BALB/c鼠和裸鼠呼吸道合胞病毒感染比较研究

目的 比较普通BALB/c鼠和裸鼠呼吸道合胞病毒(RSV)感染免疫及炎症反应特点.方法 BALB/c鼠和裸鼠感染RSV后不同时间空斑形成试验检测肺组织病毒滴度,计数支气管肺泡灌洗液(BALF)白细胞总数和分类,HE染色分析肺组织病理学改变,免疫组化检测肺组织F4/80+细胞和CD49b+细胞.ELISA检测BALF中TNF-α、IFN-r、IL-12和IL-10浓度.结果 BALB/c鼠和裸鼠感染RSV后肺组织病毒滴度在第3天达峰值,感染裸鼠带毒时间更长,在感染后各天病毒滴度明显高于BALB/c鼠(P＜0.05),肺组织病理改变也更重.感染BALB/c鼠和裸鼠BALF白细胞总数明显升高,分类以淋巴细胞为主.感染裸鼠与感染BALB/c鼠比较,肺组织检测到更多的F4/80+巨噬细胞和CD49b+NK细胞(P＜0.05),BALF中TNF-α、IL-12和IL-10水平更高(P＜0.05).结论 RSV感染裸鼠与BALB/c鼠比较,病毒复制水平更高,时间更持久,炎症反应更重.单核巨噬细胞和NK细胞是RSV感染重要的免疫细胞和炎症细胞,炎症反应强度并不一定与T细胞免疫应答平行.     

T cell redistribution kinetics after secondary infection of BALB/c mice with respiratory syncytial virus.

BALB/c mice were infected intranasally with live respiratory syncytial virus (RSV) and reinfected 4 weeks later. At regular intervals thereafter groups of animals were killed and T cell subsets were determined in blood, spleen and bronchoalveolar lavage (BAL) with flow cytometry employing T cell subset-specific MoAbs. Total lymphocyte counts in the peripheral blood decreased 1-3 days after infection, returning to preinfection levels on day 8 (P = 0.0111). Simultaneously, a marked increase of lymphocytes was noted in the BAL, reaching a maximum at day 8 (P < 0.0001). Both CD4+ and CD8+ T cells decreased in the blood on day 1-3 (P < 0.0097 and P = 0.003 respectively), and increased in the BAL progressively towards a maximum at day 8 (P < 0.0001). In BAL, CD4+ cells increased 35-fold and CD8+ cells 27-fold during the first week after reinfection. On the other hand, in the spleen a significant decline of CD4+ and CD8+ cells was noted 1 day post-infection (P = 0.0002). It is concluded that a strong T cell redistribution response among systemic and mucosal tissues occurs after reinfection with RSV. The kinetics of this response differ both quantitatively and qualitatively from the T cell response after primary infection. The magnitude of cell traffic is more pronounced in blood, spleen and BAL than after primary infection. CD4+ T cells are more intensively distributed to the lungs than after primary infection.     

Gamma interferon controls Eimeria vermiformis primary infection in BALB/c mice.

Neutralization of endogenous gamma interferon by treatment with a rat monoclonal antibody caused enhancement of infection with the protozoon Eimeria vermiformis in naive BALB/c mice. The effect was dose dependent and was apparent when a monoclonal antibody was given at 2 h before infection or up to 7 days postinfection, but it decreased with increasing time postinfection between days 4 and 7. The titers of parasite-specific antibodies in the serum were not significantly affected by the injection of monoclonal antibodies. Treatment during priming did not prevent the development of resistance to challenge, and treatment at the time of challenge did not abrogate established immunity. The results indicate that gamma interferon is involved in the control of primary infection with E. vermiformis in BALB/c mice but not in the expression of immunity to challenge.     

Primary respiratory syncytial virus infection in mice

A mouse model of respiratory syncytial virus (RSV) infection is described. A high-titered, large-volume inoculum results in replication of RSV to a high titer in lungs of BALB/c mice. Mice older than 15 weeks of age are more susceptible to RSV infection. Titers up to 10(6.9) plaque-forming units (pfu)/gram lung can be attained in 32-week-old mice. Older mice experience a clinical illness manifested by ruffled fur, reduced activity, and weight loss. Lung histology of older mice infected with RSV shows bronchiolitis and increased number of lymphocytes and macrophages in alveolar spaces compared with that of mice less than 8 weeks old. This model will serve as the basis for investigating immunodeterminants of recovery and protection from RSV infection.     

