| Objective:The human lung adenocarcinoma cells A549(human A549)and pig alveolar macrophages PAM(pig PAM)are infected with two strains for H3N2 subtype of SIV,one is SW2783 reassorted with pam/ 09 H1N1 virus in internal genes and another is SW650 unreassorted with pam / 09 H1N1 virus in internal genes.Both SW viruses are used to study the virus properties and the expressions of mi RNA93 and mi RNA192 after virus infection.The study will provides some clues to explore the regularity of swine influenza virus infection passing across species.Methods:1.SW2783 and SW650 strains isolated and identified in our laboratory were used to infect human A549 and pig PAM cells respectively.CPE,HA and TCID50 tests were performed after human A549 and pig PAM cell lines were infected with SW2783 and SW650 respectively.The differences of replication,virulence and changes for viruses were compared after SW2783 and SW650 were used to infect human A549 and pig PAM cells.2.Expressions of mi RNA 93 and mi RNA 192 were detected by q RT-PCR.3.Expression of viral NP,HA after human A549 cells infected were detected by Western blot.4.Expression of IFN-? secreted by the human A549 cells was detected by ELISA.Results:1.From the results of TCID50 and HA,we inferred that the infection of SW2783 in human A549 and pig PAM cells showed a gradual adaptation process,suggesting that SW2783 could have a potential to go across species to infect humans,while SW650 did not show adaptability to tested cells,which demonstrated the trend of decreased infection ability,even the lost of ability to infect cells,suggesting that the ability of SW650 to be used to infect human A549 and pig PAM cells is limited;CPE experiment also showed the similar trend,CPE began to appear at 24 h,but the most obvious result was appeared at 48 h,a lot of cells died at 72 h.It was speculated that there were some special mi RNAs during the adaptive infection and they would affect the ability of viruses to replication and infection in the host cells.2.The results of quantitative RT-PCR were showed as the following: After SW2783 was used to infect human A549 cells,the expressions of mi RNA192 and mi RNA93 in the infection group were highest at 48 h after infection,and the difference was statistically significant(p=0.0020;p=0.0050);Compared with negative control group,after SW650 was used to infect human A549 cells,the expression of mi RNA93 was highest at 72 h,and the difference was statistically significant(p=0.0007).The expression of mi RNA192 was highest at 48 h,and the difference was statistically significant(p=0.0020).After that,mi RNA93 and mi RNA192 inhibitors were added,the expressions of mi RNA93 and mi RNA192 in the inhibitor group were significantly lower than those in the infection group.3.The results of Western blot were showed as the following: After SW2783 was used to infect human A549 cells,the expressions of NP and HA were highest at 24 h,and the difference between the infected group and the negative control was statistically significant(p=0.0000;p=0.0000);Compared with the negative control group,after SW650 was used to infect human A549 cells,the expressions of NP and HA were highest at 48 h,the difference was statistically significant(p=0.0000;p=0.0000);After mi RNA93 inhibitor was added,SW2783 was used to infect human A549 cells for 48 hs,the expressions of NP and HA in the inhibitor infection group were all up-regulated compared with inhibitor control and the difference was statistically significant(p=0.0006;p=0.0000).After mi RNA192 inhibitor was added,SW2783 was used to infect human A549 cells for 48 hs,there was no significant difference in the expression of NP between the inhibitor infection group and the inhibitor control,but compared with protein expression,the former was significantly down-regulated,and the difference was statistically significant(p=0.0002).After SW650 was used to infect human A549 cells for 48 hs the NP expression in the inhibitor infection group was up-regulated compared to the inhibitor control,with a statistically significant difference(p=0.0400).For the comparison of the HA expression between the inhibitor control and the inhibitor infection group,there was not statistically significance;After mi RNA192 inhibitor was added,for the comparison between the inhibitor control and the inhibitor infection group,the expression of HA increased,NP decreased instead,the difference was significant(p=0.0000).4.The inflammatory factor detection was showed as the following: after SW2783 and SW650 were used to infect human A549 cells respectively,for the comparison of the levels of IFN-? between the inhibitor control and the uninfected group at 8h,48 h and 72 h,the levels of IFN-? were showed as an upward trend;After the SW650 was used to infect human A549 cells with adding mi RNA93 inhibitor,the level of IFN-? was significantly increased in the inhibitor group compared with the inhibitor control,and the level of IFN-? at 48 h was highest,the difference was statistically significant(p=0.0020);after SW2783 was used to infect A549 cells,the level of IFN-? was significantly increased in the inhibitor group compared with the inhibitor control,and the level of IFN-? was highest in the inhibitor group at 48 h,and the difference was statistically significant(p=0.0004).Conclusions:1.SW2783 is one of strains for H3N2 swine influenza virus which has a killing effect on human A549 cells and pig PAM cells,indicating that the virus has the affinity to infect the respiratory tract cells of mammals and has a potential to infect humans;SW650 is one of strains for swine influenza virus which does not reassort with the H3N2 subtype of the pdm/09 H1N1 in internal genes and it has limited ability to infect human A549 cells and pig PAM cells.2.mi RNA93 is involved in the NP synthesis of SW2783 and SW650,while mi RNA93 is only involved in the HA synthesis of SW2783,but mi RNA93 has no obvious effect on the HA synthesis of SW650;mi RNA192 can positively regulate the HA synthesis of SW650,but it can negatively regulate the HA protein and NP synthesis of SW2783.3.Down-regulation of mi RNA93 can target the RNA of influenza virus,to increase virus replication and to enhance virus virulence,and it can promote host cells infected by viruses to secrete inflammatory factor IFN-?. |
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