Intranasal vaccination with ebola virus GP amino acids 258-601 protects mice against lethal challenge.

Ebola virus (EBOV) disease (EVD) leads to lethal hemorrhagic fever with a case fatality rate as high as 90%, thus posing a serious global public health concern. However, while several vaccines based on the EBOV glycoprotein have been confirmed to be effective in animal experiments, no licensed vaccines or effective treatments have been approved since the first outbreak was reported in 1976. In this study, we prepared the extracellular domain of the EBOV GP protein (designated as N20) by prokaryotic expression and purification via chromatography. Using CTA1-DD (designated as H45) as a mucosal adjuvant, we evaluated the immunogenicity of N20 by intranasal administration and the associated protective efficacy against mouse-adapted EBOV challenge in mice. We found that intranasal vaccination with H45-adjuvanted N20 could stimulate humoral immunity, as supported by GP-specific IgG titers; Th1 cellular immunity, based on IgG subclasses and IFN-γ/IL-4 secreting cells; and mucosal immunity, based on the presence of anti-EBOV IgA in vaginal lavages. We also confirmed that the vaccine could completely protect mice against a lethal mouse-adapted EBOV (MA-EBOV) challenge with few side effects (based on weight loss). In comparison, mice that received N20 or H45 alone succumbed to lethal MA-EBOV challenge. Therefore, mucosal vaccination with H45-adjuvanted N20 represents a potential vaccine candidate for the prevention of EBOV in an effective, safe, and convenient manner.

Epitope-based recombinant diagnostic antigen to distinguish natural infection from vaccination with hepatitis A virus vaccines.

Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine.

[The Preparation of Epitope-based Recombinant Rubella Virus Diagnostic Antigen].

To prepare an epitope-based recombinant Rubella virus (RV) recombinant diagnostic antigen(designated 'H29') and preliminarily evaluate its antigenicity. With Glutathione S-Transferase (GST) located at the N-terminal, and the His tag at the C-terminal, the epitope-based RV recombinant diagnostic antigen (designated'H29') was expressed in Escherichia coli (E.coli) and purified by affinity and anion exchange chromatography. Based on the antigenicity of H29 identified by Western blot (WB), we constructed and evaluated a novel early diagnostic ELISA for RV infection. The soluble H29 protein with a high homogeneity was obtained; the WB analysis demonstrated that the H29 protein could bind to a monoclonal antibody for RV-E1 and GST antigens, as well as detect RV acute-phase serum. Using the novel ELISA, the serum from 48 cases with positive RV infection,48 cases with negative RV infection, and 48 healthy people was detected, displaying the excellent consistency. Using prokaryotic expression and chromatography purification, the epitope-based recombinant RV-IgM diagnostic antigen was obtained with excellent antigenicity, which could be applied for the serological detection of the early infection with RV.

Protective effect of enterovirus­71 (EV71) virus­like particle vaccine against lethal EV71 infection in a neonatal mouse model.

Enterovirus-71 (EV71) is a viral pathogen that causes severe cases of hand, foot and mouth disease (HFMD) among young children, with significant mortality. Effective vaccines against HFMD are urgently required. Several EV71 virus-like particle (VLP) vaccine candidates were found to be protective in the neonatal mouse EV71 challenge model. However, to what extent the VLP vaccine protects susceptible organs against EV71 infection in vivo has remained elusive. In the present study, the comprehensive immunogenicity of a potential EV71 vaccine candidate based on VLPs was evaluated in a neonatal mouse model. Despite lower levels of neutralizing antibodies to EV71 in the sera of VLP-immunized mice compared with those in mice vaccinated with inactivated EV71, the VLP-based vaccine was shown to be able to induce immunoglobulin (Ig)G and IgA memory-associated cellular immune responses to EV71. Of note, the EV71 VLP vaccine candidate was capable of inhibiting viral proliferation in cardiac muscle, skeletal muscle, lung and intestine of immunized mice and provided effective protection against the pathological damage caused by viral attack. In particular, the VLP vaccine was able to inhibit the transportation of EV71 from the central nervous system to the muscle tissue and greatly protected muscle tissue from infection, along with recovery from the viral infection. This led to nearly 100% immunoprotective efficacy, enabling neonatal mice delivered by VLP-immunized female adult mice to survive and grow with good health. The present study provided valuable additional knowledge of the specific protective efficacy of the EV71 VLP vaccine in vivo, which also indicated that it is a promising potential candidate for being developed into an EV71 vaccine.

