Primary sheep fetal fibroblasts (SFFCs) have emerged as a valuable resource for investigating the molecular and pathogenic mechanisms of orf viruses (ORFV). However, their utilization is considerably ...restricted due to the exorbitant expenses associated with their isolation and culture, their abbreviated lifespan, and the laborious procedure.
In our investigation, the primary SFFCs were obtained and immortalized by introducing a lentiviral recombinant plasmid containing the large T antigen from simian virus 40 (SV40). The expression of fibronectin and vimentin proteins, activity of SV40 large T antigen, cell proliferation assays, and analysis of programmed cell death revealed that the immortalized large T antigen SFFCs (TSFFCs) maintained the same physiological characteristics and biological functions as the primary SFFCs. Moreover, TSFFCs demonstrated robust resistance to apoptosis, extended lifespan, and enhanced proliferative activity compared to primary SFFCs. Notably, the primary SFFCs did not undergo in vitro transformation or exhibit any indications of malignancy in nude mice. Furthermore, the immortalized TSFFCs displayed live ORFV vaccine susceptibility.
Immortalized TSFFCs present valuable in vitro models for exploring the characteristics of ORFV using various techniques. This indicates their potential for secure utilization in future studies involving virus isolation, vaccine development, and drug screening.
This study reviews the FMDV receptor-binding domain, integrin receptors, and heparan sulfate receptors to provide references for studies regarding the mechanisms underlying FMDV infection.
BACKGROUNDPeste des petits ruminants (PPR), caused by the PPR virus (PPRV), is an acute and fatal contagious disease that mainly infects goats, sheep, and other artiodactyls. Peripheral blood ...mononuclear cells (PBMCs) are considered the primary innate immune cells.OBJECTIVESPBMCs derived from goats were infected with PPRV and analyzed to detect the relationship between PPRV replication and apoptosis or the inflammatory response.METHODSQuantitative real-time polymerase chain reaction was used to identify PPRV replication and cytokines expression. Flow cytometry was conducted to detect apoptosis and the differentiation of CD4+ and CD8+ T cells after PPRV infection.RESULTSPPRV stimulated the differentiation of CD4+ and CD8+ T cells. In addition, PPRV induced apoptosis in goat PBMCs. Furthermore, apoptosis and the inflammatory response induced by PPRV could be suppressed by Z-VAD-FMK and Z-YVAD-FMK, respectively. Moreover, the virus titer of PPRV was attenuated by inhibiting caspase-1-dependent apoptosis and inflammation.CONCLUSIONSThis study showed that apoptosis and the inflammatory response play an essential role in PPR viral replication in vitro, providing a new mechanism related to the cell host response.
BACKGROUNDPeste des petits ruminants (PPR) is an infectious disease caused by the peste des petits ruminants virus (PPRV) that mainly produces respiratory symptoms in affected animals, resulting in ...great losses in the world's agriculture industry every year. Single-domain variable heavy chain (VHH) antibody fragments, also referred to as nanobodies, have high expression yields and other advantages including ease of purification and high solubility. OBJECTIVESThe purpose of this study is to obtain a single-domain antibody with good reactivity and high specificity against PPRV. METHODSA VHH cDNA library was established by immunizing camels with PPRV vaccine, and the capacity and diversity of the library were examined. Four PPRV VHHs were selected, and the biological activity and antigen-binding capacity of the four VHHs were identified by western blot, indirect immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) analyses. ELISA was used to identify whether the four VHHs were specific for PPRV, and VHH neutralization tests were carried out. ELISA and western blot analyses were used to identify which PPRV protein was targeted by VHH2. RESULTSThe PPRV cDNA library was constructed successfully. The library capacity was greater than 2.0 × 10⁶ cfu/mL, and the inserted fragment size was approximately 400 bp to 2000 bp. The average length of the cDNA library fragment was about 1000 bp, and the recombination rate was approximately 100%. Four single-domain antibody sequences were selected, and proteins expressed in the supernatant were obtained. The four VHHs were shown to have biological activity, close affinity to PPRV, and no cross-reaction with common sheep diseases. All four VHHs had neutralization activity, and VHH2 was specific to the PPRV M protein. CONCLUSIONSThe results of this preliminary research of PPRV VHHs showed that four screened VHH antibodies could be useful in future applications. This study provided new materials for inclusion in PPRV research.
Porcine circovirus 2 (PCV2) is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as ...porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS). The capsid (Cap) protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs) in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli ), because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2.
In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO). The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs.
we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection.
As a zoonotic infectious disease, orf outbreaks have been reported in China in recent years. However, molecular epidemiology analysis has not been performed for Chinese orf virus (ORFV) strains. ...Here, we have identified 13 ORFVs from goats and sheep in China between 2009 and 2011. Thirty-four complete
B2L
sequences were used to construct a phylogenetic tree to elucidate the molecular epidemiology of ORFV in China. Nucleotide sequences of
B2L
genes of clinical samples and attenuated vaccine strains were aligned and compared. Three genotypes were found by molecular epidemiology analysis. Amino acid substitutions were dispersed among
B2
polypeptides from wild and attenuated ORFV strains.
