| Duck hepatitis A virus（DHAV）is a single-stranded positive-stranded small RNA virus that can cause a very high lethality rate in ducklings,which leads to great economic losses.Oligoadenylate synthetase ubiquitin-like（OASL）is the only member of the oligoadenylate synthetase（OAS）gene family found in avians,belonging to interferon-stimulated genes（ISGs）.It plays an important role in antiviral defense and stimulating adaptive immunity.In this paper,by analyzing the prevalence of DHAV in China and the protection rate of the currently used DHAV vaccine,mastering the epidemiological data of DHAV and evaluating the effectiveness of vaccine prevention and control,we intend to reduce the economic losses caused by the virus to the waterfowl breeding industry,to explore the relationship between OASL,IFN-α and tissue virus copies number in duck embryo tissues and duck embryo hepatocytes,to provide theoretical basis and relevant information for the study of duck’s innate immune antiviral mechanism.1 Meta-analysis of the prevalence of DHAV infectionThrough the established screening process,25 literatures that met the criteria from 14 provinces were collected for meta-analysis,with a total sample of 689,549 cases.After the data was corrected for statistics,it was estimated that the national DHAV incidence rate was 12%（95%CI:3%-20%）,mortality was 11%（95%CI:2%-19%）,incidence rates trended downward over time,with DHAV-1 serotype strains accounting for 38%（95%CI:21%-56%）of the samples tested and DHAV-3 serotype strains accounting for 49%（95%CI:31%-68%）of the samples tested,it is speculated that the incidence of this serotype virus has been effectively controlled due to the popularization of DHAV-1 vaccine,and the incidence of DHAV-3 has no cross protection with DHAV-1,therefore,the proportion of DHAV-3 in the test samples increased.2 Meta-analysis of DHAV vaccine protection efficacyThrough the established screening process,14 studies and patents on the protective efficiency of DHAV-1 and DHAV-3 vaccines were obtained.The meta-analysis showed that the survival rate of ducks in the experimental group immunized with duck hepatitis virus vaccine was significantly higher than that without immunization[RR=12,95%CI（6-26）,P<0.01].The control group was vaccinated according to the standard immunization program.The age of the immunized ducks and the valence of the vaccine did not affect the protection efficiency of the ducklings,and they could provide sufficient antibody levels.Analysis of vaccine data sources showed that it can provide immune protection for ducklings in clinical practice,but the protective effect of vaccine immunization under small-scale experimental conditions is still different from that of large-scale clinical immunization,suggesting that vaccine development needs to be accelerated with the variation of virus strains.3 DHAV-3 infection activates the endogenous expression of OASL in duck embryosFluorescence quantitative PCR was established to detect DHAV-3 by absolute quantitative method,and the minimum detection limit was 6.8×102 copies/20μL.15-day-old SPF duck embryos were induced with 103ELD50 DHAV-3,and hearts,liver,stomach,intestine,thigh muscle tissue were collected at 6 h,12 h,24 h,and 48 h after inoculation,OASL,IFNα mRNA relative expression and DHAV-3 copy number were measured by real-time PCR.The experimental results showed that compared with 0 hours,the OASL transcription in the liver was significantly increased at 24 h（P<0.05）,and the OASL transcription in the liver,heart,stomach,intestine,and muscle at 48 h was significantly increased（P<0.01）,and reached the maximum in the heart.The expression level of IFN-α was followed by the liver,and the relative expression changes in the stomach,intestine and muscle were much lower than those in the first two tissues;IFN-α expression in the heart and liver had no significant difference in the first 12 h,and it started to rise at 24 h and reached peak at 48 h.It reached the maximum value（P<0.01）,and the expression level in the stomach,intestine and muscle increased slowly,but the relative expression level was lower than that in the heart and liver.The virus copy number in each tissue reached a peak at 24 h,and then decreased.The difference in the transcription level of OASL in different tissues is similar to that of IFN-α,which suggested that OASL and downstream IFN-α were closely related in various tissues of duck embryos infected with DHAV-3,provided a theoretical basis for the natural antiviral immunity of ducks.4 DHAV-3 infection activates the endogenous expression of OASL in duck embryo hepatocytesThe duck embryo hepatocytes were isolated and cultured and identified by glucose-6phosphateand and glycogen staining.103TCID50 DHAV-3 monolayer duck embryo hepatocytes were induced,and the cells were collected at 6 h,12 h,24 h,36 h,48 h,and 72 h after inoculation;siRNA was transfected into the monolayer duck embryo hepatocytes to silence the OASL target gene,103TCID50 DHAV-3 inoculate monolayer duck embryo hepatocytes silencing target genes after 24 h,cells were collected at 24 h and 48 h after inoculation,and the relative expression of OASL,IFN-α mRNA and DHAV-3 copy number were measured.The experimental results showed that compared with DEHC without DHAV-3 inoculate,the expression of OASL in the experimental group was significantly up-regulated at 6 h,and reached the maximum value at 48 h,with a very significant difference（P<0.01）;The initial stage of 6-36 h increased slowly,the difference was not significant,the expression of IFN-αincreased at 48 h,the difference was significant（P<0.05）,and the difference was extremely significant at 72 h（P<0.01）;In the early stage of infection,the proliferation was slow for 24 h,followed by rapid proliferation,reaching a maximum copy number at 48 h,followed by a decrease in viral copy number.siRNA could effectively silence the mRNA expression of DEHC OASL at 48 hours（P<0.05）.Compared with the control group,the expression of IFNa inoculate by DHAV-3 in siRNA treated DEHC was significantly reduced at 48 h（P<0.01）.DHAV-3 significantly increased at 24-48 h（P<0.01）.Experiments above showed that knockdown of OASL in duck embryo hepatocytes leads to decreased IFN-α transcription and increased DHAV-3 replication.It demonstrated that duOASL,like most OAS family members,also has antiviral functions. |
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