Immune responses to HBsAg conjugated to protein D of non-typeable Haemophilus influenzae in mice.

BACKGROUND: Hepatitis B vaccine that contains an aluminum hydroxide adjuvant induces apoptotic death of Hepa 1-6 cells. Difficult-to-degrade chemical additives in vaccines effectively enhance vaccine immunogenicity, but also affect the host tissue. Identification of bio-molecules that are readily degraded and compatible in vivo as an adjuvant is important for vaccine research. The hapten-carrier effect suggests that stimulation of helper T (Th) cells by carrier adjuvants is feasible. Protein D (PD) of non-typeable Haemophilus influenzae covalently conjugated to some polysaccharide vaccines has been confirmed to convert T-cell independent (TI) antigens into T-cell dependent (TD) antigens, and elicit strong T-cell responses ultimately. Herein, we would substitube PD for aluminum hydroxide adjuvant in Hepatitis B vaccine. METHODS AND RESULTS: Truncated PD (amino acids 20-364) was expressed in Escherichia coli and purified by (NH4)2SO4 precipitation and DEAE chromatography. After evaluation of antigenicity by western blotting, PD was covalently conjugated to yeast-derived recombinant HBsAg by cross-linking with glutaraldehyde. Intramuscular immunization with the conjugate induced higher level of HBsAg-specific antibody than did HBsAg alone (p < 0.05), and was comparable to commercial Hepatitis B vaccine. During the surveillance period (days 35-105), anti-HBs titers were hold high. Moreover, the conjugated vaccine enhanced Th1 immune responses, while Th2 responses were also activated and induced an antibody response, as determined by IFN-γ ELISPOT and IgG1/IgG2a ratio assays. CONCLUSIONS: Recombinant truncated PD covalently conjugated to HBsAg antigen enhanced the immunogenicity of the antigen in mice simultaneously by humoral and cellular immune response, which would facilitate therapeutic hepatitis B vaccines.

Epidemiology of hepatitis E virus in China: results from the Third National Viral Hepatitis Prevalence Survey, 2005-2006.

In China, hepatitis E virus (HEV) is prevalent and causes disease, but its epidemiological profile is not well understood. We used a commercial enzyme-linked immunosorbent assay to detect total antibodies to hepatitis E virus in 15,862 serum samples collected during the Third National Viral Hepatitis Prevalence Survey. The results were analyzed to calculate estimates of HEV seroprevalence and to examine the effects of some putative risk factors. The seroprevalence of HEV in the general Chinese population during the period from 2005 through 2006 was 23.46% (95% confidence interval [CI], 18.41%-28.50%). The farming population, the age group of 15-60 year olds, and those living in the Midwest or Mideast region and in Xinjiang province had the highest seroprevalence estimates. The prevalence of HEV is high in China. The seroprevalence rate of HEV shows an unbalanced distribution among areas with different geographic location and economic development levels. The characteristics of the distribution associated may be due to the route of HEV transmission (via contaminated water or animal reservoirs). Within the same region, the seroprevalence of HEV is generally increased with age.

[Construction of recombinant CHO cell strain for high expression of HBsAg].

OBJECTIVE: To overexpress hepatitis B virus S gene in CHO cells cultured in serum-free media. METHOD: Plasmid was constructed by cloning of HBV S gene and then it was transfected into CHO cells. After cell screen, the positive clones were identified and isolated into a serum-free media followed by the serological and morphological characterization of the expression product. RESULT: CHO cell strains which can express HBsAg efficiently and stably were obtained. Spherical and filamentous HBsAg could be detected under electronic microscope. The titer of the expression product was up to 1:5000. CONCLUSION: Serum-free media cultured CHO cell strain for overexpression of HBsAg was successfully constructed and the expression product was high antigenic.

[Antibody detection of hepatitis E virus in human population of different national in China].

