Construction And Application Of Reverse Genetic Platform Of B Influenza Virus

Objectives:We construct the reverse genetics system of influenza B virus.Study on the factor which can influence the characteristics of virus based on the reverse genetics system.Methods:Bidirectional promoter vector pHW2000 which is usually used for influenza type A was used to construct reassortment influenza B virus. Eight plasmids containing each gene segment of the virus was contracted.293T cells cultured with MDCK cells were transfected with eight plasmids, then the reassortment influenza B virus was rescued successfully after propagated in embryonated chicken eggs or MDCK cells. The representative strains insolated in 2013-2015 were selected based on the genetic characterization of the viruses, which included wild type virus and inter-lineage reassortment virus. We determined the number of virus particles contained in the virus liquid by TCID50 method. Then diluted the liquid to contain the same number of virus particles to infect MDCK cells, and collected the virus under each time point. Using TCID50 method to test the virus tier of each time point, then use time point as X-axis, the titer as Y-axis to obtain the growth curve of virus. We determined neuraminidase kinetic constants of all virus. With the constructed reverse genetic system of influenza B virus, different gene combinations of the type B influenza viruses were constructed. The growth characteristics of the reassortment viruses were determined, and the factors affecting the growth characteristics of virus were analyzed.Results:We successfully constructed the reverse genetic system of influenza B, and further used this system to rescue reassortment virus with different genetic constitution. The growth curves of most wild type virus and reassortment virus between year 2013 and 2015 are similar. It reached a peak after 48 hours post infection, and maintained to 72 hours, then the virus titer declined gradually. The Km value of virus is between 16 and 40. From the results of growth curve of reassortment virus, it showed that the replacement of single gene fragment had no significant effect on the growth characteristics of the virus, however the growth characteristic of reassortment virus can be reduced by replacing the segments in different combinations.Conclusions:Our results demonstrate that the influenza B virus can be rescued by pHW2000 reverse genetic system borrowed from influenza A virus, and it can be further used for the study on growth characteristics of influenza B virus; Multiple genes have combined effects on the growth characteristics of influenza B virus.