[The etiological identification of an aseptic encephalitis outbreak (ten cases) in a hospital of Xiamen city, 2011].

OBJECTIVE: To identify the etiology of an aseptic encephalitis outbreak (ten cases) in a hospital of Xiamen city from 11 to 17 May, 2011. METHODS: A total of ten patients' throat swabs, anal swabs and cerebrospinal fluid were collected and detected by RT-PCR for pan-enterovirus. The samples containing detectable pan-enterovirus were tested by PCR with genotype-specific general primers located in VP1 region of enterovirus genotype A, B and C (HEV-A, B and C). The PCR products of VP1 segment were purified and sequenced, and phylogenetic analysis was performed. Meanwhile, the pathogens in those samples were isolated in Vero cell culture. Homologous analysis of VP1 sequences were carried out for the cultured virus samples and the original clinical samples to identify the outbreak etiology. RESULTS: Among the ten cases, seven cases were positive for pan-enterovirus nucleic acid. When tested by genotype-specific PCR, the throat and anal swab samples from those 7 patients were positive with HEV-B VP1 primers. Meanwhile, the HEV-B VP1 segments were sequenced and phylogenetic analyzed, which indicated the seven cases were all infected by enterovirus Echo 30. The sequences from those samples had homology of 95.3% - 97.1% with the epidemic strains in Zhejiang, 2004. Out of the seven cases, the sequences of XM2, XM3, XM4, XM8 throat swab samples and XM3, XM6 throat samples showed 99.4% - 100.0% homology which were different from the sequence of XM1, and the homology was 92.8% - 93.4%. Furthermore, the viruses were isolated using Vero cells from XM1, XM2, XM3, XM4 and XM8 throat swab samples, and the VP1 sequence showed more than 99.9% homology with the original specimens. CONCLUSION: The local outbreak of aseptic encephalitis was caused by Echo 30 of enterovirus genotype B, and the epidemic strains may have different genetic background.

[HEV capsid protein interacts with CYP 2A6 and decreases its coumarin 7-hydroxylation activity].

E2 is a recombinant hepatitis E virus capsid protein including its main antigenic determinants but lacking of the particle assembling domain. P239 was the C-terminal extending protein of E2 and could self-assemble to form virus like particles, which might serve as mimicry of virions both structurally and antigenically. We previously used yeast two-hybrid system to screen proteins interacting with E2 based on a human hepatocyte cDNA library. One candidate was identified as the segment (aa388-437) of cytochrome P450 2A6 protein, which is predominantly expressed in liver and important for metabolization. Some studies have demonstrated that hepatitis virus infection may altered cell metabolic clearance of coumrarin which were rapidly matebolised by CYP2A6. In this research, we demonstrated that the protein interaction between HEV capsid proteins and CYP2A6 by pull-down and co-immunoprecipitation. It was also found that their interaction could decrease the CYP2A6 catalytic activity when p239 was incubated within the CYP2A6-transfected Huh7 cells. These results suggested that CYP2A6 might be related to the pathological process when HEV invaded host cells.

[Recombinant HEV caspid protein p239 specifically attached on HepG2 cells and blocked the infection of wild-type HEV on liver cells].

By using Western blot and immunofluorescence assays, the recombinant HEV capsid protein p239 was found specifically attached to the HepG2 cell surface and entered to the cytoplasm with the increase of incubation temperature. Pre-mixture of wild-type HEV with p239 blocked the infectivity of the virus on primary cultured human hepatocytes and HepG2 cells, indicating that p239 and HEV competed the same targeting site on these cells. These data provide evidence that p239 has a similar cell surface structure with wild-type HEV.

Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus.

AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier. METHODS: One to 6 tandem copies of HBV preS1 (21-47) fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E.coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric particles was detected with immuno-capture PCR. RESULTS: Recombinant antigens CI, CII, CIII carrying 1-3 copies of HBV preS1 (21-47) individually could form virus-like particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preS1 (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CI, CII, CIII could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CI, CII and CIII) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CI, CII and CIII were able to capture HBV virions in immuno-capture PCR assay in vitro. CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.