Eukaryotic expression of GP5 and M protein of porcine reproductive and respiratory syndrome virus and immunogenicity evaluation / 生物工程学报

In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.

The I226R protein of African swine fever virus inhibits the cGAS-STING-mediated innate immune response / 生物工程学报

This study aimed to explore the mechanism of how African swine fever virus (ASFV) I226R protein inhibits the cGAS-STING signaling pathway. We observed that I226R protein (pI226R) significantly inhibited the cGAS-STING-mediated type Ⅰ interferons and the interferon-stimulated genes production by dual-luciferase reporter assay system and real-time quantitative PCR. The results of co-immunoprecipitation assay and confocal microscopy showed that pI226R interacted with cGAS. Furthermore, pI226R promoted cGAS degradation through autophagy-lysosome pathway. Moreover, we found that pI226R decreased the binding of cGAS to E3 ligase tripartite motif protein 56 (TRIM56), resulting in the weakened monoubiquitination of cGAS, thus inhibiting the activation of cGAS and cGAS-STING signaling. In conclusion, ASFV pI226R suppresses the antiviral innate immune response by antagonizing cGAS, which contributes to an in-depth understanding of the immune escape mechanism of ASFV and provides a theoretical basis for the development of vaccines.

Establishment of RIG-I knockout 293T cell line and its effect on the replication of influenza B virus / 生物工程学报

The CRISPR/Cas9 gene editing technology directs Cas9 protein to recognize, bind and cleave the target site specifically by using artificial single-guide RNA (sgRNA), through non-homologous end joining or homologous end-recombinant repair mechanisms of cells, which can be engineered to knockout or knock-in of genomes. RIG-I is a pattern recognition receptor that recognizes the 5'-triphosphate-containing RNA in the cytoplasm and activates IRF3/7 and NF-κB by interacting with the downstream signaling molecule MAVS, thus initiating the expression of type I interferons and inflammatory factors. Previous studies found that influenza B virus (IBV) can up-regulate the expression of RIG-I. In the present study, to explore whether RIG-I is the major receptor for IBV to active the antiviral innate immune response and its effect on IBV replication, RIG-I gene in 293T cells was knocked out by CRISPR-Cas9 system, and a stable RIG-I knockout 293T (RIG-I(-/-) 293T) cell line was screened by puromycin pressure. The results of Western blotting showed that RIG-I was not expressed in this cell line after IBV or Sendai virus (SeV) infection, indicating that the RIG-I(-/-) 293T cell line was successfully constructed. The transcription levels of interferons, inflammatory factors and interferon-stimulated genes in RIG-I(-/-) 293T cells which were infected by IBV decreased significantly compared with those in wild-type 293T cells. Moreover, the phosphorylation of p65 and IRF3 were not detected in IBV or SeV infected RIG-I(-/-) 293T cells. It is indicated that the expression of cytokines mainly depends on the RIG-I-mediated signaling pathway at the early stage of IBV infection. Furthermore, the multi-step growth curves of IBV in the wild type and RIG-I(-/-) 293T cells showed that RIG-I inhibited the replication of IBV. Collectively, the RIG-I knockout 293T cell line was successfully constructed. We found that RIG-I is the main receptor for IBV to active the antiviral innate immune response and is critical for inhibiting IBV replication, which lays the foundation for further study of IBV infection mechanism.

Research advance of influenza B viruses / 生物工程学报

Influenza B virus (IBV) is a segmented negative-strand RNA virus, which often causes local outbreak or seasonal epidemic along with influenza A virus (IAV) in the world. It is pathogenic to children, teenagers and elderly people and has a higher mortality rate in children and adolescents, so it poses a serious threat to public health and health. IBV is more likely to cause complications than IAV and the disease burden of IBV even exceeds IAV in the epidemic season. Recently, especially after winter of 2017, IBV has become the dominant strain in many areas of our country and seriously affects people's health. In view of this, this article reviews the structure, epidemiology, immunology and prevention of IBV, aiming at enhancing public's perceptions of the virus and providing reference for making strategies for prevention and control of influenza B.