Protection of mice from respiratory Sendai virus infections by recombinant vaccinia viruses.

Mechanisms of protection of mice from Sendai virus, which is exclusively pneumotropic and causes a typical respiratory disease, by immunization with recombinant vaccinia viruses (RVVs) were investigated. Although the RVV carrying a hemagglutinin-neuraminidase gene of Sendai virus (Vac-HN) propagated in the noses and lungs of mice by either intranasal (i.n.) or intraperitoneal (i.p.) inoculation, no vaccinia virus antigens were detected in the mucosal layer of upper and lower airways of the i.p.-inoculated mice. The mice immunized i.n. with Vac-HN or Vac-F (the RVV carrying a fusion protein gene of Sendai virus) demonstrated the strong resistance to Sendai virus challenge both in the lung and in the nose, whereas the i.p.-immunized mice showed almost no resistance in the nose but showed a partial resistance in the lung. Titration of Sendai virus-specific antibodies in the nasal wash (NW), bronchoalveolar lavage (BAL), and serum collected from the Vac-F-immunized mice showed that the NW from the i.n.-immunized mice contained immunoglobulin A (IgA) antibodies but no IgG and the BAL from the mice contained both IgA and IgG antibodies. On the other hand, neither IgA nor IgG antibodies were detected in the NW from the i.p.-immunized mice and only IgG antibodies were detected in the BAL, although both i.n.- and i.p.-immunized mice exhibited similar levels of serum IgG, IgA, and neutralizing antibodies. The resistance to Sendai virus in the noses of i.n.-immunized mice could be abrogated by the intranasal instillation of anti-mouse IgA but not of anti-IgG antiserum, while the resistance in the lung was not significantly abrogated by such treatments. These results demonstrate that IgA is a major mediator for the immunity against Sendai virus induced by the RVVs and IgG is a supplementary one, especially in the lung, and that the RVV should be intranasally inoculated to induce an efficient mucosal immunity even if it has a pantropic nature.     

F0-containing noninfectious Sendai virus can initiate replication in mouse lungs but requires a relatively long incubation period.

The replication of LLC-MK2-grown noninfectious Sendai virus, containing exclusively fusion (F) glycoprotein precursors, was examined in the mouse lung to study the accessibility of virus inoculated intranasally to the virus activator present in the lung. When mice were intranasally inoculated with various doses of the virus after in vitro activation with trypsin, the 50% mouse infectious dose (MID50) was determined to be 0.7 cell-infectious units (CIU) per mouse, indicating that one infectious unit of Sendai virus is enough to initiate replication in the mouse lung and that the present experimental system is highly sensitive. On the other hand, in mice inoculated with virus not treated with trypsin, virus replication in the lung was recognized even in mice inoculated with samples containing no infectious virus, and the MID50 was determined to be 67.5 CIU per mouse (here, CIU were assayed after in vitro trypsin treatment). When mice were infected with 20 MID50 of trypsin-treated infectious and untreated noninfectious viruses (an approximately 100-fold greater amount of noninfectious virus than of infectious virus was used), the noninfectious virus was found to require 2 more days of incubation than the infectious virus, and many of the F proteins synthesized in the lungs of mice infected with the F0-containing virus were present in the cleaved form. In addition, the infection of mice with noninfectious virus was strongly suppressed by aprotinin, a serine protease inhibitor. These results indicate that Sendai virus can initiate replication in the mouse lung even with the F0-containing noninfectious virus and strongly suggest that this infection process is mediated by cleavage activation of the F0 proteins of inoculated viruses by a serine protease(s) present in the lumen of the mouse respiratory tract but that activation of the noninfectious virus is an inefficient process.     

Protection of mice from wild-type Sendai virus infection by a trypsin-resistant mutant, TR-2.

A trypsin-resistant mutant of Sendai virus, TR-2, which could be activated by chymotrypsin but not by trypsin or the protease present in mouse lung, was inoculated intranasally into mice after being activated in vitro. TR-2 hardly brought about clinical illness or lung lesions in mice; the protease present in the lung could not activate the progeny virus, and the infection terminated after one-step replication. Nevertheless, the immunoglobulin A antibody against wild-type Sendai virus was produced in the respiratory tracts as well as the serum immunoglobulin G antibody, and the mice were protected from the challenge of the wild-type Sendai virus. On the basis of these results, TR-2 may provide a new model of live vaccine for paramyxoviruses; its availability as a live vaccine is also discussed.     

