Anti-PRV Monoclonal antibody, clone 1A8

Pseudorabies virus (PRV) can spread along the peripheral nerves near the site of infection in the animals, and gradually migrates into the central nervous system, where it leads to the development of brain lesions. The aim of this study was to investigate the dynamics of microglia after PRV inoculation. A mouse model inoculated with PRV was established to study the interactions between PRV and microglia, microglial recruitment, and polarization effects. The mice were subcutaneously inoculated with different doses of PRV-Bartha K61 vaccine strain. The obtained results showed that mouse mortality rates increased with the applied doses of virus, and brain lesions, located in the brain tail and brain stem, were observed in each investigated group. Inflammatory cells were shown to infiltrate through the vasculature into perivascular cuff, and the number of microglia was increased as well. Mouse group treated with a medium infection dose demonstrated a high survival rate while developing serious brain lesions, and therefore, this dose was selected for further experiments. Immunohistochemistry, flow cytometry, and confocal laser scanning microscopy were used to analyze PRV-microglia interactions. After PRV inoculation, proliferating cell nuclear antigen (Pcna) and Iba1 double-positive cells were observed in the brain lesions, together with the activated microglia, suggesting that PRV can induce microglial proliferation and activation. Furthermore, 5-bromo-deoxy-uridine (BrdU) labeling demonstrated that microglial cells did not proliferate in situ and the proliferating cells originated from peripheral blood monocytes, mainly from the inflammatory monocytes (Ly6C(high)). In addition, microglia polarized into both M1 and M2 phenotypes by PRV infection. The results obtained in this study may help understand the development of pseudorabies infection and help improve the treatment, by recruiting and enhancing immune response.

This study described a successful application of the Quality by Design (QbD) approach to pseudorabies virus (PRV) production process development in a fixed-bed bioreactor using the serum-free medium (SFM). The innovated tube-fixed-bed bioreactor was used as a scale-down model of the fixed-bed bioreactor for process development. Risk analysis was performed using Ishikawa diagram combined with failure mode effects analysis (FMEA). The comparative experiment was performed to screen proper medium for adherent African green monkey kidney (Vero) cells from three commercially available SFMs (VP-SFM, ProVERO-1 and Vero-A). The Vero-A medium showed as an outstanding one for further study. The PRV titer in harvest medium was consider as Critical Quality Attribute (CQA) and the Critical Process Parameters (CPPs) [time of infection (TOI), multiplicity of infection (MOI) and initial inoculation cell density] ranked high with risk priority number (RPN) were taken into design of experiment (DoE) methodology. Then prediction model of PRV production process was established and a robust PRV production process was explored. Under the robust setpoint conditions, the Xcell 1 L laboratory-scale fixed-bed bioreactor yielded PRV titer up to 7.87 log(10) TCID50/mL at 3 dpi, which was comparable with that in the tube-fixed-bed bioreactor. Combination of the tube-fixed-bed bioreactor and QbD approach could further accelerate the development of a robust virus production process.