Sendai Virus Infection In Mice Research Articles

Sendai virus C protein affects macrophage function, which plays a critical role in modulating disease severity during Sendai virus infection in mice.

Sendai virus (SeV) accessory protein C limits the generation of double-stranded RNAs, defective interfering RNAs, or both, during viral transcription and replication, thereby limiting interferon-β production. Our recent in vitro analyses on murine macrophage cell lines demonstrated that this protein also contributes to restricting macrophage function, including the production of nitric oxide (NO) and inflammatory cytokines in addition to interferon-β, in infected macrophages. This study showed that depletion of airway macrophages by clodronate-loaded liposomes led to the development of severe viral pneumonia in recombinant C gene-knockout SeV (SeV∆C)-infected mice, but did not modulate disease severity in wild-type SeV-infected mice. Furthermore, the severe disease observed in macrophage-depleted, SeV∆C-infected mice was associated with exacerbated virus replication in the lungs, leading to severe airway inflammation and pulmonary edema, indicating lung injury. These results suggested that the antimacrophage activity of SeV C protein might play a critical role in modulating lung injury and associated diseases caused by SeV.

Distinctive and critical roles for cellular immunity and immune-inflammatory response in the immunopathology of Sendai virus infection in mice

Respiratory viral infections result in severe pulmonary injury, to which host immune response may be a significant contributor. At present, it is not entirely clear the extent to which lung injury is a necessary consequence of host defense. In this report, we use functional genomics approach to characterize the key roles of cellular immunity and immune-inflammatory response in the immunopathology of Sendai virus infection in resistant C57BL/6J and susceptible DBA/2J mice. Infected mice manifested an immune-inflammatory response characterized by the pulmonary influx of neutrophils and mononuclear cells. DBA/2J mice mounted a vigorous immune response, with significant up-regulation of cytokine/chemokine genes in two successive waves through the course of infection. Whereas, C57BL/6J mice displayed an efficient immune response with less severe pathology and clusters of immune-inflammatory responsive genes were exclusively up-regulated on day 4 in this strain. Overall, DBA/2J mice exhibited a dysregulated hyper-inflammatory cytokine/chemokine cascades that does not limit viral spread resulting in a predisposition to severe lung pathology. This response is similar to severe human respiratory paramyxovirus infections, which will serve as a model for the elucidation of hyper-immune inflammatory response that result to severe immunopathology in respiratory viral infections.

Multigenic control of resistance to Sendai virus infection in mice

Experimental infection of mice with Sendai virus (SeV) is frequently used as a model of viral pathogenesis of human respiratory disease. To understand the differences in host response to SeV among mice strains, we carried out genetic mapping studies in DBA/2 (D2) (susceptible) and C57BL/6 (B6) (resistant) mice. F 1, F 2, and N 2 backcrossed mice were generated and examined for their disease resistance and susceptibility. For the determination of virulence, percentage body weight loss and survival time were used as phenotypes. We, then, carried out a genome wide scan on 108 backcrossed mice for linkage with percentage body weight loss as phenotype. A major quantitative trait locus (QTL) showing significant linkage was mapped to the distal portion of Chr 4 ( SeV1). In addition, two other QTLs showing suggestive statistical linkage were also detected on Chr 8 and 14. We, further, performed genome scan for interactions with least squares analysis of variance of all pairs of informative makers in backcrossed progenies. We identified a highly significant epistatic interaction between D3Mit182 and D14Mit10, then denoted as SeV2 and SeV3, respectively, and the latter was the same locus showing a suggestive level on Chr 14 in QTL analysis. Considered genotypes of these three loci, we could account for more than 90% of genetic effect on the differential response to SeV infection between B6 and D2 mice. These findings revealed a novel gene interactions controlling SeV resistance in mice and will enable the identification of resistance genes encoded within these loci.

