Passive serum antibody causes temporary recovery from influenza virus infection of the nose, trachea and lung of nude mice.

BALB/c normal and nude mice were infected with a non-lethal mouse-passaged A/PC/1/73 (H3N2) influenza virus in order to assess the role of T cells on the course of disease of the nose, trachea and lung. The tracheal epithelium of both mouse strains was desquamated by 3 days after infection. Although normal regeneration began, nude mice never completed that regeneration whereas normal mice had fully regenerated tracheas by Day 14. This failure to complete the recovery was also evident from the continued virus shedding by the nude mouse. In order to assess the role of serum antibody on recovery from infection, ferret, goat or mouse antibody to H3N2 influenza virus was passively administered to nude mice after infection. It resulted in a transient decrease in virus shedding from the nose, trachea and lung, and complete but temporary regeneration of the tracheal epithelium. However, later in the course of the infection, when serum antibody levels were no longer detectable, the tracheal epithelium of these animals redesquamated and large amounts of virus were again shed from nose, trachea and lungs. We conclude that: (i) desquamation of the ciliated epithelium of the trachea is not T-cell dependent; and (ii) serum antibody can contribute to temporary recovery from infection, but by itself is insufficient for permanent recovery of the nose, trachea or lung.     

Serum antibody prevents lethal murine influenza pneumonitis but not tracheitis.

This paper reports studies showing the effects of serum antibody upon influenza infection at two different sites: the trachea and lung. Tracheal desquamation, pulmonary consolidation, death, and virus shedding were examined after infection of mice with a lethal A/Port Chalmers/1/73 (H3N2) influenza virus. Immune serum administered intraperitoneally before infection prevented death and pulmonary consolidation and also significantly lowered lung virus shedding as compared with controls receiving normal serum. However, this protection did not extend to the ciliated epithelium of the trachea because serum antibody did not prevent desquamation of the trachea or significantly decrease viral yield from the trachea. These results indicate that serum antibody is protective against severe pulmonary parenchymal disease but not for disease of the ciliated epithelium.     

The role of cellular susceptibility in the declining severity of respiratory influenza of ferrets with age

A comparison was made, both in vivo and in organ culture, between newborn (1-day-old) and suckling (15-day-old) ferrets of lower respiratory tract tissue infected with a virulent strain (clone 7a) of influenza virus. Newborn ferrets were killed by influenza virus following intranasal inoculation but suckling ferrets were almost as resistant as adult ferrets. In newborn ferrets there was a rapid, severe and progressive infection of lung tissue with infection of alveolar cells as well as those of bronchial and bronchiolar epithelium (assessed by monitoring virus infectivity and by fluorescent antibody staining). In suckling ferrets, as previously shown for adult animals, the lung infection was less severe, less persistent and confined to the epithelium of bronchi with only a small bronchiolar involvement and even less alveolar cell infection. These differences observed in vivo were repeated in organ cultures obtained from various areas of the lung. i.e. alveolar and airway epithelial cells of newborn ferrets exhibited a greater susceptibility than those of older ferrets. Thus, it appears that one factor determining the greater susceptibility of the lower respiratory tract of newborn ferrets is a greater inherent susceptibility of alveolar and airway epithelial cells to infection with influenza virus. Other factors may also be involved and have yet to be investigated.     

The role of cellular susceptibility in the declining severity of respiratory influenza of ferrets with age.

A comparison was made, both in vivo and in organ culture, between newborn (1-day-old) and suckling (15-day-old) ferrets of lower respiratory tract tissue infected with a virulent strain (clone 7a) of influenza virus. Newborn ferrets were killed by influenza virus following intranasal inoculation but suckling ferrets were almost as resistant as adult ferrets. In newborn ferrets there was a rapid, severe and progressive infection of lung tissue with infection of alveolar cells as well as those of bronchial and bronchiolar epithelium (assessed by monitoring virus infectivity and by fluorescent antibody staining). In suckling ferrets, as previously shown for adult animals, the lung infection was less severe, less persistent and confined to the epithelium of bronchi with only a small bronchiolar involvement and even less alveolar cell infection. These differences observed in vivo were repeated in organ cultures obtained from various areas of the lung. i.e. alveolar and airway epithelial cells of newborn ferrets exhibited a greater susceptibility than those of older ferrets. Thus, it appears that one factor determining the greater susceptibility of the lower respiratory tract of newborn ferrets is a greater inherent susceptibility of alveolar and airway epithelial cells to infection with influenza virus. Other factors may also be involved and have yet to be investigated.     

