Identification Of Key Viral Proteins For Efficient Entry Of Pseudorabies Virus Variant Into Nerve Cells

Pseudorabies(PR)is an animal contagious disease caused by pseudorabies virus(PRV),which has caused huge economic losses to the pigindustry in our country and even worldwide.With the extensive vaccination of the Bartha-K61 strain vaccine,pseudorabies has been effectively controlled word widely.But under immune pressure,PRV continuously undergoes adaptive evolution to the environment.It is precisely because of the continuous accumulation of this evolution that the PRV variant strain appeared in our country in 2011,which can escape the immune protection of the Bartha-K61 strain.The PRV variant strain and the previously epidemic PRV strain belongto different evolvement branches and the PRV variant strain possesses significantly differenced biological characteristics.The PRV variant strain not only significantly enhanced the pathogenicity to domestic pigs,mice,sheep and other animals,but also showed extremely serious neurological symptoms and nerve tissue damage,causinghuge economic losses to the pigindustry in our country.However,key scientific issues such as the mechanism underlyingthe increased pathogenicity to the host and increased tropism to the host nervous system of PRV variant strains have not been resolved yet.Therefore,the identification of key viral factors that increase the pathogenicity of PRV variant strain is particularly important.This study firstly compared the basic characteristics of PRV variant strain and classic strain in infected nerve cells.The results showed that PRV variant strain can produce more progeny viruses in the early stage of infected nerve cells.This experimental phenomenon showed both in the cultured nerve cells and primary nerve cells.However,this discrepancy between the two strains will shrink with the increase of the infection dose and the extension of the infection time.Subsequently,to explore the reasons for the increase in viral titers of the progeny of PRV variant strain,we further compared the life cycle of the PRV variant strain and classic strain infectingnerve cells.Through independent parallel experiments,we found that the adsorption and internalization efficiency of variant strain is significantly higher than that of the classic strain.The adsorption efficiency and the internalization efficiency of the PRV variant strain on nerve cells N2 a are about 5 times and 3 times higher than that of the PRV classic strain separately.Combiningthe comparison of axonal transport and genome replication stages,we found that the difference between the PRV variant strain and the classic PRV strain in the life cycle of infectingnerve cells is mainly concentrated in the invasion stage.In order to identify the key viral factors that lead to the enhanced efficiency of PRV variants invadingnerve cells,we performed a functional evaluation of the mutated PRV invasion key viral protein gB/gC/gD,and found that the mutated gB/gC/gD proteins all contributed to the enhanced nerve cell invadingefficiency of PRV variant strain,and it also has a significant synergistic enhancement effect.The affinity between the gB/gC/gD proteins from the variant strain and its correspondingreceptor was significantly enhanced,indeed the affinity between the gC/gD proteins from the PRV variant strain and heparan sulfate and mouse Nectin-1 protein was higher than that of the PRV classic strain about 5 times;the mutated gB/gD proteins synergistically promote the penetration efficiency of the PRV variant strain duringthe internalization process.Furthermore,we verified the above results by evaluatingthe nerve cell invasion efficiency of various gB/gC/gD gene interchanged chimeric viruses,and finally considerate the mutated gB/gC/gD proteins as the key viral factor that causes the increased nerve cell invadingefficiency of PRV variant strain.In general,this study found that PRV variant strain has a significantly enhanced efficiency of nerve cell invasion;all the mutated gB/gC/gD proteins contributed to the enhanced efficiency of PRV variant strain invadingnerve cells by synergistic effect.This study provides a reference for analyzingthe mechanism of enhanced virulence of PRV variant strain,and also lays a solid foundation for elucidatingthe neurotropic or neurovirulence enhancement of PRV variant strain.At the same time,this study has a potential application value,and a new PRV variant strain vaccine can be designed based on the key mutation sites.The study will provide new insight into the development of new PRV vaccines.