Evaluation of Extracellular Subviral Particles of Dengue Virus Type 2 and Japanese Encephalitis Virus Produced by Spodoptera frugiperda Cells for Use as Vaccine and Diagnostic Antigens

New or improved vaccines against dengue virus types 1 to 4 (DENV1 to DENV4) and Japanese encephalitis virus (JEV), the causative agents of dengue fever and Japanese encephalitis (JE), respectively, are urgently required. The use of noninfectious subviral extracellular particles (EPs) is an inexpensive and safe strategy for the production of protein-based flavivirus vaccines. Although coexpression of premembrane (prM) and envelope (E) proteins has been demonstrated to produce EPs in mammalian cells, low yields have hindered their commercial application. Therefore, we used an insect cell expression system with Spodoptera frugiperda-derived Sf9 cells to investigate high-level production of DENV2 and JEV EPs. Sf9 cells transfected with the prM and E genes of DENV2 or JEV secreted corresponding viral antigens in a particulate form that were biochemically and biophysically equivalent to the authentic antigens obtained from infected C6/36 mosquito cells. Additionally, equivalent neutralizing antibody titers were induced in mice immunized either with EPs produced by transfected Sf9 cells or with EPs produced by transfected mammalian cells, in the context of coimmunization with a DNA vaccine that expresses EPs. Furthermore, the results of an enzyme-linked immunosorbent assay (ELISA) using an EP antigen derived from Sf9 cells correlated significantly with the results obtained by a neutralization test and an ELISA using an EP antigen derived from mammalian cells. Finally, Sf9 cells could produce 10- to 100-fold larger amounts of E antigen than mammalian cells. These results indicate the potential of Sf9 cells for high-level production of flavivirus protein vaccines and diagnostic antigens.Dengue virus types 1 to 4 (DENV1-4) and Japanese encephalitis virus (JEV), the causative agents of dengue fever and Japanese encephalitis (JE), respectively, are globally important human pathogens (10) for which new or improved vaccines are urgently required. DENV1-4 cause dengue fever and dengue hemorrhagic fever in tropical areas and many subtropical areas. An estimated 50 million to 100 million dengue cases occur annually, with 2.5 billion people at risk of infection (11). However, there is no approved vaccine for dengue diseases, and the development of such a vaccine is urgently needed (12). JEV is the single largest cause of childhood viral encephalitis in the world, with an estimated 50,000 cases annually. Mortality rates can reach 30% among confirmed cases, and as many as one-third of survivors suffer from permanent and severe psychoneurological sequelae (13, 39). Although inactivated vaccines are used internationally for JE, they are too expensive for widespread use in most developing countries (3), and therefore, more cost-effective alternatives are needed.Neutralizing antibodies are important in host protection against dengue diseases and JE (10, 34). For JE, previously used mouse brain-derived (16, 44) and more recently used Vero cell-derived (20, 25, 35) inactivated vaccines can efficiently induce neutralizing antibody responses. However, these protein-based vaccines are produced from infectious agents, and their production therefore requires biosafety level 2 or 3 containment facilities and complex purification protocols, thus increasing the cost of the vaccine. Vaccine production without infective procedures can be achieved using genetic engineering techniques (29, 55).DENV1-4 and JEV are members of the genus Flavivirus in the family Flaviviridae (37). The envelope (E) protein is the major component of the envelopes of flavivirus virion particles and possesses most of the neutralizing epitopes (46). The other protein on the envelopes of mature virions is the membrane (M) protein, which is synthesized as the precursor membrane (prM) protein in infected cells. Cells expressing flavivirus prM and E proteins are known to secrete nucleocapsid-free subviral extracellular particles (EPs), which are similar to slowly sedimenting hemagglutinin (SHA) particles secreted from flavivirus-infected cells (47). EPs of JEV synthesized in mammalian expression systems have been evaluated for their immunogenicity and/or protective efficacy in mice (15, 24, 43). Two of these studies (24, 43) demonstrated that the EPs induced neutralizing antibodies at levels comparable to those induced by an inactivated JE vaccine. In our laboratory, mammalian cell lines continuously expressing EPs of dengue type 2 virus (DENV2) (26) or JEV (27) have been generated and designated D cells and F cells, respectively. The EPs contained an E protein that was antigenically and biochemically equivalent to the authentic E protein, and the EPs were immunogenic and protective in mice. However, the yields of viral antigens produced from D and F cells were low and would not meet the requirements for commercial vaccine production. Increasing the levels of viral antigen production from transfected cells would reduce the cost of vaccine preparation.Recently, insect cell expression systems have been increasingly used in various fields of medical sciences (1, 6, 17, 53). In general, insect cells are easier to cultivate than mammalian cells, because they often do not require serum supplementation in the culture medium or incubation under CO₂. In addition, insect cells can be adapted to suspension culture, allowing cultures to be simply scaled up. Furthermore, various techniques have been developed for high-density culture of insect cells; for example, in one study, the immobilization of insect cells within biomass support particles achieved a density of approximately 3 × 10⁷ cells/cm³ (50). Thus, the insect cell expression system can be a simple and inexpensive strategy for vaccine antigen production.In addition to their use as vaccine antigens, EPs derived from mammalian cells could be used as serodiagnostic antigens (27, 32). The production of serodiagnostic antigens may also encounter problems when the antigens are sourced directly from infectious agents. Currently, numerous commercial assays utilizing several different formats, such as the immunochromatography test and the IgM capture enzyme-linked immunosorbent assay (ELISA), are available for the diagnosis of DENV and JEV infections (4, 49). These commercial tests use viral antigens derived from transfected or infected cultured cells. Thus, the application of insect cell-derived EPs as diagnostic antigens would be an attractive alternative.In this study, we produced EPs of DENV2 and JEV in a transient expression system using the Sf9 cell line, which was derived from the pupal ovarian tissue of the fall armyworm, Spodoptera frugiperda. These proteins were evaluated for vaccine and diagnostic antigens, mainly by direct comparison with mammalian-cell-derived EPs. The EPs produced from Sf9 cells were immunogenic in mice and useful as antigens for ELISA. In addition, Sf9 cells produced larger amounts of antigen than CHO cells, suggesting the potential applicability of insect cells for the production of DENV2 and JEV antigens for vaccines and serodiagnostic tests.     