OBJECTIVE: To investigate the seroprevalence of HEV infection in different national human population in Han, Hui and Zang in China. METHODS: EIA was used for detecting anti-HEV IgG of the serum samples. All samples were collected in 2006-2008 in Sichuan, Beijing, Heilongjianin, Sandong, Gansuo, Ningxia and Qinghai areas. RESULTS: The total positive rate of anti-HEV IgG was 17.97% (1878/ 10448), 24.32% (1794/7376) in Han national, 3.59% (81/2258) of Hui national and 0.37% (3/814) of Zang national, respectively. The positive rate of Han human at different age group, was 5.19% of < or = 10 year, 11.64% of 11-20 year, 20.08% of 21-30 year, 34.17% of 31-40 year, 41.75% of 41-50 year, 48.58% of 51-60 year, 57.43% of > or = 61 year. The positive rate of Hui human at different age group, was 3.11%, 3.96%, 2.11%, 3.98, 2.52%, 4.57% and 6.67%, respectively. Three positive of Zang human was between 21-60 year. CONCLUSIONS: The HEV infection in Han national population was higher than the Hui and Zang national, significantly. The HEV infection was correlation with age significantly, the infection rate was increased with age.

[The assemblage, purification and characterization of EV71 VLPs expressed in baculovirus].

To construct a recombinant expression plasmid Bacmid-P1-3CD containing the P1 and 3CD genes of enterovirus 71(EV71), the P1 and 3CD genes were cloned into the same baculovirus shuttle vector (Bacmid). Recombinant AcMNPV-P1-3CD was obtained by transfecting the Bacmid-P1-3CD into the insect cell line of S f9. With the IFA and Western-blot methods for identification of expression products confirmed that the target protein was expressed in interior of infected S f9 cells. Electron microscopy showed that the structural protein capsid P1 was cut by virus-encoded protease 3CD and assembled into EV71 virus like particles (VLPs) about 27nm diameter. Different values of MOI and time points of expression were compared to explore the optimal expression condition, and the results showed that the time point could be a more important factor. Then we used S f9 cells with serum-free medium in CellSTACK-10 Culture Chambers to produce EV71 VLPs in the confirmed condition. After purification of VLPs by density gradient centrifugation, we observed on SDS-PAGE profile the purified sample contained three major proteins whose molecular masses corresponded to those of VP1 (39kD), VP0 (34kD) and VP3 (26kD) as well as the intact structure, which can be greatly used for further study in protein structure and genetic engineering vaccine research.

[The investigation of hepatitis D virus infection situation in the human with HBsAg in Foshan of Guangdong province].

OBJECTIVE: To investigate the seroprevalence of hepatitis D virus in Foshan of Guangdong province, to provide the data for the study about it in China. METHODS: ELISA kits from two different companies were used for detecting anti-HDV IgG of all the serum samples, and then RT-PCR was carried out about the selected serum to ensure the results. All the serum samples were collected in 2011 in The First People's Hospital of Foshan. RESULTS: The results from two ELISA kits and RT-PCR were identical. Eight samples were positive. CONCLUSIONS: The seroprevalence rate of HDV in Foshan is higher than that in China. It has no statistically significant difference between female and male. Morever, the older with HBsAg are susceptible to HDV.

[Hepatitis C viruses infection situation in the human population of six provinces in China].

OBJECTIVE: To investigate the seroprevalence of hepatitis C viruse infection in the human population in six regions of Beijing, Heilongjiang, Shandong, Ningxia, Gansu and Sichuan in China. METHODS: ELISA was used for detecting anti-HCV IgG of the serum samples. All sample were collected in 2006-2008 in six areas. RESULTS: 9538 samples were detected. The total positive rate of anti-HCV was 0.39% (37), 0.23% (3/1328) in Beijing, 0.74% (12/1629) in Heilongjiang, 0.26% (5/1962) in Shandong, 0.1% (1/1000) in Ningxia, 0.44% in Gansu (9/2 037) and Sichuan (7/1582), respectively. The 37 positive samples at the sex were 19 (51.35%) of man and 18 (48.65%) of female. At the age group were 1 (2.70%) of < 10 years old, 5 (13.51%) of 10-19 years old, 4 (10.81%) of 20-29 years old, 6 (16.22% ) of 30-39 years old, 9 (24.32%) of 4049 years old, 12 (32.43%) of > or = 50 years old. The positive samples were detected anti-HAV-IgG, anti-HEV-IgG and HBsAg/ HBcAb /HBeAb. The positive Number was 35 (94.59%), 10 (27.03%) and 2 (5.41%) respectively. CONCLUSIONS: HCV infection rate in the population was 0.1% -0.74%, 1/3 of > or = 50 years old in HCV positive. Hepatitis C virus co-infection with HAV, HEV and HBV.

