Biological Characteristics Of Infectious Laryngotracheitis Virus Epidemic Strains And The Molecular Mechanisms Of Host Proteins Affecting Its Replication

Chicken infectious laryngotracheitis virus(Infectious Laryngotracheitis Virus,ILTV)belongs to?herpesvirus subfamily and herpesviridae family,which can cause infectious laryngotracheitis in chickens.Infectious laryngotracheitis was first reported in the United States in 1925.Since it was also found in China in the 1960s,and it has occurred in many provinces.It is one of the most important diseases endangering the poultry industry.The attenuated vaccine is mainly used to prevent the disease in domestic and abroad,but the virulence of vaccine strain is generally strong,the harm of latent infection and the disadvantage of being unable to distinguish vaccine strain from a wild strain,and the lack of basic research on ILTV restricts the improvement of prevention and control technology of infectious laryngotracheitis.In this study,according to the key host protein affecting ILTV replication and its mechanism,signal peptidase complex subunit 3(SPCS3)was identified for the first time as one of the key host proteins affecting ILTV replication in cells.It was found that SPCS3 protein can cleave the gL protein of ILTV.The main results of this study are as follows:1.The growth characteristics and pathogenicity of three strains of ILTV:SH2016,SH2017 and GD2018 were studied.All three ILTV strains could proliferate in the chicken liver cancer cells(LMH).120 hours after infection with ILTV,the titer of SH2016 reached10^3.08TCID₅₀,SH2017 and GD2018 reached 10^3.42TCID₅₀ and 10^5.08TCID₅₀respectively,Among them,GD2018 was more adapted to LMH cell growth than the other two strains;Inoculation of 9-11 day old SPF chicken embryos via the chorionic allantoic membrane method,collection of allantoic membranes 168h after inoculation resulted in a titer of 10^3.25TCID₅₀for SH2016 and 10^2.67 TCID₅₀ and 10^4.33 TCID₅₀for SH2017 and GD2018,respectively,with GD2018 being more adapted to grow on allantoic membranes;Harvesting allantoic fluid,SH2016 titers up to 10^2.67TCID₅₀,SH2017 and GD2018 up to10^0.33 TCID₅₀ and 10^0.5 TCID₅₀respectively,SH2017 and GD2018 do not grow and SH2016can replicate in the allantoic cavity.Inoculation of 9?11-day old SPF chicken embryos by means of the allantoic cavity and collection of the allantoic membrane 168h after inoculation,with titres up to 10^3.25 TCID₅₀ for SH2016,10^2.58 TCID₅₀ and 10^1.83TCID₅₀ for SH2017 and GD2018 respectively.Harvesting allantoic fluid,SH2016 titrated up to 10^4.58TCID₅₀,SH2017 and GD2018 up to 10^1.25 TCID₅₀ and 10^0.67TCID₅₀ respectively,SH2017and GD2018 barely grew,while SH2016 was more adapted to proliferate in the allantoic cavity.In addition,three strains of ILTV were used to infect 8-week-old SPF chickens.SH2016 showed the most obvious clinical signs and was the only strain that caused death in chickens,and;the three strains of ILTV only replicated in the laryngeal tracheal tissue of chickens and were not detected in other organs;GD2018 had the highest antibody potency at 14 d post-challenge and SH2016 had the highest antibody potency at 21 d post-challenge.2.The genomes of three strains of ILTV were sequenced by high-throughput sequencing,and the whole genome sequences were spliced,and the protein sequences were predicted according to the genome structure.The genomic lengths of the three ILTV strains were as follows:SH2016 was 153.805kb,SH2017 was 153.024kb and GD2018 was153.855 kb.The GC contents of SH2016,SH2017 and GD2018 was 48.16%,48.06%and48.10%,respectively.The genomic structure of the three strains of ILTV consists of four regions:UL,US,IRs and TRs.According to the phylogenetic analysis of the nucleic acid sequence and 10 amino acid sequences of the virus genome,the phylogenetic tree of the genomic DNA showed that SH2016 was most closely related to the Russian isolate Rus/Ck/Tatarstan/1009/1643,but far away from SH2017 and GD2018,SH2017 was closely related to the American isolate LT Blen,and GD2018 was closely related to the