Expression of the Nucleoprotein of the Puumala Virus from the Recombinant Semliki Forest Virus Replicon: Characterization and Use as a Potential Diagnostic Tool

Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), μ-capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.     

Antigenic Properties and Diagnostic Potential of Baculovirus-Expressed Infectious Bursal Disease Virus Proteins VPX and VP3

The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX and VP3 genes in insect cells. VPX and especially VP3 were expressed at high levels in insect cells and simple to purify. The immunogenicity of both proteins was similar to that of the native virus. VPX was able to elicit neutralizing antibodies but VP3 was not. Purified VPX and VP3 were tested in an indirect ELISA with more than 300 chicken sera. There was an excellent correlation between the results of the ELISA using VPX and those of the two commercial kits. VP3 did not perform as well as VPX, and the linear correlation was significantly lower. A comparison with the standard reference technique, seroneutralization, showed that the indirect ELISA was more sensitive. Therefore, VPX-based ELISA is a good alternative to conventional ELISAs that use whole virions.     

Diagnostic Potential of Toscana Virus N Protein Expressed in Escherichia coli

The nucleocapsid (N) protein of the Toscana (TOS) virus was expressed in Escherichia coli by using a pET15b vector. The recombinant protein was purified by affinity chromatography and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The recombinant antigen was reactive with positive human sera, and the reactivity correlated very well (r = 0.9) with that of a whole-virus antigen when tested by EIA with 30 TOS virus-positive and 30 TOS virus-negative serum samples. The results demonstrate that the recombinant N protein can be easily produced in a procaryotic system and used for diagnostic assays for TOS virus immunity.     

