruses. As a representative oncogenic alphaherpesvirus, Marek’s disease virus(MDV) causes an important immunosuppressive and rapid-onset neoplastic disease of poultry, namely Marek’s disease(MD). Vaccinations can efficiently prevent the onset of MD lymphomas and other clinical disease, often heralded as the first successful example of vaccination-based control of cancer. MDV infection is also an excellent model for research into virally-induced tumorigenesis. Recently, great progress has been made in understanding the functions of ncRNAs in MD biology.Herein, we give a review of the discovery and identification of MDV-encoded viral miRNAs, focusing on the genomics,expression profiles, and emerging critical roles of MDV-1 miRNAs as oncogenic miRNAs(oncomiRs) or tumor suppressor genes involved in the induction of MD lymphomas. We also described the involvements of host cellular miRNAs, lincRNAs, and circRNAs participating in MDV life cycle, pathogenesis, and/or tumorigenesis. The prospects, strategies, and new techniques such as the CRISPR/Cas9-based gene editing applicable for further investigation into the ncRNA-mediated regulatory mechanisms in MDV pathogenesis/oncogenesis were also discussed, together with the possibilities of future studies on antiviral therapy and the development of new efficient MD vaccines.

Human PRV Infection in China: An Alarm to Accelerate Eradication of PRV in Domestic Pigs

Pseudorabies,also known as Aujeszky's disease,is one of the most economically important viral diseases in pigs and is lethal to other susceptible animals(Ren et al.2020).The causative agent,pseudorabies virus(PRV),is an enveloped virus with a large double-stranded DNA genome encoding at least 70 proteins(Wong et al.2019).PRV belongs to the family Herpesviridae,subfamily Alphaherpesvirinae,genus Varicellovirus and infects multiple animals,such as pigs,dogs,cats,rabbits,cattle,sheep,goats,minks.

Structural prediction of porcine sialoadhesin V-set Ig-like domain sheds some light on its role in porcine reproductive and respiratory syndrome virus(PRRSV) infection

Porcine reproductive and respiratory syndrome(PRRS) is characterized by reproductive failures in sows and respiratory diseases in pigs of all ages. PRRS virus(PRRSV) is its causative agent and has caused huge economic losses in the swine industry. Porcine sialoadhesin(pSn) is a putative receptor of PRRSV. Previous studies have shown that a pSn V-set Ig-like domain is signi ficant in PRRSV infection. However, its structural details are not fully known, hindering our deep understanding of PRRSV infection. In this study, we successfully cloned, expressed and puri fied the p Sn V-set Ig-like domain in Drosophila S2 cells. Then we tried to crystallize the target protein and predicted its structure. This will establish the foundation for the further structural study of p Sn, deepen our understanding of the invasion mechanism of PRRSV,and support the structural information for the development of clinical drugs and vaccines against PRRSV.

Efficient purification of cell culture-derived classical swine fever virus by ultrafiltration and size-exclusion chromatography

Large-scale production of cell culture-based classical swine fever virus(CSFV)vaccine is hampered by the adverse reactions caused by contaminants from host cell and culture medium.Hence,we have developed an efficient method for purifying CSFV from cell-culture medium.Pure viral particles were obtained with two steps of tangential-flow filtration(TFF)and size-exclusion chromatography(SEC),and were compared with particles from ultracentrifugation by transmission electron microscopy(TEM),infectivity and recovery test,and real time fluorescent quantitative PCR(FQ-PCR).TFF concentrated the virus particles effectively with a retention rate of 98.5%,and 86.2%of viral particles were obtained from the ultrafiltration retentate through a Sepharose 4 F F column on a biological liquid chromatography system.CSFV purified by TFF-SEC or ultracentrifugation were both biologically active from 1.0×10–4.25 TCID50·mL^(–1) to 3.0×10^(–6.25) TCID50·mL^(–1),but the combination of TFF and SEC produced more pure virus particles than by ultracentrifugation alone.In addition,pure CSFV particles with the expected diameter of 40–60 nm were roughly spherical without any visible contamination.Mice immunized with CSFV purified by TFF-SEC produced higher antibody levels compared with immunization with ultracentrifugation-purified CSFV(P<0.05).The purification procedures in this study are reliable technically and feasible for purification of large volumes of viruses.

Non-proteolytic ubiquitination of OTULIN regulates NF-κB signaling pathway

NF-κB signaling regulates diverse processes such as cell death,inflammation,immunity,and cancer.The activity of NF-κB is controlled by methionine 1-linked linear polyubiquitin,which is assembled by the linear ubiquitin chain assembly complex(LUBAC)and the ubiquitin-conjugating enzyme UBE2L3.Recent studies found that the deubiquitinase OTULIN breaks the linear ubiquitin chain,thus inhibiting NF-κB signaling.Despite the essential role of OTULIN in NF-κB signaling has been established,the regulatory mechanism for OTULIN is not well elucidated.To discover the potential regulators of OTULIN,we analyzed the OTULIN protein complex by proteomics and revealed several OTULIN-binding proteins,including LUBAC and tripartite motif-containing protein 32(TRIM32).TRIM32 is known to activate NF-κB signaling,but the mechanism is not dear.Genetic complement experiments found that TRIM32 is upstream of OTULIN and TRIM32-mediated NF-κB activation is dependent on OTULIN.Mutagenesis of the E3 ligase domain showed that the E3 ligase activity is essential for TRIM32-mediated NF-κB activation.Further experiments found that TRIM32 conjugates polyubiquitin onto OTULIN and the polyubiquitin blocks the interaction between HOIP and OTULIN,thereby activating NF-κB signaling.Taken together,we report a novel regulatory mechanism by which TRIM32-mediated non-proteolytic ubiquitination of OTULIN impedes the access of OTULIN to the LUBAC and promotes NF-κB activation.

Marek’s disease virus-encoded microRNAs: genomics, expression and function

MicroRNAs (miRNAs) are the recently discovered small non-coding RNA molecules that have post-transcriptional regulatory functions in many important biological processes. A large number of miRNAs have been found to be encoded by viral genomes, especially in herpesviruses. Previous research regarding miRNAs encoded by herpesviruses, including Marek’s disease virus (MDV), has demonstrated their involvement in lytic replication, latent infection, T-lymphocyte transformation and tumorigenesis. MDV is an oncogenic alphaherpesvirus, with the ability to induce tumors in natural hosts; however, formation of these tumors can be prevented by immunization with attenuated or nonpathogenic forms of the virus. Marek’s disease is considered to be a good biomedical model for investigating the biology, genetics, and immunology of tumorigenesis. In this paper, we review the discovery and identification of MDV-encoded miRNAs, along with their genomics, expression profiles, and currently known functions. We also discuss the prospects and techniques possibly applicable to the further investigation of the biological roles of MDV-encoded miRNAs.

Erratum to:Marek's disease virus-encoded microRNAs:genomics,expression and function

In the original version of the article the fourth and fifth name above "Mid-cluster" in Figure 2 are incorrect. The correct names are M1-5p and M1-3p*.