Acquisition of different transcriptional shear mRNA and biological function of porcine interleukin 18 binding protein in PRRSV infection.

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.IMPORTANCEPRRSV-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.

Evolution Characterization and Pathogenicity of an NADC34-like PRRSV Isolated from Inner Mongolia, China.

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.

Concurrent Gene Insertion, Deletion, and Inversion during the Construction of a Novel Attenuated BoHV-1 Using CRISPR/Cas9 Genome Editing.

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.

Recombinant Bovine Herpesvirus Type I Expressing the Bovine Viral Diarrhea Virus E2 Protein Could Effectively Prevent Infection by Two Viruses.

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.

Subunit vaccine based on glycoprotein B protects pattern animal guinea pigs from tissue damage caused by infectious bovine rhinotracheitis virus.

Infectious bovine rhinotracheitis (IBR) is caused by Bovine herpesvirus type 1 (BoHV-1), which seriously threatens the global cattle industry. Only vaccination to improve immunity is the most direct and effective means to prevent IBR. Attempts are being made to use subunit vaccines, deleted or recombinant viral vaccines to reduce or eradicate IBR. For investigating the immunological characteristics of glycoprotein B subunit vaccine in pattern animal guinea pigs, the partial glycoprotein B (gB) of BoHV-1 with dominant antigenic characteristic was selected. A recombinant prokaryotic expression vector pET-32a-gB with the truncated gB gene was constructed, expressed, identified and the purified proteins were used to immunize guinea pigs. The immune effect of the subunit vaccine was assessed by monitoring clinical symptoms, viral load, antibody secretion, and histopathological changes. The results indicated that guinea pigs immunized with the gB subunit vaccine produced high levels of anti-gB antibodies and virus-neutralizing antibodies. The gB subunit vaccine significantly reduced viral shedding and lung tissue damage after IBRV challenge. The animals inoculated the gB subunit vaccine also had less virus reactivation. Its protective effect on viral shedding and tissue damage was similar to that of inactivated BoHV-1 vaccine. This work is a proof-of-concept study of subunit vaccine-induced protection against BoHV-1. And it is expected to be a candidate vaccine for the prevention of IBR.

Recent advances in the study of NADC34-like porcine reproductive and respiratory syndrome virus in China.

Since porcine reproductive and respiratory syndrome virus (PRRSV) was first described in China in 1996, several genetically distinct strains of PRRSV have emerged with varying pathogenicity and severity, thereby making the prevention and control of PRRS more difficult in China and worldwide. Between 2017 and 2021, the detection rate of NADC34-like strain in China increased. To date, NADC34-like strains have spread to 10 Chinese provinces and have thus developed different degrees of pathogenicity and mortality. In this review, we summarize the history of NADC34-like strains in China and clarify the prevalence, genomic characteristics, restriction fragment length polymorphisms, recombination, pathogenicity, and vaccine status of this strain in China. In so doing, this study aims to provide a basis for the further development of prevention and control measures targeting the NADC34-like strain.