Evolutionary pattern of human respiratory syncytial virus (subgroup A): cocirculating lineages and correlation of genetic and antigenic changes in the G glycoprotein.

The genetic and antigenic variability of the G glycoproteins from 76 human respiratory syncytial (RS) viruses (subgroup A) isolated during six consecutive epidemics in either Montevideo, Uruguay, or Madrid, Spain, have been analyzed. Genetic diversity was evaluated for all viruses by the RNase A mismatch cleavage method and for selected strains by dideoxy sequencing. The sequences reported here were added to those published for six isolates from Birmingham, United Kingdom, and for two reference strains (A2 and Long), to derive a phylogenetic tree of subgroup A viruses that contained two main branches and several subbranches. During the same epidemic, viruses from different branches were isolated. In addition, closely related viruses were isolated in distant places and in different years. These results illustrate the capacity of the virus to spread worldwide, influencing its mode of evolution. The antigenic analysis of all isolates was carried out with a panel of anti-G monoclonal antibodies that recognized strain-specific (or variable) epitopes. A close correlation between genetic relatedness and antigenic relatedness in the G protein was observed. These results, together with an accumulation of amino acid changes in a major antigenic area of the G glycoprotein, suggest that immune selection may be a factor influencing the generation of RS virus diversity. The pattern of RS virus evolution is thus similar to that described for influenza type B viruses, expect that the level of genetic divergence among the G glycoproteins of RS virus isolates is the highest reported for an RNA virus gene product.     

抗H9亚型禽流感病毒血凝素单克隆抗体的制备和鉴定

目的:获得分泌抗H9亚型禽流感病毒(AIV)血凝素单克隆抗体的杂交瘤细胞。方法:以H9N2亚型AIV为免疫原,免疫6～8周龄雌性BALB/c小鼠,取其脾细胞与骨髓瘤细胞Sp2/0-Ag-14,在PEG4000的作用下进行细胞融合,通过血凝抑制(HI)试验筛选分泌抗H9亚型AIV血凝素单克隆抗体的杂交瘤细胞。结果:经过连续3～4次克隆化,获得能稳定分泌抗H9亚型AIV血凝素的单克隆抗体细胞系6株,分别命名为1B2、1C10、1G2、2B7、2E3和5E11。6株细胞培养上清HI效价为24～28,腹水HI效价为210～213。除1G2为IgM外,其余5株均为IgG1。Western blotting结果显示,1B2、1C10、2B7和2E3能与AIVH9蛋白在Mr为75000处反应,表明其是针对AIVH9亚型血凝素蛋白的单抗。特异性试验表明该6株单抗均只与H9亚型AIV发生特异性HI反应,而不与其他14个HA亚型的AIV及新城疫病毒、传染性支气管炎病毒发生交叉反应,显示出良好的特异性。结论:制备了针对H9亚型禽流感病毒血凝素的单克隆抗体,为禽流感的快速诊断和病毒的抗原性分析等奠定了基础。     

Subtype-specific conformational differences within the V3 region of subtype B and subtype C human immunodeficiency virus type 1 Env proteins

The V3 region of the human immunodeficiency virus type 1 gp120 Env protein is a key domain in Env due to its role in interacting with the coreceptors CCR5 and CXCR4. We examined potential subtype-specific V3 region differences by comparing patterns of amino acid variability and probing for subtype-specific structures using 11 anti-V3 monoclonal antibodies (V3 MAbs). Differences between the subtypes in patterns of variability were most evident in the stem and turn regions of V3 (positions 9 to 24), with the two subtypes being very similar in the base region. The characteristics of the binding of V3 MAbs to Env proteins of the subtype B virus JR-FL and the subtype C virus BR025 suggested three patterns, as each group of MAbs recognized a specific conformation- or sequence-based epitope. Viruses pseudotyped with Env from JR-FL and BR025 were resistant to neutralization by the V3 MAbs, although the replacement of the Env V3 region of the SF162 virus with the JR-FL V3 created a pseudotyped virus that was hypersensitive to neutralization. A single mutation in V3 (H13R) made this chimeric Env selectively resistant to one group of V3 MAbs, consistent with the mAb binding properties. We hypothesize that there are intrinsic differences in V3 conformation between subtype B and subtype C that are localized to the stem and turn regions and that these differences have two important biological consequences: first, subtype B and subtype C V3 regions can have subtype-specific epitopes that will inherently limit antibody cross-reactivity, and second, V3 conformational differences may potentiate the frequent evolution of R5- into X4-tropic variants of subtype B but limit subtype C virus from using the same mechanism to evolve X4-tropic variants as efficiently.     