Masking of the contribution of V protein to sendai virus pathogenesis in an infection model with a highly virulent field isolate

Sendai virus V protein is not essential for virus replication in cultured cells but is essential for efficient virus replication and pathogenesis in mice, indicating that the V protein has a luxury function to facilitate virus propagation in mice. This was discovered in the Z strain, an egg-adapted avirulent laboratory strain. In the present study, we reexamined the function of Sendai virus V protein by generating a V-knockout Sendai virus derived from the Hamamatsu strain, a virulent field isolate, which is an appropriate model for studying the natural course of Sendai virus infection in mice. We unexpectedly found that the V-knockout virus propagated efficiently in mice and was as virulent as the wild-type virus. Switching of the functionally important V unique region demonstrated that this region of the Hamamatsu strain was also functional in a Z strain background. It thus appears that the V protein is nonsense in a field isolate of Sendai virus. However, the V protein was required for virus growth and pathogenesis of the Hamamatsu strain in mice when the virulence of the virus was attenuated by introducing mutations that had been found in an egg-adapted, avirulent virus. The V protein therefore seems to be potentially functional in the highly virulent Hamamatsu strain and to be prominent if virus replication is restricted.

Functionally heterogeneous CD8(+) T-cell memory is induced by Sendai virus infection of mice.

It has recently been established that memory CD8(+) T cells induced by viral infection are maintained at unexpectedly high frequencies in the spleen. While it has been established that these memory cells are phenotypically heterogeneous, relatively little is known about the functional status of these cells. Here we investigated the proliferative potential of CD8(+) memory T cells induced by Sendai virus infection. High frequencies of CD8(+) T cells specific for both dominant and subdominant Sendai virus epitopes persisted for many weeks after primary infection, and these cells were heterogeneous with respect to CD62L expression (approximately 20% CD62L(hi) and 80% CD62L(lo)). Reactivation of these cells with the antigenic peptide in vitro induced strong proliferation of antigen-specific CD8(+) T cells. However, approximately 20% of the cells failed to proliferate in vitro in response to a cognate peptide but nevertheless differentiated into effector cells and acquired full cytotoxic potential. These cells also expressed high levels of CD62L (in marked contrast to the CD62L(lo) status of the proliferating cells in the culture). Direct isolation of CD62L(hi) and CD62L(lo) CD8(+) T cells from memory mice confirmed the correlation of this marker with proliferative potential. Taken together, these data demonstrate that Sendai virus infection induces high frequencies of memory CD8(+) T cells that are highly heterogeneous in terms of both their phenotype and their proliferative potential.

Enumeration of Antigen-Presenting Cells in Mice Infected with Sendai Virus

Substantial progress has been made in understanding Ag presentation to T cells; however, relatively little is known about the location and frequency of cells presenting viral Ags during a viral infection. Here, we took advantage of a highly sensitive system using lacZ-inducible T cell hybridomas to enumerate APCs during the course of respiratory Sendai virus infection in mice. Using lacZ-inducible T cell hybridomas specific for the immunodominant hemagglutinin-neuraminidase HN421-436/I-Ab and nucleoprotein NP324-332/Kb epitopes, we detected APCs in draining mediastinal lymph nodes (MLNs), in cervical lymph nodes, and also in the spleen. HN421-436/I-Ab- and NP324-332/Kb-presenting cells were readily detectable between days 3 and 9 postinfection, with more APCs present in the MLN than in the cervical lymph nodes. Interestingly, no infectious virus was detected in lymphoid tissue beyond day 6, suggesting that a depot of noninfectious viral Ag survives, in some form, for 2-3 days after viral clearance. Fractionation of the MLN demonstrated that APC frequency was enriched in dendritic cells and macrophages but depleted in the B cell population, suggesting that B cells do not form a large population of APCs during the primary response to this virus.

Augmentation of protective immune responses against viral infection by oral administration of schizophyllan

An oral administration of fungal polysaccharide schizophyllan has augmented protective immune responses to Sendai virus infection in mice and the rodshaped DNA virus of Penaeus japonicus (RV-PJ) infection in Kuruma shrimps. When schizophyllan was administered orally at a dose of 50 or 100 mg/kg body weight per day, the survival rates after virus challenge were significantly higher than those of the control groups. High phagocytic activities were observed in the haemocytes of the schizophyllan-fed shrimps.These results suggest that schizophyllan confers effective protection against viral infection by increasing antiviral immune responses, and that it could be used to boost immunity to virus infection in animals or in invertebrates.