Characterization of Toxoplasma gondii SAG2 Expressed in Insect Cells by Recombinant Baculovirus and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

A baculovirus carrying the SAG2 gene of Toxoplasma gondii was constructed, and recombinant SAG2 protein (S-rSAG2) was expressed in insect cells. S-rSAG2 was recognized by sera from cats and pigs infected with T. gondii. Mice immunized with S-rSAG2 produced high titers of specific immunoglobulin G2a (IgG2a) and IgG1 antibodies. In an indirect fluorescent antibody test, all mouse antisera against S-rSAG2 reacted strongly to the natural parasites, but those against rSAG2 expressed in Escherichia coli (E-rSAG2) only showed very weak reaction, although no markedly difference was found in the reaction to denatured antigen, T. gondii lysate, in Western blot analysis. The results suggest that S-rSAG2 is better than E-rSAG2 in both antigenicity and immunogenicity. Enzyme-linked immunosorbent assay (ELISA) with S-rSAG2 could differentiate clearly between sera from 30 specific-pathogen-free cats and 4 experimentally infected cats. Serum samples from domestic cats in Japan were tested by the ELISA and compared with a latex agglutination test (LAT) and ELISA with E-rSAG2. Of 187 samples, all 35 LAT-positive sera had strong reactions to S-rSAG2 and E-rSAG2. Of the 152 LAT-negative sera, 18 were positive in the ELISA with S-rSAG2, whereas only 2 were positive in the ELISA with E-rSAG2. Although there were significant correlations among the three methods, the ELISA with S-rSAG2 was more sensitive than the others, which could be attributed to the fact that S-rSAG2 shares some common conformational structure with the native antigen. The results suggest that S-rSAG2 would be a useful reagent for the detection of T. gondii infection in cats.     

Fluorescent Probing of Lymphocyte and Erythrocyte Plasma Membranes in Persistence of Tick-Borne Encephalitis Virus: Monitoring of Structural Changes

The structure of lymphocyte and erythrocyte plasma membrane in patients with long persistence of tick-borne encephalitis virus was studied using fluorescent probes pyrene and 1-anilinonaphthalene-8-sulfonate. The authors analyze the clear-cut disorders in the membranes of both lymphocytes and erythrocytes in viral tick-borne-encephalitis from the viewpoint of generalized involvement of cell membranes in infectious process.     

Growth,purification and characterization of Semliki Forest Virus in Ehrlich Ascites tumor cell suspensions

Summary The growth of Semliki Forest Virus (SFV) in suspension cultures of Ehrlich Ascites (EA) cells and its purification is described. Large volumes of virus material were concentrated by filtration with DIAFLO XM-300 membrane and precipitation with ammonium sulfate. A combination of protamine sulfate treatment, centrifugation of the virus onto a 50 per cent sucrose cushion, and sedimentation through a 5–30 per cent sucrose density gradient was employed.The purified virus particles were homogeneous as revealed by electron microscopy, by moving boundary electrophoresis, and by polyacrylamide gel electrophoresis. Virus suspensions containing 1 mg/ml of protein had a hemagglutinin titer of 1:12,000 when measured with 0.25 per cent goose red blood cells.With 6 Figures     

Altered IL-1β and IL-2 mRNA Expression in Murine Splenic Cells After Concomitant Stimulation by Semliki Forest Virus and Lipopolysaccharide

Co-infection with virus and bacteria happens frequently and often results in an exacerbated clinical course of the disease, possibly due to mechanisms including altered cytokine production. In the present study, the authors investigated the combined effects of avirulent Semliki Forest virus (SFV-A7) and bacterial lipopolysaccharide (LPS) on the interleukin-1β (IL-1β) and IL-2 gene expression in murine splenic cells. The authors found that 10 ng/ml of LPS in the culture medium induced expression of IL-1β but not IL-2, while infection with SFV-A7 did not induce either of these two cytokines. However, when SFV-A7 and LPS were applied together, a synergistic increase of both IL-1β and IL-2 was observed. Further experiments showed that addition of SFV-A7 3 h before LPS enhanced, whereas addition of the virus 3 h after the LPS inhibited, IL-1β gene expression. These results indicate that an interaction of virus and Gram-negative bacteria can result in an altered cytokine gene expression.     

