Early identification and retrieval or deletion of human lymphocyte subpopulations responding to influenza virus or respiratory syncytial virus challenge

Differences in immune responses of human mononuclear leukocytes (MNL) have been demonstrated following exposure in vitro to influenza virus or respiratory syncytial virus (RSV). In the current studies, we sought to identify early differences in reactive subpopulations that emerge from within the heterogeneous resting MNL pool after challenge. MNL were sham-exposed or exposed to influenza virus or RSV, separated, and retrieved by countercurrent centrifugal elutriation after 3 d. Exposure to influenza virus caused a relative decline in the number of large MNL, but an increase in small lymphocytes. Large cells that remained included primitive lymphoblasts, rare plasma cells, and typical lymphocytes of progressively greater volume. Exposure to RSV increased the number of large MNL, but diminished the number of small lymphocytes. The subpopulation of large cells consisted of atypical and large granular lymphocytes. Furthermore, deletion of the latter large, reactive lymphocytes led to abrogation of an RSV-specific proliferative response upon subsequent challenge. Thus, the specific and different subpopulations reactive after infectious virus challenge could be identified, retrieved, and manipulated without dependence ona priori, phenotypic markers.     

Molecular epidemiology of human respiratory syncytial virus

Human respiratory syncytial virus (HuRSV) is the majorviral cause of severe lower respiratory tract disease in babies and infants with epidemics occurring annually in the winter in temperate climates. Analysis of the antigenic and genetic variability of HuRSV isolates has shown that there are two groups of the virus and that each group can be further subdivided into a number of genotypes in which the attachment protein shows the greatest variability together with progressive change. Epidemics are made up of multiple genotypes whose proportions vary from year to year. The various genotypes cocirculate with very similar viruses distributed world-wide.     

TT virus loads and lymphocyte subpopulations in children with acute respiratory diseases

TT virus (TTV) produces chronic plasma viremia in around 90% of healthy individuals of all ages and has, therefore, been proposed as a commensal human virus. We recently demonstrated that in children hospitalized for acute respiratory diseases high TTV loads were associated with severe forms of disease. Here, we report that in such children TTV loads showed an inverse correlation with the percentage of circulating total T and helper T cells and a direct correlation with the percentage of B cells. Thus, florid TTV replication might contribute to lymphocyte imbalances and, possibly, immunosuppressive effects, thus resembling related animal viruses.     

Purification of human respiratory syncytial virus fusion glycoprotein

Human respiratory syncytial virus (RSV) fusion glycoprotein (F) elicits neutralizing antibodies to RSV and has therefore attracted much attention as a suitable candidate antigen in the development of gene-based vaccines against RSV infections. However, a major obstacle in vaccine development has been the problem of antigen purification. To address this problem, we have developed a new method that combines sucrose gradient ultracentrifugation and a two-step chromatographic process, to purify RSV F from RSV particles propagated in HEp-2 cells. Analysis of the fractions produced using this method showed recovery of a functional homodimer with a molecular weight of 140 kDa, and 54% preservation of the original F.     

Animal models of human respiratory syncytial virus disease

Infection with the human pneumovirus pathogen, respiratory syncytial virus (hRSV), causes a wide spectrum of respiratory disease, notably among infants and the elderly. Laboratory animal studies permit detailed experimental modeling of hRSV disease and are therefore indispensable in the search for novel therapies and preventative strategies. Present animal models include several target species for hRSV, including chimpanzees, cattle, sheep, cotton rats, and mice, as well as alternative animal pneumovirus models, such as bovine RSV and pneumonia virus of mice. These diverse animal models reproduce different features of hRSV disease, and their utilization should therefore be based on the scientific hypothesis under investigation. The purpose of this review is to summarize the strengths and limitations of each of these animal models. Our intent is to provide a resource for investigators and an impetus for future research.     

Co-infection of human metapneumovirus with adenovirus or respiratory syncytial virus among children in Japan

Human metapneumovirus (hMPV) is one of the etiological agents of acute respiratory tract infections. From June 2005 to May 2006, we collected 185 clinical specimens from children in Osaka City, Japan, and detected 41 hMPV RNA. Of the 41 specimens, four (9.8%) also contained other viruses (3 with adenovirus [AdV] and 1 with respiratory syncytial virus [RSV]). The clinical symptoms of patients coinfected with AdV were indistinct from those of patients mono-infected with hMPV. The symptoms of the one patient co-infected with RSV were clinically severe. Further research is needed to clarify the effect of hMPV on other respiratory viruses or vice versa.     

