Immunogenicity and protective potency of Norovirus GII.17 virus-like particle-based vaccine.

OBJECTIVES: Noroviruses (NoVs) are major cause of acute viral gastroenteritis in worldwide, and the lack of a cell culture system that must be considered the virus like particles (VLPs) are used as an effective vaccine development. MATERIALS AND METHODS: In the present study, we investigated the expression of the major capsid protein (VP1) of the Genogroup II, genotype 17 (GII.17) NoV, using recombinant baculovirus system in insect cells, as well as a saliva binding blockade assay to detect their protective potency. RESULTS: Our results showed that GII.17 VLPs could be successfully generated in sf9 insect cells, and electron microscopic revealed that GII.17 VLPs appeared as spherical particles with a - 35 nm diameter. Immunized mice with purified VLPs produced GII.17 specific sera and could efficiently block GII.17 VLPs binding to the saliva histo-blood group antigens (HBGAs). CONCLUSIONS: Together, these results suggested that GII.17 VLPs represent a promising vaccine candidate against NoV GII.17 infection and strongly support further preclinical and clinical studies.

Excretion of infectious hepatitis E virus into milk in cows imposes high risks of zoonosis.

UNLABELLED: Hepatitis E virus (HEV) represents the main cause of acute hepatitis worldwide. HEV infection in immunocompromised patients involves a high risk for the development of chronic hepatitis. Because HEV is recognized as a zoonotic pathogen, it is currently believed that swine is the primary reservoir. However, this is not sufficient to justify the strikingly high seroprevalence of HEV in both developing and Western countries. Thus, this study aimed to identify new zoonotic sources that bear a high risk of transmission to humans. We collected fecal, blood, and milk samples of cows in a typical rural region of Yunnan Province in southwest China, where mixed farming of domestic animals is a common practice. HEV RNA was quantified by quantitative real-time polymerase chain reaction, and the whole genome was sequenced. HEV infectivity was assessed in rhesus macaques. We found a high prevalence of active HEV infection in cows as determined by viral RNA positivity in fecal samples. Surprisingly, we discovered that HEV is excreted into milk that is produced by infected cows. Phylogenetic analysis revealed that all HEV isolates from cow/milk belong to genotype 4 and subtype 4h. Gavage with HEV-contaminated raw and even pasteurized milk resulted in active infection in rhesus macaques. Importantly, a short period of boiling, but not pasteurization, could completely inactivate HEV. CONCLUSION: Infectious HEV-contaminated cow milk is recognized as a new zoonotic source that bears a high risk of transmission to humans; these results call attention to understanding and establishing proper measurement and control of HEV zoonotic transmission, particularly in the setting of mixed farming of domestic animals. (Hepatology 2016;64:350-359).

Hepatitis E virus infection activates signal regulator protein α to down-regulate type I interferon.

Hepatitis E virus (HEV) is a major cause of enterically transmitted acute hepatitis worldwide. However, the mechanism of HEV replication is unclear. Type I interferon is the first defense line of host against viral infection. Signal regulator protein α (SIRP-α) plays an important role in negative regulation of innate immunity. In the present study, HEV infection significantly activated the expression of SIRP-α and down-regulated phosphorylation of IRF3, consequently resulted in suppression of type I interferon (IFN-ß). In conclusion, HEV exploited SIRP-α to negative regulated IFN-ß of the host innate immune system to promote viral infection. It suggested that interfering with the functions of SIRP-α should be considered as a potential therapeutic approach to the prevention and treatment of HEV infection.

[Construction of Recombinant Full-length Hepatitis E Virus Fused with EGFP and Assessment of Infectivity].

The purpose of this study was to construct recombinant full-length hepatitis E virus(HEV)fused with enhanced green fluorescent protein(EGFP),and assess its infectivity in A549 cells. Two fragments from the full-length HEV genome and the EGFP gene were amplified by PCR. The EGFP gene was inserted downstream of the HEV ORF2 and then cloned into the pGEM® -7Zf(+)vector containing the T7 and SP6RNA polymerase promoters, producing pGEM-HEV-EGFP. The construction of the pGEM-HEV-EGFP recombinant plasmid was confirmed by restriction enzyme digest and sequencing. The pGEM-HEV-EGFP recombinant plasmid was transfected into A549 cells to assess infectivity using Lipofectamine. EGFP expression was observed at 24hpost-transfection,and expression of the HEV ORF2 was detected by immunofluorescence, confirming the presence of the HEV ORF2 and EGFP fusion protein. Cytopathic effects were observed at day seven post-transfection. The infectivity of pGEM-HEV-EGFP was confirmed by the presence of fluorescence after three continuous passages. The recombinant pGEM-HEV-EGFP vector was successfully constructed and effectively infected A549 cells, which will facilitate future studies on the mechanisms of HEV infection and pathogenesis.