[The characterization analysis of HAV recombinant antigen was expressed by vaccinia virus vector].

OBJECTIVE: To find a suitable cell line for hepatitis A antigen expressed by vaccinia virus vector and to find a way of inactivation and preservation of the HAV recombinant antigen. Methods Series of cell lines such as K4,143, HEL, Hep-2 and Vero were inoculated with vaccinia virus that can express HAV recombinant antigen. ELISA was used to determine the contents of expression antigen. The characterization of the HAV antigen expressed by vaccinia virus was then analyzed after it was treated with different methods. RESULTS: The expression of HAV recombinant antigen in K4,143 and HEL cell lines was a little more than expression in Hep-2 and Vero cell lines. The antigenicity is obviously higher when HAV recombinant antigen was inactivated by beta-propiolactone other than it was inactivated by formalin. It was best to preserve the prepared HAV recombinant antigen under -40 degrees C condition. CONCLUSIONS: The application of vaccinia virus vector in hepatitis A antigen preparation was very useful and promising.

General epidemiological parameters of viral hepatitis A, B, C, and E in six regions of China: a cross-sectional study in 2007.

BACKGROUND: Viral hepatitis is a serious health burden worldwide. To date, few reports have addressed the prevalence of hepatitis A, B, C, and E in China. Therefore, the general epidemiological parameters of viral hepatitis remain unknown. PRINCIPAL FINDINGS: In this cross-sectional study, we performed a serological prevalence analysis of viral hepatitis A, B, C, and E in 8,762 randomly selected Chinese subjects, which represented six areas of China. The overall prevalence of anti-Hepatitis C virus antibody (anti-HCV) was 0.58%, which was much lower than was estimated by WHO. The prevalences of Hepatitis B virus surface antigen (HBsAg), anti-Hepatitis B virus surface protein antibody (HBsAb), and anti-Hepatitis B virus core protein antibody (HBcAb) were 5.84%, 41.31%, and 35.92%, respectively, whereas in the group of subjects less than 5 years old, these prevalences were 1.16%, 46.77%, and 8.69% respectively, which suggests that the Hepatitis B virus (HBV)-carrier population is decreasing, and the nationwide HBV vaccine program has contributed to the lowered HBV prevalence in the younger generation in China. Meanwhile, a large deficit remains in coverage provided by the national HBV immune program. In addition, our data suggested the possibility that HBsAb may not last long enough to protect people from HBV infection throughout life. The overall prevalence of anti-Hepatitis A virus antibody (anti-HAV) and anti-Hepatitis E virus antibody (anti-HEV) were as high as 72.87% and 17.66%, respectively. The indices increased with age, which suggests that a large proportion of Chinese adults are protected by latent infection. Furthermore, the pattern of HEV infection was significantly different among ethnic groups in China. CONCLUSIONS: Our study provided much important information concerning hepatitis A, B, C, and E prevalence in China and will contribute to worldwide oversight of viral hepatitis.

[Antibody detection of hepatitis E virus in some human population, swine and chicken in Beijing in China].

OBJECTIVE: To investigate the seroprevalence of HEV infection in human population, swine and chicken in Beijing region. METHODS: EIA was used for detecting anti-HEV IgG of the serum samples. All samples were collected in 2006-2007 in Beijing areas. RESULTS: The anti-HEV IgG was detected positive in 21.52% of human (260/1208), 46.88 % (15/32) of swine, but was negative in chickens (0/24). The positive rate of human at different age group, was 5.60% (14/250) of 11-20 year, 20% (42/210) of 21-30 year, 24.03% (62/258) of 31-40 year, 26.44% (78/295) of 41-50 year, 32.82% (64/195) of 51-60 year. The male (29.51%) was higher than the female (21.70%). CONCLUSION: The HEV infection was correlation with age and sex significantly. The infection rate was increased with age, the positive rate in swine was more double than the human population.

