Nonspecificity of the phenomenon of influenza virus phagocytosis by murine macrophages

Peritoneal macrophage cultures from intact mice and those immune to influenza virus A/PR/8/34 (HON1) were infected with homologous virus or influenza virus A/England/42/72 (H3N2) whereupon virus was isolated from chick embryos. It was established that in intact macrophages, both viruses duplicated similarly. Macrophages immune to virus HON1 equally disintegrated both in homologous virus and heterologous influenza virus H3N2.     

Varying immunological effects in mice of intranasal infection with different strains of influenza virus

Intranasal infection of CBA/Ca mice with a sublethal dose of A/2 Japan influenza virus 305/57 decreased the blastogenic response to concanavalin A and phytohemagglutinin, and less to lipopolysaccharide andEscherichia coli bacteria. This depression of the blastogenic responses could be transferred from infected donor mice by intravenous injection of 4×10⁷ spleen cells to otherwise untreated syngenic recipient mice. Similar infections with A/Victoria 3/75 and A/Texas 1/77 influenza virus strains caused less depressing effects. Less consistent results were seen with NMRI mice. No impairment of the antibody responses to unrelated protein antigen could be noted after such intranasal influenza infection. In contrast, the IgE antibody response was particularly increased after infection with Texas virus. Some deleterious effects of Victoria and Texas virus infections on the delayed hypersensitivity response to picryl chloride were seen in CBA mice but not in NMRI mice. This immune suppression by virus infection was not reflected by the defense against intraperitoneal infection withListeria monocytogenes andE. coli. In contrast, a small increase in resistance toListeria infection was recorded. The results of this study lend little support to the hypothesis that influenza infection impairs the immunological defense against a following bacterial infection, but may result in allergy.     

Influenza virus in human exhaled breath: an observational study

Background

Recent studies suggest that humans exhale fine particles during tidal breathing but little is known of their composition, particularly during infection.

Methodology/Principal Findings

We conducted a study of influenza infected patients to characterize influenza virus and particle concentrations in their exhaled breath. Patients presenting with influenza-like-illness, confirmed influenza A or B virus by rapid test, and onset within 3 days were recruited at three clinics in Hong Kong, China. We collected exhaled breath from each subject onto Teflon filters and measured exhaled particle concentrations using an optical particle counter. Filters were analyzed for influenza A and B viruses by quantitative polymerase chain reaction (qPCR). Twelve out of thirteen rapid test positive patients provided exhaled breath filter samples (7 subjects infected with influenza B virus and 5 subjects infected with influenza A virus). We detected influenza virus RNA in the exhaled breath of 4 (33%) subjects–three (60%) of the five patients infected with influenza A virus and one (14%) of the seven infected with influenza B virus. Exhaled influenza virus RNA generation rates ranged from <3.2 to 20 influenza virus RNA particles per minute. Over 87% of particles exhaled were under 1 µm in diameter.

Conclusions

These findings regarding influenza virus RNA suggest that influenza virus may be contained in fine particles generated during tidal breathing, and add to the body of literature suggesting that fine particle aerosols may play a role in influenza transmission.     

Induction of homotypic and heterotypic T- and B-cell immunity with influenza A virus in mice

Infection of mice with live influenza A virus induces cytolytic T lymphocytes (CTL) as well as B cells capable of reacting with target cells infected with the appropriate virus subtypes. In Balb/c mice CTL reveal a broad cross-reactivity against all influenza A substrains known. In contrast B-cell responses are restricted to virus subtypes which are identical in regard to the hemagglutinin (HA) of the sensitizing virus. Reinfection with homologous live influenza virus within 6–7 months results in no or in a drastically diminished B-cell response as compared to a priming situation and fails to induce CTL. Inability to induce secondary immunity to homologous influenza virus was correlated with the presence of circulating antibodies specific for the sensitizing virus subtype. Cross-boosting with heterologous live influenza A virus induces homotypic and heterotypic CTL and B-cell immunity with characteristics of secondary responses. Preparations of inactivated intact influenza virus are unable to reactivate CTL memory in vivo but induce B-cell activity. B-cell responses stimulated by this procedure are restricted to the boosting virus. Attenuated viruses, which are produced by recombination of wild strains with cold-adapted strains, are also efficient in stimulating in vivo CTL memory if used for cross-boosting.     