Experimental acute respiratory Burkholderia pseudomallei infection in BALB/c mice

Burkholderia pseudomallei is the causative agent of melioidosis, which is considered a potential deliberate release agent. The objective of this study was to establish and characterise a relevant, acute respiratory Burkholderia pseudomallei infection in BALB/c mice. Mice were infected with 100 B. pseudomallei strain BRI bacteria by the aerosol route (approximately 20 median lethal doses). Bacterial counts within lung, liver, spleen, brain, kidney and blood over 5 days were determined and histopathological and immunocytochemical profiles were assessed. Bacterial numbers in the lungs reached approximately 10⁸ cfu/ml at day 5 post-infection. Bacterial numbers in other tissues were lower, reaching between 10³ and 10⁵ cfu/ml at day 4. Blood counts remained relatively constant at approximately 1.0 × 10² cfu/ml. Foci of acute inflammation and necrosis were seen within lungs, liver and spleen. These results suggest that the BALB/c mouse is highly susceptible to B. pseudomallei by the aerosol route and represents a relevant model system of acute human melioidosis.     

Protection of BALB/c mice from respiratory syncytial virus infection by immunization with a synthetic peptide derived from the G glycoprotein.

A synthetic peptide homologous to amino acids 174-187 of the G glycoprotein of the A2 strain of human respiratory syncytial (RS) virus (G/174-187) was shown to induce protection from live virus challenge of BALB/c mice after immunization with three doses of 50 micrograms of peptide coupled to keyhole limpet hemocyanin. Immunized mice showed high levels of circulating RS-specific antibodies as detected by ELISA assay; however, no neutralizing antibodies were found. Moreover, an important short-term cytotoxic T-cell response was observed with lymphocytes isolated from the lungs but not from the spleen of immunized mice. This response was lost 24 weeks after immunization; however, mice remained protected against challenge with live RS virus. In addition, a monoclonal antibody that specifically binds to peptide G/174-187 was found efficient in conferring passive protection from challenge: this data further supports our results on the importance of the 174-187 region in protection. Another peptide, spanning amino acids 144 to 159, was shown to induce neutralizing antibodies but did not confer protection.     

Role of eosinophils in the pathogenesis of Mycobacterium bovis BCG infection in gamma interferon receptor-deficient mice

A profound eosinophil infiltration of granulomas is observed in the lungs of Mycobacterium bovis bacillus Calmette Guérin-infected gamma interferon receptor-deficient mice. Blockade of eosinophil proliferation and recruitment into the lung by treatment with anti-interleukin-5 monoclonal antibody marginally reduced mycobacterial growth within the lung but did not affect dissemination of the infection to other tissues.     

Decreased bacterial clearance from the lungs of mice following primary respiratory syncytial virus infection

Virus respiratory infections often precede bacterial pneumonia in healthy individuals. In order to determine the potential role of respiratory syncytial virus (RSV) in bacterial secondary infections, a mouse sequential pulmonary infection model was developed. Mice were exposed to RSV then challenged with Streptococcus pneumoniae (StPn). Exposure of BALB/c mice to 10(6)-10(7) plaque forming units (pfu) of virus of RSV significantly decreased StPn clearance 1-7 days following RSV exposure. This finding was not restricted to StPn alone: exposure to RSV followed by Staphylococcus aureus (SA) or Pseudomonas aeruginosa(PA) resulted in similar decreases in bacterial clearance. Both bronchoalveolar lavage (BAL) cell counts and pulmonary histopathology demonstrated that RSV-StPn exposed mice had increased lung cellular inflammation compared to mice receiving StPn or RSV alone. The effect of RSV infection on bacterial clearance was dependent on the mouse genetic background: C57BL/6J mice (relatively resistant to RSV infection) demonstrated a modest change in StPn clearance following RSV exposure, whereas FVBN/J mice (similar to the BALB/cJ mice in RSV susceptibility) demonstrated a similar degree of RSV-associated decrease in StPn clearance 7 days following RSV exposure. Neutrophils from the RSV-StPn sequentially exposed BALB/cJ mice were functionally altered-produced greater levels of peroxide production but less myeloperoxidase (MPO) compared to mice receiving StPn alone. These data demonstrate that RSV infection decreases bacterial clearance, potentially predisposing to secondary bacterial pneumonia despite increased lung cellular inflammation, and suggest that functional changes occur in the recruited neutrophils that may contribute to the decreased bacterial clearance.     

