Platform establishment of the Cre-loxP recombination system for genetic manipulation of the Lumpy skin disease virus.

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in Asia, including China. Genetic manipulation of the LSDV is essential for the elucidation of the pathogenic mechanism and biological function of the LSDV-encoded protein. In this study, we established a platform for the Cre-loxP recombination system under a modified early-late H5 promoter of the VACV for quick construction of the recombinant LSDV virus. The recombinant virus, LSDV-EGFP-ΔTK, was purified and obtained using serial limited dilution and picking the single cells methods. Using the lentiviral package system, a Cre recombinase enzyme stable expression MDBK cell line was established to supply the Cre recombinase for the reporter gene excision. A genetically stable, safe TK gene-deleted LSDV (LSDV-ΔTK) was constructed using homologous recombination and the Cre-loxP system. It was purified using limited dilution in the MDBK-Cre cell line. Establishing the Cre-loxP recombination system will enable sequential deletion of the interested genes from the LSDV genome and genetic manipulation of the LSDV genome, providing technical support and a platform for developing the attenuated LSDV vaccine.

Identification of the murine osteoblastic cell MC3T3-E1 as a permissive cell line in response to lumpy skin disease virus.

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in China. Screening suitable cells for LSDV replication is vital for future research on pathogenic mechanisms and vaccine development. Previous comparative studies have identified that the rodent-derived BHK21 is a highly susceptible cell model to LSDV infection. Using western blot, indirect immune-fluorescence assay, flow cytometry, and transmission electron microscopy methods, this study is the first to identify the murine osteoblastic cell line MC3T3-E1 as a novel permissive cell model for LSDV infection. The establishment of MC3T3-E1 as a suitable infectious cell model enhances our understanding of the species range and cell types of the permissive cells and nonpermissive that support LSDV replication. It is helpful to accelerate future research on the pathogenesis, clinical application, and vaccine development of LSDV.

Phylogenetic and pathogenic characterization of lumpy skin disease virus circulating in China.

The genomic characterization of emerging pathogens is critical for unraveling their origin and tracking their dissemination. Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in Asia including China. Although the first Lumpy skin disease (LSD) outbreak was reported in 2019, the origin, transmission, and evolutionary trajectory of LSDV in China have remained obscure. The viral genome of a circulating LSDV strain in China, abbreviated LSDV/FJ/CHA/2021, was sequenced using the next-generation sequencing technique. The morphology and cytoplasmic viral factory of these LSDV isolates were observed using transmission electron microscopy. Subsequently, the genomic characterization of this LSDV isolate was systematically analyzed for the first time using the bioinformatics software. The current study revealed that several mutations in the genome of LSDV isolates circulating in China were identified using single nucleotide polymorphisms (SNPs) analysis, an instrument to evaluate for continuous adaptive evaluation of a virus. Furthermore, phylogenomic analysis was used to identify the lineage using the whole genome sequences of 44 LSDV isolates. The result revealed that the isolates from China were closely similar to that of the LSDV isolates from Vietnam, which are divided into a monophyletic lineage sub-group I. The SNPs and Simplot analysis indicate no significant occurrence of the recombinant event on the genome of LSDV isolates in China. Notably, the live virus challenge experiment demonstrated that the pathogenic characterization of this LSDV isolate belongs to a virulent strain. Collectively, we gain the first insight into the evolutionary trajectory, spatiotemporal transmission, and pathogenic characterization of circulating LSDV in China. This study provides a unique reference for risk assessment, guiding diagnostics, and prevention in epizootic and non-epizootic areas.

The comparative study revealed that the hTERT-CSF cell line was the most susceptible cell to the Lumpy skin disease virus infection among eleven cells.