[Molecular Epidemiology of Astrovirus Infection Among Hospitalized Infants in Kunming City, China, 2013～2014].

We investigated the infection and molecular-epidemiologic characteristics of human astrovirus (HAstV) of hospitalized infants in Kunming City from the year 2013 to 2014.Infection and genotype of HAstV of 63 samples of diarrheal feces and 42 controls were analyzed by reverse transcription-polymerase chain reaction(RT-PCR).The complete genome sequence of a HAstV strain was amplified and sequenced. The positive rate of HAstV in 63 feces samples was 41.27%(26/63).The main circulating genotype of HAstV was HAstV1.Only 1sample was positive for HAstV in 42controls(2.38%).A complete genome sequence of the HAstV strain was identified as HAstV1 by phylogenetic analyses. These data provide an important theoretical basis for the control of viral diarrhea in infants in Kunming City.

Pregnancy serum facilitates hepatitis E virus replication in vitro.

Hepatitis E virus (HEV) infection causes high mortality in pregnant women. However, the pathogenic mechanisms of HEV infection in pregnant women remain unknown. In this study, the roles of pregnancy serum in HEV infection were investigated using an efficient cell culture system. HEV infection was exacerbated by supplementing with pregnancy serum, especially theat in third trimester of pregnancy. Oestrogen receptors (ER-α and ER-ß) were activated in cells supplemented with pregnancy serum and were significantly inhibited during HEV infection. Type I IFN, especially IFN-ß, showed delayed upregulation in HEV-infected cells supplemented with the serum in the third trimester of pregnancy, which indicated that delayed IFN-ß expression may facilitate viral replication. Results suggested that pregnancy serum accelerated HEV replication by suppressing oestrogen receptors and type I IFN in the early stage of infection.

Complete Genome Sequence of the Salmonella enterica Serovar Paratyphi A Bacteriophage LSPA1 Isolated in China.

The bacteriophage LSPA1 was isolated from hospital sewage (Kunming, China), and lytic activity was demonstrated against the Salmonella enterica serovar Paratyphi A CMCC50973 strain. This bacteriophage has a 41,880-bp double-stranded DNA (dsDNA) genome encoding 58 coding sequences (CDSs) and belongs to the family Siphoviridae.

Simian foamy virus prevalence in Macaca mulatta and zookeepers.

The simian foamy virus (SFV) has been reported to be transmissible among humans occupationally exposed to nonhuman primates. Nevertheless, epidemiological and genotypic data on the SFV in Macaca mulatta and zookeepers in China are limited. In the present study, SFV proviral DNA was detected in 74 blood samples from M. mulatta and 12 saliva specimens from zookeepers by nested polymerase chain reaction. A total of 29 blood samples from M. mulatta (29/74, 39.19%) and two saliva specimens from zookeepers (2/12, 16.67%) were positive. The phylogenetic analysis indicated that these SFV strains shared the highest homology with Macaca fascicularis (93.4%). The two SFV strains infected human beings, and shared the highest homology of 98.6% with each other as well as 90.8-99.5% with M. mulatta. The investigation revealed the high prevalence of the SFV in M. mulatta in China and its zoonotic transmission to humans.

[The effect of various antigen compounds of tuberculosis composite antigen vaccine on the immune function in mice].

OBJECTIVE: To study the immune function of mice immunized by different combinations of antigen 85b (Ag85b), fusion protein culture filtered protein 10 (CFP-10), early secreted antigenic target 6 kDa protein (ESAT-6) and heat shock protein X (Hsp X) with combined adjuvants of Bacille Calmette-Guerin (BCG) CpG and aluminum. METHODS: According to antigen combinations, 48 BALB/c mice were divided into 8 groups: (1) group A: Ag85b + CFP-10/ESAT-6 + HspX + adjuvant; (2) group B: CFP-10/ESAT-6 + HspX + adjuvant; (3) group C: Ag85b + HspX + adjuvant; (4) group D: Ag85b + CFP-10/ESAT-6 + adjuvant; (5) group E: Ag85b + adjuvant; (6) group F: CFP-10/ESAT-6 + adjuvant; (7) group G: HspX + adjuvants; (8) control group: saline (6 mice per group). The mice were subcutaneously immunized 3 times. One week after the third subcutaneous immunization, spleens were collected for enzyme-linked immunospot (ELISPOT) assay to detect IFN-γ and IL-4 secretion, and for the lymphocyte proliferation assay to observe antigen-specific lymphocyte proliferation. Serum samples were separated for enzyme-linked immunosorbent assay (ELISA) to detect the titers of antigen-specific IgG, IgG(1) and IgG(2a) antibodies. RESULTS: The amount of IFN-γ spots in Group E [median(quartile), 122.8 (78.4 - 184.4)] was significantly more than that in group C [14.3 (6.5 - 14.6)] and the control group [0.5 (0.5 - 1.3)] (u = 0.0, P < 0.01). The amount of IL-4 spots in Group D stimulated with Ag85b and CFP-10/ESAT-6 [173.5 (78.8 - 233.4), 132.8 (50.3 - 159.4)] were significantly more than those in the control group [0.5 (0.5 - 1.3), 5.3 (2.9 - 6.5)] (u = 0.0, P < 0.01). The level of stimulation index of lymphocyte proliferation in Group A, C, D, E (2.42 ± 0.50, 2.18 ± 0.37, 2.86 ± 0.51, 2.70 ± 0.15) was significantly higher than that of the control group (1.11 ± 0.13) (F = 20.96, P < 0.01). The level of antigen-specific IgG, IgG(1), IgG(2a) antibody titers induced by Hsp X [lg(antibody dilution degree), 3.90 - 5.21] was significantly higher than those induced by Ag85b (3.30 - 4.51) and CFP-10/ESAT-6 (3.10 - 4.05) (F = 63.8 - 70.4, P < 0.01). CONCLUSIONS: With the use of adjuvants, different antigen combinations showed different influences on the immune function in mice. A combination of 3 antigens did not elicit the best immune effect, suggesting that the interaction among antigens may affect their immunity.