[Hepatitis viruses infection situation in human population of the Gansu province].

OBJECTIVE: To investigate the seroprevalence of hepatitis viruses in human population of Ganso province. METHODS: ELISA was used for detecting anti-HAY IgG, HBsAg/HBsAb, anti-HCV IgG and anti-HEV IgG of the serum samples. All sample were collected in four areas of KL, LT, HN and ZhL of Gansu province in 2008. RESULTS: 1977 samples were detected, The positive rates of anti-HAY, HBsAg/HBsAh, anti-HCV, and anti-HEY are 84.57% (1672/1977), 4.81% (95/1977) and 28.73% (568/1977), 0.46% (9/1977),and 13.10% (259/1977), respectively. The positive rate at different age group, for anti-HAY was 43.21% of c 9 years old, 80.23% of 10-19 years old, 93.02% of 20-29 years old, 95.55% of 30-39 years old, 95.60%-97.52% of 40-60 years old. For HBsAg/HBsAb were 2.09% or 29.27%, 3.02% or 21.81%, 6.20% or 36.43%, 5.93% or 31.16%, 6.43% or 3l.43%, 7.32% or 24.88%, 3.33% or 28.89% at the same age group, respectively, for anti-HCV, was 0.59% of 30-39 years old, 1.79% of 40-49 years old, 0.98% of 50-59 years old. For HEY-IgG was 3.48% of < or =9 years old, 6.05% of 10-19 years old, 8.53% of 20-29 years old, 19.29%-21.67% of 30- > or =60 years old. CONCLUSIONS: The inoculation againt HAY and HBY is enhanced in the youngster population. HBsAg carrier and UCY infection is decreasing. The HEY infection in the Hid people is lower than the Han nationality.

[Antibody detection and sequencing analysis of hepatitis E virus in human population, swine and chicken in Sichuan region in China].

OBJECTIVE: To investigate the seroprevalence of HEV infection and genotype. METHODS: ELISA were used for detecting anti-HEV IgG of the serum samples, the nested reverse transcriptase PCR (RT-nPCR) was used for detecting HEV RNA in patient serum and swine bile samples. All samples were collected in 2005-2007 in some districts in Sichuan province. The primers used for genotyping were the ORF2 region of HEV genome. RESULTS: The anti-HEV IgG was detected positive in childrens 6.10% (41/672), adults 42.26% (280/ 661), swines 88.89% (32/36), chickens negative (0/59). 1 case of 15 serum samples of anti-HEV IgM positive and 3 of 54 swine bile samples were positive for HEV RNA by RT-PCR.Sequence analysis of 4 isolates has 92.1% to 98.6% nucleotide sequence homology. These isolates from human and swine were identified closely related to Ch-T21 strain 90.1%-96.9% sequence homology, which belonged to HEV genotype 4B. CONCLUSIONS: The swine were the risk factors in the spread of hepatitis E virus.

[Expression of human IL-12 in mammalian cell and study on its biological activities].

OBJECTIVE: To obtain recombinant human interleukin 12 by genetic engineering, and to explore possibility of its clinical application in treatment of tumor and chronic hepatitis. METHODS: Bicistronic expression vector P35-IRES-P40 was constructed for the simultaneous translation of IL-12 p35 and p40 cDNA subunit through internal ribosomal entry sites (IRES). pCI-dhfr-P35-IRES-P40 vector was constructed for expression in CHO-DHFR- cells. Positively cloned cells were screened by means of ELISA. Pools of clones with increased expression of IL-12 could be generated by selection in methotrexate. To determine the biological activities of rhIL-12, PHA-activated lymphoblasts proliferation assay and IFN-gamma induction assay were used in this study. RESULTS: Genetically engineered cells expressing hIl-12 were obtained and all the cell lines showed the stabile expression of rhIL-12 in high efficiency and good growth properties. CONCLUSION: rhIL-12 have good biological activities, it can stimulate activation and proliferation of T cells and induce production of IFN-gamma.