Lumpy skin disease virus (LSDV) is a rapidly emerging pathogen in Asia, including China. Improving the propagation of LSDV is important for diagnostics and vaccine production. Our study identified and compared the LSDV susceptibility of eleven standard cells using western blot, indirect immune-fluorescence assay, quantitative PCR, and 50 % tissue culture infectious dose. Our finding revealed that the LSDV strain could infect five cell lines and show a cytopathic effect. Furthermore, the hTERT-CSF cell line had the highest level of virus in the five cell models, followed by BHK-21, MDBK, Vero, and hTERT-ST. Hence, hTERT-CSF could be used as a candidate cell line for basic and applied research, clinical application, and LSDV vaccine development, providing a vital reference in LSDV and other viruses.

Establishment of a fetal cow (Bos Borus) skin fibroblasts cell line with immortalized characterization through human telomerase reverse transcriptase (hTERT) ectopic expression.

The ectopic introduction of the human telomerase reverse transcriptase (hTERT) is an effective way to establish an immortalized cell line. Here, hTERT was obtained by RT-PCR, and the eukaryotic expression plasmid and lentivirus shuttle plasmid of hTERT was successfully constructed by the homologous recombination method. The stable expression of hTERT in fetal cow skin fibroblasts (CSF) was established using the lentivirus package system. The hTERT-CSF proliferate and have immortalized characteristics. Meanwhile, the chromosome analysis identified that the number and structure of the hTERT-CSF genome maintain stable. The indirect immunofluorescence, western blot, and flow cytometry showed that the hTERT gene had been successfully integrated into the primary genome of bovine skin and stably expressed. The viral infection experiment first identifies the hTERT-CSF as a vulnerable cell model responding to the Lumpy skin disease virus (LSDV). Establishing hTERT-CSF provides an important cell model for basic and applied research, clinical application, and vaccine development. It provides an essential reference for the future's rapid establishment of other immortalized cell lines.

Molecular characterization, expression, and functional identification of TANK-binding kinase 1 (TBK1) of the cow (Bos taurus) and goat (Capra hircus).

The role of TANK-binding kinase 1 (TBK1) of humans and mice in innate immunity is well elucidated. Still, the molecular characterization and biological function of the TBK1 gene in herbivorous animals are less studied. Here, the open reading frame (ORF) of TBK1 of the cow and goat was firstly cloned and successfully expressed. The Phylogenetic tree analysis reveals that the TBK1 gene of goats and cows is similar to chicken and mute swans, respectively. Some evolutionary distances of the TBK1 gene were still present among different species. A slightly subcellular distribution difference was observed among full-length and truncated TBK1 of goats and cows. Dual-luciferase reporter assay has shown that the full-length TBK1 of goats and cows plays a vital role in the induction of IFN-ß production. The viral infection experiment showed that the over-expression of the full-length TBK1 gene of the cow and goat significantly suppresses intracellular viral replication of the Lumpy skin disease virus (LSDV) in infected cells. Our study showed that TBK1 in the cows and goats is a crucial immunoregulatory for IFN-ß production during viral infection, contributing to a better understanding of innate immunity in the herbivorous animal.

Screening of host genes regulated by ID1 and ID3 proteins during foot-and-mouth disease virus infection.

Foot-and-mouth disease virus (FMDV) is an important pathogen that harms cloven-hoofed animals and has caused serious losses to livestock production since its discovery. Furthermore, inhibitor of DNA binding (ID) proteins have been thoroughly studied in tumorigenesis, differentiation and metastasis, but its role in viral infection is rarely known. In this study, three gene knockout cell lines ID1 KO, ID3 KO, ID1/3 KO were obtained based on BHK-21 cells. We found that ID1 and ID3 genes single or double knockout promote the replication of FMDV. Moreover, compared with negative control cells during virus infection, there were 551 up-regulated genes and 1222 down-regulated genes in the ID1 KO cell line; 916 up-regulated genes and 1845 down-regulated genes in the ID3 KO cell line; 810 up-regulated genes and 1566 down-regulated genes in ID1/3 KO cell line. Further genes expression patterns verification results also showed a good correlation between the data of RT-qRCR and RNA-seq. These findings provide a basis for studying the relevant mechanisms between host genes and ID genes during FMDV infection.

Development of a Potential Penside Colorimetric LAMP Assay Using Neutral Red for Detection of African Swine Fever Virus.