Seroepidemiology and molecular characterization of hepatitis E Virus in Macaca mulatta from a village in Yunnan, China, where infection with this virus is endemic.

BACKGROUND: Hepatitis E virus (HEV) infection is a significant public health concern and has been identified as a zoonotic infection. OBJECTIVES: Since no reports have characterized the epidemiological and genotypic features of HEV infections in Macaca mulatta (rhesus macaques) from Yunnan, China, where swine HEV infections are endemic, we aimed to investigate these characteristics. MATERIALS AND METHODS: Seroepidemiological and molecular characterization of HEV in both Macaca mulatta and pigs from the Yunnan province of China were conducted using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-nested PCR (RT-nPCR). Four hundred and eighty-two stool samples (320 from Macaca mulatta and 162 from pigs) and 92 serum samples (all from Macaca mulatta) were collected for the detection of HEV RNA and anti-HEV antibodies (IgG/IgM). RESULTS: Thirty-three rhesus macaques (35.87%) were positive for HEV IgG. Of these, 3 were also positive for HEV IgM. Four different strains of swine HEV RNA were detected in pigs; however, we failed to detect any in Macaca mulatta. CONCLUSIONS: Results indicate that Macaca mulatta may not be a natural reservoir of HEV.

[Cloning and characterization of the chloramphenicol resistant gene of Serratia marcescens strain KMR-3].

Chloramphenicol-resistant gene was cloned and analyzed by constructing genomic DNA library of Serratia marcescens KMR-3. It showed that cloned chloramphenicol-resistant gene encoded a protein product of 397 amino acids. The protein belonged to PRK10473 protein, and it showed 92% similarity to drug resistance transporter, Bcr/CflA subfamily of Serratia proteamaculans 568. Regulation elements including promoter, terminator, Shine-Dalgarno (SD) sequence and transcription start site also were identified.

[The analysis of promoters and the effective expression of exogenous proteins in a cold-adapted bacterium].

Based on the constructed promoter probe vectors that could replicate both in E. coli and in a cold-adapted bacterium, several candidate promoters were isolated and their activities were evaluated by RT-PCR. The transcription initiation sites and core sequence of promoters were determined by primer extension analysis. A low-temperature expression vector was constructed by using the strongest promoter and a thermolabile alpha-amylase gene was successfully overproduced under control of this promoter at low temperature (7 degrees C), while the secreted alpha-amylase amounted up to 35% of the total extracellular proteins. The expression system is expected to be useful for the production of thermolabile exogenous proteins at low temperatures.

[Construction of promoter probe vector for a cold-adapted bacterium, Acinetobacter sp. DWC6].

In order to build a protein expression system in a cold-adapted bacterium Acinetobacter sp. DWC6, a promoter probe vector was constructed based on the plasmid pBR322. A fragment containing the promoter of the beta-lactamase gene (the ampicillin resistance gene) in pBR322 was eliminated and replaced by a fragment comprizing a kanamycin resistance gene amplified from pJRD215. DNA fragment harboring in the Acinetobacter species specific ori was also inserted into the plasmid pBR322 to construct a promoter probe vector named pBAP1, which could replicate both in E. coli and in Acinetobacter sp. DWC6. The promoter selection library was constructed by randomly inserting genomic DNA fragment of Acinetobacter sp. DWC6 at upstream of reported gene, and target promoters were screened from genomic library on ampicillin selection plates. The function of pBAP1 and isolated promoters were determined by detection of the ampicillin sensitivity and the expression level of beta-lactamase in the host cell.