Construction and properties of pseudorabies virus recombinants with altered control of immediate-early gene expression.

To investigate how altered control of expression of the essential immediate-early (IE) gene of pseudorabies virus influences virus replication and virulence, we replaced the IE promoter with the tissue-specific promoters of the bovine cytokeratin IV gene (CKIV), the bovine cytokeratin VIb gene (CKVIb), or the inducible promoter of Drosophila heat shock gene HSP70. We compared expression of the IE gene of the wild-type virus and recombinant viruses in different cell types and at different temperatures and found that IE expression had become cell type or temperature dependent. When a recombinant virus was titrated on nonpermissive cells or was titrated at nonpermissive temperatures in vitro, the plating efficiency was reduced by more than 99%. Mice were inoculated subcutaneously (s.c.), intraperitoneally (i.p.), or intranasally (i.n.) with a dose equal to 100 times the 50% lethal dose of the wild-type virus. After inoculation with temperature-sensitive recombinant N-HSP, two (s.c.), two (i.p.), and four (i.n.) of five mice died. However, at this dose, recombinant N-CKIV, which contains a promoter specific for stratified epithelial tissue of the tongue mucosa, was not lethal when inoculated s.c. or i.p. but killed four mice when inoculated i.n. Recombinant N-CKVIb, which contains a promoter specific for the suprabasal layers of the epidermis, was not lethal after inoculation by any of the three routes. In explant cultures of nasal mucosa of pigs, replication of N-CKIV and N-CKVIb was not markedly reduced in the epithelium. However, in contrast to results obtained with wild-type virus, infection of the stroma was not observed. We conclude that the replicative ability and virulence of pseudorabies virus can be influenced by altering control of expression of the IE gene.     

Influence of Statolon on Resistance of Mice to Influenza

Various interferon inducers are known to elicit protection against lethal or infecting doses of certain viral agents. Because of the relatively high morbidity rate of influenza and its seasonal occurrence, we wished to determine whether statolon-induced interferon might be effective in controlling this disease. Mice were treated intraperitoneally with statolon and challenged with influenza A(2) virus by the intranasal route. Although interferon was present in the serum at the time of virus administration, no change in mortality rate was observed. There was, however, a significant increase in the mean survival time of treated animals. Similar results were obtained when Newcastle disease virus was used as the interferon inducer. To determine the effect of the route of challenge, other mice were treated with statolon or Newcastle disease virus and inoculated with mengovirus by the intranasal or intraperitoneal route. The results demonstrated that the treated mice were protected to similar degree against challenge by either route. It is suggested that the relative ineffectiveness of interferon in protecting mice against influenza is due to an intrinsic characteristic of the virus itself rather than the type of interferon induced or the route of virus challenge.     

Essentially pure murine interferon-alpha/beta primes poly rI:rC and Sendai virus-induced interferon production in mice

Intramuscular (i.m.) injection of mice with 2000 IU/g of essentially pure murine interferon-alpha/beta (MuIFN-alpha/beta) 3 h before the induction of IFN by an intraperitoneal (i.p.) inoculation of 10 haemagglutinating units (HAU) per g Sendai virus or 3 micrograms/g polyriboinosinic and polyribocytidylic acid complex (poly rI:rC) elicited a primed IFN response in both cases. Antiserum to MuIFN-alpha/beta neutralized both the priming and antiviral activities of the IFN preparation used. Comparison of the kinetics of primed and unprimed IFN production by Sendai virus indicated that the early (2-4 h) period of IFN production was affected.     