African swine fever (ASF) has caused huge economic losses to the swine industry worldwide. Since there is no commercial ASF vaccine available, an early diagnosis is extremely important to prevent and control the disease. In this study, ASF virus (ASFV) capsid protein-encoding gene (p72) was selected and used to design primers for establishing a one-step visual loop-mediated isothermal amplification (LAMP) assay with neutral red, a pH-sensitive dye, as the color shift indicator. Neutral red exhibited a sharp contrast of color change from faint orange (negative) to pink (positive) during LAMP for detection of ASFV. The designed primer set targeting highly conserved region of the p72 gene was highly specific to ASFV and showed no cross-reactivity with other swine viruses. The detection limit for the one-step visual LAMP developed was 10 copies/reaction based on the recombinant plasmid containing the p72 gene of ASFV. More importantly, the developed one-step visual LAMP showed high consistency with the results of the real-time polymerase chain reaction (qPCR) method recommended by World Organization for Animal Health (OIE). Furthermore, the results demonstrate that the colorimetric detection with this LAMP assay could be directly applied for the whole blood and serum samples without requiring genome extraction. Based on our results, the developed one-step visual LAMP assay is a promising penside diagnostic tool for development of early and cost-effective ASF monitoring program that would greatly contribute to the prevention and control of ASF.

ID1 inhibits foot-and-mouth disease virus replication via targeting of interferon pathways.

Inhibitor of DNA-binding 1 (ID1) protein has been studied intensively for its functions in tumorigenesis and maintenance of stem cell-like properties, but its roles in virus infection are less understood. In the present study, we have clearly shown that the foot-and-mouth disease virus (FMDV) promotes ID1 degradation via Cdh1-mediated ubiquitination to facilitate its replication. Mechanistic investigations reveal Forkhead Box O1 (FOXO1) as an ID1 partner, which suppresses interferon regulatory factors 3 expression and interferon (IFN) production. Further investigation identified that ID1 suppresses FOXO1 transcription activity through HDAC4-mediated deacetylation, promoting IFN production and antiviral immune response. These studies establish a prominent role for ID1 in suppressing FDMV replication, which may be extended to other viruses.

MiR-4334-5p Facilitates Foot and Mouth Disease Virus Propagation by Suppressing Interferon Pathways via Direct Targeting ID1.

Emerging evidence indicates that the host microRNAs (miRNAs) are important intracellular regulators and play pivotal roles in intricate host-pathogen interaction networks. In our previous studies, ssc-microRNA-4334-5p (miR-4334-5p) was identified as a differentially expressed miRNA in microarray-based miRNAs profiling experiment, but whether miR-4334-5p regulates foot and mouth disease virus (FMDV) propagation is less understood. Here, we demonstrated that miR-4334-5p expression level was up-regulated shortly after FMDV infection, transfection of miR-4334-5p mimics promoted, while inhibitor transfection suppressed FMDV replication correspondingly. Further bioinformatic analysis and experimental study suggested ID1 was the direct target of miR-4334-5p, suppressing FMDV replication by regulating interferon (IFN) pathways. These findings shed light on microRNAs-ID1-interferon axis in regulating FMDV replication.

MicroRNA-4331-5p promotes FMDV replication through inhibiting interferon pathways in PK-15 cells.

MicroRNAs play vital roles in regulating the battle between pathogens and host cells during viral challenging. MiR-4331 aggravates transmissible gastroenteritis virus (TGEV) -induced mitochondrial damage, also suppresses transcription of TGEV gene 7 via targeting cellular CDCA7. Otherwise, miR-4331-5p affects H1N1/2009 influenza A virus replication by targeting viral HA and NS. However, whether microRNA ssc-miR-4331-5p (miR-4331-5p) regulates foot and mouth virus (FMDV) replication remains unclear. To explore the role of miR-4331-5p in FMDV infection, we detected the expression level of miR-4331-5p in porcine kidney (PK-15) cells. The results showed that FMDV infection directly upregulates miR-4331-5p expression, while transfection of mimics or inhibitor of miR-4331-5p promotes or inhibits FMDV replication. Further investigation clearly showed that miR-4331-5p increases FMDV replication through inhibiting type I interferon pathways. These data demonstrate that miR-4331-5p plays an important role in regulating FMDV replication.

