Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development.

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Virome characterization of diarrheic red-crowned crane (G. japonensis).

BACKGROUND: The red-crowned crane is one of the vulnerable bird species. Although the captive population has markedly increased over the last decade, infectious diseases can lead to the death of young red-crowned cranes while few virological studies have been conducted. METHODS: Using a viral metagenomics approach, we analyzed the virome of tissues of the dead captive red-crowned crane with diarrhea symptoms in Dongying Biosphere Reserve, Shandong Province, China and feces of individual birds breeding at the corresponding captive breeding center, which were pooled separately. RESULTS: There is much more DNA and RNA viruses in the feces than that of the tissues. RNA virus belonging to the families Picornaviridae, and DNA viruses belonging to the families Parvoviridae, associated with enteric diseases were detected in the tissues and feces. Genomes of the picornavirus, genomovirus, and parvovirus identified in the study were fully characterized, which further suggested that infectious viruses of these families were possibly presented in the diseased red-crowned crane. CONCLUSION: RNA virus belonging to the families Picornaviridae, and DNA viruses belonging to the families Genomoviridae and Parvoviridae were possibly the causative agent for diarrhea of red-crowned crane. This study has expanded our understanding of the virome of red-crowned crane and provides a baseline for elucidating the etiology for diarrhea of the birds.

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance.

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the detection cost, an intelligent high-throughput AST/phage screening system, including a 96-dot matrix inoculator, image acquisition converter, and corresponding software, was developed according to AST criteria and the breakpoints of resistance (R) formulated by the Clinical & Laboratory Standards Institute (CLSI). AST and statistics of minimum inhibitory concentration (MIC) distributions (from R/8 to 8R) of 1,500 Salmonella strains isolated from poultry in Shandong, China, against 10 antimicrobial agents were carried out by the intelligent high-throughput AST/phage screening system. The Lar index, meaning "less antibiosis, less resistance and residual until little antibiosis", was obtained by calculating the weighted average of each MIC and dividing by R. This approach improves accuracy in comparison with using the prevalence of resistance to characterize the antimicrobial resistance (AMR) degree of highly resistant strains. For the strains of Salmonella with high AMR, lytic phages were efficiently screened from the phage library by this system, and the lysis spectrum was computed and analyzed. The results showed that the intelligent high-throughput AST/phage screening system was operable, accurate, highly efficient, inexpensive, and easy to maintain. Combined with the Shandong veterinary antimicrobial resistance monitoring system, the system was suitable for scientific research and clinical detection related to AMR.

3Cpro of FMDV inhibits type II interferon-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation.

Foot-and-mouth disease virus (FMDV) has developed various strategies to antagonize the host innate immunity. FMDV Lpro and 3Cpro interfere with type I IFNs through different mechanisms. The structural protein VP3 of FMDV degrades Janus kinase 1 to suppress IFN-γ signaling transduction. Whether non-structural proteins of FMDV are involved in restraining type II IFN signaling pathways is unknown. In this study, it was shown that FMDV replication was resistant to IFN-γ treatment after the infection was established and FMDV inhibited type II IFN induced expression of IFN-γ-stimulated genes (ISGs). We also showed for the first time that FMDV non-structural protein 3C antagonized IFN-γ-stimulated JAK-STAT signaling pathway by blocking STAT1 nuclear translocation. 3Cpro expression significantly reduced the ISGs transcript levels and palindromic gamma-activated sequences (GAS) promoter activity, without affecting the protein level, tyrosine phosphorylation, and homodimerization of STAT1. Finally, we provided evidence that 3C protease activity played an essential role in degrading KPNA1 and thus inhibited ISGs mRNA and GAS promoter activities. Our results reveal a novel mechanism by which an FMDV non-structural protein antagonizes host type II IFN signaling.

Mechanisms and applications of probiotics in prevention and treatment of swine diseases.

