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.

Hepatitis B Immunoprophylactic Failure and Characteristics of the Hepatitis B Virus Gene in Mother-Infant Pairs in Parts of China.

OBJECTIVE: To determine the hepatitis B immunoprophylactic failure rate in infants born to hepatitis B virus (HBV) infected mothers and to characterize HBV genes. METHODS: HBV-serological testing was conducted for pregnant women and infants. The complete genomes of 30 HBV isolates were sequenced, and genetic characteristics were analyzed using MEGA 5 software. RESULTS: The immunoprophylactic failure rate for infants who had completed the scheduled hepatitis B vaccination program was 5.76% (32/556). High sequence homology (99.8%-100%) was observed in 8 of the 10 mother-infant pairs. We identified 19 subgenotype C2 strains, 9 subgenotype B2 strains, and 2 subgenotype C1 strains. Three serotypes were detected: adr (19/30), adw (9/30), and ayw (2/30). The frequency of amino acid mutation of the 'a' determinant region was 16.67% (5/30), including that of Q129H, F134Y, S136Y, and G145E. We detected 67 amino acid mutations in the basal core promoter, precore, and core regions of the genome. CONCLUSION: The immunoprophylactic failure rate in infants born to HBV-infected mothers is low in the regions of China examined during this study. Moreover, HBV mutation in the 'a' determinant region could not account for immunoprophylactic failure for all infants.

The Regulatory Roles of ncRNA Rli60 in Adaptability of Listeria monocytogenes to Environmental Stress and Biofilm Formation.

Listeria monocytogenes is a facultative anaerobic Gram-positive bacterium. It is well adapted to external environments and able to infect both humans and animals. To understand the impacts of ncRNA Rli60 on the adaptability of L. monocytogenes to environmental stresses and biofilm formation, a rli60 deletion strain of L. monocytogenes (LM-Δrli60) was constructed using splicing by overlap extension PCR (SOE-PCR) and homologous recombination and then compared it with wild-type strain L. monocytogenes EGD-e in the aspects of adaptability to environmental stresses by measuring their growth under stresses of different temperatures, and acidic, alkaline, hypertonic and alcoholic conditions, and capability of biofilm formation by using crystal violet staining, as well as the transcriptional levels of genes (gltB and gltC) related to the biofilm formation by real-time quantitative PCR (qRT-PCR). The results showed that (1) the growth of LM-Δrli60 strain was significantly slower under environmental stresses of low temperature (30 °C), high temperature (42 °C), as well as alkaline and alcoholic conditions, (2) the amount of biofilm formed by LM-Δrli60 was attenuated, and (3) the transcriptional levels of gltB and gltC genes at 24 h and 48 h in LM-Δrli60 revealed a significant reduction. Overall, the results confirmed that ncRNA Rli60 plays important roles in regulating the adaptability of L. monocytogenes to environmental stresses and biofilm formation possibly through impacting the expression of gltB and gltC genes.

The roles of noncoding RNA Rli60 in regulating the virulence of Listeria monocytogenes.

BACKGROUND: Listeria monocytogenes (LM) is an important zoonotic foodborne pathogen. Noncoding RNA (ncRNA) has an important role in regulating its virulence. As a member of ncRNA, however, the function of Rli60 in regulating LM virulence remain unclear. The aim of this study was to investigate the role of Rli60 in regulating LM virulence. METHODS: Using a homologous recombination method, a LM EGD-e rli60 gene deletion strain (LM-Δrli60) was constructed and compared with a LM EGD-e strain in the following respects: (1) adhesiveness, invasion ability, intracellular survival, proliferation, and transcription of virulence genes in the mouse macrophage cell line RAW264.7; (2) 50% lethal dose (LD50) to the BALB/c mouse; and (3) the amount in the mouse liver and spleen and the effects on pathology of mouse liver, spleen, and kidney after inoculation. RESULTS: The LM-Δrli60 strain had a significantly higher adhesion rate and lower invasion rate with significantly lower intracellular survival and proliferation rates in the RAW264.7 cell line, compared to the LM EGD-e strain. Inoculation with LM-Δrli60 strain significantly affected the transcription of virulence genes. The LD50 of LM-Δrli60 to BALB/c mouse was increased by 2.12 logarithmic magnitude, which indicated that the virulence in LM-Δrli60 is significantly decreased (p < 0.05). The amount of LM-Δrli60 in the liver and spleen was significantly lower than the amount of LM EGD-e in these organs (p < 0.05). The pathological damage due to LM-Δrli60 infection in the mouse liver, spleen, and kidney was lower than the damage due to LM EGD-e infection. CONCLUSION: This study confirmed that the rli60 deletion could significantly affect LM virulence, adhesion, invasion, survival, and proliferation. This suggests that Rli60 has an important role in regulating LM virulence.

