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 on a priori, phenotypic markers.     

Immunomodulatory effects of sensory nerves during respiratory syncytial virus infection in rats

Respiratory syncytial virus (RSV) infection is associated with exaggerated neurogenic inflammation in the airways. This study sought to determine whether irritation of the mucosal sensory fibers affects the recruitment of lymphocytes and monocytes to RSV-infected airways. Pathogen-free rats were inoculated with RSV or with virus-free medium and were injected 5 days later with capsaicin to stimulate airway sensory nerves. Bronchoalveolar lavage was performed 1, 5, or 10 days after nerve stimulation, and samples were analyzed by differential cell count and flow cytometry. Without nerve stimulation, RSV caused a minimal increase in the number of lymphocytes and monocytes above pathogen-free control levels. After nerve stimulation, numerous lymphocytes, predominantly CD4+ T cells, and monocytes were recruited in the airways of infected rats, whereas no difference was found in pathogen-free controls. RSV induced overexpression of the neurokinin 1 (NK1) receptor for substance P on discrete lymphocyte subpopulations within the bronchial-associated lymphoid tissue (BALT), and treatment with a specific NK1 receptor antagonist abolished the recruitment of both lymphocytes and monocytes to infected airways. Our data suggest that airborne irritants stimulating mucosal sensory fibers during RSV infection exert important immunomodulatory effects by attracting to the infected airways selected lymphocyte subpopulations from the local BALT as well as monocytes.     

The Effect of Virus Particle Size on Chemiluminescence Induction by Influenza and Sendai Viruses in Mouse Spleen Cells

Suspensions of orthomyxo- and paramyxoviruses are composed of pleomorphic particles ranging from large filaments to small spheres. Influenza and Sendai viruses were separated according to size by gel filtration and the induction of luminol-dependent chemiluminescence (CL) by particles of similar size was studied in suspensions of mouse spleen cells known to contain phagocytes. CL reflects the generation by the cells of reactive oxygen species. CL induction decreased with particle size for both viruses. Compared with small spheres, large influenza filaments were approximately 10 times as efficient in activating cellular light emission while the ratio between large and small Sendai viruses was 3:1. Small Sendai virus particles were also less efficient in lysing red cells and had lower neuraminidase activity. By contrast, with influenza virus, only neuraminidase and not the hemolytic activity decreased with the virus size. When influenza virus filaments were broken into smaller particles by sonication, the capacity to induce chemiluminescence dropped markedly while the hemolytic and hemagglutinating activities increased and neuraminidase activity remained unaltered. These results suggest that the presentation of influenza virus hemagglutinin and neuraminidase glycoproteins in a large particle, leading to extensive receptor crosslinking, may be an important factor in the efficient activation of CL by filamentous influenza virus. We suggest that radical generation as reflected in cellular CL may relate to the toxic in vivo effects that contribute to the pathogenesis of influenza and infections with paramyxoviruses.     

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement

Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2‐YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2‐YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2‐YFP fluorescence were affected in fibrillarin‐silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed.     

Heterogeneity of mouse lymphocytes in interferon production upon influenza virus challenge in culture

Heterogeneity of mouse lymphocytes with respect to interferon production upon influenza virus challenge in culture was studied. Spleen cells produced much more interferon than thymocytes or mesenteric lymph node cells. Spleen cells mainly responsible for interferon production belonged to the hydrocortisone-sensitive population. When spleen lymphocytes were separated into seven fractions by centrifugation on a serum albumin density gradient, they were found to differ greatly in interferon producing capacity; a small fraction of intermediate density, representing a few percent or less of the total lymphocytes, produced markedly high levels of interferon.     