Probiotics can improve animal health by regulating intestinal flora balance, improving the structure of the intestinal mucosa, and enhancing intestinal barrier function. At present, the use of probiotics has been a research hotspot in prevention and treatment of different diseases at home and abroad. This review has summarized the researchers and applications of probiotics in prevention and treatment of swine diseases, and elaborated the relevant mechanisms of probiotics, which aims to provide a reference for probiotics better applications to the prevention and treatment of swine diseases.

Novel p22 and p30 dual-proteins combination based indirect ELISA for detecting antibodies against African swine fever virus.

Introduction: African swine fever virus (ASFV) infection is one of the most complex and fatal hemorrhagic viral diseases, causing a devastating loss to the swine industry. Since no effective vaccine is available, prevention and control of ASFV heavily depends on early diagnostic detection. Methods: In this study, a novel indirect ELISA was established for detecting antibodies against ASFV using dual-proteins, p22 and p30. Recombinants p22 and p30 were expressed and purified from E.coli vector system by recombined plasmids pET-KP177R and pET-CP204L. p22 and p30 were mixed as antigens for developing the indirect ELISA. Results: Through optimizing coating concentrations of p30 and p22, coating ratio (p30: p22 = 1:3), and serum dilution (as 1:600), the established ELISA performed higher specificity, sensitivity, and repeatability against ASFV-positive serum. Furthermore, 184 clinical serum samples from suspected diseased pigs were verified the established ELISA in clinical diagnosis. The results showed that compared with two commercial ELISA kits, the established ELISA possessed higher sensitivity and almost uniform coincidence rate. Conclusion: The novel indirect ELISA based on dual-proteins p30 and p22 performed a valuable role in diagnostic detection of ASFV, providing a broad insight into serological diagnostic methods of ASFV.

Illumina high-throughput sequencing for the genome of emerging fowl adenovirus D species and C species simultaneously.

In recent years, clinical cases of inclusion body hepatitis (IBH) and hydropericardium syndrome (HPS) have been emerging and increasing in chicken flocks worldwide. Mixed infections with 2 or more fowl adenovirus (FAdV) serotypes were common in these cases. Herein, we collected a clinical sample that was positive for FAdV from 40-day-old broilers with IBH and HPS symptoms in Shandong province of China and determined the complete genome of FAdVs on the Illumina HiSeq4000 platform. The results showed that the sample contained 2 FAdV strains of D species and C species and named SD1763-1 and SD1763-2 respectively. The genome of SD1763-1 strain was 43,913 nt in length, with a G+C content of 53.51%, whereas SD1763-2 strain was 43,721 nt in length, with a G+C content of 54.87%. Sequence alignment and phylogenetic analysis revealed that strain SD1763-1 was clustered together with serotype 2/11 of FAdV-D, and SD1763-2 was clustered together with FAdV-4. There is no recombination between the genomes of the 2 viruses of FAdV-D and FAdV-C in the present study. This is the first report of obtaining 2 genomic sequences of FAdV strains simultaneously by direct use of deep sequencing in one clinical individual chicken sample, which provided direct evidence for mixed infections of adenovirus serotypes in the clinic and enriched the genome data to explore the geographic biomarkers and virulence signatures of the genus Aviadenovirus.

Complete genome and pathogenesis of a novel recombinant Senecavirus A isolate in P.R. China.

Senecavirus A (SVA), formerly called Seneca Valley virus (SVV) was first isolated from the USA in 2002. This study isolated an SVA strain from a pig herd in Shandong Province, PR China and designated it SVA-CH-SDGT-2017. The full-length genome, excluding the poly(A) tails of the SVA isolates, was 7280 nucleotides long, with the genomic organization resembling and sharing high nucleotide identities of 90.7-96.9 % with other previously reported SVA isolates. To investigate the pathogenicity of the SVA isolates, experimental infections of pigs were performed. The SVA strains successfully infected the pigs, as evidenced by the presence of virus shedding and robust serum neutralizing antibody responses. In addition, the contact-exposed experiment showed that the virus shedding of the contact-exposed pigs was approximately a 100-fold reduced compared to that of the inoculated group, indicating that the virus is capable of transmission to pigs. Our findings provide useful data for studying the pathogenesis and transmission of SVA in pigs.