[Comparison of ELISA diagnostic kits used in China for hepatitis C virus IgG antibody detection].

OBJECTIVE: To compare the commercial diagnostic reagent available in China for hepatitis C virus ( HCV) IgG antibody detection in order to provide some useful information for HCV prevention. METHODS: The HCV-IgG detection reagents produced by six different Enterprises named A, B, C, D, E and F were chosen and applied to detect 160 HCV specious sera samples. HCV-IgG reagent from ABBOTT was adopted as gold-standard and the samples in gray zone were determined by RIBA method finally. RESULTS: 160 sera samples comprised 88 positive samples and 72 negative samples. The total conformity ranged from 88.13% to 98.13% and the Youden indexes ranged from 0.74 to 0.96 when the reagents from six different Enterprises were compared with gold-standard. The highest conformity was 98.13%, observed in D reagent with the highest Youden index of 0.96. CONCLUSION: The total conformity rates were more than 88% when the HCV-IgG antibody detection reagents from six different Enterprises were compared with ABBOTT reagent. It was highly conformable. However, some reagent proved to be less conformable in negative samples detection.

[Value of combined detection of GP73 and alpha-fetoprotein in diagonosis of hepatocellular carcinoma].

OBJECTIVE: To explore the diagnostic value of the measurement of serum Golgi protein 73 (GP73) in the diagnosis of hepatocellular carcinoma (HCC). METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum GP73 in the 81 cases of HCC, 71 cases of chronic hepatitis or cirrhosis (CH/LC) and 65 cases of healthy blood donors, and to evaluate the sensitivity and specificity in the diagnosis of HCC through the ROC curves. RESULTS: The average levels of serum GP73 in HCC, CH/LC and Normal groups were (152.67 +/- 33.59) ng/ml, (93.15 +/- 20.02) ng/ml and (58.95 +/- 17.29) ng/ml(o) After calculating through the ROC curves, 120 ng/ml was set as the optimal cut-off point, GP73 has a sensitivity of 77.80% and a specificity of 78.00%. CONCLUSIONS: GP73 as a serum marker in the diagnosis of HCC had a higher sensitivity than AFP, and the combined detection of GP73 and AFP could improve HCC diagnosis.

[Investigation of the relationship between apolipoprotein gene polymorphism and hepatitis B virus infection in China].

OBJECTIVE: To explore the gene polymorphisms of ApoAI-75 Msp1, ApoB Msp1, ApoCIII Sst1, LRP5, and ApoE genotypes in two pairs of semi different modes of hepatitis B for HBV markers. METHODS: The patients are divided into 9 groups. There were a total of 720 cases, 80 patients in each group, The patients was carried out by SnaPshot method (single-base multilocus micro-sequencing), and different genotypes of each locus were conducted by the method of sequencing in order to support the final evidence of the accuracy of test results. RESULTS: There was association between gene polymorphisms of ApoAI-75Msp1 and ApoE and different modes of two pairs of semi-hepatitis B (P < 0.05), while there wasn't any association between gene polymorphisms of ApoB-Msp1, ApoCIII-Sst1, LRP5 and different modes of two pairs of semi-hepatitis B (P > 0.05). CONCLUSION: The gene polymorphism of ApoAI-75Msp1 and ApoE was associated with the different modes of HBV markers.

[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.

[Establishment and application of TaqMan real-time RT-PCR for the detection of hepatitis E virus].