Influenza virus inoculum volume is critical to elucidate age‐dependent mortality in mice

The elderly exhibit increased mortality to influenza viral infection for unclear reasons. Mice are frequently used to model how aging impacts disease. Several studies have shown that aged mice exhibit an increased mortality to influenza virus, but two recent studies demonstrated the opposite. These two studies administered the virus intranasally in 20 µL, whereas the other studies used a viral inoculum in at least 30 µL. To determine whether the volume of the inoculum could explain the conflicting reports, we infected young and aged mice via intranasal instillation of 40 µL or 20 µL containing 1 x 10⁴ plaque‐forming units (PFU) of H1N1 influenza virus. We found that intranasal administration of 40 µL but not 20 µL of inoculum resulted in age‐dependent mortality in mice. Compared to aged mice infected with 40 µL inoculum, those infected with 20 µL inoculum showed reduced levels of live virus and IFN‐β in the lung 3 days postinfection. Furthermore, aged mice administered 40 µL of Evans blue intranasally displayed increased dye retention in their bronchoalveolar lavage fluid compared to those administered 20 µL of Evans blue. Our data demonstrate that the inoculating volume of virus is critical for adequate delivery of influenza virus to the lung and thus for efficient infection of aged mice. These findings shed light on discrepant results in the literature regarding aged mice and influenza infection, and establish that mice can be used to examine how aging impacts the response to this biomedically important infection.     

Activity of recombinant human alpha interferon against influenza virus infection in mice

The in vivo antiviral activity of recombinant human leukocyte hybrid interferon, HuIFN-alpha AD, was examined. Results showed that this material in highly purified form did not protect mice against a lethal dose of influenza virus, although administration of natural MuIFN-alpha/beta to mice infected with a lethal dose of influenza virus had a marked protective effect. The effect of alveolar macrophages treated with IFN on influenza virus replication was examined in vitro. The antiviral activity of alveolar macrophages treated with HuIFN-alpha AD was lower than that of MuIFN-alpha/beta. It is concluded that HuIFN-alpha AD is effective in direct inhibition of influenza virus, but not in indirect inhibition mediated by alveolar macrophages or in protection of mice from influenza virus infection.     

新型H7N9病毒在同居小鼠中的传播途径

目的进一步了解新型H7N9流感病毒的致病性、传播能力以及通过何种途径进行传播。方法 H7N9病毒感染小鼠后与同居小鼠合笼,研究同居小鼠的临床变化指征、病毒复制情况、病毒在组织中的分布以及病理变化。以同居小鼠分泌物接种其他小鼠,观察同居小鼠通过何种途径传播病毒。结果 H7N9病毒可以在肺组织、肠组织和脑组织中复制,并可以在同居小鼠中传播。H7N9病毒感染小鼠其咽、眼分泌物以及粪便均具有感染性,其中尤以咽拭子的传播风险最高。结论 H7N9病毒可以不通过适应就感染小鼠,并引起小鼠间传播。被感染小鼠分泌物具有感染性。     

Some aspects of the adaptation of influenza virus onto mice

Summary The author describes the adaptation onto mice of an influenza A strain, isolated byMulder from the tracheal mucosa of a man, who died from an acute fulminant staphylococcal-pneumonia and discusses the nature of the adaptation phenomenon.Communication of the Workteam of Acute Respiratory Diseases of the Institute for Preventive Medicine.     