Monoclonal antibody-based immunohistochemical diagnosis of Malaysian Nipah virus infection in pigs

Formalin-fixed, paraffin wax-embedded tissues of three Malaysian farm pigs naturally infected with Nipah virus were used to investigate the value of anti-Nipah virus mouse monoclonal antibodies (Mabs) and rabbit polyclonal antibody for immunohistochemical diagnosis. Mabs 11F6 and 12A5 gave intense immunolabelling in lung tissue that had been fixed in 10% neutral buffered formalin for about 4 years, whereas the reactivity of Mabs 13A5 and 18C4 and polyclonal antibody was reduced significantly by long-term formalin fixation. Immunohistochemical examination of Malaysian farm pig samples with Mab 11F6 confirmed the affinity of Nipah virus for respiratory epithelium, renal glomerular and tubular epithelium, meningeal arachnoidal cells, and systemic vascular endothelium and smooth muscle. In addition, Nipah virus antigens were identified in laryngeal epithelial cells, Schwann cells of peripheral nerve fascicles in the spleen, and endothelial cells in the atrioventricular valve. The study demonstrated the value of Mabs 11F6 and 12A5 for the immunohistochemical diagnosis of Nipah virus infection in pigs.     

A comparison in germ-free mice of the pathogenesis of Sendai virus and mouse pneumonia virus infections

The poathogenesis of Sendai virus and pneumonia virus of mice (PVM) was studied using the immunoperoxidase technique on paraffin lung sections. The pathology of Sendai virus corresponds to that of a bronchopneumonia, with virus demonstrated by immunoperoxidase in the bronchial epithelium, and sometimes in macrophages, for a period of 2--9 days post-infection. Pneumonia virus of mice produces an interstitial pneumonia with virus demonstrated in the bronchial epithelium but also in the alveolar walls and alveolar macrophages. This virus can be demonstrated between 2 and 7 days post-infection. This technique was used to demonstrate PVM in the case of a natural outbreak of this disease and may eventually become a routine technique for the screening of lung tissue for respiratory viruses.     

Effects of nitrogen dioxide exposure on Clara cell proliferation and morphology.

The present study was performed to relate Clara cell proliferation and Clara cell morphology to duration and intensity of nitrogen dioxide (NO2) exposure as a model of lung injury in rats. Male Sprague-Dawley rats were exposed to 5, 10 and 20 ppm NO2 for three (short term exposure) and 25 days (long term exposure). Epithelial proliferation was immunohistochemically assessed by BrdU-incorporation into the bronchial and bronchiolar epithelium. Clara cell specific 10 kD protein was detected using an antibody versus recombinant protein. Following short term exposure Clara cells showed a significant alteration of their morphology: the cells lost the apical intraluminal projections, and the damaged epithelium was covered by a layer of CC10 reactive material. After long term exposure the Clara cell morphology was indistinguishable from that of unexposed controls. In all short term exposure groups cellular proliferation was higher in the bronchiolar epithelium than in the bronchial epithelium. Whereas short term exposure to 5 ppm NO2 sufficed to induce a maximum epithelial proliferation of the bronchiolar epithelium, long term exposure to 5 ppm NO2 yielded no significant increase in the epithelial proliferation, a finding reflecting the adaptation of the airway epithelium to oxidative stress. Long term exposure to 10 and 20 ppm NO2 resulted in a dosilinear increase of cell proliferation in the bronchial and bronchiolar epithelium. Double labelling of CC10 and BrdU showed that BrdU uptake and hence proliferation were restricted to Clara cells. We conclude that changes of the Clara cell morphology are closely related to the extent of inhalative stress and functional status of the Clara cell. The results are in keeping with the assumption that the Clara cell exerts progenitor cell functions in the airway epithelium.     

Persistence of pneumonia virus of mice and Sendai virus in germ-free (nu/nu) mice.