Analysis of murine B-cell epitopes on Eastern equine encephalitis virus glycoprotein E2

The Eastern equine encephalitis virus (EEEV) E2 protein is one of the main targets of the protective immune response against EEEV. Although some efforts have done to elaborate the structure and immune molecular basis of Alphaviruses E2 protein, the published data of EEEV E2 are limited. Preparation of EEEV E2 protein-specific antibodies and define MAbs-binding epitopes on E2 protein will be conductive to the antibody-based prophylactic and therapeutic and to the study on structure and function of EEEV E2 protein. In this study, 51 EEEV E2 protein-reactive monoclonal antibodies (MAbs) and antisera (polyclonal antibodies, PAbs) were prepared and characterized. By pepscan with MAbs and PAbs using enzyme-linked immunosorbent assay, we defined 18 murine linear B-cell epitopes. Seven peptide epitopes were recognized by both MAbs and PAbs, nine epitopes were only recognized by PAbs, and two epitopes were only recognized by MAbs. Among the epitopes recognized by MAbs, seven epitopes were found only in EEEV and two epitopes were found both in EEEV and Venezuelan equine encephalitis virus (VEEV). Four of the EEEV antigenic complex-specific epitopes were commonly held by EEEV subtypes I/II/III/IV (1-16aa, 248-259aa, 271-286aa, 321-336aa probably located in E2 domain A, domain B, domain C, domain C, respectively). The remaining three epitopes were EEEV type-specific epitopes: a subtype I-specific epitope at amino acids 108–119 (domain A), a subtype I/IV-specific epitope at amino acids 211–226 (domain B) and a subtype I/II/III-specific epitope at amino acids 231–246 (domain B). The two common epitopes of EEEV and VEEV were located at amino acids 131–146 and 241–256 (domain B). The generation of EEEV E2-specific MAbs with defined specificities and binding epitopes will inform the development of differential diagnostic approaches and structure study for EEEV and associated alphaviruses.     

Serotypes and subtypes of Neisseria meningitidis serogroup B strains associated with meningococcal disease in Canada, 1977-1989

Typing of Neisseria meningitidis serogroup B disease isolates was carried out using a panel of serotype-and subtype-specific monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (ELISA). Three hundred and sixty-two strains isolated from 1977 to 1986 were typed using five serotyping and seven subtyping reagents and outer membrane vesicles as antigens. Serotype 2b accounted for 30% of the disease isolates. The most common subtype was P1.2, which occurred on 18.5% of all strains or 48.6% of the serotype 2b strains. Of the 362 strains typed, 135 (37.3%) were serotyped and 122 (33.7%) were subtyped. Overall, 185 (51.1%) of the strains could be assigned a serotype and (or) subtype. Strains (221) isolated during the years 1987-1989 were typed using a panel of 6 serotyping and 12 subtyping reagents by whole-cell ELISA. Strains of serotypes 4 (21.7%) and 15 (20.8%) were the most common and carried a wide variety of subtypes. The most common subtypes were P1.2 (11.8%) and P1.16 (9.5%). Of the 221 strains analyzed, 132 (59.7%) were assigned a serotype and 123 (55.7%) a subtype and with all 18 MAbs, 192 (86.9%) of the strains were serotyped and (or) subtyped. Two different MAbs to the four epitopes 2a, 15, P1.2, and P1.16 gave discordant reactions of 0.3, 6.6, 2.6, and 2.2%, respectively, when used to analyze over 300 strains of N. meningitidis.     