Antigenic Characterization of Hantaan and Seoul Virus Nucleocapsid Proteins Expressed by Recombinant Baculovirus: Application of a Truncated Protein, Lacking an Antigenic Region Common to the Two Viruses, as a Serotyping Antigen

Hantaan virus (HTN) and Seoul virus (SEO) are members of the genus Hantavirus in the family Bunyaviridae and are causative agents of hemorrhagic fever with renal syndrome. The complete and truncated nucleocapsid proteins (NP) of HTN and SEO were expressed by a recombinant baculovirus system. Antigenic characterization of the NP using monoclonal antibodies (MAbs) indicated that the binding sites for the serotype-specific MAbs were located between amino acids (aa) 155 and 429. A Western blot assay indicated that the serotype-specific epitopes were conformation dependent. An indirect immunofluorescence antibody (IFA) assay with the truncated NP (aa 155 to 429) was able to distinguish convalescent-phase sera from HTN and SEO patients. However, the antibody titers with the truncated NP were lower than those with the whole NP. The truncated NP of SEO (aa 155 to 429) could be used as an enzyme-linked immunosorbent assay (ELISA) antigen, but the truncated NP from HTN lost its reactivity when used for ELISA. The IFA assay using baculovirus-expressed truncated NP as an antigen is a rapid, simple, and safe test for distinguishing between HTN and SEO infections by serotype.     

Development of a Western Blot Assay for Detection of Bovine Immunodeficiency-Like Virus Using Capsid and Transmembrane Envelope Proteins Expressed from Recombinant Baculovirus

A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.     

Enzyme-Linked Immunosorbent Assays Using Recombinant Envelope Protein Expressed in COS-1 and Drosophila S2 Cells for Detection of West Nile Virus Immunoglobulin M in Serum or Cerebrospinal Fluid

Humans infected with West Nile virus (WNV) develop immunoglobulin M (IgM) antibodies soon after infection. The microtiter-based assays for WNV IgM antibody detection used by most state public health and reference laboratories utilize WNV antigen isolated from infected Vero cells or recombinant envelope protein produced in COS-1 cells. Recombinant antigen produced in COS-1 cells was used to develop a WNV IgM capture enzyme immunoassay (EIA). A supplementary EIA using WNV envelope protein expressed in Drosophila melanogaster S2 cells was also developed. Both assays detected WNV IgM in the sera of experimentally infected rhesus monkeys within approximately 10 days postinfection. Human sera previously tested for WNV IgM at a state public health laboratory (SPHL) were evaluated using both EIAs. Among the sera from 20 individuals with laboratory-confirmed WNV infection (i.e., IgM-positive cerebrospinal fluid [CSF]) that were categorized as equivocal for WNV IgM at the SPHL, 19 were IgM positive and one was negative by the new EIAs. Of the 19 IgM-positive patients, 15 were diagnosed with meningitis or encephalitis; the IgM-negative patient was not diagnosed with neurological disease. There was 100% agreement between the EIAs for the detection of WNV IgM. CSF samples from 21 individuals tested equivocal for WNV IgM at the SPHL; all 21 were positive in both bead assays, and 16 of these patients were diagnosed with neurological disease. These findings demonstrate that the new EIAs accurately identify WNV infection in individuals with confirmed WNV encephalitis and that they exhibit enhanced sensitivity over that of the microtiter assay format.     

乙型肝炎病毒多表位抗原基因真核表达载体的构建及其在哺乳动物细胞中的表达

将自行设计、合成的乙型肝炎病毒多表位抗原基因BPT ,克隆入真核表达载体pCDNA3 1,构建重组质粒pCDNA3 1/BPT。后者经脂质体法转染小鼠BALB/c 3T3细胞 ,G4 18加压筛选出阳性转染细胞 ,并用间接免疫荧光法鉴定其在小鼠BALB/c 3T3细胞的表达。该重组质粒经胫骨前肌注射小鼠 ,10 0 μg/次每只小鼠 ,间隔 2周加强一次 ,共 3次。免疫诱发了特异性抗体和淋巴细胞增殖反应 ,并用PCR法在小鼠的心脏、肝脏及肺组织中检测到pCDNA3 1/BPT ,这为研究HBV多表达抗原的核酸疫苗提供了初步的资料     

Development and Evaluation of Recombinant Nucleocapsid Protein Based Diagnostic ELISA for Detection of Nipah Virus Infection in Pigs

The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.     