The pathogenicity and persistence of pneumonia virus of mice (PVM) and Sendai virus has been studied using germ-free nu/nu mice. PVM was found to infect cells of the bronchial epithelium (and the alveolar wall) of the lungs of germ-free nu/nu mice using the immunoperoxidase technique. The virus was located in the bronchial epithelium for 11 days before elimination, but persisted in the alveolar wall for the duration of the experiment (20 days). After Day 10 a humoral antibody response to PVM was observed which persisted, although at a low level (1 in 40), by haemagglutination-inhibition (HI) testing. Sendai virus in nu/nu mice also infected cells of the bronchial epithelium and this persisted for the duration of the experiment (27 days). The persistence of virus in the bronchial epithelium in relation to lack of humoral antibody is discussed with reference to local secretory antibody production, especially since this does not occur with PVM.     

Distribution of rotavirus antigen in intestinal lymphoid tissues: potential role in development of the mucosal immune response to rotavirus.

Groups of suckling BALB/c mice were inoculated with murine rotavirus (MRV) at 5 days of age and sequentially tested for the presence of MRV antigen (Ag) in intestinal villus epithelium (VE), Peyer's patch (PP), mesenteric lymph node (MLN), spleen, liver and lung, using indirect immunofluorescence techniques (IF). MRV Ag was first detected in VE 24 h after oral administration of the virus and remained in VE for as long as 10 days post-inoculation (pi). MRV Ag was observed in the epithelium overlying PP at 3-7 days pi and in subepithelial and interfollicular areas in the PP between 3 and 20 days pi. MRV Ag was also detected in MLN during the same period of time. Small numbers of MRV-Ag-containing cells were detected in the lamina propria (LP) of intestinal villi at 10 days pi. Most MRV-Ag-containing cells in PP and MLN were Ia-positive but negative for Lyt-1, Lyt-2 and MAC-1 cell surface markers. MRV-Ag-containing cells were negative for surface or cytoplasmic immunoglobulin. Intestinal antibody response to MRV indicated by the presence of MRV-specific IgA plasma cells in LP was first detectable 10 days pi. Highest density of MRV-specific plasma cells was observed in the duodenum, with a similar distribution to that of MRV-Ag-containing cells in PP. These observations suggest that MRV-Ag uptake is largely limited to the PP associated epithelium and the virus Ag is subsequently transported to the underlying lymphoid follicles and MLN. These findings suggest a close relationship between the availability of MRV Ag in the lymphoid follicles at different locations and the subsequent development of local antibody responses in different segments of small intestine.     

Pneumotropism of Sendai Virus in Relation to Protease-Mediated Activation in Mouse Lungs

The pneumotropism of Sendai virus in mice was studied in relation to the activation and replication of the virus in the lung. Inactive Sendai virus grown in LLC-MK₂ cells, which possessed an uncleaved precursor glycoprotein, F, and was noninfectious to tissue culture cells, neither grew nor caused pathological changes in the lung of mice. When trypsin treatment was made which cleaved F into F₁ and F₂ subunits, the virus became activated so that it could initiate replication in the bronchial epithelium of the lung. In this case, the progeny virus was produced in the activated form and multiple-cycle replication occurred successively. A parallel relationship was found between the degree of the viral replication and that of clinical signs of the respiratory disease, body weight loss, and histopathological changes in the lung. A protease mutant, TR-2, which was able to be activated only by chymotrypsin but not by trypsin, could also initiate replication in the bronchial epithelium, when activated by chymotrypsin before inoculation into mice. The progeny virus, however, remained inactive, and the replication was limited to a single cycle, which resulted in the limited lung lesion. The overall results suggest that some activating mechanism for the progeny virus of wild-type Sendai virus exists in the lung of mice and the principle (activator) responsible for this phenomenon has a character similar to trypsin. The possible location of the activator is discussed.     

Histopathology of fatal adenovirus infection of the respiratory tract in young children

Type 7 adenoviruses were isolated from lung tissue obtained at the necropsies on five children aged 8 to 15 months. The clinical and radiological findings were those of bronchiolitis and pneumonia. Four children had an extensive necrotizing bronchitis and bronchiolitis. The surviving bronchial epithelium, and all bronchial epithelium in the fifth case, was proliferating. There was evidence that this proliferation was a virus-induced effect and not reparative. There was virus tropism to alveolar lining cells with a pneumonia showing mononuclear cellular reaction, necrosis, and hyaline membranes. Distinctive necrotizing lesions were found in the bronchial glands of all cases. Two contrasting types of intranuclear inclusions were present in all lesions and their significance and inter-relationships are discussed. These pathological changes are specific for infections by a limited number of adenovirus serotypes.     