Expression, purification, and characterization of recombinant human interleukin 24 in Escherichia coli.

Interleukin-24 (IL-24) can induce apoptosis of a broad range of tumor cells, and this function of IL-24 is independent of classic tumor suppressor genes, such as p53, Rb and p16. Here, we report the expression, purification and preparation of a recombinant IL-24 protein (rIL-24) without post-translational modifications, which may selectively induce apoptosis of tumor cells in vitro. We found that non-fusion rIL-24 was not able to be expressed by vectors pET11c, 28a, and 22b in Escherichia coli. To obtain recombinant non-fusion IL-24 protein, the encoding region for IL-24 was cloned between KpnI and BamHI in pET32a. The Trx (Thioredoxin)/IL-24 fusion proteins were expressed in the form of inclusion bodies in E. coli host strain BL21 (DE21). The expression level was more than 30% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >96% were obtained. Trx/IL-24 proteins were digested by enterokinase (EK) to both Trx and rIL-24 fragments which then were separated by cation exchange chromatography. Cell proliferation experiments proved that the rIL-24 (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the rIL-24 may have cancer therapeutic applications.

[Construction and identification of ureB gene vaccine of H. pylori].

AIM: To clone ureB gene of H.pylori and construct its gene vaccine. METHODS: ureB gene was amplified by PCR from genome of H. pylori 11637 strain and subcloned into pMD18-T vector. The vector digested with restriction enzyme (Sal I and Bgl II) was inserted into pTCAE and transformed into E. coli DH5alpha. The positive recombinant plasmid identified by digesting with restriction enzyme (Sal I and Xho I) and sequencing named pT-ureB. The pT-ureB was transfected into CHO cells by electroporation method. The expression of UreB protein was detected by Western blot. RESULTS: The pT-ureB was obtained by cloning and recombinant DNA technique. The Western blot analysis showed that the expression of UreB protein (M(r)approximately 62,000) was detected in culture supernatant of CHO cells following transfection with pT-ureB. CONCLUSION: UreB DNA vaccine of H. pylori was successfully constructed. The expression of UreB protein can be detected in culture supernatants of transfected CHO cells.

Restriction fragment length polymorphism of adhesin gene hpaA from different Helicobacter pylori strains of Chongqing, China.

AIM: To assess the variability of adhesin gene hpaA between different Helicobacter pylori (H pylori) strains with PCR-restriction fragment length polymorphism (RFLP). METHODS: Twelve different H pylori strains were chosen to amplify the 710-bp segments of gene hpaA. These strains were NCTC11637, SS1; Chongqing clinical isolates CCS9801, CCS9802, CCS9803, CCS9806, CCS9809, CCS9810, CCS9813, which were gained from patients of gastritis; Mongolia gerbil adapted H pylori strains (abbreviation MG), which were gained from the following steps: gastric mucosal specimens of Mongolia gerbils infected by clinical isolate CCS9803 were cultured and detected, the positive H pylori strains were named as the first generation of Mongolia gerbil adapted H pylori strains (abbreviation MG1) and then were subcultured with healthy Mongolia gerbil to generate MG2, in turn to gain the ninth generation (abbreviation MG9). All hpaA segments, obtained from 12 different H pylori strains, were digested by HhaI and HaeIII individually and analyzed by agarose gel electrophoresis. RESULTS: In all 12 strains, the 710-bp PCR products were successfully amplified and products were cloned to pMD18-T vector respectively, then the recombinant plasmids were digested simultaneously with NcoI and XhoI to recover the small fragments. The objective fragments from 12 different H pylori strains digested with Hae III could be seen as 4 types of bands and 5 types with Hha I. According to the hpaA RFLP patterns, the 12 H pylori strains could be divided into 5 groups: group I, NCTC11637 and SS1; group II, CCS9809, which RFLP type digested with HaeIII was the same as strains of group I, but HhaI RFLP showed difference compared with the other groups; group III, CCS9810; group IV, CCS9803; group V: CCS9801, CCS9802, CCS9806, CCS9813, MG1, MG3 and MG9. The sequence data of 12 hpaA segments were analyzed by DNAsis software and it was observed that: (1) The homologies of base pair and amino acid sequence between strains NCTC11637, SS1, CCS9809 were 99.6% and 98.9%, respectively; (2) The homology of base pair and amino acid sequence between CCS9803 and CCS9810 was 97.7% and 99.1%; (3) That of the rest strains, CCS9801, CCS9802, CCS9806, CCS9813, MG1, MG3, MG9 reached 99.4% and 98.4%; (4) The base pair homologies between all hpaA fragments of different sources were higher than 94.6%, therefore the correspondence of deduced amino acid sequence was higher than 96.8% between each other. CONCLUSION: The gene hpaA from different H pylori strains revealed variation, and this might provide an effective method for molecular epidemiological survey of H pylori.