Dengue-1 virus envelope glycoprotein gene expressed in recombinant baculovirus elicits virus-neutralizing antibody in mice and protects them from virus challenge

In order to test the feasibility of baculovirus (Autographa californica nuclear polyhedrosis virus, AcNPV) expression vectors for making immunogens against dengue-1 (DEN-1) virus, a portion of the envelope (E) glycoprotein gene of DEN-1 virus was cloned and expressed. The recombinant baculovirus contains 107 nucleotides from the 3' terminus of the DEN-1 matrix (M) gene, which encodes a hydrophobic signal peptide and extends through the first 1, 245 nucleotides of E, terminating 243 nucleotides before the 3' terminus of E. When the recombinant virus was grown in Spodoptera frugiperda cells, about 1 mg of E antigen was made per 10(9) cells. Recombinant E antigen reacted with E protein-specific monoclonal antibodies and stimulated production of DEN-1 virus neutralizing antibody in BALB/c mice. Mice immunized with recombinant E antigen or with heat-inactivated DEN-1 virus were protected significantly against lethal DEN-1 virus challenge. A dose/response effect was observed, with increasing amounts of recombinant antigen leading to increased survival. These results demonstrate the utility of baculovirus for producing immunogens against DEN-1 virus.     

α-Thalassaemia and globin gene rearrangements in French Polynesia

Abstract: The prevalence of α-thalassaemia and various globin gene rearrangements was determined in 1992 individuals living on 11 islands in French Polynesia. The gene frequencies for α+ -thalassaemia (almost exclusively the -α^3.7III deletion form) range from 5.3% to 19.2%. Haematological indices on 177 heterozygotes and 27 homozygotes for the -α^3.7 III variant showed considerable overlap with indices of normal individuals; although there was a broad correlation of average indices with α-globin genotype, individual values were a poor indication of genotype. A non-deletion form of α+ -thalassaemia (αα^Th), triplicated α genes (ααα) and single zeta gene (-ζ) chromosomes were present at low frequencies (< 1%), whereas triplicated γ gene (γγγ) and triplicated ζ (ζζζ) arrangements were more common (1.1–16.3%). α⁰-thalassaemia, probably introduced from Southeast Asia in the early part of this century, was observed in a number of individuals of Chinese and Chinese/Polynesian ancestry. Because of the high frequency of α+ -Thalassaemia on some islands, it therefore seems likely that haemoglobin H disease (resulting from the interaction between α⁰ and α+ -thalassaemia) must occur in parts of French Polynesia.     

Diversity of envelope glycoprotein from human immunodeficiency virus type 1 of recent seroconverters in Thailand

The envelope-coding sequence of human immunodeficiency virus type 1 (HIV-1) was determined for 11 Thai seroconverters between 1995 and 1996. On the basis of the env sequences, all subjects were infected with HIV subtype E. Compared with the interpatient protein diversity among HIV-1 Thai reference sequences from 1990 to 1992 (4.4%), the diversity among the 1995-1996 seroconverters was approximately double (9.5%). The tetrapeptide tip of the V3 loop was invariant for 10 of the 11 seroconverters, and identical to that observed in sequences derived from the 1990-1992 group. However, in the V3 region, sequences from 2 of the 11 subjects demonstrated more than 5 amino acid changes relative to the reference strains. This may represent the "aging" of the HIV epidemic seen in other endemic regions. These findings may have substantial implications for vaccine development and evaluation for both HIV antibody and cytotoxic T lymphocyte repertoire recognition.     

Immunological responses to envelope glycoprotein 120 from subtypes of human immunodeficiency virus type 1.

The outer envelope glycoprotein (gp120) from subtypes A-E of HIV-1 was purified using a specific high mannose-binding lectin, Galanthus nivalis agglutinin. All isolates were grown in peripheral blood lymphocyte cells in order to avoid selection in cell lines. A comparison of the reactivities of the envelope proteins was made using sera from patients infected with the different subtypes. In this study, the B and C subtype envelope glycoproteins showed the strongest immunological reactivity, when reacted with sera from patients infected with the same subtype of virus. On the other hand, sera of patients infected with subtype A or C virus had the strongest and broadest reactivities, to envelope glycoproteins of many subtypes. The purified gp120 proteins from all five subtypes stimulated mononuclear cells from HIV-1 (subtype B)-infected patients, indicating conserved T cell-activating epitopes. The immunological reactivities indicate that strong antigenicity does not always predict the broadest immunogenicity of an envelope glycoprotein. Glycoprotein 120 from foreign subtypes may serve to induce strong cross-reactive immune responses.     

Oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein.

The envelope (env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of two noncovalently associated subunits, gp120 and gp41, that are formed gradient sedimentation, polyacrylamide gel electrophoresis, gradient sedimentation, polyacrylamide gel electrophoresis, and chemical cross-linking, we show that gp160 is synthesized as a monomer and subsequently forms stable homodimers. The molecule remains dimeric after cleavage to gp120/gp41 but is less stable to detergent solubilization and centrifugation. Analysis of wild-type and mutated env proteins indicated that interactions between the ectodomain regions of adjoining gp41 subunits are important for dimer formation and stability. A higher-order oligomeric form was also recovered, probably a tetramer consisting of two noncovalently associated dimers. The proposed subunit composition of the HIV-1 env protein is identical to that previously observed for the paramyxovirus envelope proteins F and HN.     

Neutralization of pseudotyped vesicular stomatitis virus expressing hepatitis C virus envelope glycoprotein 1 or 2 by serum from patients

Infection with hepatitis C virus (HCV) generally progresses to chronic disease, although a minority of patients appear to clear viremia spontaneously. In this investigation, serum samples were analyzed for virological parameters, serum alanine aminotransferase (ALT) levels, and neutralizing antibody response against pseudotyped vesicular stomatitis virus (VSV) generated using chimeric envelope glycoprotein 1 (E1) or 2 (E2) of HCV. Testing of sequential serum samples that were collected beginning at the onset of acute-phase disease demonstrated intermittent viremia, elevated ALT levels, and detectable neutralization activity against VSV in 9 of 10 patients. Serum neutralization activity did not exhibit a correlation with the genotype of the infecting HCV or with virus load. On the other hand, patients with chronic HCV infection consistently had detectable amounts of virus present but no significant variation in ALT levels, and serum samples from a majority (>90%) of patients failed to show detectable neutralization activity.     

河南省九株狂犬病毒的糖蛋白基因序列分析

目的 了解河南省狂犬病毒与人用、兽用狂犬病疫苗株在糖蛋白基因核苷酸和氨基酸序列水平上的差异,为有效控制狂犬病疫情提供初步的科学依据.方法 以反转录-半套式聚合酶链反应(RT-heminested-PCR)扩增2006年12月分离自河南省信阳市的9株狂犬病毒街毒株,经纯化、克隆、测序后获得9条糖蛋白基因全长序列,采用生物信息学软件构建基因系统发育树,对糖蛋白基因序列和氨基酸序列进行分析.结果 9株狂犬病毒糖蛋白在核苷酸及氨基酸序列水平上彼此的同源性分别为97.6%～98.9%和99.2%～99.8%;9株病毒与CTN疫苗的同源性最高,其核苷酸及氨基酸同源性分别为85.6%～93.0%和91.9%～92.9%;9株病毒与其他疫苗株相比,其核苷酸及氨基酸同源性分别为80.4%～83.3%和87.7%～92.5%;与已知的基因1型狂犬病毒比较,9株病毒糖蛋白氨基酸序列发生了若干位点的氨基酸取代.结论 9株河南省狂犬病毒街毒株均属基因1型,CTN疫苗株可能为目前我国河南省所流行的狂犬病提供较好的保护效果.     

2012年1株南美洲输入登革1型病毒的基因组特征

目的 分析2012年1株从南美洲输入登革1型病毒的基因组特征,探索登革病毒的传播规律.方法 测定DF1203毒株的全长基因组序列,分析该毒株与97株南美洲登革1型毒株的遗传联系,同时比较该毒株与全球最相似毒株的遗传联系.结果 DF1203毒株的基因组全长10 735个核苷酸,编码区全长10 179个核苷酸.种系发生分析显示,该毒株与南美洲登革1型毒株存在一定差异,BLAST分析表明DF1203与东南亚毒株存在密切遗传联系,尤其与近年柬埔寨毒株在种系发生树上处于同一分支.结论 虽然DF1203的患者来自南美洲苏里南,毒株遗传背景分析却显示与东南亚毒株存在亲密遗传关系.由此表明连续有效的登革病毒监测,尤其是检测分离株的部分或全部基因组序列,对于研究登革病毒传播路径、遗传进化,具有重要意义.     