Regional T- and B-cell responses in influenza-infected ferrets.

Ferrets were infected with A/Port Chalmers/72 influenza virus and the T- and B-cell responses in the spleen, in lymph nodes draining the upper and lower respiratory tract, and in lung washings were examined in vitro. Lymphocyte responses were measured by using a hemolytic plaque assay for B cells and a proliferation assay for T cells. Virus and antibody levels were measured in respiratory tract washings, and antibody titers were measured in sera from infected animals. Individual B cells secreting specific antibody to A/Port Chalmers/72 virus were detected in regional lymph node and spleen preparations as early as 3 days and as late as 43 days after infection. T-cell assays showed an in vitro response of lymph node cells to A/Port Chalmers/73 virus from day 6 to day 43. Virus was isolated from the respiratory tract up to 7 days after infection. Serum hemagglutination-inhibiting antibody was first detectable on day 6, with maximum titers reached by day 10. These results demonstrated that antibody production and a cellular immune responses were detectable at regional sites at a time when virus was still present and before serum antibody was measured.     

Protective effect of serum antibody on respiratory infection of influenza C virus in rats

Summary The effects of serum antibody on the replication of influenza C virus in the nose and lung were evaluated in rats challenged with the virus by the intranasal and endotracheal routes, respectively. Convalescent rat serum administered intraperitoneally prior to infection suppressed virus replication significantly in both the nasal and pulmonary tissues. Resistance achieved was however much greater in the lung than in the nose. Rats with a serum neutralizing antibody titer of 1:800 showed almost complete resistance to pulmonary virus infection, and virus yield from the lung was reduced 10- to 100-fold in animals with the antibody titer of 1:80–160 or less. In contrast, significant decrease in virus shedding from the nose was observed only in animals with a serum antibody titer of 1:800 or greater. The effect of adoptive transfer of monoclonal antibodies (MAbs) to haemagglutinin-esterase (HE) glycoprotein and matrix (M) protein on pulmonary virus replication was also examined. Anti-HE MAbs with neutralization activity prevented virus shedding from the lung almost completely whereas non-neutralizing anti-HE MAb and anti-M Mab showed no inhibitory effect.     

Effects of a viral laryngotracheitis on the epithelial barrier of chicken airways

The effects of a virus infection on the barrier function of tracheal epithelium were compared to the effects of a chemical agent (methacholine) which selectively increases membrane permeability, and both were compared to controls. The disruption of the airway epithelium induced by the virus infection caused an increased permeation of horseradish peroxidase (HRP) through this barrier. Methacholine enhanced HRP uptake from the airway lumen to the blood as compared to controls. Visualization of HRP in the tracheal epithelium by transmission electron microscopy correlated with the radioimmunoassay measurements in the blood. Serial anti-HRP antibody titers were measured by a competitive binding technique. The antigen permeation induced by methacholine was associated with an enhanced anti-HRP antibody production. The larger increase in antigen permeation seen with the viral infection was associated with depressed anti-HRP titers. It was concluded that viral disruption of the airway epithelial barrier may contribute to an increased uptake of orally inhaled antigens. The relationship, however, between the increased antigen penetration consequent to the viral infection and the development of allergy remains unclear.     

Production of passive immunity in neonatal ferrets following maternal vaccination with killed influenza A virus vaccines.

Neonatal ferrets may be passively immunized following maternal vaccination with formalin-inactivated influenza A virus vaccine, but the level of protection from partial to complete depends upon the number of doses used to vaccinate the mother, the presence or absence of aluminum hydroxide adjuvant, whether or not the mothers were 'primed' by prior infection with a serologically heterologous type A virus, and the age of the neonate at challenge. Neonates were completely protected up to 2 weeks of age, but susceptibility returned to nasal epithelium at 5 weeks and to lung at 7 weeks. Mothers immunized up to 9 months previously also partially or completely protected their offspring, this correlating with the maternal serum haemagglutination-inhibition (HI) antibody titre at the time of neonatal challenge, not the duration of immunity.     