Distinctive Kinetics of the Antibody-Forming Cell Response to Sendai Virus Infection of Mice in Different Anatomical Compartments

The single-cell ELISPOT assay was used to determine the frequency and isotype commitment of virus-specific antibody-forming cells (AFC) at different anatomical locations following intranasal Sendai virus infection of C57BL/6 and 129/Sv mice. AFC responses in the mediastinal and cervical lymph nodes showed sharp increases and declines, first of IgM AFC, peaking about 7 days after infection, and then of IgG and IgA AFC, peaking about 10 days after infection. A wave of IgM AFC preceding the other isotypes was less evident in the spleen, where peak frequencies of AFC occurred 14 days after infection. Virus-specific AFC appeared in the bone marrow with a unique kinetic pattern, increasing in frequency gradually over the first 3 weeks after infection to a plateau that remained constant. Circulating IgM and IgG achieved significant titers approximately a week after infection; IgM titers were transient, but IgG levels increased sharply and remained high, reflecting the longevity of the bone marrow AFC response. Strain differences in isotype bias were noted, particularly preferential switching to the γ2a gene in 129/Sv mice. The B-cell response to acute respiratory viral infection thus exhibits features that are distinct from the primary response to nonreplicating antigens.

Effect of the synthetic lipid A-related compound, DT-5461, on resistance to Sendai virus infection in mice

DT-5461 enhanced host resistance to Sendai virus infection in mice. Intranasal (i.n.) administration of 200 μg of DT-5461 per mouse 3 days before infection was the most effective administration route, dose and timing. DT-5461 enhanced the cytotoxicity of murine natural killer (NK) cells. In addition, DT-5461 activated murine peritoneal macrophages, resulted an augmented of cytotoxicity and the induction of tumor necrosis factor-α (TNF-α). Therefore, these immunomodulating activities presented by DT-5461 caused protection against Sendai virus infection.

Stimulation of host-defense mechanism with synthetic adjuvants and recombinant cytokines against viral infection in mice.

The efficacy of synthetic immunoadjuvants and recombinant cytokines for the potentiation of host-resistance against virus infection was investigated using mouse models infected with Sendai virus and herpes simplex type 1 virus (HSV). The synthetic MDP derivative, MDP-Lys(L18), and recombinant cytokines, IL-1 beta, IFN-gamma, G-CSF and GM-CSF were shown to be effective for the stimulation of nonspecific protection against Sendai virus infection in mice. Both MDP-Lys(L18) and GM-CSF were effective for the protection against HSV infection in cyclophosphamide (CY)-treated mice. B30-MDP was suggested to be useful as an immunoadjuvant for the potentiation of antigenicity of recombinant or component vaccines.

Prophylactic activity of dihydro-heptaprenol, a synthetic polyprenol derivative, against Sendai virus infection in mice

The effect of a chemically synthesized polyprenol derivative, dihydroheptaprenol (DHP), on the non-specific resistance of mice to Sendai virus infection was investigated. The mice that received 200 micrograms of DHP intranasally twice, at 3 days and 1 day before the infection, showed a significant protection against Sendai virus infection. Treatment of mice twice even with as much as 2000 micrograms of DHP through the subcutaneous route, however, had no protective effect against infection. Excess interferon and tumour necrosis factor production in intranasally DHP-treated mice was seen 1 day after the infection when compared with Sendai virus alone controls or with DHP alone controls. Variance analysis of these findings indicates a prophylactic activity of DHP in pulmonary viral infections.

Protection of Mice against Sendai Virus Pneumonia by Non‐Neutralizing Anti‐F Monoclonal Antibodies

Nine monoclonal antibodies (MAbs) directed to F protein of Sendai virus were obtained and characterized for their protective ability against Sendai virus infection in mice. None of the MAbs showed hemagglutination-inhibition (HI), hemolysis-inhibition (HLI), or neutralization (NT) activities in vitro when assayed by standard methods. Some of the MAbs, however, showed complement-requiring NT (C-NT) and complement-requiring hemolysis (C-HL) activities when assayed in the presence of complement. Passive immunization experiments revealed that the MAbs with higher C-NT and C-HL activities showed protective activity against Sendai virus pneumonia in mice, and that some MAbs with IgG1 isotype having neither C-NT nor C-HL activity also showed the protective activity. Digestion of the MAbs with pepsin which split immunoglobulin molecules into F(ab')2 and Fc fragments greatly suppressed the protective activity. These results suggest that not only complement-mediated immunological responses such as immune virolysis but also antibody-dependent cellular cytotoxicity (ADCC) and/or immune phagocytosis, in which complement system is not necessarily involved, play an important role in the protection of mice from Sendai virus infection.

