The Construction And Biological Characterisics Analysis Of Replication-incompetent 2009 Pandemic A/H1N1 Influenza Virus

It has been one hundred years from the influenza pandemic in 1918,influenza was threating the human life and health constantly,and bringing a huge economic loss.The human urge to know the mechanisms of influenza virus replication and transmission,and to develop a universal influenza vaccine.In the late 1960s,the molecular biology techniques progressed rapidly.Gene recombinant technology has been widely applied.Based on the gene recombination technology,influenza reverse genetics technology developed rapidly.The development of influenza reverse genetics technology provides a strong technical support to explore the mechanism in influenza virus replication transmission,pathogenicity and to develop influenza vaccine.In the 1990s',scientist constructed some replication incompetent influenza viruses,that only can replicate in its specific host cells,or else it only execute a single round of infection.By reverse genetics technology,we generated a replication-incompetent PB2-D from the UIXD P18-2(UI182),which is a mouse adaption strain from 2009 H1N1 influenza virus A/Changchun/01/2009(H1N1).Besides that,some specific host cells and transgenic animal models were constructed also.This was a feasibility exploration for the application of replication-incompetent virus.We will carry out our topic in the following 4 parts:1.Construction of transgenic cells what is essential for the replication-incompetent viruses' package and replication.The mammalian expression plasmids with PB1,PB2,PA,NP and M1 genes of UI182 were transfected into the 293T cells and the MDCK cells with lipo3000 and screened by the antibiotics,to construct transgenic cell lines.Transgenic cell lines were passaged and identified by fluorescence observation,reverse transcription PCR,Western blotting hybridization.The results indicated that we have successfully constructed 5 293T transgenic cell lines and 5 MDCK transgenic cell lines,293T-PB1,293T-PB2,293T-PA,293T-NP,293T-M1,and MDCK-PB1,MDCK-PB2,MDCK-PA,MDCK-NP,MDCK-M1.2.Construction of the replication incompetent H1N1 influenza virus.PB2-D.Based on the infectious clones of UI182,we introduced stop mutations into the coding region of the infectious clones of PB1,PB2,PA,NP and M1 to construct UI182-PB1-D,UI182-PB2-D,UI182-PA-D,UI182-NP-D,and UI182-M1-D.One replication incompetent influenza virus was been successfully rescued,we named it PB2-D.The recombinant virus was identified by transmission electron microscope observation,hemagglutination test,reverse transcription PCR and sequencing.The morphological identification results show that the recombinant virus PB2-D has a classic morphological appearance of influenza virus.And PB2-D can cause hemagglutination in cock red cells which is an important characteristic of influenza virus.The PCR products of the PB2-D genome were consistant with its parental UI182 except the point mutations that inserted artificially.3.Evaluation of the biological characteristics of PB2-D and the application.Its replication capacity in MDCK cells.MDCK-PB2 cells,embryonated eggs and mice were analyzed.The results showed that PB2-D could replicate in MDCK-PB2 cells but not MDCK cells,with a similar replication kinetics compared to its parental UI182.When inoculated in the embryonated eggs and mice,PB2-D couldn't replicate.The replication incompetent virus H5-PB2-D and H7-PB2-D were constructed and successfully applied in the detection of serum antibodies in stray dogs.4.Construction of transgenic mice that express PB2 of the influenza virus UI182.We transferred the UI182 PB2 gene into the zygotes by the prokaryotic injection.Through amplifing the PB2 gene from the tail genome by PCR,2 positive one were identified,one was male and one was female.The results of animal live imaging showed that the reporter gene was effectively expressed in the two positive transgenic mice(GO).After mating with the wild type mouse,some positive mice(G1)were selected and be analyzed by the reverse transcript PCR and western blot.The results indicated that PB2 gene can be transcripted into mRNA and translated into protein in the G1 mice.So,we successfully constructed a transgenic line of mice that expressed PB2 of influenza virus UI182.When we inoculated the PB2-D in the PB2 transgenic mice,the viruses titer was 103.03TCID50/mL in the lung of trnagenic mice at 3 days post inoculation.Unlike transgenic cells,the PB2-D replicates less efficiently in transgenic mice.We successfully constructed one replication incompetent H1N1 virus PB2-D.The structure characteristics,pathogenicity and proliferative capability of this virus were analyzed.Analysis results shown that the PB2-D have a classic morphological appearance of influenza viruses,and it only replicate in MDCK-PB2 but not in MDCK,embryonated eggs and mice.Because of those characteristics,the replication incompetent recombinant influenza virus is suitable for the study about highly pathogenic avian influenza virus.In order to enrich the application range of replication incompetent recombinant influenza virus,we constructed transgenic mice that expressed UI182 PB2,although the PB2-D replicated not efficient in the transgenic mice.The results of this study are very important for the application of the replication incompetent influenza viruses in exploring the mechanism of high pathogenic influenza viruses transmission and pathogenicity.