[Evaluation of the ELISA diagnostic kits for hepatitis E virus antibody in the reference serum, the suspect patients of hepatitis E and normal persons' sera].

OBJECTIVE: To evaluate the quality of the ELISA diagnostic kits for detecting hepatitis E virus (HEV) specific IgG antibody. METHODS: Five diagnostic kits (WT, GD, HM, GeneLabs and KH) for anti-HEV IgG were assayed by detecting HEV IgG in the HEV diagnostic reference sera from 24 positive cases and 30 negative cases. 42 cases clinical suspect patients with hepatitis E from Ditan hospital in Beijing in 1994 to 1995 and 230 normal persons' sera from Chengdu city, Sichuan province in September in 2005. RESULTS: The conformity rates of positive and negative sera was 95.83% (23) and 96.67% (29) for WT kit, 87.50% (21) and 100% (30) for GD kit, 87.50% (21) and 96.67% (29) for HM kit, 66.67% (16) and 100% (30) for GeneLabs kit, 45.83% (11) and 100% (30) for KH kit for detecting anti-HEV positive and negative reference sera, respectively. The five kits were used to detect anti-HEV IgG among 42 clinical suspect patients with hepatitis E and 230 normal persons' sera, The positive rate was 97.62% (41) and 31.74% (73) by WT, 100% (42) and 17.39% (40) by GD kit, 97.62% (41) and 23.91% (55) by HM kit, 90.48% (38) and 7.83% (18) by GeneLabs kit, 90.48% (38) and 3.48% (8) by KH, respectively. CONCLUSION: The positive rates of all the 5 kits were over 90% and had a very high conformity in detecting anti-HEV IgG in clinical patient with hepatitis E, positive rates of 3.48% to 31.74% were found in detecting the normal persons' sera. The research showed insufficiency of the ELISA kits for epidemiological survey of HEV infection in population.

[Expression of the hepatitis Delta antigen in prokaryotic cell and evaluation of its application as an EIA diagnostic reagent].

BACKGROUND: To construct the pRSETB-HDAg recombinant expression plasmid and to obtain soluble hepatitis D virus antigen with high biological and antigenic activity. METHODS: HDAg gene fragment was inserted into fusion expression pRSET B vector that includes T7 promoter and a polyhistidine tag. The recombinant plasmid was transformed into host bacterium BL21 after induction with IPTG. The expression supernatant was purified by chelating affinity chromatography and the recombinant HDAg antigenic activity was detected by EIA. RESULTS: EIA detection using the recombinant HDAg showed strong positive reaction with hepatitis D patients sera. The positive rates of the EIA, compared with HDAg from USA and Hua Mei EIA kit in detecting 26 cases of anti-HDV positive reference sera, were 100%, 96.15% and 100%, respectively. CONCLUSION: Recombinant plasmid for HDAg with good antigenicity was successfully constructed and could be used as hepatitis D antibody detection reagent.

[Expression and antigenicity analysis of hepatitis G virus NS5 gene].

BACKGROUND: To determine the antigenicity of HGV NS5 recombinant proteins expressed in E.coli. METHODS: HGV NS5a,NS5b and core/NS5b fusion genes were cloned into pThioC vector. Three expression plasmids were transformed into JM109(DE3) competent cells then expressed with induction by IPTG. Western blot and ELISA were used to determine the antigenicity after the three recombinant proteins were purified. RESULTS: After identification by restriction enzyme and sequencing, it was confirmed that the expressed was target proteins espected. Purified expression proteins were found strongly immunoreactive among anti HGV positive sera by Western blot and ELISA. Compared with mixed recombinant antigen (including core, NS5a synthetic peptide and NS3 recombinant proteins), in the 22 positive sera detected with mixed antigen, 68%(15/22), 90%(20/22) and 73%(16/22) were positive by P5a,P5b and Pc?5b antigens; In the 70 negative samples with mixed antigen, 7%(5/70), 1%(1/70) and 6%(4/70) were positive by P5a, P5b and Pc?5b antigens. The positive alone was found among RTPCR positive specimen using these recombinant antigens. CONCLUSIONS: NS5 gene expressed in E.coli?which couldn't be covered with other regions of antigens was one of the essential epitopes to HGV immunologic diagnosis.