Bovine respiratory disease complex (BRDC) is a comprehensive disease in cattle caused by various viral and bacterial infections. Among them, bovine herpesvirus type I (BoHV−1) and bovine viral diarrhea virus (BVDV) play important roles and have caused huge financial losses for the cattle industry worldwide. At present, vaccines against BRDC include trivalent attenuated BoHV−1, BVDV−1, and BVDV−2 live vaccines, BoHV−1 live attenuated vaccines, and BoHV−1/BVDV bivalent live attenuated vaccines, which have limitations in terms of their safety and efficacy. To solve these problems, we optimized the codon of the BVDV−1 E2 gene, added the signal peptide sequence of the BoHV−1 gD gene, expressed double BVDV−1 E2 glycoproteins in tandem at the BoHV−1 gE gene site, and constructed a BoHV−1 genetics-engineered vectored vaccine with gE gene deletion, named BoHV−1 gE/E2−Linker−E2⁺ and BoHV−1 ΔgE. This study compared the protective effects in BoHV−1, BoHV−1 ΔgE, BoHV−1 gE/E2−Linker−E2⁺, and BVDV−1 inactivated antigen immunized guinea pigs and calves. The results showed that BoHV−1 gE/E2−Linker−E2⁺ could successfully induce guinea pigs and calves to produce specific neutralizing antibodies against BVDV−1. In addition, after BoHV−1 and BVDV−1 challenges, BoHV−1 gE/E2−Linker−E2⁺ can produce a specific neutralizing antibody response against BoHV−1 and BVDV−1 infections. Calves immunized with this type of virus can be distinguished as either vaccinated animals (gE^-) or naturally infected animals (gE⁺). In summary, our data suggest that BoHV−1 gE/E2−Linker−E2⁺ and BoHV−1 ΔgE have great potential to prevent BVDV−1 or BoHV−1 infection.

Bovine herpesvirus type I (BoHV-1) is an important pathogen that causes respiratory disease in bovines. The disease is prevalent worldwide, causing huge economic losses to the cattle industry. Gene-deficient vaccines with immunological markers to distinguish them from wild-type infections have become a mainstream in vaccine research and development. In order to knock out the gE gene BoHV-1, we employed the CRISPR/Cas9 system. Interesting phenomena were observed at the single guide RNA (sgRNA) splicing site, including gene insertion, gene deletion, and the inversion of 5′ and 3′ ends of the sgRNA splicing site. In addition to the deletion of the gE gene, the US9 gene, and the non-coding regions of gE and US9, it was found that the US4 sequence, US6 sequence, and part of the US7 sequence were inserted into the EGFP sgRNA splicing site and the 3′ end of the EGFP sequence was deleted. Similar to the BoHV-1 parent, the BoHV-1 mutants induced high neutralizing antibodies titer levels in mice. In summary, we developed a series of recombinant gE-deletion BoHV-1 samples using the CRISPR/Cas9 gene editing system. The mutant viruses with EGFP⁺ or EGFP⁻ will lay the foundation for research on BoHV-1 and vaccine development in the future.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen that causes severe abortions in sows and high piglet mortality, resulting in huge economic losses to the pig industry worldwide. The emerging and novel PRRSV isolates are clinically and biologically important, as there are likely recombination and pathogenic differences among PRRSV genomes. Furthermore, the NADC34-like strain has become a major epidemic strain in some parts of China, but the characterization and pathogenicity of the latest strain in Inner Mongolia have not been reported in detail. In this study, an NADC34-like strain (CHNMGKL1-2304) from Tongliao City, Inner Mongolia was successfully isolated and characterized, and confirmed the pathogenicity in pigs. The phylogenetic tree showed that this strain belonged to sublineage 1.5 and had high homology with the strain JS2021NADC34. There is no recombination between CHNMGKL1-2304 and any other domestic strains. Animal experiments show that the CHNMGKL1-2304 strain is moderately virulent to piglets, which show persistent fever, weight loss and high morbidity but no mortality. The presence of PRRSV nucleic acids was detected in both blood, tissues, nasal and fecal swabs. In addition, obvious pathological changes and positive signals were observed in lung, lymph node, liver and spleen tissues when subjected to hematoxylin–eosin (HE) staining and immunohistochemistry (IHC). This report can provide a basis for epidemiological investigations and subsequent studies of PRRSV.