Delayed-Type Hypersensitivity (DTH) Response to Dengue Virus in Mice: Effect of Route of Sensitization and Splenectomy

This study was designed to determine the role of the sensitization route and the spleen in the development of delayed-type hypersensitivity (DTH) to dengue virus in mice. DTH was measured by footpad swelling response. Strong but transient DTH was produced in cyclophosphamide (CY) pretreated mice sensitized subcutaneously (s.c.) or intravenously (i.v.) with dengue virus type 4. Subcutaneous inoculation of virus in incomplete Freund's adjuvant (IFA) further enhanced the DTH elicited. The time course of DTH generated by s.c. and i.v. sensitization were similar with the peak reactivity seen on day 6 after sensitization. Poor DTH was observed in mice given an i.p. inoculation even when CY and/or IFA were used. Intracerebral (i.c.) inoculation also sensitized mice poorly. Splenectomized mice showed enhanced DTH response when compared to intact mice. In contrast to intact mice, pretreatment of splenectomized mice with CY did not alter the DTH level. Splenectomized mice inoculated s.c. with virus in IFA showed poorer DTH than mice sensitized with virus alone.     

Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis

The role of natural killer cells in the temporal development of coxsackievirus B3-induced myocarditis in adolescent CD-1 male mice was examined. Inoculation of purified CVB3m induced maximum NK cell activity in the splenic populations at 3 days postinoculation (p.i.) as assessed by lysis of YAC-1 cells; maximum virus titers in heart tissues were also found at day 3 p.i. Mice depleted of NK cells after injection of anti-asialo GM1 antiserum i.v. had decreased NK cell activity, increased CVB3m titers in heart tissues, and exacerbated myocarditis. Although lesion number was not increased in heart tissues of the latter mice, lesions in these mice exhibited increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3m only. No alteration in interferon titers were observed in CVB3m-infected mice treated with anti-asialo GM1 antiserum as compared with normal CVB3m-infected mice. Measurements of splenic NK cell activity in mice inoculated with doses of 10(2) to 10(8) PFU of CVB3m per mouse or UV-irradiated virus suggest that replication of CVB3m is required for NK cell activation. An amyocarditic variant of CVB3m (ts5R) was shown to replicate in heart tissues and to elicit NK cell activity comparable to that elicited by CVB3m. Therefore, the data suggest that NK cell activation depends on virus replication and that these cells provide some protection against CVB3m-induced myocarditis by limiting virus replication in heart tissues.     

Pneumopathogenicity in mice of a Sendai virus mutant, TSrev-58, is accompanied by in vitro activation with trypsin.

The pneumopathogenicity of a trypsin-sensitive revertant of Sendai virus, TSrev-58, which was derived from a trypsin-resistant mutant, TR-5, was examined in mice. In comparison with TR-5, the revertant had a single amino acid substitution at residue 116 (Ile----Arg) on F protein, which was the cleavage site, and had the same trypsin sensitivity as the wild-type virus. However, TSrev-58 still had a single amino acid difference from the wild-type virus at residue 109 (Asn----Asp) (M. Itoh, H. Shibuta, and M. Homma, J. Gen. Virol. 68:2939-2943, 1987). Nevertheless, the present study revealed that TSrev-58 had the same pneumopathogenicity in mice as the wild-type virus. This result indicates that the activating protease of Sendai virus present in the lungs of mice is quite similar to trypsin and also that the in vitro trypsin sensitivity of Sendai virus can be a good marker of pneumopathogenicity in mice.     

Systemic priming-boosting immunization with a trivalent plasmid DNA and inactivated murine cytomegalovirus (MCMV) vaccine provides long-term protection against viral replication following systemic or mucosal MCMV challenge

We previously demonstrated that vaccination of BALB/c mice with a pool of 13 plasmid DNAs (pDNAs) expressing murine cytomegalovirus (MCMV) genes followed by formalin-inactivated MCMV (FI-MCMV) resulted in complete protection against viral replication in the spleen and salivary glands following sublethal intraperitoneal (i.p.) challenge. Here, we found that following intranasal (i.n.) challenge, titers of virus in the lungs of the immunized mice were reduced approximately 1,000-fold relative to those for mock-immunized controls. We next sought to extend these results and to determine whether similar protection levels could be achieved by priming with a pool of three pDNAs containing three key plasmids (IE1, M84, and gB). We found that the three-pDNA priming elicited IE1- and M84-p65-specific CD8+ T lymphocytes and, following FI-MCMV boost, high levels of virion-specific immunoglobulin G (IgG) and virus-neutralizing antibodies. When mice were i.n. challenged 4 months after the last boost, titers of virus in the lungs of immunized mice were reduced 1,000- to 2,000-fold from those for controls during the peak of viral replication. Additionally, titers of virus were either at or below the detection limits for the salivary glands, liver, and spleen of the majority of the immunized mice. Following sublethal i.p. challenge, virus was undetectable in all of the above target organs of the immunized mice. Virion-specific IgA in the lungs was consistently detected by day 6 post-i.n. challenge for the immunized mice and by day 14 for controls. These results demonstrate the immunity and high levels of protection of the priming-boosting vaccination against both systemic and mucosal challenge.     