北京地区病毒性脑炎患者病原体的分离和初步鉴定

１９９１年夏秋季，我们从４１例北京儿童医院临床诊断为病毒性脑炎（非乙脑）患者的７５份急性期血和／或脑脊液中分离到３３株病毒。对其中１２株进行电镜观察，电镜下均见大小相同的球形病毒颗粒，毒粒的直径约为４４．７８±１．３５ｕｍ，无囊膜，具双层蛋白外壳，视野内多见空心颗粒。超薄切片显示病毒在感染细胞的胞浆内发生，直径约为４７．６±２．３ｕｍ。正常细胞中未见病毒颗粒。对其中３株病毒进行理化性状分析，多次试验均显示该病毒抵抗５＇－碘脱氧尿苷，抵抗乙醚，耐酸，能在地鼠肾ＢＨＫ－２１和白纹伊蚊Ｃ６／３６细胞上增殖并出现细胞病变。该病毒与本室制备的披膜病毒科甲组、乙脑病毒和布尼亚病毒科的组特异性免疫腹水均不反应。脑内接种３日龄乳小白鼠可引起不规律的发病和死亡。上述结果表明，该病毒是一类４５ｕｍ左右、无囊膜、耐酸的ＲＮＡ病毒。     

Swiss Army Survey in Switzerland to Determine the Prevalence of Francisella tularensis, Members of the Ehrlichia phagocytophila Genogroup, Borrelia burgdorferi Sensu Lato, and Tick-Borne Encephalitis Virus in Ticks

 A total of 6071 Ixodes ricinus ticks were collected on Swiss Army training grounds in five regions of Switzerland. The aim of the survey was to assess the prevalence of ticks infected with the human pathogens Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and the European tick-borne encephalitis virus. TaqMan PCR (PE Biosystems, USA) and TaqMan RT-PCR (PE Biosystems) analyses were performed on DNA and RNA extracted from pools of ten ticks grouped by gender. Here, for the first time, it is shown that ticks may harbor Francisella tularensis in Switzerland, at a rate of 0.12%. Furthermore, 26.54% of the ticks investigated harbored Borrelia burgdorferi sensu lato, 1.18% harbored members of the Ehrlichia phagocytophila genogroup, and 0.32% harbored the European tick-borne encephalitis virus. A new instrumentation was applied in this study to carry out and analyze more than 2300 PCR reactions in only 5 days. Furthermore, the results reveal that people working in outdoor areas, including army personnel on certain training grounds contaminated with ticks containing tick-borne pathogens, are at risk for different tick-borne diseases.     

Varicella-Zoster Virus Glycoproteins E and I Expressed in Insect Cells Form a Heterodimer That Requires the N-Terminal Domain of Glycoprotein I

Varicella-zoster virus (VZV) glycoproteins E and I (gE and gI), which are major components of the virion envelope, form a noncovalently linked complex. To understand their properties and functions, we expressed and purified soluble forms of gE and gI in the baculovirus system. Extracellular domains of gE and gI were cloned into baculoviruses, using either native or insect-derived signal peptides. Each recombinant virus yielded soluble protein in culture medium although a higher level of secretion was achieved with insect-derived signal peptides in recombinant gE baculoviruses. A soluble gE–gI complex was formed by co-infecting insect cells with recombinant gE and gI baculoviruses and detected by immunoprecipitation followed by Western blotting analyses. By gel filtration and cross-linking studies, we showed that the VZV gE–gI complex expressed in insect cells is a heterodimer. Interestingly, two recombinant gI proteins in which signal peptides were replaced with insect-derived signal peptides did not associate with gE. Amino-terminal sequencing and site-specific mutational studies showed that the replacement of only the signal peptides did not prevent complex formation but alterations in the processed amino-terminus of gI abrogated its ability to complex with gE. These findings indicate that the mature amino-terminus of gI is required for gE–gI complex formation by the external domains of VZV gE and gI.     