Sendai virus infection of mice with protein malnutrition

Sendai virus pneumonia was produced in BALB/c mice fed protein-deficient diets in an effort to understand the severity of viral pneumonia in infants in developing countries. Animals on the deficient diet became clinically malnourished, and some aspects of cellular immunity were altered. In protein-deprived animals, the 50% lethal dose of intranasally administered Sendai virus was over 1,000-fold lower, pulmonary virus titers were higher, the infection was prolonged, and lung infection was established at a lower inoculum than in normal animals.

Macrophage activation and host augmentation against Sendai or herpes simplex virus (HSV) infections with synthetic polypeptides in mice

Poly- L-Lys (mean mol. wt; 12,000), poly- L-Arg (5000) and poly- L-Orn were found to activate peritoneal macrophages effectively in vivo in 14 synthetic homo polypeptides. The ability of sequential poly( L-Arg- L-X) (5000) to activate macrophages was less than that of poly- L-Arg. Neither ( L-Arg) 12 nor ( L-Arg) 6 by themselves activated macrophages, but poly- D-Arg (5000) did, as also did poly- L-Arg; this suggests that the polycationic character of poly- L-Arg plays a role in the activation of macrophages. The intranasal administration of poly- L-Lys, - L-Arg, - L-Orn, - D-Arg, all of which activated macrophages, augmented host resistance against Sendai virus infection in mice. The protection afforded by poly- L-Arg seemed to depend on its mol. wt: the order of protection was poly- L-Arg>( L-Arg) 12>( L-Arg) 6. The intranasal administration of poly- L-Arg 3 days before the infection was effective, while that 1 day before infection was not. There was no difference between the groups in the titer of interferon produced by the infection of Sendai virus given with poly- L-Arg either 3 days before or 1 day before the infection. The administration of poly- L-Arg 3 days before the infection caused a significant decrease of the virus titer in the lung 6 days after the infection when compared with the control or the mice given 1 day before. The intravenous administration of 2-chloroadenosine (2-Cl-Ade), which is a selective inhibitor of macrophages, into the mice which had received poly- L-Arg intranasally 3 days before the infection caused a significant decrease in the survival rate of the mice, indicating that the macrophages activated with poly- L-Arg are likely to be an important element in affording the protection. Subcutaneous administration of poly- L-Arg revealed protective activity against systematic infection with herpes virus-type 1.

Immunological activities of multiprenylacetyl derivatives of muramyldipeptides

The immunological properties of six kinds of multiprenylacetic acids and six kinds of multiprenylacetyl muramyldipeptide (MDP) derivatives were examined by using experimental models in mice and guinea-pigs. All the multiprenylacetyl MDP derivatives, particularly TMD-232, showed potent adjuvant activity on the circulating antibody formation against bacterial α-amylase in mice, and induction of delayed-type hypersensitivity to monoazobenzenearsonate N- acetyl- l-tyrosine in guinea-pigs. All multiprenylacetic acid preparations tested in this study, however, showed no adjuvant activity in these immune systems. Both TMD-17 and TMD-232 entrapped into multilamellar vesicles showed potent host stimulation activity against Sendai virus infection in mice.

Induction of tumoricidal macrophages and production of cytokines by synthetic muramyl dipeptide analogues

The ability of various synthetic muramyl dipeptide (MDP) derivatives to induce the production of interleukin-1 (IL-1) and colony stimulating factor (CSF) in vitro and in vivo and to induce cytotoxic macrophages was studied. 6-O-L18-MDP(Me) and MDP-Lys(L18), which were potent inducers of IL-1 and CSF production and of cytotoxic macrophages, had protective activity against Sendai virus infection in mice. In contrast, 1-O-L18-(6-O-P)-MDP(Me) and 2-N-L18-MDP exhibited weak or no ability to induce IL-1 and CSF production and no induction of tumoricidal macrophages, and did not protect against infection of Sendai virus. MDP derivatives, except 2-N-L18-MDP, efficiently rendered macrophages cytotoxic against target cells in the presence of murine recombinant interferon-gamma in vitro. The derivatives that induced cytokines and cytotoxic macrophages appeared to produce anti-viral activity.

