Objective To search for the neutralizing epitopos on hepatitis E virus (HEV) capsid besides the known neutralizing epitope (aa459-606). Methods By analysis of several strains of monoclonal antibodies against HEV capsid and their recognized epitopes, the neutralizing activity of epitope (aa394-458) at N-terminus was compared with that of an immunodominant neutralizing epitope (an459-606). Re-suits The research showed a novel potential neutralizing epitope in aa423-437 of HEV ORF2 though detec-ting and comparing the characteristics of several antibodies and corresponding determinations. The epitope is a linear non-immunodominant epitope which is different from the other neutralizing epitope in aa459-606.And the amino acids sequence of this novel epitope is conservative. Conclusion ORF2 aa423-437 is a no-vel potential neutralizing leaner epitope of HEV. It is believed that the present work adds fundamental knowl-edge to our understanding of HEV capsid domain and contributes to the prevention and control of this dis-ease.

Objective To identify the protein interacting with hepatitis E virus(HEV) recombi-nant capsomeric particles(P239). Methods Protein interacting with HEV was analyzed by the pull-down, MALDI-TOF-MS, co-immunoprecipitation (Co-IP) and CONFOCAL. Results A protein interacting with HEV recombinant particle (P239) was identified as HSP90 by MALDI-TOF-MS. The interaction between HSP90 and P239 was further confirmed by Co-IP. The protein level and localization of HSP90 and P239 in HepG2 were detected. The total quantity of HSP90 didn't change, and the movement of HSP90 from plasma membrane to perinuclei region with P239 was observed. Conclusion HSP90 may play an important role in the trafficking of P239. It suggests that HSP90 participate in the transportation of HEV after infection, which may contribute to the prevention and control of the disease.

Objective To express the recombinant caspid of genotype 4 hepatitis E virus(HEV) ORF2. Methods HEV recombinant capsid protein D66 was expressed in E. coli, using the ORF2 fragment (aa368-606, obtained from swine bile) of genotype 4 HEV. Results The recombinant capsid proteins D66 self-assemble to be particle with a radius of 13 nm through dimeric form in neutral solution. Coated particles reacted well with sera obtained from patients during acute or recovered phase of HEV infection. Immunofluorescence and immnoblot assay suggested that D66 bound and penetrated HepG2 cell lines, and the process of attachment was blocked by sera collected from patients during acute or recovered phase of HEV infection.Conclusion Recombinant D66 particles simulate the structure at the surface of genotype 4 HEV well and specifically adhere and penetrate the host cells, which lays the foundation for the investigation of the molecular mechanism of genotype 4 HEV infection.

The lack of effective in vitro infection model for hepatitis E virus (HEV) has greatly hindered the quantitative analysis of neutralizing titers of anti-HEV antibodies and human sera, thus impeding further studies of HEV-stimulated antibody responses and the immunological mechanisms. In order to improve this situation, the infection of HepG2 cells that are inefficient for HEV replication was continuously monitored until the viral load reached the limit of detection on day 13, the results of which confirmed the feasibility of using this cell line to establish the infection model. Then, neutralization assays of five anti-HEV murine monoclonal antibodies and serum samples collected from four HEV vaccine recipients (collected before and after vaccination) were performed by 96 multi-channel parallel infections, nucleic acid extraction, and qPCR. The results showed that the cell model can be applied for quantitative evaluation of the neutralizing capacity of different antibodies and antiserum samples from HEV vaccine recipients. In this study, we have successfully established a high-throughput in vitro HEV replication model, which will prove to be useful for the evaluation of HEV vaccines and studies of HEV epitopes.
Animals ; Antibodies, Viral ; analysis ; immunology ; Hepatitis Antibodies ; analysis ; immunology ; Hepatitis E ; immunology ; virology ; Hepatitis E virus ; chemistry ; immunology ; physiology ; High-Throughput Screening Assays ; methods ; Humans ; Mice ; Mice, Inbred BALB C ; Neutralization Tests ; methods ; Virus Replication

