Study On Immune Response Characteristics Of Swine Influenza Virus GX18ca Strain

The pathogeny of swine influenza virus?SIV?belongs to genus Influenza-virus A of the Orthomyxoviridae family,which can cause symptoms such as high fever,cough,mental fatigue and breathing difficulties in swine herds.The disease has a high morbidity rate and a low mortality rate,so it did not arise people's attention to pig vaccination.Both human and avian influenza viruses can be transmitted to pigs since cells of the porcine respiratory tract contain both both human and avian influenza virus receptors,,and swine influenza virus can also be transmitted to humans and birds.Therefore,the protection of swine influenza is of great significance in preventing the spread of influenza viruses across species,preventing the global pandemic of influenza,and maintaining public health.Currently,vaccination is the main method to control and prevent the epidemic and spread of influenza.The commercial vaccines are mainly inactivated vaccines,which have high safety and strong specificity,and also have shortcomings such as long production cycle,complicated production process,and it could not induce an effective immune response.Immunologically cold-adapted attenuated influenza vaccine is superior to inactivated vaccine.Immunization through the nasal route can induce mucosal immune,humoral immune and cellular immune response,induce broadly cross-reactive protection against heterologous viruses.In our study,the cold adaptation and temperature sensitivity of the swine influenza virus EA H1N1subtype GX18ca strain were identified by EID₅₀ comparison at the appropriate temperature and the limited temperature.According to the whole genome sequencing,compared with the parental wild-type strain,GX18ca strain had amino acid changes at six sites,which were distributed on PB2,PB1,HA and NA genes.Moreover,the mutation sites were different from those of previously reported cold-adapted donor strains.The mouse infectivity test found that after the GX18ca strain was challenged,there was no virus replication in the turbinate of the mouse,and the virus titer in the lung was significantly lower than the parental wild-type strain.The paraffin sections of HE stained of the lung showed no obvious pathological changes.This shows that the GX18ca strain is highly attenuation and safety in mice.The GX18ca should also be considered for live vaccine candidate strain and we conducted a challenge protection test to evaluate its safety and effectiveness.Based on epidemiological investigation and genetic evolution analysis,A/swine/Liaoning/972/2016?H1N1?and A/Sichuan 1/2009?H1N1?of the representative strains of EA H1N1 and 2009/H1N1 subtypes were selected as challenge strains.It is preliminarily judged that there are differences in antigenicity between the A/swine/Guangxi/18/2011?H1N1?,A/swine/Liaoning/972/2016?H1N1?and A/Sichuan/1/2009?H1N1?according to the serological results.The results showed that the cold-adapted strain GX18ca generally induce broadly cross-reactive protection against A/swine/Liaoning/972/2016?H1N1?strain within the same lineage,and complete protection against the heterologous lineage influenza virus A/Sichuan/1/2009.We immunized mice with a dose of 10⁶EID₅₀/mL.The serological test results showed that the serum HI and neutralizing antibody titers against heterologous influenza viruses were low,however,ELISA test results show that the vaccine can induce systemic humoral immunity and local mucosal immunity,and the cross-reactive activity of mucosal antibody is stronger than systemic reactions.We used flow cytometry to detect the number of relevant specific T cells.The results showed that GX18ca strain can induce a large number of specific T cells against heterologous strains and stimulate cellular immune responses.We used EA H1N1 subtype swine influenza virus GX18ca strain as a model virus and explored its immune protection effect was preliminarily.These data in the present study demonstrate this strain has good safety and immunogenicity,and can provide cross-immunization protection against EA H1N1subtype and H1N1/2009 subtype influenza virus,In addition,it provides alternative strategies for the development of new influenza attenuated vaccines and insight understanding of the immune protection mechanism of cold-adapted attenuated vaccines.