OBJECTIVE: To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis E virus (HEV). METHODS: According to the references, primers-probe sets which were located in ORF2, the conservative part of HEV genome were designed and therefore we established a HEV TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HEV RNA in clinical samples. RESULTS: The HEV Real-time RT-PCR assay established in this study were able to detect HEV RNA with a detection limit of 10 copies/reaction. When the detection of a same sample was repeated for several times, coefficients of variation (CV) was all less than 1.53%. Our data also suggested that there were 1.87 x 10(6)-8.12 x 10(9) RNA copies in 1 ml of the clinical samples. CONCLUSION: The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HEV RNA. It was applied successfully in the pathogen detection of clinical samples.

[Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

OBJECTIVE: To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. METHODS: Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software. RESULTS: SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene. CONCLUSION: Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

[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.

[Genetic analysis of HAV strains isolated from patients with acute hepatitis in Hebei Shijiazhuang of China].

OBJECTIVE: To characterize the hepatitis A virus (HAV) wild type strains circulating in Hebei Shijiazhuang of China during 2005-2007, to provide the bases for further investigation of the sources of HAV infection. METHODS: The VP1/P2A junction regions were detected by RT-PCR from HAV IgM positives serum samples during 2005 and 2007, the 34 RT-PCR positive samples were sequenced and subjected to phylogenetic analysis by Neighbor Joining (NJ) method. RESULTS: All the detected HAV strains were identified as sub-genotype I A, the homology of nucleotide sequence in the VP1-2A imation region ranged from 95%-100%, the amino acid sequences of HAV strains almost had no difference. CONCLUSION: There are different HAV strains existing in Hebei Shijiazhuang of China, same HAV strain may exist in different areas; or in one area, identical or different HAV strains may be detected. This work provides the bases for further investigation of the sources of HAV infection and also for effectively control measures to prevent the spread of the disease.

[Genotyping of hepatitis A virus prevalent strains in Xinjiang Hetian of China in 2006].

OBJECTIVE: To analysis the genotypes of wild type hepatitis A virus circulated in Xinjiang Hetian of China in 2006. METHODS: The Vp1-2A region of HAV genome was amplified and sequenced from serum samples collected in Xinjiang Hetian of China in 2006, and subjected to phylogenetic analysis by Neighbor Joining (NJ) method. RESULTS: The nucleotide sequence differences in the VP1-2A region among Xinjiang Hetian HAV strains ranged from 0%-3.9%, all belonged to sub-genotype 1A. Genetically similar strains were identified among Xinjiang Hetian 2006 and Xinjiang Yili 2005 of China isolates. Only 0-2 amino acid differences were found among the Xinjiang Hetian HAV isolates in the VP1-2A region. CONCLUSION: There were different HAV strains existing in the investigated areas, these strains may have different transmission pathways for the spread of the disease. The results indicate the usefulness of molecular epidemiological methods in studying changes in the circulating HAV strains and in tracing transmission routes, and also for effectively control measures to prevent the spread of the disease.

[Development of an extraction and concentration method for the detection of hepatitis A virus in different samples].

OBJECTIVE: To develop an extraction and concentration method for the detection of hepatitis A virus (HAV) in shellfish, water, serum and saliva samples by nested RT-PCR. METHODS: HAV were artificially inoculated into the above samples, calm sample was extracted using glycine buffer pH9.5, PEG precipitation; water sample was PEG precipitated directly; then all the samples including serum and saliva samples were extracted using Trizol regent, followed by nested RT-PCR detection using primers from HAV VP1-2A region. RESULTS: The detection limit for HAV in cultured cell lysis was 0.1TCID50; in water, serum or salva sample was 1TCID50 respectively, in calm sample was 1-10 TCID50. HAV RNA was detected in water and sera samples collected from the HAV outbreak region, sequenced and analysis. CONCLUSION: The method developed here is convenient, specific and capable of detecting low levels of HAV in different samples, would be useful for diagnostic laboratories in order to perform HAV analysis in cases of foodborne infections or for molecular epidemiology investigation of HAV outbreaks.

Anatomic study of maximum intensity projection of the membranous labyrinth and the internal auditory meatus - MRI scan in 16 Chinese adults.