Molecular epidemiology and evolution of human respiratory syncytial virus and human metapneumovirus

Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are ubiquitous respiratory pathogens of the Pneumovirinae subfamily of the Paramyxoviridae. Two major surface antigens are expressed by both viruses; the highly conserved fusion (F) protein, and the extremely diverse attachment (G) glycoprotein. Both viruses comprise two genetic groups, A and B. Circulation frequencies of the two genetic groups fluctuate for both viruses, giving rise to frequently observed switching of the predominantly circulating group. Nucleotide sequence data for the F and G gene regions of HRSV and HMPV variants from the UK, The Netherlands, Bangkok and data available from Genbank were used to identify clades of both viruses. Several contemporary circulating clades of HRSV and HMPV were identified by phylogenetic reconstructions. The molecular epidemiology and evolutionary dynamics of clades were modelled in parallel. Times of origin were determined and positively selected sites were identified. Sustained circulation of contemporary clades of both viruses for decades and their global dissemination demonstrated that switching of the predominant genetic group did not arise through the emergence of novel lineages each respiratory season, but through the fluctuating circulation frequencies of pre-existing lineages which undergo proliferative and eclipse phases. An abundance of sites were identified as positively selected within the G protein but not the F protein of both viruses. For HRSV, these were discordant with previously identified residues under selection, suggesting the virus can evade immune responses by generating diversity at multiple sites within linear epitopes. For both viruses, different sites were identified as positively selected between genetic groups.     

Differential type I interferon induction by respiratory syncytial virus and influenza a virus in vivo

Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-alpha/beta induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-alpha/beta production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-alpha/beta in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.     

Monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein.

Six monoclonal antibodies directed against respiratory syncytial virus proteins were produced. Each was characterized by immunoprecipitation and indirect immunofluorescence. One was directed against the nucleocapsid protein. NP 44, two were directed against a 37,000-dalton protein, two were directed against the major envelope glycoprotein, GP 90, and one was directed against the 70,000-dalton envelope protein, VP 70. Indirect immunofluorescence stain patterns of infected HEp-2 cells defined GP 90 and VP 70 as viral proteins expressed on the cell surface, whereas NP 44 and the 37,000-dalton protein were detected as intracytoplasmic inclusions. One of the anti-GP 90 antibodies neutralized virus only in the presence of complement but did not inhibit cell-cell fusion. The anti-VP 70 antibody neutralized virus without complement and inhibited cell-cell fusion of previously infected HEp-2 cells, thus identifying VP 70 as the fusion protein.     

Recombinant respiratory syncytial virus bearing a deletion of either the NS2 or SH gene is attenuated in chimpanzees

The NS2 and SH genes of respiratory syncytial virus (RSV) have been separately deleted from a recombinant wild-type RSV strain, A2 (M. N. Teng and P. L. Collins, J. Virol. 73:466-473, 1998; A. Bukreyev et al., J. Virol. 71:8973-8982, 1997; and this study). The resulting viruses, designated rA2DeltaNS2 and rA2DeltaSH, were administered to chimpanzees to evaluate their levels of attenuation and immunogenicity. Recombinant virus rA2DeltaNS2 replicated to moderate levels in the upper respiratory tract, was highly attenuated in the lower respiratory tract, and induced significant resistance to challenge with wild-type RSV. The replication of rA2DeltaSH virus was only moderately reduced in the lower, but not the upper, respiratory tract. However, chimpanzees infected with either virus developed significantly less rhinorrhea than those infected with wild-type RSV. These findings demonstrate that a recombinant RSV mutant lacking either the NS2 or SH gene is attenuated and indicate that these deletions may be useful as attenuating mutations in new, live recombinant RSV vaccine candidates for both pediatric and elderly populations. The DeltaSH mutation was incorporated into a recombinant form of the cpts248/404 vaccine candidate, was evaluated for safety in seronegative chimpanzees, and can now be evaluated as a vaccine for humans.     