The pathogenesis of respiratory syncytial virus infection in infant ferrets.

The infant ferret is susceptible to respiratory syncytial virus infection in both the upper and lower respiratory tracts. In the nose, viral replication is restricted to the surface respiratory epithelium in the nasal passages and turbinates. In the lungs, viral replication is of a lower order of magnitude and is localized in the alveolar cells. The pattern of viral replication in nasal tissues is independent of the age of the animal at infection, whereas the pattern in lung tissues shows a striking age dependence, with viral replication progressively decreasing as a function of age. Thes age dependence appears to be due to an intrinsic age-related mechanism yet to be defined. We feel that the infant ferret is an acceptable model for the study of respiratory syncytial virus disease and that the study of age dependence observed in ferrets may allow elucidation of the mechanisms involved in the age dependence seen in humans.     

The pathogenesis of respiratory syncytial virus infection in cotton rats.

The cotton rat is susceptible to respiratory synctial virus infection in both the upper and lower portions of the respiratory tract. Virus replicates to high titer in the nose and lungs and to relatively low titer in the trachea. Immunofluorescence studies demonstrated viral antigen in the nasal epithelium and the bronchial and bronchiolar epithelium but not in the trachea or the alveolar cells of the lungs. Histopathologic changes included a desquamative, exudative rhinitis of moderate severity and a mild proliferative bronchiolitis. Serum neutralizing antibody developed in all animals by the ninth day after infection, reaching extremely high titer in several instances. Unlike the previously described response of experimentally infected infant ferrets, cotton rats are uniformly susceptible to pulmonary infection throughout life, thereby offering a model for long-term pulmonary studies heretofore not available.     

Cellular immunity,but not gamma interferon,is essential for resolution of Babesia microti infection in BALB/c mice

A new strain of Babesia microti (KR-1) was isolated from a Connecticut resident with babesiosis by hamster inoculation and adapted to C3H/HeJ and BALB/c mice. To examine the relative importance of humoral and cellular immunity for the control of B. microti infection, we compared the course of disease in wild-type BALB/c mice with that in BALB/c SCID mice, JHD-null (B-cell-deficient) mice, and T-cell receptor alphabeta (TCRbeta(-/-)) or gamma interferon (IFN-gamma) (IFN-gamma(-/-)) knockout mice following inoculation with the KR-1-strain. SCID mice and TCRalphabeta knockouts sustained a severe but nonlethal parasitemia averaging 35 to 45% infected erythrocytes. IFN-gamma-deficient mice developed a less severe parasitemia but were able to clear the infection. In contrast, in six of eight JHD-null mice, the levels of parasitemia were indistinguishable from those in the wild-type animals. These data indicate that cellular immunity is critical for the clearance of B. microti in BALB/c mice but that disease resolution can occur even in the absence of IFN-gamma.     

Differential induction of gamma interferon in Legionella pneumophila-infected macrophages from BALB/c and A/J mice

Gamma interferon (IFN-gamma), a pleiotropic cytokine, is now known to be produced by macrophages as well as by NK cells, gammadelta cells, and activated T cells. The autocrine biological functions of IFN-gamma on the macrophage include the upregulation of major histocompatibility complex MHC class II and the activation to an antiviral state. In this study, the production of IFN-gamma by macrophages was demonstrated to correspond to antibacterial activity. Legionella pneumophila replicates intracellularly in thioglycolate (TG)-elicited macrophages (TG-macrophages) from A/J mice, while TG-macrophages from BALB/c mice restrict bacterial growth after an initial period of growth. BALB/c TG-macrophages were shown to express IFN-gamma mRNA at 24 and 28 h, which corresponded to the initiation of anti-L. pneumophila activity. Moreover, IFN-gammaneutralization by antibody treatment of the cultures resulted in increased L. pneumophila growth in the macrophages. In contrast, no IFN-gamma mRNA was expressed in TG-macrophages from A/J mice, where L. pneumophila grew unrestricted. As would be expected, IFN-gamma treatment decreased bacterial growth. An IFN-gamma-mediated antibacterial activity was, however, inducible in A/J macrophages by the addition of interleukin-12 following L. pneumophila infection. Thus, autocrine IFN-gamma is involved in anti-L. pneumophila activity associated with different growth patterns and appears to be important during intracellular infection.     