Recombinant Influenza Virus Carrying the Respiratory Syncytial Virus (RSV) F85-93 CTL Epitope Reduces RSV Replication in Mice

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants worldwide. Despite decades of research, there is still no registered vaccine available for this major pathogen. We investigated the protective efficacy of a recombinant influenza virus, PR8/NA-F_85–93, that carries the RSV CD8⁺ T cell epitope F_85–93 in its neuraminidase stalk. F_85–93-specific cytotoxic T lymphocytes (CTLs) were induced in mice after a single intranasal immunization with PR8/NA-F_85-93 virus, and these CTLs provided a significant reduction in the lung viral load upon a subsequent challenge with RSV. To avoid influenza-induced morbidity, we treated mice with matrix protein 2 (M2e)-specific monoclonal antibodies before PR8/NA-F_85-93 virus infection. Treatment with anti-M2e antibodies reduced the infiltration of immune cells in the lungs upon PR8/NA-F_85-93 infection, whereas the formation of inducible bronchus-associated lymphoid tissue was not affected. Moreover, this treatment prevented body weight loss yet still permitted the induction of RSV F-specific T cell responses and significantly reduced RSV replication upon challenge. These results demonstrate that it is possible to take advantage of the infection-permissive protection of M2e-specific antibodies against influenza A virus to induce heterologous CD8⁺ T cell-mediated immunity by an influenza A virus vector expressing the RSV F_85-93 epitope.     

Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance.     

流感病毒感染介导的免疫病理损伤研究进展

流感病毒感染(如暴发性流行或高致病性禽流感H5N1感染)可以造成广泛的病理损伤及严重的并发症,其肺部病理损伤以肺水肿及广泛的炎性渗出为特点,并伴有大量的中性粒细胞、巨噬细胞、淋巴细胞浸润及促炎因子和趋化因子的产生．组织学及病理学研究表明,过度的宿主应答反应是介导病理损伤的主要原因之一,而这些在流感病毒感染过程中介导组织损伤的免疫分子与细胞,在病毒的有效清除过程中同样至关重要．主要对甲型流感病毒感染过程中免疫系统的多种效应成分如何引发及加重病理性损伤等有害方面加以综述．为深入了解流感病毒感染防御机制及寻找并设计出既无害又能有效地治疗流感病毒感染的策略提供理论指导．     

The Effect of Virus Particle Size on Chemiluminescence Induction by Influenza and Sendai Viruses in Mouse Spleen Cells

Suspensions of orthomyxo- and paramyxoviruses are composed of pleomorphic particles ranging from large filaments to small spheres. Influenza and Sendai viruses were separated according to size by gel filtration and the induction of luminol-dependent chemiluminescence (CL) by particles of similar size was studied in suspensions of mouse spleen cells known to contain phagocytes. CL reflects the generation by the cells of reactive oxygen species. CL induction decreased with particle size for both viruses. Compared with small spheres, large influenza filaments were approximately 10 times as efficient in activating cellular light emission while the ratio between large and small Sendai viruses was 3:1. Small Sendai virus particles were also less efficient in lysing red cells and had lower neuraminidase activity. By contrast, with influenza virus, only neuraminidase and not the hemolytic activity decreased with the virus size. When influenza virus filaments were broken into smaller particles by sonication, the capacity to induce chemiluminescence dropped markedly while the hemolytic and hemagglutinating activities increased and neuraminidase activity remained unaltered. These results suggest that the presentation of influenza virus hemagglutinin and neuraminidase glycoproteins in a large particle, leading to extensive receptor crosslinking, may be an important factor in the efficient activation of CL by filamentous influenza virus. We suggest that radical generation as reflected in cellular CL may relate to the toxic in vivo effects that contribute to the pathogenesis of influenza and infections with paramyxoviruses.     

Human lymphocyte apoptosis after exposure to influenza A virus

Infection of humans with influenza A virus (IAV) results in a severe transient leukopenia. The goal of these studies was to analyze possible mechanisms behind this IAV-induced leukopenia with emphasis on the potential induction of apoptosis of lymphocytes by the virus. Analysis of lymphocyte subpopulations after exposure to IAV showed that a portion of CD3(+), CD4(+), CD8(+), and CD19(+) lymphocytes became apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling positive). The percentage of cells that are infected was shown to be less than the percentage of apoptotic cells, suggesting that direct effects of cell infection by the virus cannot account fully for the high level of cell death. Removal of monocytes-macrophages after IAV exposure reduced the percent of lymphocytes that were apoptotic. Treatment of virus-exposed cultures with anti-tumor necrosis factor alpha did not reduce the percentage of lymphocytes that were apoptotic. In virus-exposed cultures treated with anti-FasL antibody, recombinant soluble human Fas, Ac-DEVD-CHO (caspase-3 inhibitor), or Z-VAD-FMK (general caspase inhibitor), apoptosis and production of the active form of caspase-3 was reduced. The apoptotic cells were Fas-high-density cells while the nonapoptotic cells expressed a low density of Fas. The present studies showed that Fas-FasL signaling plays a major role in the induction of apoptosis in lymphocytes after exposure to IAV. Since the host response to influenza virus commonly results in recovery from the infection, with residual disease uncommon, lymphocyte apoptosis likely represents a part of an overall beneficial immune response but could be a possible mechanism of disease pathogenesis.     