可用于辅助蛋白功能研究的人呼吸道合胞病毒双顺反子微型基因组的构建及拯救

【目的】构建可表达增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的人呼吸道合胞病毒(human respiratory syncytial virus,RSV)双顺反子微型基因组cDNA克隆及重组质粒,并进行拯救,以探讨并验证RSV的聚合酶蛋白或辅助蛋白在RSV反向遗传学操作中的作用。【方法】利用全基因合成和分子生物学相结合的方法,在获得可分别表达EGFP和无生物活性蛋白的单顺反子微型基因组质粒pUC57-RSV-EGFP和pUC57-RSV-ORF1的基础上,进一步克隆至pBR322B载体,获得编码EGFP及无生物活性蛋白的双顺反子微型基因组质粒,经限制性内切酶和核酸序列分析正确后,与可表达4种辅助蛋白的辅助质粒共转染至BHK-T7细胞,通过荧光显微镜观察EGFP的表达以及RT-qPCR对EGFP mRNA的转录水平进行定量分析。【结果】成功构建了编码EGFP和无生物活性蛋白的RSV双顺反子微型基因组质粒pBR322B-RSVⅡ-EGFP,经与编码4种辅助蛋白的辅助质粒共转染至BHK-T7细胞,发现4种辅助蛋白对EGFP的表达具有不同的功能活性。【结论】以可表达EGFP报告基因的RSV双顺反子微型基因组重组质粒,实现了对4种辅助蛋白的功能验证,其中M2-1蛋白在双顺反子微型基因组拯救过程中具有转录延长的生物学活性。     

Expression and purification of human respiratory syncytial virus recombinant fusion protein

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia coli BL21A using the pET28a vector at 37 °C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. coli and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies.     

Expression and characterization of a multivalent human respiratory syncytial virus protein

Respiratory syncytial virus (RSV) has been recognized as one of the most common causes of severe respiratory tract infection in infants worldwide. As yet, a safe and effective vaccine has not been developed to protect humans from RSV. The F and G surface proteins have been widely investigated due to their potential to induce protective immunity. In addition, the M2 protein has been shown to be important in inducing a T-cell response. Our project involved the cl oning of the immunodominant regions of the RSV F, M2 and G proteins into a bacterial vector, pET-32a(+). The recombinant RFM2G protein was expressed in Escherichia coli and purified using His Bind columns. The purified rRFM2G protein was analyzed by poly-acrylamide gel electrophoresis and Western blotting. The predicted structure of the recombinant protein built by the Swiss PDB Viewer program suggested a rod shape with a distinct swollen head and neck which was confirmed by transmission electron microscopy and atomic force microscopy. BALB/c female mice were immunized with either RSV, rRFM2G alone, or rRFM2G in combination with flagellin as a mucosal adjuvant. Serum was collected on days 0, 14, 28 and 49 to assess the immune response by Enzyme-linked immunosorbent assay. Intranasal immunization of mice with the rRFM2G protein yielded significantly high serum IgG titers. Co-administration of the rRFM2G protein with flagellin did not augment the serum antibody response.     

Antigen mimicry involving measles virus hemagglutinin and human respiratory syncytial virus nucleoprotein.

Intergenic antigenic relationships between measles virus and respiratory syncytial (RS) virus-specific structural components were studied by using monoclonal antibodies. Of 75 monoclonal antibodies against these components, only one, an anti-measles virus hemagglutinin monoclonal antibody, cross-reacted. Immunofluorescence analysis of measles virus- and RS virus-infected cells with this monoclonal antibody showed qualitatively different staining patterns which indicated that the antigen involved in cross-reaction was the RS virus nucleoprotein or phosphoprotein. A radioimmunoprecipitation assay showed the antigen to be the nucleoprotein.     

Response of vaccinated and unvaccinated bighorn sheep (Ovis canadensis canadensis) to experimental respiratory syncytial virus challenge

Five Rocky Mountain bighorn sheep (Ovis canadensis canadensis), approximately 5 mo old and without detectable antibody titers to respiratory syncytial virus (RSV), were assigned to two groups to study the effects of RSV challenge inoculation in vaccinated (n = 3) and unvaccinated (n = 2) bighorns. The three lambs vaccinated with a modified live bovine RSV vaccine developed a detectable antibody response to the vaccine. Vaccinated and unvaccinated lambs challenged with an ovine isolate of RSV developed increased levels of neutralizing antibody, but clinical signs of disease were not observed. Neutralizing antibody titers to RSV remained higher (2-4-fold) in vaccinated lambs over time when compared to unvaccinated lambs.