Pathogenesis of experimental Ebola Zaire virus infection in BALB/c mice.

Guinea-pigs and non-human primates have traditionally been used as animal models for studying Ebola Zaire virus (EBO-Z) infections. The virus was also recently adapted to the stage of lethal virulence in BALB/c mice. This murine model is now in use for testing antiviral medications and vaccines. However, the pathological features of EBO-Z infection in mice have not yet been fully described. To identify sites of viral replication and characterize sequential morphological changes in BALB/c mice, adult female mice were infected with mouse-adapted EBO-Z and killed in groups each day for 5 days post-infection. Tissues were examined by light microscopy, immunohistochemistry, electron microscopy and in-situ hybridization. As in guinea-pigs and non-human primates, cells of the mononuclear phagocytic system were the earliest targets of infection. Viral replication was observed by day 2 in macrophages in lymph nodes and spleen. By the time of onset of illness and weight loss (day 3), the infection had spread to hepatocytes and adrenal cortical cells, and to macrophages and fibroblast-like cells in many organs. Severe lymphocytolysis was observed in the spleen, lymph nodes and thymus. There was minimal infection of endothelial cells. All of these changes resembled those observed in EBO-Z-infected guinea-pigs and non-human primates. In contrast to the other animal models, however, there was little fibrin deposition in the late stage of disease. The availability of immunodeficient, "gene-knockout" and transgenic mice will make the mouse model particularly useful for studying the early steps of Ebola pathogenesis.     

Genetic susceptibility to respiratory syncytial virus infection in inbred mice

Differences in the severity of respiratory syncytial virus (RSV)-induced lower respiratory disease in infants have been attributed to multiple environmental and genetic factors. To identify the genetic factor(s) influencing RSV susceptibility, we examined RSV infection in eight inbred mouse strains. Lung RSV titers differed significantly between mouse strains: the RSV titers were 15-fold higher in AKR/J (permissive) mice compared with C57BL/6J (resistant) mice at 4 days after inoculation. This strain-specific difference in RSV titers suggested that susceptibility to RSV infection was attributable to genetic differences between strains. To examine the mode of inheritance of RSV susceptibility, F1 and backcross (F1 x AKR/J) progeny were infected and RSV titers determined. RSV titers in the F1 progeny were similar to those found in the resistant (C57BL/6J) parent, suggesting resistance was inherited as a dominant trait. The distribution of RSV titers in backcross progeny were discordant with that predicted for a single gene effect, suggesting susceptibility was influenced by more than one gene. These data suggest that RSV susceptibility is a multigenic trait that should be amenable to resolution by genomic analysis.     

Production of interferon gamma in respiratory syncytial virus infection of humans is not associated with interleukins 12 and 18

In order to understand early events in the immune response to respiratory syncytial virus (RSV) infection, we studied the presence of various chemokines and cytokines in respiratory secretions of human infants with RSV infection. Interferon gamma (IFNgamma) was present in 30/39 (76.9%) subjects tested, but the IFNgamma-inducing cytokines interleukin (IL)12 and IL18 were detectable in 6/40 (15%) and 11/38 (28.9%) subjects, respectively. Quantities of IL12 and IL18 did not correlate with those of IFNgamma. IL18, but neither IFNgamma nor IL12 was found in significantly greater concentrations in subjects with mild, nonhypoxic forms of bronchiolitis than in those with upper respiratory illness alone or hypoxic bronchiolitis. The findings suggest that IFNgamma may be induced independently of the activities of IL12 and IL18 during RSV infection. Immune responses characterized by relatively greater release of IL18 may be associated with milder forms of bronchiolitis.     