Basal Level p53 Suppresses Antiviral Immunity against Foot-and-Mouth Disease Virus.

Tumor suppressor protein p53 (p53) is a master transcription factor that plays key roles in cell cycle arrest, apoptosis, senescence, and metabolism, as well as regulation of innate immunity during virus infection. In order to facilitate their replication and spreading, viruses have evolved to manipulate p53 function through different strategies, with some requiring active p53 while others demand reduction/inhibition of p53 activity. However, there are no clear-cut reports about the roles of p53 during the infection of foot-and-mouth disease virus (FMDV), the causative agent of a highly contagious foot-and-mouth disease (FMD) of cloven-hoofed animals. Here we showed that p53 level was dynamically regulated during FMDV infection, being degraded at the early infection stage but recovered to the basal level at the late stage. Cells depleted of p53 showed inhibited FMDV replication and enhanced expression of the immune-related genes, whereas overexpression of p53 didn't affect the viral replication. Viral challenge assay with p53 knockout mice obtained similar results, with viral load decreased, histopathological changes alleviated, and lifespan extended in the p53 knockout mice. Together, these data demonstrate that basal level p53 is required for efficient FMDV replication by suppressing the innate immunity.

Host microRNA miR-1307 suppresses foot-and-mouth disease virus replication by promoting VP3 degradation and enhancing innate immune response.

MicroRNAs (miRNAs) play important regulatory roles during interactions between virus pathogens and host cells, but whether and how they work in the case of foot-and-mouth disease virus (FMDV) is less understood. Based on a microarray-based miRNA profiling in the porcine kidney cell line PK-15, we identified 36 differentially expressed host miRNAs at the early stage of FMDV infection, among which miR-1307 was significantly induced. Functional characterization demonstrated that miR-1307 attenuated FMDV replication. Further experiments proved that miR-1307 specifically promoted the degradation of the viral structural protein VP3 indirectly through proteasome pathway. Moreover, innate immune signaling was activated and expression of immune responsive genes was significantly enhanced in the miR-1307-overexpressing clones. Together, our data demonstrated that miR-1307 suppresses FMDV replication by destabilizing VP3 and enhancing host immune response. Importantly, subcutaneous injection of miR-1307 agomir delayed the FMDV-induced lethality in suckling mice, exhibiting its therapeutic potential to control foot-and-mouth disease (FMD).

Expression of the VP1 protein of FMDV integrated chromosomally with mutant Listeria monocytogenes strain induced both humoral and cellular immune responses.

Live vector-based vaccine is a modern approach to overcome the drawbacks of inactivated foot-and-mouth disease (FMD) vaccines such as improper inactivation during manufacture. Listeria monocytogenes (LM), an intracellular microorganism with immune-stimulatory properties, is appropriate to be utilized as a live bacterial vaccine vector. FMDV-VP1 protein has the capability to induce both cellular and humoral immune responses since it is considered the most immunogenic part of FMDV capsid and has the most of antigenic sites for viral neutralization. The codon-optimized vp1 gene was ligated to the integrative pCW702 plasmid to construct the target cassette. The antigen cassette was integrated successfully into the chromosome of mutant LM strain via homologous recombination for more stability to generate a candidate vaccine strain LM△actAplcB-vp1. Safety evaluation of recombinant LM△actAplcB-vp1 revealed it could be eliminated from the internal organs within 3 days as a safe candidate vaccine. Mice groups were immunized I.V. twice with the recombinant LM△actAplcB-vp1 at an interval of 2 weeks. Antigen-specific IgG antibodies and the level of CD4+- and CD8+-specific secreted cytokines were estimated to evaluate the immunogenicity of the candidate vaccine. The rapid onset immune response was detected, strong IgG humoral immune response within 14 days post immunization and augmented again after the booster dose. Cellular immunity data after 9 days post the prime dose indicated elevation in CD4+ and CD8+ secreted cytokine level with another elevation after the booster dose. This is the first report to explain the ability of attenuated mutant LM to be a promising live vector for FMDV vaccine.

