| Fowl adenovirus serotype 4(FAdV-4)is a linear double-stranded DNA with a genome length of 43-46 kb,which is 8-10 kb longer than that of human adenovirus.Genus-common genes encode viral structural proteins and participate in viral DNA replication.They are located in the center of the genome.Most of the genera-specific genes involved in virus-host interactions are located at the end of the genome.The functions of FAdV genus-specific genes have not been revealed,except for ORF1(d UTPase),ORF8(Gam-1)and ORF22.FAdV-4 is the predominant causative agent of hepatitis-hydropericardium syndrome(HHS)in chickens,Since July 2015,outbreaks of HHS caused by a novel genotype of FAdV-4 have been reported in China,causing severe economic losses to the poultry industry.We constructed an infectious clone of the novel FAdV-4 and established a FAdV-4-based vector system.Here,we attempted to systematically investigate the effects of genus-specific genes on the virus replication with reverse genetics approaches.To study the influence of viral replication of non-structural protein genes on the right side of the genome,restriction endonucleases Hind III,Eco RV,Xba I,Bsi WI,Eag I and Spe I can divide this region into 5 fragments,which combine 2 of the 6 restriction sites A series of FAdV-4 mutants can be constructed,and a total of six recombinant viruses of XHE-CX19A,XEX-CX19A,XXB-CX19A,XBE-CX19A,XES-CX19A,and XHB-CX19A can be constructed.Among them,XHE-CX19A,XEX-CX19A,XXB-CX19A,and XHB-CX19A can replicate and proliferate normally in LMH cells.Through a one-step growth curve,it can be seen that both the recombinant virus and the parental virus FAdV4-GFP can reach higher titers.The two recombinant viruses XEX-CX19A and XXB-CX19A even reached 1x10~8 IU/m L.Virus plaque experiments showed that deleting the Hind III-Eco RV fragment weakened virus replication,and deleting the Eco RV-Xba I fragment enhanced virus replication.Deleting the Xba I-Bsi WI fragment did not significantly affect the growth of the virus.Even if the 3 fragments between Hind III-Bsi WI were deleted at the same time,the virus could still growth,but the ability of the virus to replicate and proliferate is further weakened.The other 2 strains of recombinant viruses XBE-CX19A and XES-CX19A showed no fluorescence aggregation or very little fluorescence aggregation after transfection.With the extension of culture time,these fluorescence aggregations did not increase or even disappear.In order to describe the activities of XBE-CX19A and XES-CX19A more accurately,the separated cells were collected on the 3 day and 5 day of transfection,and the cells and culture medium were collected on the 7 day to determine the virus titer.The final result showed that the transfection from them The progeny virus can be rescued in LMH cells,but the virus production is extremely low,and it is difficult to maintain the progeny virus growth.To study the effect of specific ORFs on virus replication and proliferation,delete GAM1 to construct XGAM1-CX19A,and on this basis introduce two frameshift mutations to further prevent the expression of ORF22,construct X22G-CX19A,and delete the coding sequences of ORF16 and ORF17 to construct X1617-CX19A,3strains of recombinant viruses can be rescued successfully and grow normally in LMH cells.Through the virus plaque experiment,it can be seen that the proliferation ability of XGAM1-CX19A is weakened,and the proliferation ability of X22G-CX19A is further weakened.This indicates that GAM1 and ORF22 have a synergistic effect on the growth of FAdV-4,but both are not essential genes,while the proliferation ability of X1617-CX19A is similar to that of the parental virus.In order to understand whether GAM1 is an important gene,a helper plasmid pc DNA3-GAM1 was constructed.The results showed that the virus titer of XBE-CX19A increased by 35 times after the helper plasmid was added,while the titer of XES-CX19A virus increased by 260 times.The growth has a certain promoting effect.Promoter experiments show that ORF16-17 and ORF28-29 have promoter activity.Flow cytometry experiments also show that p KFA4RP-GC has a strong ability to start to the right.The start activity is 48%of the CMV promoter,and p KFA4RP2-GC is to the right.The promoter activity is 7%of the CMV promoter.At the same time,the transcription activity of the two promoters to the left is also detected,but it is negligible.According to the chicken embryo experiment,the parental virus FAdV4-GFP initially died on the 10th day,and the death rate reached 100%at the end of the experiment.Compared with the parental virus,the recombinant virus XHE-CX19A died3 days later,and the death rate was 28.6%.This result indicates that the deletion of ORFF22 and ORF20a will slightly affect the virulence of the virus,while the chicken embryos in the XGAM1-CX19A group did not die,it shows that although GAM-1 is a non-essential gene for virus replication in vitro,it significantly reduces the level of replication in vivo.The liver was collected to determine the titer.In the XHE-CX19A group,there was no significant difference in virus production between dead chicken embryos and viable chicken embryos.The average virus yield of FAdV4-GFP group was about 7 times that of XHE-CX19A group,while the virus yield of XHE-CX19A group was about 4 orders of magnitude of that of XGAM1-CX19A group.Through the transcriptome experiments of FAdV4-GFP and FAdV4-CX19A,it can be seen that at 8h after infection,the transcript of GAM1 exceeds 200 TPM,and the gene-specific genes increase less than twice in the next 4 h.At 18 h,GAM1 has more than 500 transcripts,FAdV4-CX19A has similar transcripts,and there is no significant difference between the two viruses.The results show that FAdV-4 is a genus-specific gene that is transcribed at the same time,and no early genes that can specifically control the transcription of other genes have been found.In summary,the replication of avian adenovirus serotype 4 is related to genus-specific genes,but none of the genes are necessary. |
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