Increased pathogenesis and inflammation of airways from respiratory syncytial virus infection in T cell deficient nude mice

Respiratory syncytial virus (RSV) infection is ubiquitous and leads to various outcomes between immunocompetent and immunocompromised individuals. This study aimed to compare RSV infection and inflammatory responses between immunocompetent BALB/c mice and immunodeficient nude mice. RSV titers in both infected BALB/c mice and nude mice peaked on the third day post-inoculation, but the nude mice had longer lasting and higher levels of viral replication. RSV infection induced a more severe grade of pulmonary histopathology and larger numbers of leukocytes in airways of nude mice than that of BALB/c mice. RSV infection increased pulmonary macrophages and natural killer (NK) cells in both strains of mice. Furthermore, infected nude mice had larger numbers of pulmonary macrophages and NK cells than infected BALB/c mice. Whereas the RSV infected BALB/c mice secreted more tumor necrosis factor -alpha (TNF-alpha), interleukin-12 (IL-12), interferon-gamma (IFN-gamma) and IL-10 than control BALB/c mice, the infected nude mice had higher levels of TNF-alpha, IL-12 and IL-10 than the infected BALB/c mice. The inflammation induced by RSV infection did not correspond with the immune response of T cells. Macrophages and NK cells were potent immunocytes and inflammatory cells in RSV infection especially when T lymphocytes were deficient. Therefore, nude mice may be a good model for severe and persistent RSV infection in immunocompromised hosts.     

Isotype distribution and specificity of the antibody response to primary Moloney murine sarcoma virus infection in BALB/c mice

The development and isotype distribution of Moloney murine leukemia virus (M-MuLV)-specific serum antibodies following primary inoculation with Moloney murine sarcoma/leukemia virus (M-MuSV/M-MuLV) in adult BALB/c mice have been investigated using an enzyme-linked immunosorbent assay (ELISA). The primary antibody responses to M-MuSV/M-MuLV consisted of the IgM, IgG2a, IgG2b, and IgG3 isotypes; no M-MuLV-specific serum IgG1 or IgA antibodies were detected. The detectable antibody response was biphasic, with an early peak of virus-specific titers seen between 10 and 15 days after inoculation and a second peak seen in regressor sera. Pooled regressor sera contained IgM, IgG2a, and IgG2b antibodies which bound to M-MuLV-expressing lymphoma cells. Immunoelectron microscopy with regressor sera showed IgG bound both to infected cell surfaces and to mature viral particles, while IgM bound only to infected cell surfaces. These findings were supported by immunoprecipitation analyses which demonstrated binding of the M-MuLV-specific antibodies to both virion-associated and cell-associated antigens encoded by the gag and env genes.     

Monoclonal antibodies protect against respiratory syncytial virus infection in mice.

Twenty-five monoclonal antibodies (Mab) to respiratory syncytial virus (RSV) and two to hepatitis B virus were inoculated intravenously into mice. Twenty-four hours later the mice were challenged intranasally with RSV. Eleven of 14 Mab against fusion protein and four out of six Mab against a larger glycoprotein (GP84) significantly reduced the titre of RSV in the lungs when mice were killed 5 days later. Five Mab against three other RSV proteins and two Mab against hepatitis B virus had no significant effect on RSV infection. These results indicated that serum IgG against one epitope on the fusion protein and another on the larger glycoprotein (GP84) will completely protect mice against challenge. These epitopes are primary candidates for an RSV vaccine produced by techniques of gene cloning and peptide synthesis.     

Pathogenesis of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is recognized as the most important cause of serious lower respiratory tract illness in infants and young children worldwide causing repeat infections throughout life with serious complications occurring in the elderly and immune compromised patient. The level of disease pathogenesis associated with RSV infection is balanced between virus elimination and the nature of the immune response to infection. The innate and adaptive immune responses to RSV infection are not fully elucidated; however, significant progress has been made in understanding the virus-host relationship and mechanisms associated with disease pathogenesis. This review summarizes important aspects of these findings, and provides current perspective on processes that may contribute to RSV disease pathogenesis.     

呼吸道合胞病毒感染的研究进展

呼吸道合胞病毒是在世界范围内引起婴幼儿呼吸道感染的最常见病原微生物,它不仅可以引起呼吸系统的一些常见症状和体征,而且在肺外器官如中枢神经系统、心血管系统、内分泌系统等各器官也可引起一些尚未被人们普遍认识的肺外表现,而这些临床特点的重要性也愈来愈受到人们的重视.