Transcript Profiling Identifies Early Response Genes against FMDV Infection in PK-15 Cells.

Foot-and-mouth disease (FMD) is a highly contagious disease that results in enormous economic loses worldwide. Although the protection provided by vaccination is limited during early infection, it is recognized as the best method to prevent FMD outbreaks. Furthermore, the mechanism of host early responses against foot-and-mouth disease virus (FMDV) infection remains unclear. In our study, a pig kidney cell line (PK-15) was used as a cell model to reveal the mechanism of early pig responses to FMDV infection. Four non-treated control and four FMDV-treated PK-15 cells were sequenced with RNA-seq technology, and the differentially expressed genes (DEGs) were analyzed. The results showed that 1212 DEGs were in the FMDV-infected PK-15 cells, including 914 up-regulated and 298 down-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in the tumor necrosis factor (TNF), cytokine-cytokine receptor interaction, NOD-like receptor, toll-like receptor, NF-κB, and the chemokine signaling pathways. To verify the results of the DEGs, 30 immune-related DEGs (19 up-regulated and 11 down-regulated) were selected for Quantitative Reverse Transcriptase polymerase chain reaction (RT-qPCR) verification. The results showed that RT-qPCR-measured genes exhibited a similar pattern as the RNA-seq analyses. Based on bioinformatics analysis, during FMDV early infection, we found that a series of cytokines, such as interleukins (IL6), chemokines (CXCL2, CCL20 and CCL4), and transcription factors (ZFP36, FOS, NFKBIA, ZBTB3, ZNF503, ZNF283, dymeclin (DYM), and orthodenticle homeobox 1 (OTX1)) were involved in the battle between FMDV and the host. Combined with their features and functions, we propose inflammation as the main early mechanism by which the host responds to FMDV infection. These data provide an additional panel of candidate genes for deciphering the mechanisms of a host's early response against FMDV infection.

Active evolution of memory B-cells specific to viral gH/gL/pUL128/130/131 pentameric complex in healthy subjects with silent human cytomegalovirus infection.

Human cytomegalovirus (HCMV) can cause life-threatening infection in immunosuppressed patients, and in utero infection that may lead to birth defects. No vaccine is currently available. HCMV infection in healthy subjects is generally asymptomatic, and virus persists as latent infection for life. Host immunity is effective against reactivation and super-infection with another strain. Thus, vaccine candidates able to elicit immune responses similar to those of natural infection may confer protection. Since neutralization is essential for prophylactic vaccines, it is important to understand how antiviral antibodies are developed in natural infection. We hypothesized that the developmental path of antibodies in seropositive subjects could be unveiled by interrogating host B-cell repertoires using unique genetic signature sequences of mAbs. Towards this goal, we isolated 56 mAbs from three healthy donors with different neutralizing titers. Antibodies specific to the gH/gL/pUL128/130/131 pentameric complex were more potent in neutralization than those to gB. Using these mAbs as probes, patterns of extended lineage development for B-cells and evidence of active antibody maturation were revealed in two donors with higher neutralizing titers. Importantly, such patterns were limited to mAbs specific to the pentamer, but none to gB. Thus, memory B-cells with antiviral function such as neutralization were active during latent infection in the two donors, and this activity was responsible for their higher neutralizing titers. Our results indicated that memory B-cells of neutralizing capacity could be frequently mobilized in host, probably responding to silent viral episodes, further suggesting that neutralizing antibodies could play a role in control of recurrent infection.

Protection of a novel epitope-RNA VLP double-effective VLP vaccine for foot-and-mouth disease.