Effect of human immunodeficiency virus type-1 envelope glycoprotein gp160 on cytokine production from cord-blood T cells

We have recently demonstrated that the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp160 enhances the in vitro differentiation of hematopoietic myeloid progenitor cells derived from cord blood by inducing secretion of colony-stimulating factor(s) (CSF) in T cells, presumably through the interaction of gp160 with CD4 molecules. In this study, we investigated the gp 160-induced humoral CSFs in cord blood by enzyme-linked immunosorbent assay (ELISA) and by polymerase chain reaction on reverse-transcribed mRNA (RT-PCR). We demonstrate that gp160 can induce interleukin (IL)-3, IL-6, and granulocyte-macrophage CSF (GM-CSF) protein secretion only in purified cord-blood T cells (CB-T) and not in detectable amounts in whole cord blood cells (WCB); cytokine mRNA induction occurred in purified CB-T and WCB, but was significantly greater in the former. Treatment of gp160 with soluble CD4 (sCD4) abolished the secretion of all three cytokines in CB-T cells, which suggests that interaction of gp160 with CD4 molecules is required for the secretion of these cytokines from CB- T cells. However, in WCB cells, sCD4 treatment of gp160 resulted in inhibition of only IL-3 and GM-CSF mRNA, whereas IL-6 secretion was enhanced. Purified cord-blood monocytes secreted only IL-6 in response to gp160, and the gp160-induced IL-6 secretion by monocytes was also further increased by gp160 + sCD4 complex. Furthermore, monocyte culture supernatants suppressed gp160-induced IL-3 secretion from CB-T cells. These findings indicate that (1) CB-T cells are a potent source of gp160-induced hematopoietic cytokines, and (2) that different mechanisms are involved in the induction of IL-6 by gp160 in the T- and non-T-cell fractions of cord blood. The ability of HIV gp160 to induce hematopoietic CSFs in cord blood may be important in HIV pathogenesis.     

Possible dengue sequential infection: dengue spread in a neighbourhood during the 1996/97 dengue-2 epidemic in French Polynesia

A DEN-2 epidemic occurred in French Polynesia from August 1996 to April 1997 after 7 years of DEN-3 circulation. The susceptible population constituted all expatriates and Polynesians under 21. In August 1996, two successive DEN-2 cases occurred in Teroma, a Tahitian neighbourhood close to the international airport of Tahiti. A serological prospective study of persons < 21 years living in Teroma was conducted. The study population was bled in September 1996, October 1996 and June 1997. Analysis of dengue spread in Teroma confirmed that dengue transmission occurs primarily in the house, thus vector control campaigns should incorporate focal insecticide spraying and systematic daily use of insecticide in houses. The evolution in time of the disease demonstrated that among a susceptible population, prevalence and incidence rates are related to the time of exposure, and consequently to age. Comparison of dengue incidence or dengue prevalence between populations therefore requires adjusted age rates. Most studies did not adjust for age, leading to the conclusion that DHF is more frequent during secondary than during primary dengue infection. Prospective studies taking into account the time of dengue exposure are necessary to confirm the sequential infection hypothesis.     

贵州省狂犬病病毒糖蛋白基因序列特征分析

目的 分析贵州省狂犬病病毒的糖蛋白基因（G基因）序列特征,为狂犬病的有效预防和控制提供科学依据。方法 以RT- PCR扩增贵州省近年来不同地区的人和犬狂犬病病毒阳性脑组织标本狂犬病病毒G基因序列,对扩增产物测序后采用生物信息学软件进行序列分析。结果 贵州省狂犬病毒阳性标本经RT-PCR扩增、测序与拼接后得到8份狂犬病病毒阳性样本的G基因序列。同源性分析显示,8份狂犬病病毒阳性样本的G基因序列在核苷酸及氨基酸水平上彼此的同源性分别为87.4%～99.9%和83.3.%～100%,与狂犬病毒基因1型病毒株G基因的核苷酸和氨基酸序列同源性最高（分别为86.5%～87.0%和83.3%～100%）。进化树分析显示,贵州省狂犬病病毒G基因的亲缘进化关系较近,且与狂犬病毒基因1~7型中的基因1型代表毒株的亲缘进化关系最近;除GZ01和GZ09外,其余狂犬病毒G基因与国内湖北、湖南、安徽、广西、江苏和上海毒株相距较近。除GZ09与国外的马来西亚和泰国代表毒株亲缘进化关系最近外,其与毒株与印度尼西亚毒株亲缘进化关系最近。结论 本研究从狂犬病病毒G基因水平证明贵州省狂犬病毒株属于基因1型,揭示了我省贵州省狂犬病毒与国内外已报道毒株的亲缘进化关系,该研究结果将为贵州省狂犬病的有效预防和控制提供科学依据。     

