Construction of an infectious cloning system of porcine reproductive and respiratory syndrome virus and identification of glycoprotein 5 as a potential determinant of virulence and pathogenicity.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection of pigs causes a variety of clinical manifestations, depending on the pathogenicity and virulence of the specific strain. Identification and characterization of potential determinant(s) for the pathogenicity and virulence of these strains would be an essential step to precisely design and develop effective anti-PRRSV intervention. In this study, we report the construction of an infectious clone system based on PRRSV vaccine strain SP by homologous recombination technique, and the rescue of a chimeric rSP-HUB2 strain by replacing the GP5 and M protein-coding region from SP strain with the corresponding region from a highly pathogenic strain PRRSV-HUB2. The two recombinant viruses were shown to be genetically stable and share similar growth kinetics, with rSP-HUB2 exhibiting apparent growth and fitness advantages. Compared to in cells infected with PRRSV-rSP, infection of cells with rSP-HUB2 showed significantly more inhibition of the induction of type I interferon (IFN-ß) and interferon stimulator gene 56 (ISG56), and significantly more promotion of the induction of proinflammatory cytokines IL-6, IL-8, ISG15 and ISG20. Further overexpression, deletion and mutagenesis studies demonstrated that amino acid residue F16 in the N-terminal region of the GP5 protein from HUB2 was a determinant for the phenotypic difference between the two recombinant viruses. This study provides evidence that GP5 may function as a potential determinant for the pathogenicity and virulence of highly pathogenic PRRSV.

Molecular Signatures in Swine Innate and Adaptive Immune Responses to African Swine Fever Virus Antigens p30/p54/CD2v Expressed Using a Highly Efficient Semliki Forest Virus Replicon System.

African swine fever virus (ASFV) causes a devastating viral hemorrhagic disease in domestic pigs and Eurasian wild boars, posing a foremost threat to the swine industry and pig farming. The development of an effective vaccine is urgently needed, but has been hampered by the lack of an in-depth, mechanistic understanding of the host immune response to ASFV infection and the induction of protective immunity. In this study, we report that immunization of pigs with Semliki Forest Virus (SFV) replicon-based vaccine candidates expressing ASFV p30, p54, and CD2v, as well as their ubiquitin-fused derivatives, elicits T cell differentiation and expansion, promoting specific T cell and humoral immunity. Due to significant variations in the individual non-inbred pigs in response to the vaccination, a personalized analysis was conducted. Using integrated analysis of differentially expressed genes (DEGs), Venn, KEGG and WGCNA, Toll-like receptor, C-type lectin receptor, IL17 receptor, NOD-like receptor and nucleic acid sensor-mediated signaling pathways were demonstrated to be positively correlated to the antigen-stimulated antibody production and inversely correlated to the IFN-γ secreting cell counts in peripheral blood mononuclear cells (PBMCs). An up-regulation of CIQA, CIQB, CIQC, C4BPA, SOSC3, S100A8 and S100A9, and down-regulation of CTLA4, CXCL2, CXCL8, FOS, RGS1, EGR1 and SNAI1 are general in the innate immune response post-the second boost. This study reveals that pattern recognition receptors TLR4, DHX58/DDX58 and ZBP1, and chemokines CXCL2, CXCL8 and CXCL10 may play important roles in regulating this vaccination-stimulated adaptive immune response.

Suspension culture of Marek's disease virus and evaluation of its immunological effects.

Marek's disease virus (MDV) is a cell-associated α-herpesvirus of chickens. It is difficult to grow MDV in suspension culture. Therefore, MDV vaccines are currently produced using adherent primary chicken embryo fibroblasts, and on a large scale this is labour-intensive and costly. In this study, the CVI988 strain of MDV was inoculated into chicken fibroblast cell line UMNSAH/DF-1 (DF-1) cultured by microcarrier suspension for the proliferation experiment. Moreover, the effects of culture conditions, such as inoculation method, multiplicity of infection (MOI), microcarrier concentration, and pH value, on the proliferation of MDV were investigated. The results demonstrated that the maximum viral load of 64.76 ± 2.64 × 106 PFU/flask in a working volume of 100 ml could be obtained using synchronous cell seeding and inoculation method at an MOI of 0.02 and a microcarrier concentration of 5 g/l at pH 7.2. At the same time, the CVI988/DF-1 vaccines prepared by the microcarrier culture process and the traditional adherent cell culture process (CVI988/Rispens) were compared through bird experiments. We found a protective rate of 94.4% using the CVI988/DF-1 vaccine with specific pathogen-free chickens that was equivalent to that of the commercial vaccine CVI988/Rispens (protection rate of 94.1%). In this study, the MDV CVI988/DF-1 vaccine prepared by the microcarrier suspension culture of DF-1 cells could provide effective immune protection for specific pathogen-free chickens, providing a reference for the prevention and control of MD and further development of a large-scale bioreactor for producing the MD vaccine.