Russian isolate Rus/Ck/Penza/2013/2701 and the Italian isolate 4784/80.In the phylogenetic tree of gC and gL proteins,which in the three ILTV strains were remarkably conserved in the same branch;In the phylogenetic tree of gD,gE and gG proteins,SH2016and GD2018 shared high homology in the same branch,but lower homoglogy with SH2017;In the phylogenetic tree of of gB,gH,gM,gI and TK proteins,SH2017 had extensive homology to GD2018,but lower homology with SH2016.Ten amino acid mutation sites of three strains of ILTV virus were compared,and it was found that except for the same gC protein sequence of the three strains of ILTV,the other 9 virus proteins had different amino acid sites.Whether these protein differences were related to the difference of ILTV replication ability and pathogenicity of the three strains needs to be further studied.3.The whole chicken genome CRISPR/Cas9 knockout library was constructed,and the whole genomic range of LMH cells was scanned one by one,and the gene knockout cell library was constructed.Then the ILTV was used to infect the cell library,and the surviving cells were sequenced with high throughput to determine the knockout host genes in the surviving cells.The results of the first sequencing showed that three non-coding RNA(ncRNA)genes and SPCS3(signal peptide complex subunit-3)genes may be associated with viral replication.The subsequent three repetitive experiments showed that SPCS3gene was the gene with the highest knockout rate in living cells,so it is speculated that the host protein SPCS3 may be one of the key genes affecting ILTV replication.4.Comparing the growth of ILTV in LMH cells and SPCS3 truncated LMH cell lines(named 1-6 and 1-14,respectively),the virus titer of ILTV decreased by 10^2.625 times in 1-6 cell lines and 10^3.75 times in 1-14 cell lines,indicating that SPCS3 protein truncation can indeed inhibit ILTV replication.The viral proteins with signal peptides and multiple transmembrane structures in ILTV were predicted by online biology software,and 9 viral proteins with single signal peptide and 4 eukaryotic expression plasmids with multiple transmembrane structures were successfully constructed.After transfection,8 viral proteins were successfully expressed.The differences of expression of viral proteins in LMH cells and 1-14 cell lines were compared.There is a significant difference in the molecular weight of ILTV envelope glycoprotein L(gL)protein between the two cells,indicating that SPCS3truncation affects its ability to splice gL protein,which may be the main reason for affecting ILTV replication.5.In order to understand the effect of SPCS3 truncation on other alphaherpesvirus,alpha herpesviruses of duck enteritis virus(DEV)and human herpes simplex virus type 1(HSV-1)were used to infect LMH cells and 1-14 cell lines respectively,and their growth differences were compared.The results showed that the virus titer of DEV in 1-14 cell lines was 10² times lower than that in LMH cells,but there was no difference in the growth of HSV-1 between the two cells.Furthermore,the eukaryotic expression plasmids of gL protein of DEV and HSV-1 were constructed.After transfection into LMH cell and 1-14cell line,the molecular weight of gL protein of the two viruses had no significant change,but the expression of gL protein of DEV in 1-14 cell line was significantly lower than that in LMH,while the expression of gL protein of HSV-1 in 1-14 cell line had no significant change.It is further suggested that truncated SPCS3 may affect the stability of gL protein,thus affectingDEV replication.This study enriched the biological information of the whole-genome sequence of ILTV strains in China and clarified the biological characteristics of three ILTV epidemic strains.Through CRISPR/cas9 high-throughput screening technology,a host protein molecule associated with gallid alphaherpesvirus 1 replication was screened,and the mechanism of action of the host protein that plays a key role in ILTV replication was refined,trying out a new perspective for the study of the molecular mechanism of ILTV pathogenesis.