Human cytotoxic T cells stimulated by antigen on dendritic cells recognize the N, SH, F, M, 22K, and 1b proteins of respiratory syncytial virus.

We examined the human cytotoxic T-cell repertoire of nine adults to 9 of the 10 proteins of respiratory syncytial (RS) virus. Peripheral blood mononuclear cells from normal adults were stimulated with RS virus in vitro. The resulting polyclonal cultures were tested for lysis of B-lymphoblastoid cell lines infected with recombinant vaccinia viruses expressing each of nine individual RS virus proteins. The use of peripheral blood dendritic cells to present antigen gave more easily reproducible results over a shorter culture period than conventional methods. The six RS virus proteins most strongly recognized were the nucleoprotein N (nine of nine donors with greater than 10% above background lysis; P = 0.0004), the surface proteins SH (six of nine donors; P = 0.002) and F (five of nine donors; P = 0.008), the matrix proteins M (five of nine donors; P = 0.004) and 22K (three of nine donors; P = 0.01) and the nonstructural protein 1b (six of nine donors; P = 0.004). There was no significant recognition of the major surface glycoprotein G (two of nine donors), the internal phosphoprotein P (one of nine donors), or the nonstructural protein 1c (one of nine donors). Recognition was major histocompatibility complex class I restricted, but no association between major histocompatibility complex phenotype and protein specificity of T cells was seen. Recognition of F and 22K appeared to be associated with recent infection indicated by increased levels of anti-RS virus immunoglobulin G antibody in serum measured by enzyme-linked immunosorbent assay. Since cytotoxic T-cell recognition of RS virus proteins has been demonstrated to be important in the clearance of virus from infected hosts, the N, M, SH, 1b, F, and 22K proteins should be considered potential vaccine components.     

Antigenic and Functional Analyses of Glycoprotein of Rabies Virus Using Monoclonal Antibodies

Thirty-five monoclonal antibodies (MAbs) against glycoprotein (G protein) of the RC-HL strain of the rabies virus have been established. Using these MAbs, two antigenic sites (I and II) were delineated on the G protein of the RC-HL strain in a competitive binding assay. Of these, 34 MAbs recognized the epitopes on site IL Site II was further categorized into 10 subsites according to their patterns in a competitive binding assay. Each site II-specific MAb showed 5 to 23 nonreciprocal competitions. The reactivities of 35 MAbs to rabies and rabies-related viruses in an indirect immunofluorescent antibody test showed that six MAbs in group A binded to rabies and rabies-related viruses and eight MAbs in group E reacted only with rabies viruses, considering that the former represent the genus-specific of Lyssavirus and the latter are rabies virus-specific. From biological assays, 28 of the 35 MAbs showed neutralization activity, 31 showed hemagglutination inhibition (HI) activity, and 18 showed immunolysis (IL) activity. The MAbs recognizing neutralization epitopes fell into at least three groups: those exhibiting both HI and IL activity, those showing only HI activity, and those showing neither HI nor IL activity. All IL epitopes overlap with HA epitopes. Five of the nine MAbs which reacted with the antigen treated by sodium dodecyl sulfate in ELISA were not reduced, or reduced only slightly, in the titer. None of the MAbs reacted with 2-mercaptoethanol-treated antigen. Only one MAb that recognized site I reacted with the denatured G protein in a Western blotting assay, indicating that its epitope is linear. These results suggest that almost all of the epitopes on the G protein of the rabies virus are conformation-dependent and the G protein forms a complicated antigenic structure.     