A ligand-binding pocket in the dengue virus envelope glycoprotein

Dengue virus is an emerging global health threat. Its major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. A crystal structure of the soluble ectodomain of E from dengue virus type 2 reveals a hydrophobic pocket lined by residues that influence the pH threshold for fusion. The pocket, which accepts a hydrophobic ligand, opens and closes through a conformational shift in a beta-hairpin at the interface between two domains. These features point to a structural pathway for the fusion-activating transition and suggest a strategy for finding small-molecule inhibitors of dengue and other flaviviruses.     

Immunogenicity of the envelope glycoprotein of avian sarcoma virus.

The expression of type E-specific glycoprotein in chick-helper factor-positive [chf(+)] chickens is not restricted to a certain stage of embryogenesis but persists in postembryonic life. This finding prompted an investigation of the immunogenicity of the viral envelope glycoprotein of exogenous avian sarcoma virus in chf(+) and chf(-) chickens. Sensitization of both classes of chickens resulted in the induction of detectable antibody reactivity to determinants of the glycoprotein that were type-specific as well as group-specific. Because group-specific determinants are present on type E-specific glycoprotein, these results link immunity to exogenous viral envelope glycoprotein in chf(+) chickens with autoreactivity. A model is proposed to rationalize the induction of reactivity in this system.     

New description of Toxoplasma gondii genotypes from French Polynesia

We report here the first isolation and genotyping of two human Toxoplasma gondii strains from French Polynesia. The parasites had new and atypical genotypes, and were responsible for asymptomatic congenital toxoplasmosis. Both genotypes were divergent from the common strains isolated in Europe, North America, South America, Africa and China.     

Hepatitis C virus E1 envelope glycoprotein interacts with apolipoproteins in facilitating entry into hepatocytes

Our previous studies demonstrated that hepatitis C virus (HCV) envelope glycoproteins 1 and 2 (E1 and E2) display distinct reactivity to different cell-surface molecules. In this study, we characterized the interaction of E1 and E2 with apolipoproteins in facilitating virus entry. The results suggested a higher neutralization of vesicular stomatitis virus (VSV)/HCV E1-G pseudotype infectivity by antibodies to apolipoprotein E (ApoE) than apolipoprotein B (ApoB), with VSV/HCV E2-G pseudotype infectivity remaining largely unaffected. Neutralization of cell-culture-grown HCV infectivity by antiserum to ApoE and, to a lesser extent, by ApoB further verified their involvement in virus entry. HCV E1, but not E2, displayed binding with ApoE and ApoB by enzyme-linked immunosorbent assay. Binding of E1 with apolipoproteins were further supported by coimmunoprecipitation from human hepatocytes expressing E1. Rabbit antiserum to a selected E1 ectodomain-derived peptide displayed ～ 50% neutralization of E1-G pseudotype infectivity. Furthermore, E1 ectodomain-derived synthetic peptides significantly inhibited the interaction of E1 with both the apolipoproteins. Investigation on the role of low-density lipoprotein receptor (LDL-R) as a hepatocyte surface receptor for virus entry suggested a significant reduction in E1-G pseudotype plaque numbers (～ 70%) by inhibiting LDL-R ligand-binding activity using human proprotein convertase subtilisin/kexin type 9 and platelet factor-4, whereas they had a minimal inhibitory effect on the E2-G pseudotype. CONCLUSION: Together, the results suggested an association between HCV E1 and apolipoproteins, which may facilitate virus entry through LDL-R into mammalian cells.