| Porcine reproductive and respiratory syndrome(PRRS)is an infectious disease characterized by sow abortion,piglet respiratory disorders,and immunosuppression caused by the PRRS virus(PRRSV),which is classified as a second-class infectious disease in China.PRRS is one of the important diseases affecting the global pig industry,and it still exists widely in the global pig herd.Industry experts prefer to prevent the disease in advance rather than treat it after it appears,and most vaccines used in the market are traditional live attenuated vaccines and inactivated vaccines,but these two traditional vaccines have some insurmountable shortcomings in effectiveness and safety.Up to now,there is no specific drug for the treatment of this disease.In our laboratory,a monoclonal hybridoma cell line secreting PRRSV-GP3 protein was obtained,and its clone number was "Mab-PN9cx3".In vitro studies showed that Mab-PN9cx3 had broad-spectrum reactivity and neutralizing activity against different PRRSV strains.This study aimed to prepare the monoclonal antibody,and confirm its reactivity and neutralizing activity by in vitro tests.Subsequently,to evaluate the therapeutic effect of monoclonal antibodies on PRRSV infected piglets,and finally to determine its protective effect on heterologous PRRSV strains,which provided support for the subsequent development of new PRRSV prevention and control measures.The contents and results of this study are as follows:1.Preparation of Mab-pn9cx3 monoclonal antibody and confirmation of reactivity and neutralizing activity.The hybridoma cell line secreting Mab-PN9cx3 monoclonal antibody was injected into the abdominal cavity of mice and ascites were collected.The protein was extracted by ammonium sulfate precipitation method and then purified by Protein G Resin chromatography to obtain high purity monoclonal antibody Mab-PN9cx3.IFA test was applied to prove that Marc-145 cells infected by either PRRSV-1 or PRRSV-2 strains could both be recognized by the Mab-PN9cx3,which confirmed that the Mab-PN9cx3 possesses broad-spectrum reactivity against PRRSV.Then,the protein and transcription levels of PRRSV-N were detected by Western Blot and qPCR in cell lysates of different concentrations of Mab-PN9cx3 interacting with PRRSV-1 and PRRSV-2 viruses.The results showed that the Mab-PN9cx3 could significantly inhibit the proliferation of PRRSV in vitro.Meanwhile,the results of virus titration showed that the virus titer decreased obviously after adding the Mab-PN9cx3,and the corresponding virus titer decreased with the increase of Mab-PN9cx3 concentration,which reconfirmed that the Mab-PN9cx3 possesses broad-spectrum neutralizing activity against PRRSV in vitro.2.Evaluation of the protective effect of Mab-PN9cx3 against heterologous PRRSV infection in pigletsFour-week-old piglets were selected to be challenged with the HP-PRRSV-JXA1 strain and NADC30-HNhx-like strain respectively.After the challenge,the piglets were treated with Mab-PN9cx3 monoclonal antibodies.The clinical symptoms,body temperature,organ pathological changes after autopsy were observed and recorded,and PRRSV virus load in hilar lymph nodes were detected.The results showed that the clinical symptoms of the challenge+Mab-PN9cx3 treatment group were relieved while the symptoms of lingering fever were obvious in the challenge+non-treatment group.At the same time,no obvious pathological changes were found in the lungs and lymph tissues in the challenge+Mab-PN9cx3 treatment group after the autopsy,and only mild pathological damage was observed in the microscopic pathological sections of the lungs,while the lungs in the non-treatment challenge group and the isotype antibody control group revealed obvious sarcoidosis or consolidation,and typical interstitial pneumonia lesions could be seen in the microscopic pathological sections after H.E.staining.Immunohistochemical examination of hilar lymph nodes showed that the MabPN9cx3 could significantly inhibit the proliferation of PRRSV in the lymph tissue of piglets after Mab-PN9cx3 treatment,and had significant inhibitory effects on different genotypes of PRRSV strains.The results showed that the monoclonal antibody could reduce the clinical symptoms,as well as reduce the viral load in tissues and organs and simultaneously decrease the pathological lesions in the treatment of PRRSV infection in piglets.3.Protective mechanism of Mab-PN9cx3 against heterologous PRRSV infection in pigletsIn this study,the viral load and transcriptome analysis of PAMs cells in infected piglets were carried out to determine the activation pathway of the Mab-PN9cx3 and the genes changed by reversing PRRSV infection.The results showed that the viral load of the PAMs in the Mab-PN9cx3 treatment group was significantly lower than that in the non-treatment group on the 21st day after the challenge,which was consistent with the significant reduction of animal clinical symptoms,pathological damage,and microscopic lesions of the lung and lymphatic tissue after the Mab-PN9cx3 treatment,which showed that this was an important indicator for the Mab-PN9cx3 to protect against PRRSV challenge.However,the MabPN9cx3 has no obvious effect on the improvement of viremia after treatment.Transcriptome analysis showed that the genes whose expression profiles were reversed were rarely correlative with the pro-inflammatory cytokines,but the genes related to the steroid hormone reaction pathway were up-regulated and reversed after Mab-PN9cx3 treatment.Further analysis of PAMs transcriptome showed that a large number of cytokines related to M1 activation,such as IL-12,iNOS,TNF-α,IL-1β,and IL-6,were up-expressed.Meanwhile,a large number of pGM-CSF proteins were detected in the serum of pigs treated with Mab-PN9cx3,and it was speculated that the large amount of pGM-CSF was related to the activation of PAMs in animals towards M1,which accelerated the ability of cells to clear viruses and further activated the immune system to fight against PRRSV infection. |
PDF Full Text Request