IP-10对流感病毒致肺细胞病变作用的影响

为探讨干扰素y诱导蛋白10（IP-10）在流行性感冒病毒致肺部炎性病变中的作用,用BALB／c小鼠做动物感染模型,对只感染流感病毒和注射IP-10后感染流感病毒小鼠的肺部炎性反应进行了比较。结果显示,注射IP-10后感染流感病毒小鼠的肺部炎性反应明显重于感染对照组。研究结果表明,IP-10对流感病毒造成的肺部炎性病变的严重程度具有重要影响。     

Kinetics of DNA reparative replication in the spleen cells of CBA- and C57BL/6-strain mice induced by urethane and and the influenza virus

Different kinetics of DNA repair replication induced by urethan and influenza virus was detected in mice of varying genotypes. Inhibition of repair replication was detected in the lymphocytes of C57BL/6 mice, infected with influenza virus and treated with urethan. No inhibition of repair replication was noted in CBA mice which is characteristic of resistance to influenza virus. However, stimulation of repair replication by influenza virus was observed in these cells.     

Different functions of subsets of effector T cells in murine influenza virus infection

Enriched preparations of secondary effector T cells to influenza virus were tested for their in vivo biological function by adoptive transfer to mice 24 hr after an intranasal inoculation of infectious influenza virus. One class of cells which were Lyt 1⁺2^?3^?, I region-restricted, and could mediate DTH reaction failed to reduce lung virus titers 5 days after transfer and caused a higher mortality rate in the recipient mice than in the controls. A second class of cells which were Lyt 1^?2⁺3⁺, K,D region-restricted, and were cytotoxic and could mediate DTH activity substantially reduced lung virus titers 5 days after transfer. The influx of mononuclear cells to the lungs after adoptive cell transfer was measured by injection of [¹²⁵I]UdR 24 hr prior to harvest of lung cells, using both infected CBA and athymic BALB/ c nude (nu/nu) mice as recipients. I region-restricted cells caused increased cellular infiltration which was very marked in athymic mice. It was concluded that this reaction significantly contributed to the observed immunopathology in infected mice. Transfer of K,D region-restricted cells reduced the cellular infiltration in infected CBA mice and caused only a slight increase in infected athymic mice. The evidence supported the concept that the second class of cells exerted their protective (antiviral) effect in vivo by direct lysis of virus-infected cells rather than by liberation of lymphokines.     

Virus pneumonia (snuffling disease) in laboratory rats and wild rats due to an agent probably related to grey lung virus of mice

Summary A pneumonia-producing virus in mice has been isolated from the lungs of 39 out of 76 white laboratory rats from different colonies, and of 1 out of 2 wild rats suffering from snuffling disease. The virus was transmissible only by intranasal instillation. Contact infection of mice occurred rarely and only after prolonged contact. Since only rats of at least 4 months of age appeared to be infected, contact infection in rats also may be difficult, and early separation of young rats in small groups might, perhaps, be an effective preventive measure. The experimentally induced disease in mice tends to be chronic, as it is in rats. The experimental disease in suckling-mice, however, was usually fatal. Electron micrographs of partly purified lung suspensions showed virus-like particles having a diameter of 200–250 mμ. Chlortetracyclin and oxytetracyclin have a prophylactic and a therapeutic effect on the experimental disease. Lesions disappear after antibiotic treatment. The virus seems to be inactivated only by chlortetracyclin. Mice that have recovered by antibiotic treatment proved not immune to reinfection. The properties of the virus are highly similar to those of grey lung virus of mice. Aided by a grant from the National Health Research Council T.N.O. This paper is part of a thesis in preparation byH. Vrolijk, which has not been finished because of the decease of the author.     

Dependence of the cocarcinogenic action of the influenza virus on the nature of the influenza infection

It was shown in experiments of CC57W mice that cocarcinogenic activity of influenza A/PR8/34 virus correlates with acute or chronic pattern of infection. Prolonged persistence of the virus resulted in significant stimulation of the lung tumor incidence in infected mice. The prevention with thymosin of chronic influenza infection development in CC57W mice lead to a decrease in the incidence of lung tumors to the control level.     