The family of
contains a group of obligate intracellular bacteria that can infect a wide range of hosts. The evolutionary trend of members in this family is a hot topic, which benefits our ...understanding of the cross-infection of these pathogens. In this study, 14 whole genomes of 12
species were used to investigate the nucleotide, codon, and amino acid usage bias by synonymous codon usage value and information entropy method. The results showed that all the studied
spp. had A/T rich genes with over-represented A or T at the third positions and G or C under-represented at these positions, suggesting that nucleotide usages influenced synonymous codon usages. The overall codon usage trend from synonymous codon usage variations divides the
spp. into four separate clusters, while amino acid usage divides the
spp. into two clusters with some exceptions, which reflected the genetic diversity of the
family members. The overall codon usage pattern represented by the effective number of codons (ENC) was significantly positively correlated to gene GC3 content. A negative correlation exists between ENC and the codon adaptation index for some
species. These results suggested that mutation pressure caused by nucleotide composition constraint played an important role in shaping synonymous codon usage patterns. Furthermore, codon usage of
and
gene families adapted to that of the corresponding genome. Taken together, analyses help our understanding of evolutionary interactions between nucleotide, synonymous codon, and amino acid usages in genes of
family members.
BACKGROUNDClassical swine fever (CSF) is a severe infectious disease of pigs that causes significant economic losses to the swine industry. OBJECTIVESThis study developed a solid-phase blocking ...enzyme-linked immunosorbent assay (spbELISA) method for the specific detection of antibodies against the CSF virus (CSFV) in porcine serum samples. METHODSA spbELISA method was developed based on the recombinant E2 expressed in Escherichia coli. The specificity of this established spbELISA method was evaluated using reference serum samples positive for antibodies against other common infectious diseases. The stability and sensitivity were evaluated using an accelerated thermostability test. RESULTSThe spbELISA successfully detected the antibody levels in swine vaccinated with the C-strain of CSFV. In addition, the detection ability of spbELISA for CSFV antibodies was compared with that of other commercial ELISA kits and validated using an indirect immunofluorescence assay. The results suggested that the spbELISA provides an alternative, stable, and rapid serological detection method suitable for the large-scale screening of CSFV serum antibodies. CONCLUSIONSThe spbELISA has practical applications in assessing the vaccination status of large pig herds.
Multisystemic inflammation in pigs affected by porcine circovirus type 2 (PCV2) indicates the disordered expression of inflammatory cytokines. However, the PCV2-induced expression profile of ...inflammation cytokines and its regulating mechanism remain poorly understood. In this study, inflammatory cytokines and receptors in porcine alveolar macrophages (PAMs) after PCV2 infection were profiled in vitro by an RT
Profiler
PCR array assay. The regulatory mechanism of interleukin-1β (IL-1β) expression was investigated. Results showed that 49 of 84 inflammation cytokines and receptors were differentially expressed (
< 0.05, absolute fold change ≥2) in PAMs at different stages post-PCV2 infection. Moreover, the overexpression of single-immunoglobulin interleukin-1 related receptor (SIGIRR) or the blocking of NF-κB activation by its inhibitor markedly decreased IL-1β secretion. This finding suggested that PCV2-induced overexpression of IL-1β was associated with the downregulation of SIGIRR and the activation of NF-κB. Furthermore, the excessive activity of NF-κB in SIGIRR-knockout PAMs cell line, indicating that SIGIRR negatively regulated IL-1β production by inhibiting the activation of NF-κB. Overall, PCV2-induced downregulation of SIGIRR induction of NF-κB activation is a critical process in enhancing IL-1β production in PAMs. This study may provide insights into the underlying inflammatory response that occurs in pigs following PCV2 infection.
Abstract
Background
Porcine epidemic diarrhea virus (PEDV), an intestinal coronavirus that causes acute diarrhea and high mortality in suckling piglets, can result in high economic losses in the ...swine industry. In recent years, despite the use of China’s current vaccine immunization strategy, multiple types of PEDV strains were still found in immunized swine herds. Our research aims to explore a new rapid differentiation method to distinguish the different types of PEDV strains and assess the safety evaluation of classical attenuated vaccine strains in swine herds.
Results
In the study, a differential one-step quantitative real-time fluorescent reverse transcription recombinase polymerase amplification (real-time RT-RPA) method based on the PEDV universal real-time RT-RPA assay was established according to the ORF1 deletion sequences of three classical attenuated vaccine strains (PEDV attenuated vaccine KC189944, attenuated CV777 and DR13) and five Vero cell-adapted isolates (JS2008, SDM, SQ2014, SC1402, HLJBY), which could effectively differentiate PEDV classical attenuated vaccine strains from wild-type strains (PEDV classical wild strains and variant strains). The detection limits of PEDV RNA in the both PEDV real-time RT-RPA assays were 300 copies within 20 min at 39 °C, and the detection limits of classical attenuated vaccine strain CV777, Vero-cell-adapted isolate JS2008, and PEDV wild-type strain DX were 10
0.5
TCID
50
/100 μL, 10
1.1
TCID
50
/100 μL, and 10
1.2
TCID
50
/100 μL, respectively. Both assays were highly specific for PEDV, showing no cross-reactivity with other enteral viruses.
Conclusion
This RPA method we developed is simple, time-effective, and safe and provides a reliable technical tool for the differential diagnosis and clinical epidemic surveillance of PEDV classical attenuated vaccine strains and wild-type strains.
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