Koumiss is a traditional fermented dairy product in Inner Mongolia pastoral areas, which is deeply loved by the local people; however, there has been little research on the characteristics of probiotics. This study comprehensively explored the properties of potential probiotics in koumiss, combining in vitro assays and whole-genome sequencing. The biochemical identification and phylogenetic tree results showed that the branches of this strain were close to Lacticaseibacillus paracasei, indicating that the strain was L. paracasei. The agar diffusion assay showed that the strain could effectively inhibit the growth of pathogenic bacteria. We have also identified the CHAP structural domain at the genomic level, which may be associated with the antibacterial activity of this strain. The strain was well tolerated in a simulated gastrointestinal environment, growing well at pH = 2.5, 0.3% bile salts, and 5% NaCl while exhibiting hydrophobicity, aggregation, and antioxidant properties. In vitro experiments and genome, levels showed that resistance (resistance genes) to the antibiotics used in this study was not present in this strain. In addition, we did not observe toxic effects in acute oral administration in mice, and no virulence genes were identified at the genomic level. Therefore, the strain has the potential for probiotic development.

Interleukin-18 binding protein (IL-18BP), a natural regulator molecule of the pro-inflammatory cytokine interleukin-18 (IL-18), plays an important role in regulating the expression of the cellular immunity factor interferon-γ (IFN-γ). In a previous RNA-seq analysis of porcine alveolar macrophages (PAM) infected with the TIM and TJ strains of porcine reproductive and respiratory syndrome virus (PRRSV), we unexpectedly found that the mRNA expression of porcine interleukin 18-binding protein (pIL-18BP) in PAM cells infected with the TJM strain was significantly higher than that infected with the TJ strain. Studies have shown that human interleukin-18 binding protein (hIL-18bp) plays an important role in regulating cellular immunity in the course of the disease. However, there is a research gap on pIL-18BP. At the same time, PRRSV infection in pigs triggers weak cellular immune response problems. To explore the expression and the role of pIL-18BP in the cellular immune response induced by PRRSV, we strived to acquire the pIL-18BP gene from PAM or peripheral blood mononuclear cell (PBMC) with RT-PCR and sequencing. Furthermore, pIL-18BP and pIL-18 were both expressed prokaryotically and eukaryotically. The colocalization and interaction based on recombinant pIL-18BP and pIL-18 on cells were confirmed in vitro . Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in pigs with different PRRSV infection states to interpret the biological function of pIL-18BP in vivo . The results showed there were five shear mutants of pIL-18BP. The mutant with the longest coding region was selected for subsequent functional validation. First, it was demonstrated that TJM-induced pIL-18BP mRNA expression was higher than that of TJ. A direct interaction between pIL-18BP and pIL-18 was confirmed through fluorescence colocalization, bimolecular fluorescent complimentary (BIFC), and co-immunoprecipitation (CO-IP). pIL-18BP also can regulate pIFN-γ mRNA expression. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in different PRRSV infection states. Surprisingly, both mRNA and protein expression of pIL-18 were suppressed. These findings fill the gap in understanding the roles played by pIL-18BP in PRRSV infection and provide a foundation for further research. IMPORTANCE: PRRSV-infected pigs elicit a weak cellular immune response and the mechanisms of cellular immune regulation induced by PRRSV have not yet been fully elucidated. In this study, we investigated the role of pIL-18BP in PRRSV-induced immune response referring to the regulation of human IL-18BP to human interferon-gamma (hIFN-γ). This is expected to be used as a method to enhance the cellular immune response induced by the PRRSV vaccine. Here, we mined five transcripts of the pIL-18BP gene and demonstrated that it interacts with pIL-18 and regulates pIFN-γ mRNA expression. Surprisingly, we also found that both mRNA and protein expression of pIL-18 were suppressed under different PRRSV strains of infection status. These results have led to a renewed understanding of the roles of pIL-18BP and pIL-18 in cellular immunity induced by PRRSV infection, which has important implications for the prevention and control of PRRS.

Trefoil factor 3 (TFF3) and epidermal growth factor (EGF) exert a promotive effect on the functions of intestinal epithelial cells and offer protection to the intestinal mucosa. Lactobacillus paracasei can ameliorate intestinal mucosal damage. In this study, pPG-pTFF3/27-2, pPG-pEGF/27-2, and pPG-pTE/27-2 were constructed to express porcine TFF3, EGF, and a fusion protein (pTE). Functional assays showed they promoted Immortalized Porcine Enterocyte Cell line J2 (IPEC-J2) proliferation and migration, with pTE having a greater migratory effect. In dextran sulfate sodium (DSS)-induced colitis mice, oral administration of pPG-pTE/27-2 reduced colitis, improved mucosal integrity, increased the expression of tight-junction proteins and the serum level of Interleukin-10 (IL-10), and decreased the levels of pro-inflammatory Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), and Interleukin-1β (IL-1β). These results imply that recombinant L. paracasei 27-2 strains engineered to express pTFF3 and pEGF represent a promising approach for augmenting intestinal epithelial cell function and facilitating mucosal restitution, and they possess significant potential in the treatment of intestinal mucosal injury and inflammatory bowel disease (IBD).