Epitope Mapping of the Nonspecific Cross-Reacting Antigen Using Various Related Recombinant Proteins Expressed in Chinese Hamster Ovary Cells and Eight Distinct Monoclonal Antibodies

Antigenic epitopes of nonspecific cross-reacting antigen (NCA) recognized by 8 different monoclonal antibodies (MAbs) were analyzed in relation to the domain structures of NCA [domains N, I (A1-B1) and M] and CEA [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M]. We reconstructed cDNAs for NCA-N, NCA-N-I-M, CEA-N, CEA-N-I, CEA-N-I-II, CEA-N-I-II-III-M in a eukaryotic expression vector, pdKCR-dhfr, and expressed them in Chinese Hamster Ovary (CHO) cells. The recombinant proteins were purified by immunoadsorption and gel filtration. By solid-phase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested against eight different MAbs reactive with NCA. All 8 MAbs had been shown to recognize the protein epitopes of the NCA molecule and classified into two groups in terms of the reactivity with NCA and CEA; Group X, 5 clones reactive with both NCA and CEA; and Group Y, 3 clones reactive only with NCA. The epitopes recognized by two of five Group X MAbs were found to be present on the domain N of the NCA molecule as well as of the CEA molecule, and those of the three others were on the domain I (A1-B1) of both molecules, respectively. All three epitopes of Group Y MAbs, which were unique to NCA, were present on the domain I (A1-B1) but not on the domain N of the NCA molecule. The epitope mapping reported here helps form the basis for understanding the relation between the chemical structure and antigenic activities of the NCA molecule and may be useful to study the functions of the NCA molecule, especially those of the respective domains.     

Osteogenic Potential of Mesenchymal Stem Cells from Bone Marrow in Situ: Role of Physicochemical Properties of Artificial Surfaces

Correlation analysis demonstrated the role of inorganic parameters of the surfaces of calcium phosphate materials in the regulation of osteogenic differentiation of mesenchymal precursors. The progenitor stromal cells were isolated from syngeneic bone marrow immobilized in vitro on calcium phosphate surfaces with different structure, phasic, and elemental composition. After 45 days of subcutaneous ectopic osteogenesis in BALB/c mice, the tissues grown on these matrixes were characterized histologically. It was found that adhesion of bone marrow cells is the initial stage determining their future proliferation (conduction) over the artificial surface and the area of formed tissue plate. The success of histogenesis depends on surface roughness. The optimal roughness class was 4–5 (Russian State Standards), which enables differentiation of progenitor stromal cells under the specific microenvironmental conditions into the connective and adipose tissue cells. Differentiation of the progenitor cells into the stromal cells producing the hemopoiesis-inducing microenvironment also takes place in the foci of active hemopoiesis. Induction of osteogenic potential of the stromal precursors (osteoinduction) is determined by the ratio between calcium and phosphate atoms in surface coatings. In our experimental system, osteogenic differentiation of stromal mechanocytes was blocked only at Ca/P<0.5. __________ Translated from Kletochnye Tekhnologii v Biologii i Meditsine, Vol. **, No. 3, pp. 164–173, September, 2005     

Antigenic Diversity of Hepatitis B Virus Strains of Genotype F in Amerindians and Other Population Groups from Venezuela

The adw4 subtype of hepatitis B virus (HBV) belongs to a unique genomic group (genotype F) representing the original HBV strains from the New World. Data regarding the prevalence of this subtype among HBV carriers in South America are, however, scarce, and those concerning HBV genotype F are based on only a few samples from Latin America. In this study, serum samples were obtained from 141 hepatitis B surface antigen (HBsAg) carriers from Amerindians and urban populations from Venezuela. The HBsAg subtype was identified with monoclonal antibodies in 105 samples, and the HBV genotype was identified by reverse-phase hybridization with DNA fragments in 58 samples. The adw4 subtype was highly prevalent in the population studied (75%); among the Amerindians, the prevalence was 97%. The adw2 subtype was also present (10%), while other subtypes (ayw3 and ayw4) were only occasionally found. The HBV subtype was associated with the expected genotype in most cases (80%), and thus genotype F was highly prevalent. Sequencing of viral strains that gave genotypes unpredicted by the HBsAg subtyping confirmed seven of them as belonging to not previously described genotype-subtype associations: namely, adw2 and ayw4 within genotype F.