Objective:To construct a chemiluminescense immune quantification assay based one paired mAbs against complexed prostate specific antigen ( c-PSA).Methods:Six week-old female BALB/c mice were immunized with the commercial c-PSA antigen.After serum titer reaching a platform stage ,the spleen was immunized and fused with mouse myeloma cell lines ( Sp2/0 ) .The hybridoma were screened by indirect ELISA ,and eight generated antibodies were paired to obtain a quantitative analysis of the chemical luminescence.Results:7D6 specifically recognized c-PSA,while 1A10 recognized total PSA(t-PSA).And the paired antibody 1A10/7D6 were determined to successfully construct a chemiluminescense immune response quantitative detection method through the detection of c-PSA standard and clinical serum samples .had,positive samples have statistically significant difference ( P<0.000 1 ) with negative samples.And the correlation coefficient R 2 was 0.97 between our c-PSA quantitative results and that of the Siemens c-PSA chemiluminescense immunoassay kit .The detection linear range was 0.1-100 ng/ml,and the sensitivity was 0.005 ng/ml.Conclusion:The paired monoclonal antibodies specifically detecting c-PSA were generated and a c-PSA chemiluminescense immunoassay were developed in this study .The detection capability of our method was comparable with that of the international commercial kit .

Objective To prepare and identify the monoclonal antibodies against respiratory syn-cytial virus nucleoprotein(RSV N protein). Methods A prokaryotic expression system was used to express recombinant RSV N protein in Escherichia coli (E.coli). BALB/c mice were immunized with the recombi-nant N protein after purification. Monoclonal antibodies (McAbs) against the N protein were sorted from these BALB/c mice and then were further characterized by Western blot, ELISA and immunofluorescence. Furthermore,this study used orthogonal experiment to identify McAbs pairs,which could be used for diagno-sis. Results This study succeeded in obtaining 24 hybridoma cells that stably secreted monoclonal antibod-ies against RSV N protein. These antibodies showed good reactivity in ELISA,of which eight had strong spe-cificity in Western blot and 13 could be used in immunofluorescence. This study obtained two McAbs pairs (12F2/11H8-HRP and 12F2/15A8-HRP) that could be used in RSV detection. Conclusion This study succeeded in screening and preparation of McAbs against RSV N protein and obtaining two potential McAbs pairs for rapid detection of RSV.

Hepatitis E virus (HEV) is the major pathogen responsible for acute viral hepatitis in China. Recently, there have been new advances in the methodologies and novel discoveries in the laboratory diagnosis of HEV, including various diagnostic markers such as HEV RNA, HEV antigen, anti-HEV IgM, and anti-HEV IgG. A multi-indicator diagnostic approach should be applied across different populations, including patients with acute or chronic hepatitis E, individuals with extra-hepatic clinical symptoms, asymptomatic carriers, and specific occupational groups undergoing health assessments. The clinical significance of test results for each marker varies among these different groups.

Objective: To investigate the seroprevalence and epidemiological characteristics of hepatitis E virus (HEV) infection in pregnant women in Xiamen. Methods: Sera samples of 910 pregnant women were collected from September 2014 to June 2015 in Xiamen Huli District Maternity and Child Care Hospital. Those who intended to give birth in target hospital were included in a subgroup which was asked to collect the second serum sample. All samples were tested for anti-HEV IgM and IgG antibody by enzyme linked immunosorbent assay (ELISA). HEV RNA was tested by reverse transcription-polymerase chain reaction (RT-PCR) for the positive samples of anti-HEV IgM antibody, meanwhile, the quantitative detections for anti-HEV IgG were conducted in specimens positive for anti-HEV IgG. Results: Of the 910 pregnant women, 8 (0.88%, 95%CI 0.45%-1.73%) were anti-HEV IgM positive. HEV RNA was found in 3 cases through RT-PCR and viral load values were between 600 and 700 copies/ml; 140 (15.38%, 95%CI 13.19%-17.68%) were anti-HEV IgG positive and geometric mean concentration of the samples was 0.385 Wu/mL (95%CI 0.332-0.445 Wu/ml). The positive rate of anti-HEV IgG increased with age (P=0.004). In the subgroup, 150 pregnant women were included and followed up, 4 of those were defined as 'new HEV infection cases’ and the incidence was evaluated as 10.7/100 person-year (95%CI 3.39-25.7/100 person-year). Conclusions There were a low percentage of HEV carriers in pregnant women in Xiamen, but the risk of new primary infection in pregnant women during pregnancy was much higher than the general population, suggesting that it is necessary to expand sample size to clarify the burden of HEV infection during pregnancy.