Production of lipopolysaccharide-induced tumour necrosis factor during influenza virus infection in mice coincides with viral replication and respiratory oxidative burst

Increased morbidity and mortality occur regularly during influenza epidemics. The exact mechanisms involved are not well defined but bacterial superinfection of influenza virus infected patients is considered to play an important role. In the present study, the effect of influenza virus infection on in vivo production of turnout necrosis factor (TNF) in response to bacterial stimuli was investigated. Release of TNF in mice infected by an aerosol of influenza virus was significant after administration of bacterial lipopolysaccharide (LPS) at 72 h, whereas administration of homologous influenza virus produced only modest amounts of TNF at 96 h. Significant production of TNF was observed 48 h after intravenous administration of infectious influenza in response to LPS but not with the homologous virus. TNF induced after influenza virus infection could be blocked by a specific murine anti-TNF monoclonal antibody. Higher TNF production following aerosol influenza infection correlated with peak titres of influenza virus in the lungs of infected mice and with enhanced generation of luminoldependent chemiluminscence.     

Serial histopathological examination of the lungs of mice infected with influenza A virus PR8 strain

Avian influenza H5N1 and pandemic (H1N1) 2009 viruses are known to induce viral pneumonia and subsequent acute respiratory distress syndrome (ARDS) with diffuse alveolar damage (DAD). The mortality rate of ARDS/DAD is extremely high, at approximately 60%, and no effective treatment for ARDS/DAD has been established. We examined serial pathological changes in the lungs of mice infected with influenza virus to determine the progress from viral pneumonia to ARDS/DAD. Mice were intranasally infected with influenza A/Puerto Rico/8/34 (PR8) virus, and their lungs were examined both macro- and micro-pathologically every 2 days. We also evaluated general condition, survival rate, body weight, viral loads in lung, and surfactant proteins in serum. As a result, all infected mice died within 9 days postinfection. At 2 days postinfection, inflammation in alveolar septa, i.e., interstitial pneumonia, was observed around bronchioles. From 4 to 6 days postinfection, interstitial pneumonia with alveolar collapse expanded throughout the lungs. From 6 to 9 days postinfection, DAD with severe alveolar collapse was observed in the lungs of all of dying and dead mice. In contrast, DAD was not observed in the live infected-mice from 2 to 6 days postinfection, despite their poor general condition. In addition, histopathological analysis was performed in mice infected with a dose of PR8 virus which was 50% of the lethal dose for mice in the 20-day observation period. DAD with alveolar collapse was observed in all dead mice. However, in the surviving mice, instead of DAD, glandular metaplasia was broadly observed in their lungs. The present study indicates that DAD with severe alveolar collapse is associated with death in this mouse infection model of influenza virus. Inhibition of the development of DAD with alveolar collapse may decrease the mortality rate in severe viral pneumonia caused by influenza virus infection.     

The role of neutrophils during mild and severe influenza virus infections of mice

Neutrophils have been implicated in both protective and pathological responses following influenza virus infections. We have used mAb 1A8 (anti-Ly6G) to specifically deplete LyG6(high) neutrophils and induce neutropenia in mice infected with virus strains known to differ in virulence. Mice were also treated with mAb RB6-8C5 (anti-Ly6C/G or anti-Gr-1), a mAb widely used to investigate the role of neutrophils in mice that has been shown to bind and deplete additional leukocyte subsets. Using mAb 1A8, we confirm the beneficial role of neutrophils in mice infected with virus strains of intermediate (HKx31; H3N2) or high (PR8; H1N1) virulence whereas treatment of mice infected with an avirulent strain (BJx109; H3N2) did not affect disease or virus replication. Treatment of BJx109-infected mice with mAb RB6-8C5 was, however, associated with significant weight loss and enhanced virus replication indicating that other Gr-1(+) cells, not neutrophils, limit disease severity during mild influenza infections.     