EB病毒基因LMP1和EBNA2对支气管上皮细胞的转化

目的 观察Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）对支气管上皮细胞的转化作用。方法 用ＥＢＮＡ２和ＬＭＰＩ的真核表达质粒同时转染永生化人胚支气管上皮细胞系ＴＲ,经潮霉素Ｂ筛选得到稳定转化细胞ＴＲ／ＬＭＰＩ－ＥＢＮＡ２。结果 原位杂效和Ｗｅｓｔｅｍ ｂｌｏｔ证实ＴＲ／ＬＭＰＩ－ＥＢＮＡ２有ＥＢＮＡ２和ＬＭＰＩ的ｍＲＮＡ和蛋白表达。生长曲线,ＭＴＴ显示细胞增殖能力增强,软琼脂集落形成率ＴＲ／ＬＭＰＩ－Ｅ     

Immunohistochemical demonstration of influenza A nucleoprotein in lungs of turkeys with natural and experimental influenza respiratory disease

Influenza A virus (H1N1) and several bacteria were recovered from lungs of turkey breeder hens during a respiratory disease outbreak. Influenza A nucleoprotein was detected in the pneumonic lung tissue within macrophages and, rarely, in atrial-lining epithelium. Inconsistent recovery of pathogenic bacteria suggested that death in some turkeys resulted from acute primary viral pneumonia. In an experimental study, the gross and histologic lesions confirmed the respiratory pathogenicity of the influenza virus. The presence of intranuclear and intracytoplasmic influenza A nucleoprotein within macrophages and atrial lining epithelium of the lung, respiratory epithelium of the trachea and hypertrophied epithelial cells of the airsacs verified influenza virus replication in the respiratory system. However, the absence of mortality may suggest that secondary factors, such as bacteria, may modify the disease in natural outbreaks.     

The growth of respiratory syncytial virus in organ cultures of bovine foetal trachea

Summary Respiratory syncytial (RS) virus grown in organ cultures of bovine foetal trachea at 37° C and pH 7.2 reached maximum titres of up to 1 × 10⁵ PFU/ml between 11 and 21 days after inoculation. Virus yield was increased three fold by incubation at 33° C, but depressed by the addition of RS virus antiserum, with or without bovine complement, or by the addition of alveolar macrophages. Variation in pH or the concentration of foetal calf serum and magnesium chloride did not affect the virus yield. Virus growth did not affect ciliary activity of the cultures. Histological changes involved slight flattening of the epithelium and the appearance of phloxinophilic inclusion bodies.Fluorescent antibody staining showed more virus antigen in the peri-tracheal connective tissue than in the ciliated epithelium.The presence of non-cytopathic mucosal disease (MD) virus in RS virus infected organ cultures slightly depressed RS virus growth but did not influence ciliary activity.Thesein vitro experiments suggest that the tracheal epithelium may not be an important target in the pathogenesis of RS virus infectionin vivo.With 4 Figures     

Rapid recovery of lung histology correlates with clearance of influenza virus by specific CD8+ cytotoxic T cells.

Previous studies have shown that influenza nucleoprotein (NP)-specific cytotoxic T-cell clones do not prevent influenza infection of mice but lead to a more rapid viral clearance and recovery of the host. Here we examine the histology of the lung to see if viral clearance by cytotoxic T cells (Tc) correlates with recovery of pulmonary pathology or if it is in any way deleterious. Intransasal (i.n.) A/X31 virus infection of BALB/c mice produces lung tissue changes lasting 8-10 days in BALB/c mice, with the most severe abnormalities appearing between Days 4 and 6 (e.g. loss of epithelium, airway obliteration, peribronchiolar and perivascular cell accumulation). The transfer of Tc clone T9/13 into i.n.-infected BALB/c mice induces a transient enhanced loss of epithelium on Day 4, while by Day 6 epithelial abnormalities are much reduced in the lung compared to control infected mice. This correlates with a significant reduction in lung virus titres by Day 6; by Day 8 virus is cleared in Tc recipients and lung histology is normal. Another Tc clone (T5/5) with greater cytolytic activity resulted in significant recovery of the lung tissues by Day 4. Tc clones also resulted in enhanced perivascular infiltration of cells and variation in the infiltrating cell type. Quantification in our system required careful attention to the level of the airway assessed. These histological findings showing an enhanced tissue recovery support the previous assessment of reduced lung viral levels following the transfer of Tc cells, and show that a transient increase in lung pathology can occur.