小鼠感染合胞病毒与鼠流感病毒后肺脏病理学比较研究

目的与方法用合胞病毒(RSV)和鼠肺流感病毒(IVP)感染SPF级BALB/c小鼠,复制两种不同的小鼠病毒性肺炎动物模型,观察其临床症状,并对两种模型各自的肺部组织病理学特点进行研究。结果与结论IVP模型组与RSV模型组小鼠感染病毒后,分别在试验的2天和3天发病,临床症状均表现为精神沉郁、耸毛、卷缩、毛无光泽、活动减少、呼吸急促、咳嗽。但IVP感染模型组小鼠在发病后出现死亡,第7天其死亡率达到40%,而RSV感染模型组小鼠7日试验内无死亡病例发生。病理组织学诊断,RSV模型组小鼠为急性渗出性间质性肺炎,IVP模型组小鼠为出血性间质性肺炎。     

Expression of interleukin-4 by recombinant respiratory syncytial virus is associated with accelerated inflammation and a nonfunctional cytotoxic T-lymphocyte response following primary infection but not following challenge with wild-type virus

The outcome of a viral infection or of immunization with a vaccine can be influenced by the local cytokine environment. In studies of experimental vaccines against respiratory syncytial virus (RSV), an increased stimulation of Th2 (T helper 2) lymphocytes was associated with increased immunopathology upon subsequent RSV infection. For this study, we investigated the effect of increased local expression of the Th2 cytokine interleukin-4 (IL-4) from the genome of a recombinant RSV following primary infection and after a challenge with wild-type (wt) RSV. Mice infected with RSV/IL-4 exhibited an accelerated pulmonary inflammatory response compared to those infected with wt RSV, although the wt RSV group caught up by day 8. In the first few days postinfection, RSV/IL-4 was associated with a small but significant acceleration in the expansion of pulmonary T lymphocytes specific for an RSV CD8(+) cytotoxic T-lymphocyte (CTL) epitope presented as a major histocompatibility complex class I tetramer. However, by day 7 the response of tetramer-positive T lymphocytes in the wt RSV group caught up and exceeded that of the RSV/IL-4 group. At all times, the CTL response of the RSV/IL-4 group was deficient in the production of gamma interferon and was nonfunctional for in vitro cell killing. The accelerated inflammatory response coincided with an accelerated accumulation and activation of pulmonary dendritic cells early in infection, but thereafter the dendritic cells were deficient in the expression of B7-1, which governs the acquisition of cytolytic activity by CTL. Following a challenge with wt RSV, there was an increase in Th2 cytokines in the animals that had previously been infected with RSV/IL-4 compared to those previously infected with wt RSV, but the CD8(+) CTL response and the amount of pulmonary inflammation were not significantly different. Thus, a strong Th2 environment during primary pulmonary immunization with live RSV resulted in early inflammation and a largely nonfunctional primary CTL response but had a minimal effect on the secondary response.     

Evaluation of consistency in quantification of gene copy number by real‐time reverse transcription quantitative polymerase chain reaction and virus titer by plaque‐forming assay for human respiratory syncytial virus

The plaque‐forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque‐forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT‐qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque‐forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT‐qPCR. Two real‐time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque‐forming units. In conclusion, RT‐qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque‐forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.

     

Protection of inactivated influenza virus vaccine against lethal influenza virus infection in diabetic mice

Influenza virus infection frequently causes complications and some excess mortality in the patients with diabetes. Vaccination is an effective measure to prevent influenza virus infection. In this paper, antibody response and protection against influenza virus infection induced by vaccination were studied in mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized once or twice with inactivated influenza virus vaccine at various dosages. Four weeks after the first immunization or 1 week after the second immunization, the mice were challenged with influenza virus at a lethal dose. The result showed that the antibody responses in diabetic mice were inhibited. Immunization once with high dose or twice with low dose of vaccine provided full protection against lethal influenza virus challenge in diabetic mice, however, in healthy mice, immunization only once with low dose provided a full protection.     