CONCLUSION: Three-dimensional reconstruction of maximum intensity projection (MIP) might document objectively, stereoscopically and directly the minute structures of the membranous labyrinth and internal auditory meatus. In this study, we establish magnetic resonance imaging (MRI) measurement criteria of the inner ear in Chinese adults. OBJECTIVE: The goal of this study was to provide an anatomic basis for otolosurgery and neurosurgery in Chinese adults. MATERIALS AND METHODS: Sixteen healthy volunteer subjects were scanned by a GE-signa 1.5T MRI scanner. All original images were transferred to an MRI workstation and all the structures of the inner ear were reconstructed, rotated at various angles and measured with an MIP program. RESULTS: Anatomic structures of the membranous labyrinth and internal auditory meatus were well demonstrated in MIP images in all volunteers. All inner ear structures including utricle, saccule, cochlear duct, internal auditory meatus and three semicircular ducts produced high intensity signals.

[Molecular characteristics of inlB protein of Listeria monocytogenes wild strain and it's expression and purification].

Internalin B of Listeria monocytogenes TA wild strain was amplified by PCR method and sequenced, then subcloned into prokaryotic vector pET28a for expression. The complete length of InlB gene was 1893 bp, encoding 630 amino acids. The front 35 amino acids consisted of signal peptide. There were one cap domain with alpha-helix region, 6 leucine-rich repeat motifs, one immunoglobulin (Ig)-like domain, one B repeat sequence, 3 direct repeat GW domains and 5 N-glycosylation sites. The amino acid of leucine amounted to 10.2 percent in all amino acids of deduced sequence Compared the InlB genes with that of other 18 isolates reported in GenBank, the identities of nucleotide sequence and deduced amino acid sequence were 91.1% to approximately 99.6% and 92.3% to approximatgely 99.8%, respectively. Expressed InlB protein was detected by SDS-PAGE and confirmed by western blot. Recombinant protein was successfully purified by Ni2+ affinity chromatograph column.

[Baculovirus expression of two human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus].

OBJECTIVE: To develop human recombinant neutralizing IgG monoclonal antibodies to hepatitis A virus (HAV) by baculovirus expression system. METHODS: The heavy and light chain genes of two human-derived neutralizing Fab antibodies to HAV were cloned into baculovirus expression vector Pac-kappa-Fc and Pac-L-Fc, and further expressed in insect cells as IgG antibodies. The IgG products were purified and well characterized. RESULTS: The baculovirus expressed McAb HAFc16 fully retained the specificity of binding to hepatitis A virus and the competition with mouse anti-hepatitis A virus McAb using ELISA. The viral neutralization assay in vitro demonstrated the retention of antibody function after expression of the human antibody in insect cells. The other expressed antibody HAFc78 also has the neutralizing activity but it is directed against different epitopes of HAV when compared with HAFc16. CONCLUSION: The recombinant baculovirus/insect cells expressed human neutralizing IgG antibodies to hepatitis A virus retained all biological functions specific for hepatitis A virus. The results provided the possibility of using these antibodies to rapidly protect high risk or early exposure populations from hepatitis A virus infection.

Detection of anti-MHAV and anti-MHEV IgM in patients with sporadic acute hepatitis in Beijing between 1995-2000.

OBJECTIVE: To investigate the age range, liver function damage and prognosis of patients with sporadic acute hepatitis A and E in Beijing. METHODS: Enzyme immunoassay (EIA) was used to detect anti-HAV and anti-HEV immunoglobulin M (IgM). Serum samples were collected from the patients with sporadic acute viral hepatitis in Beijing from January 1995 to June 2000. RESULTS: The total Positive rate for anti-HAV and anti-HEV IgM was 55.2% (112) in 203 patients with acute hepatitis, of whom 22.2% (45 patients) and 33.0% (67) were positive for anti-HAV and anti-HEV respectively. The duration of anti-HEV IgM was 45-60 days and that of anti-HAV IgM was at least 2-3 months. The patients with acute hepatitis A and hepatitis E all experienced jaundice and a rising of liver enzyme, but did not develop chronic hepatitis or died. CONCLUSION: Acute hepatitis A as well as acute hepatitis E plays an important role in sporadic enterically transmitted hepatitis in Beijing.