Discovery And Clinical Application Value Of Hepatitis E Virus Secretory Antigen

Hepatitis E virus(HEV)is one of the most important causes of acute viral hepatitis around the world.In some developing countries,50%of acute viral hepatitis cases are caused by HEV.China is one of endemic areas for hepatitis E.Most hepatitis E patients present typical symptoms of acute hepatitis with a mortality rate of 1%,while the illness may cause severe or fatal disease in pregnancies with the mortality of up to 25%.HEV infection may be chronic in irnmunosuppressed patients and accelerate liver fibrosis progression.In addition,HEV infection also leads to a variety of extrahepatic symptoms.Common diagnostic markers for HEV infection include HEV IgM,HEV IgG,and HEV RNA.HEV IgG is a marker of past infection and vaccination of HEV HEV IgM is commonly used for diagnosing HEV infection in clinical practice and mainly indicates the acute phase of HEV infection.But HEV IgM will be positive for some time during the convalescent period.HEV RNA detection is an effective method for detecting pathogens and is an important marker for diagnosing current HEV infection.But RNA detection is expensive and requires specialized equipments,trained operators and testing sites.Moreover,RNA detection has high requirements for the preservation and transportation of samples.As another important marker of viral replication,HEV antigen has not been applied in the diagnosis of HEV This mainly due to the insufficient sensitivity of HEV antigen detection and results are susceptible to the levels of anti-HEV antibodies.The aim of this study was to establish a HEV antigen detection method with high sensitivity and wide application range,and to evaluate the diagnostic value of HEV antigen detection In clinic.Firstly,this study screened the best paired antibodies from 96 mAbs which recognized the HEV structural protein PORF2 and established a new HEV antigen detection method.The detection ability of the new HEV antigen detection method was 6.3 × 103 copies/mL.Compared with the previous commercial kits,the new HEV antigen detection method showed similar detection ability for different genotypes viruses and increased the detection ability of more than 10 folds.Using the new antigen detection method,this study found that the detectable pORF2 antigen in serum existed in two forms:One existed as the form of the classic virion,which bound with RNA to act as the actual capsid protein,named as pORF2C.The other newly discovered form of pORF2 did not bind to viral RNA,named pORF2S.The results of this study further revealed that the expression of two forms of pORF2 was related to two AUG start codons with identical reading frames on the orf2 gene,the first AUG codon was located at 1st nt and initiated the translation of pORF2S.The second AUG codon was located at 46th nt and initiated the translation of pORF2C.HEV pORF2S accounted for more than 99.9%of pORF2 antigen.HEV poRF2S was abundant in serum and did not bind to RNA.These determined that this marker was a new diagnostic marker,which was not associated with HEV RNA.The results of serial samples from HEV-infected rhesus monkeys showed that the periods of HEV antigen positivity was similar to the periods of HEV RNA positivity,which could effectively reflect the period of viremia and viral shedding after HEV infection.And the new antigen detection method was less susceptive to anti-HEV antibodies in the serum than the previous HEV antigen detection kits.Due to the obvious advantages of the new detection method in sensitivity and detectable period,the upgrade and replacement of previous HEV antigen detection kit was realized.Based on the specimens from acute hepatitis cohort,ths study further compared three acute phase markers of hepatitis E,inlcuding HEV IgM,HEV RNA and HEV antigen.The results showed that the sensitivity and the specificity of HEV antigen were 91.4%and 99.8%,respectively.The positive predictive value of HEV antigen was 96.0%.HEV IgM showed a slightly higher sensitivity(92.3%)than HEV antigen.While HEV IgM could persist for 32 weeks after onset,which was much longer than 3-4 weeks of well-known acute phase of HEV infection.This led to that the positive predictive value of HEV IgM was only 64.8%.Combining HEV antigen and HEV IgM could effectively increase diagnostic accuracy,with HEV RNA assisted diagnosis,95.7%of acute hepatitis E patients could be confirmed.This strategy enabled economical and effective diagnosis of acute hepatitis E.HEV investigation of qualified blood donors in Xiamen showed that the positive rates of HEV antigen and HEV RNA were both 0.28%.Seventeen donors showed positivity for both the HEV antigen and HEV RNA were follow-up.The results showed that 10 donors exhibited HEV pathogen positive for more than 70 days.Compared with typical acute hepatitis E patients,these HEV carriers had lower viral levels,but the viremic period and antigenemic period in these carriers were significantly longer and most of HEV carriers did not present with an obvious antibody response.The presence of these HEV carriers was a potential threat to blood transfusion safety.In addition,in order to expand the range of applications for HEV antigen detection,this study established a point-of?care testing strip for HEV antigen,which shortened the detection time to 15 minutes with the same sensitivity.Meanwhile,this study also established a HEV classification method based on antigen tests and realized rapid classification of HEV without sequencing.In addition,this study also found that HEV antigen was highly aggregated in the host kidney.The angtigen concentration in urine of HEV patients was 47-5556 folds that of blood,indicating that urine may be better specimens for HEV diagnosis.In summary,this study developed a new generation of HEV antigen detection kit by rescreening paired antibodies.This study found that the main target of HEV antigen detection was the secreted nonvirion-associated pORF2S,rather than the previously recognized viral capsid protein pORF2C.The discovery of pORF2S and its production mechanism provided a new target for the diagnosis of HEV and provides a new theoretical basis for understanding the clinical significance of HEV antigen.Furthermore,the new target was used in acute hepatitis diagnosis and blood donor screening in this study.This study provided a diagnostic algorithm for HEV and discovered new epidemic characteristics of HEV.These results provided an effective tool for the accurate diagnosis and pathogenesis of HEV In addition,the secreted antigen pORF2S found in this study also provided important ideas for the study of immune escape,immune tolerance,and life cycle of HEV.