Induction of immune response to influenza virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against five monoclonal antibodies (MAbs) specific for different antigenic sites on the hemagglutinin (HA) of influenza virus Mem71H-BelN (H3N1) [A/Memphis/1/71 (H3N2) x A/Bel/42 (H1N1)]. Each of the anti-Id sera was directed predominantly towards a unique (private) idiotype of the immunizing MAb, none of the five idiotypes being detectable in pooled BALB/c antisera against Mem71H-BelN virus or on most other anti-HA MAbs tested. Partial idiotypic sharing was observed, however, between certain MAbs, from different mice, having the same or similar epitope specificity for HA. When used as immunogens in BALB/c mice, two of the five anti-Id preparations induced antibodies that reacted with Mem71H-BelN virus and displayed neutralizing activity. Mice of other inbred strains responded similarly, indicating that the response was not genetically restricted by the Igh locus. From their pattern of reactivity with mutants of Mem71H-BelN virus with known single amino acid substitutions in the HA molecule, the antiviral antibodies elicited by anti-Id antibodies were shown to be directed to the same antigenic site on A/Memphis/1/71 HA as the original immunizing MAb (site A or site E, respectively). However, several of these antisera were shown to contain additional distinct subpopulations of antibodies specific for heterologous influenza A virus strains, either of the H3 subtype or of a different HA subtype (H1 or H2). Since the induction of antibodies to HA of different subtypes is not a feature of the antibody response to influenza virus itself, their induction by anti-Id antibodies merits further investigation.     

临床分离耐甲氧西林溶血性葡萄球菌的SCCmec分型及同源性分析

目的了解临床分离耐甲氧西林溶血性葡萄球菌（MRSH）的SCCmec基因型别及相同SCCmec型别菌株的同源性。方法多重PCR进行SCCmec分型,ERIC-PCR法对相同SCCmec型别菌株进行同源性分析。结果83株临床分离MRSH菌株中,SCCmecI型有23株（27．7％）,SCCmecⅡ型有10株（12．1％）,SCCmecm型有24株（28．9％）,SCCmecIV型有1株（1．2％）,I、Ⅱ混合型有8株（9．6％）,I、Ⅲ混合型有6株（7．2％）,Ⅱ、11混合型有5株（6．0％）,I、Ⅱ、Ⅲ混合型有3株（3．6％）,未分型3株（3．6％）。ERIC—PCR结果显示,23株SCCmecI型分为11型,其中A型5株,B型5株,C型3株,其余8株各为1型,2株未分型;10株SCCmecⅡ型分为6型,其中D型4株,E型2株,3株各为1型,1株未分型;24株SCCmecm型分为9型,其中F型11株,G型2株,H型2株,I型2株,5株各为1型,2株未分型。结论临床分离MRSH中,SCCmecI、Ⅲ型为多,部分菌株呈混合型别;相同SCCmec型别的部分菌株之间可能存在克隆传播。     

Antigenic and functional organization of human parainfluenza virus type 3 fusion glycoprotein.

Twenty-six monoclonal antibodies (MAbs) (14 neutralizing and 12 nonneutralizing) were used to examine the antigenic structure, biological properties, and natural variation of the fusion (F) glycoprotein of human type 3 parainfluenza virus (PIV3). Analysis of laboratory-selected antigenic variants and of PIV3 clinical isolates indicated that the panel of MAbs recognizes at least 20 epitopes, 14 of which participate in neutralization. Competitive binding assays indicated that the 14 neutralization epitopes are organized into three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB) and that the 6 nonneutralization epitopes form four sites (D, E, F, and G). Most of the neutralizing MAbs were involved in nonreciprocal competitive binding reactions, suggesting that they induce conformational changes in other neutralization epitopes. Fusion-inhibition and complemented-enhanced neutralization assays indicated that antigenic sites AB, B, and C may correspond to functional domains of the F molecule. Our results indicated that antibody binding alone is not sufficient for virus neutralization and that many anti-F MAbs neutralize by mechanisms not involving fusion-inhibition. The degree of antigenic variation in the F epitopes of clinical strains was examined by binding and neutralization tests. It appears that PIV3 frequently develops mutations that produce F epitopes which efficiently bind antibodies, but are completely resistant to neutralization by these antibodies.