Effect of muramyl dipeptide and its stearoyl derivatives on resistance to Sendai virus infection in mice

The efficacy of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), 6-O-stearoyl-MDP (L18-MDP), N alpha-acetylmuramyl-L-alanyl-D-isoglutaminyl-N epsilon-stearoyl-L-lysine (MDP-Lys-L18) and N-stearoylmuramyl-L-alanyl-D-isoglutamine (2N-L18-MDP) for augmenting host-resistance to viral infection was examined in Sendai virus infected mice. L18-MDP and MDP-Lys-L18 augmented the non-specific host-resistance to infection with Sendai virus. MDP showed a slight enhancement of host-resistance to this infection but 2N-L18-MDP was ineffective. The protective effect of MDP-Lys-L18 was seen only when the drug was administered a few days before the virus challenge. The intranasal administration of MDP-Lys-L18 was effective at 1 microgram but only slight activity was observed in mice treated intravenously or intraperitoneally even at the 100 microgram dose level. MDP-Lys-L18 treatment preceding infection augmented interferon production in the lung of the mice but MDP-Lys-L18 treatment alone induced no interferon.

Genetics of natural resistance to Sendai virus infection in mice.

The genetics of resistance to a naturally occurring respiratory infection caused by Sendai virus was examined in F1, F2, and backcross progeny of resistant C57BL/6J and susceptible DBA/2J mice and in 25 recombinant inbred strains. An intranasal inoculum of 0.1 50% tissue culture infective dose (low dose) of Sendai virus caused 0% mortality in C57BL/6J and F1 mice and 73% mortality in DBA/2J mice. An inoculum of 1.0 50% tissue culture infective dose (high dose) caused 3, 0, and 89% mortality in C57BL/6J, F1, and DBA/2J mice, respectively. Low-dose infection caused 36% mortality in F1 X DBA/2J hybrids and 0% mortality in F2 hybrids. High-dose infection caused 29 and 32% mortality in F1 X DBA/2J and F2 hybrids, respectively. Resistance was not linked to H-2 haplotype, coat color, or sex. High-dose infection caused deaths in 12 recombinant inbred strains, and the strain distribution pattern was concordant with that of a chromosome 1 marker, Sas-1, in 20 of 25 strains (P less than 0.01). Resistance therefore behaved as a simple Mendelian dominant trait which presumptively mapped to chromosome 1.

Evidence of proteolytic activation of Sendai virus in mouse lung.

A device was made to analyze the pneumotropism of Sendai virus in mouse. Minced lung blocks were prepared from the mouse intranasally infected with Sendai virus for 2 hours and cultured in a CO2 incubator. This culture system provided a suitable in vitro model of Sendai virus infection in mice in terms of the distribution of the viral antigens and histopathological findings. The progeny virus recovered from the lung culture was already activated and was accompanied by the cleavage of F glycoprotein into F1 and F2. This fact demonstrates that the activating mechanism is reversed in the lung culture as found in vivo infection of mouse lung. The viral activation and the cleavage of F glycoprotein were simultaneously inhibited by tosyllysylchloromethylketone, leupeptin, soybean trypsin inhibitor and antipain, but not by tosylamidophenylethylchloromethyl-ketone, chymostatin, pepstatin, iodoacetamide, phenylmethylsulfonylfluoride and p-chloromercuribenzoate. These results show that the activating enzyme of Sendai virus found in the lung culture was similar to trypsin. The existence of the activating enzyme may support the replication of Sendai virus in mouse lung in multiple-step and also result in the lung pathology.

Cell-mediated immunity to Sendai virus infection in mice

The development of a cell-mediated immune response to Sendai virus infection in mice was examined by the use of a 51Cr release assay of cytotoxicity. A low level of "background cytotoxicity" to Sendai virus-infected L cells was found in the spleens of uninfected CBA mice. Spleen cells from Sendai-infected mice showed an elevated level of cytotoxicity against these target cells for a period of 5 weeks, commencing 4 days after infection of the mice. A more transient response was observed in the spleens of mice infected with a serologically distinct virus, the Kunz strain of influenza. This cross-reacting, cell-mediated immune response was intermediate between that observed in unsensitized and Sendai-sensitized spleen cells. The relevance of these cell-mediated immune responses to respiratory tract virus infections is discussed.