Cellular mechanisms involved in protection and recovery from influenza virus infection in immunodeficient mice.

We investigated the role of different lymphocyte subpopulations in the host defense reaction against influenza virus infection, taking advantage of various immunodeficient mouse strains. Whereas, following immunization, wild-type animals showed complete protection against challenge with a lethal dose of A/PR8/34 (PR8) virus, mice that lack both B and T cells but not NK cells (namely, scid and RAG2(-/-) mice) did not display any protective effect in similar conditions. By contrast, J(H)D(-/-) mice devoid of B cells and immunized with virus showed a protective response after challenge with a lethal dose. The immunized J(H)D(-/-) mice that survived completely recovered from the influenza virus infection. Immunized J(H)D(-/+) mice exhibited a more complete protection, suggesting the role of specific antibodies in resistance to infection. To assess the role of natural immunity in the host defense against influenza virus, we carried out experiments with scid mice challenged with lower but still lethal doses of PR8 virus. While an increased NK activity and an increased number of NK1.1+ cells in lungs of scid mice infected with PR8 virus were noted, in vivo depletion of the NK1.1+ cells did not affect the overall survival of the mice. Our results show that specific T cells mediate protection and recovery of J(H)D(-/-) mice immunized with live virus and challenged with lethal doses of influenza virus.     

A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

Background

A subset of the virus-specific CD8+ cytotoxic T lymphocytes (CTL) isolated from the lungs of mice infected with human respiratory syncytial virus (RSV) is impaired in the ability to secrete interferon γ (IFNγ), a measure of functionality. It was suggested that the impairment specifically suppressed the host cellular immune response, a finding that could help explain the ability of RSV to re-infect throughout life.

Results

To determine whether this effect is dependent on the virus, the route of infection, or the type of infection (respiratory, disseminated, or localized dermal), we compared the CTL responses in mice following intranasal (IN) infection with RSV or influenza virus or IN or intradermal (ID) infection with vaccinia virus expressing an RSV CTL antigen. The impairment was observed in the lungs after IN infection with RSV, influenza or vaccinia virus, and after a localized ID infection with vaccinia virus. In contrast, we observed a much higher percentage of IFNγ secreting CD8+ lymphocytes in the spleens of infected mice in every case.

Conclusion

The decreased functionality of CD8+ CTL is specific to the lungs and is not dependent on the specific virus, viral antigen, or route of infection.     

转谷氨酰胺酶2抑制H1亚型流感病毒在MDCK细胞中的增殖

组织转谷酰胺酶(transglutaminase 2,TGM2)是一种普遍存在的多功能蛋白,与不同细胞的粘附和肿瘤形成有关.有证据表明,TGM2参与了宿主细胞与病毒间的相互作用,但是对于流感病毒在细胞内增殖的影响还未有报道.为了探究MDCK细胞中TGM2对H1N1亚型流感病毒增殖的影响,本研究构建了TGM2过表达和敲除...     

Priming with live respiratory syncytial virus (RSV) prevents the enhanced pulmonary inflammatory response seen after RSV challenge in BALB/c mice immunized with formalin-inactivated RSV.

To investigate enhanced disease associated with a formalin-inactivated (FI) respiratory syncytial virus (RSV) vaccine, we studied the pulmonary inflammatory response to RSV in BALB/c mice immunized with live RSV, FI-RSV, or combinations of the two. After RSV challenge, the number of granular cells, the ratio of CD4+/CD8+ lymphocytes, and the level of Th2-like cytokine mRNAs in the bronchoalveolar lavage specimens in mice immunized first with live RSV and then with FI-RSV were lower than that in FI-RSV-immunized mice and close to that in live RSV-immunized mice. These data suggest that prior live RSV infection prevents most of the enhanced inflammatory response seen in FI-RSV-immunized mice and might explain lack of enhanced disease in older FI-RSV-immunized children. A live RSV vaccine might similarly decrease the risk of enhanced disease with non-live RSV vaccines.