Foot and mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals. Previously, we found that the epitope peptide EP141-160 displayed on virus-like particles (VLP) for use as a vaccine showed high immunoreactivity and conferred partially effective protection to animals. In this study, we first combined antisense RNA with VLP as a vaccine against the foot-and-mouth disease virus (FMDV) by using a prokaryotic co-expression system. The antisense RNA against the 3D genes of FMDV was packaged into VLP with EP141-160 presented on the surface. ELISA and Western blotting proved that the epitope-RNA VLP eliciting an immune response to FMDV in mice. Furthermore, the potency of the vaccine was tested in mice and guinea pigs. The results indicated that the epitope-RNA VLP vaccine protected 40% of suckling mice and 85% (17/20) of guinea pigs from FMDV. Based on the experimental data, the epitope-RNA VLP vaccine should have value in exploring and developing vaccines against FMDV in the future.

Triple-Emitting Dumbbell Fluorescent Nanoprobe for Multicolor Detection and Imaging Applications.

The combination of different fluorescent species into one nanostructure to develop fluorescent nanoparticles with multiple emission signatures by a single wavelength excitation has become a very popular research area in the field of multiplex bioanalysis, diagnostics, and multicolor imaging. However, these novel hybrids must be elaborately designed to ensure that the unique properties of each component are conveyed, i.e., fluorescent species and nanoparticles, and are maximized without serious interactions with each other. Herein, a first triple-fluorescence dumbbell nanoprobe with large Stokes shift based on incorporating fluorescein isothiocyanate (FITC) and lanthanide complexes onto Au-Fe3O4 NPs was synthesized. This hybrid displays well-resolved triple fluorescence emission, with FITC at 515 nm, Tb(III) complex at 545 nm, and Eu(III) complex at 616 nm under a single-excitation wavelength and is used for highly selective and sensitive colorimetric detection of Cu(2+) with a detection limit of 30 nM. Under different Cu(2+) concentrations, this hybrid exhibited distinguishable multiple colors under UV light, and the color could change in the presence of different concentrations of Cu(2+). This sensor for ratio/multianalyte microscopic imaging of Cu(2+) in HeLa cells and BHK cells was also demonstrated. Target molecules, such as folic acid, can be covalently attached to the fluorescent nanoparticle surface to serve as an effective probe for simultaneous multicolor imaging folate receptor-overexpressing HeLa cell lines in vitro.

Evolutionary analysis of structural protein gene VP1 of foot-and-mouth disease virus serotype Asia 1.

Foot-and-mouth disease virus (FMDV) serotype Asia 1 was mostly endemic in Asia and then was responsible for economically important viral disease of cloven-hoofed animals, but the study on its selection and evolutionary process is comparatively rare. In this study, we characterized 377 isolates from Asia collected up until 2012, including four vaccine strains. Maximum likelihood analysis suggested that the strains circulating in Asia were classified into 8 different groups (groups I-VIII) or were unclassified (viruses collected before 2000). On the basis of divergence time analyses, we infer that the TMRCA of Asia 1 virus existed approximately 86.29 years ago. The result suggested that the virus had a high mutation rate (5.745 × 10(-3) substitutions/site/year) in comparison to the other serotypes of FMDV VP1 gene. Furthermore, the structural protein VP1 was under lower selection pressure and the positive selection occurred at many sites, and four codons (positions 141, 146, 151, and 169) were located in known critical antigenic residues. The remaining sites were not located in known functional regions and were moderately conserved, and the reason for supporting all sites under positive selection remains to be elucidated because the power of these analyses was largely unknown.

Promising MS2 mediated virus-like particle vaccine against foot-and-mouth disease.

Foot-and-mouth disease (FMD) has caused severe economic losses to millions of farmers worldwide. In this work, the coding genes of 141-160 epitope peptide (EP141-160) of VP1 were inserted into the coat protein (CP) genes of MS2 in prokaryotic expression vector, and the recombinant protein self-assembled into virus-like particles (VLP). Results showed that the CP-EP141-160 VLP had a strong immunoreaction with the FMD virus (FMDV) antigen in vitro, and also had an effective immune response in mice. Further virus challenge tests were carried out on guinea pigs and swine, high-titer neutralizing antibodies were produced and the CP-EP141-160 VLP vaccine could protect most of the animals against FMDV.