Cellular immune responses of mice to influenza virus infection

Mice infected with influenza virus develop cytotoxic T lymphocytes (CTL) specific for viral antigens prior to the appearance of virus-specific antibody-forming cells (AFCs). Effector T cells were detected at a time coincident with a precipitous decline in pulmonary virus titer. CTLs of draining lymph nodes and spleen were found to be cross-reactive among H-2 compatible cells infected with influenza type A virus subtypes. AFCs were observed to be primarily hemagglutinin specific. Virus-specific IgA-secreting AFCs were detected in mediastinal lymph nodes of infected mice.     

Inhibitory role of neutrophils on influenza virus multiplication in the lungs of mice.

The protective role of neutrophils on intranasal infection of influenza virus was investigated in 3 strains of tumor-bearing mice with neutrophilic leukocytosis. In vitro multiplication of influenza virus was inhibited by neutrophils from both normal and tumor-bearing mice, and the inhibitory effect of neutrophils was augmented by an addition of fMLP to the culture. Pulmonary virus infectivities in the early phase after infection decreased in such ICR and BALB/c mice, and virus elimination in the late phase was accelerated in the ICR mice. However, no decrease in pulmonary virus infectivity was observed in tumor-bearing C57BL/6 mice. Intranasal administration of fMLP into normal and tumor-bearing C57BL/6 mice after infection significantly inhibited the virus propagation in the lungs. The decrease in neutrophil infiltration into the lung in tumor-bearing C57BL/6 mice was confirmed from histological observations of the lung and lung lavage after infection and from analysis of the neutrophil chemotactic activity induced by fMLP. This might be responsible for the high level of pulmonary virus titer in tumor-bearing C57BL/6 mice. Phagocytic activities of alveolar macrophages and productions of neutralizing antibody were suppressed in the 3 strains of tumor-bearing mice. These observations indicated that neutrophils could be significant effector cells as a host defense mechanism against influenza virus infection in vivo, and infiltration and functional activation of neutrophils could play a significant role in virus elimination from the infected site. Furthermore, the inhibition of virus propagation by neutrophils in vitro was almost completely abrogated by an addition of ZnSO4, suggesting that calprotectin could inhibit influenza virus multiplication.     

Comparison of in vitro and in vivo methods of evaluation of the efficacy of influenza virus hemagglutinin inhibitors

One of the problem in the selection of the most effective antiviral preparations with a broad spectrum of antiviral protective activity, is the "continuity" of assays of different level of complexity so, that the most effective antiviral therapeutic, selected by in vitro assays would be the most effective in vivo. Comparative study of the efficacy of the influenza virus inhibitor in the assays of inhibition of virus binding with fetuin, inhibition of infectious focus forming units in MDCK cells, inhibition of virus yield in infected MDCK cells, and inhibition of influenza virus infectivity in mice infected by viral aerosol are presented. The value of 50% inhibiting concentration IC50 for the pare "influenza virus strain A/NIB/23/89-MA-inhibitor tetra-Aca6-6'SLN" corresponded to 6-10 microM and was invariant for three different tests--in vitro assay of inhibition of virus binding with fetuin, inhibition of yield in infected MDCK cell culture, and inhibition of virus infectivity in mice, but not for the assay of inhibition of infectious focus forming units in cell culture.     

Triazavirin prophylactic efficacy against influenza virus A (H5N1)

The experimental study of the prophylactic efficacy of Triazaverin against the experimental form of the influenza virus A (H5N1) on albino mice intranasally infected with the influenza virus A/Chicken/Kurgan/Russia/02/05 vs. the reference drugs Tamiflu, Remantadin and Arbidol showed that in doses of 1 to 100 mg/kg it was efficient in the animal protection from death. The drug was also efficient in the urgent prophylaxis. Triazaverin effectively inhibited the influenza A virus multiplication in the lungs of the albino mice.