Alteration of viral respiratory infections of mice by prior infection with mouse hepatitis virus

Pre-infection with mouse hepatitis virus (MHV) strains S, 3, or JHM reduced the ability of mice to seroconvert to PVM. Geometric mean antibody titers to PVM among MHV pre-infected mice were lower than those for control mice given only PVM, and dually infected mice seroconverted to PVM later than mice given PVM alone. PVM was not recovered from normally permissive respiratory tract tissues of MHV-S pre-infected mice. Pre-infection of DBA/2 mice with MHV-S compromised the susceptibility of these mice to lethal Sendai virus infection but did not substantially reduce the titers of infectious Sendai virus recovered from the lungs. Serologic responses to Sendai virus and lung Sendai virus titers were similar in Sendai virus-resistant C57BL/6 mice pre-infected or not with MHV-S.     

Further studies on the relative antiviral efficacies of interferons induced by poly I:C and Mengo virus.

Mouse serum interferons induced by polyI:C, vesicular stomatitis virus (VSV), reovirus, and Mengo virus were assayed in monolayers of mouse L-929 cells by the plaque-reduction method using both VSV and Mengo as challenge viruses. Titers obtained with Mengo virus as challenge were all lower than with VSV. With the interferons induced by VSV, reovirus, and ployI:C, the reductions were of the order of two- to three-fold. With Mengo virus-induced interferon the reduction was much greater (about 17-fold). This offers an explanation for the observation that, unit for unit (measured by the plaque reduction of VSV), Mengo virus-induced interferon is only about 1/10 as effective as polyI:C-induced interferon in protecting mice against lethal infection with Mengo virus. The data are consistent with the hypothesis that an interferon antagonist is produced in the serum of mice infected with Mengo virus. This antagonist, which is not produced in mice inoculated with polyI:C, or reovirus, effectively blocks the antiviral action of interferon during Mengo virus infections, both in vivo and in vitro.     

Effect of tumor necrosis factor-alpha on infection with Salmonella typhimurium in a mouse model

Mice, inoculated with Salmonella typhimurium, either intraperitoneally (i.p.) or intragastrically (i.g.), developed a systemic infection with a high death rate within a few days after inoculation. Pretreatment of the mice with moderate concentrations of i.p. administered TNF-alpha 24 h before the administration of bacteria reduced the establishment of intracellular infection in the intestinal epithelial cells, and development of bacteremia. The mortality rate was reduced, and the survival time was extended by the same treatment. This effect of TNF-alpha was more pronounced against i.g. than against i.p. inoculated bacteria. The effect was dose dependent, thus concentrations above or below the optimal dose had less effect. No synergistic effect was seen if TNF-alpha was given in combination with interferon-gamma. These results indicate, that TNF-alpha may have a physiological effect in the host defence against facultatively intracellular Gram-negative bacteria.     

Pulmonary compartmentalization of interferon and natural killer cell activity

Studies were performed to determine if natural killer (NK) activity in the mononuclear cells harvested from infected lungs was dependent on local or systemic factors. Mice were inoculated by intratracheal (it), intraperitoneal (ip), or intravenous (iv) routes with (a LD50 dose of) influenza virus A PR/8/34. At various days postinoculation cells from lungs, spleens, and peripheral blood were assayed for NK activity, and lung wash, lung homogenates, and serum were assayed for interferon. After it inoculation there was three- to fourfold increase of NK activity in the lung with little or no increase in NK activity in spleens or peripheral blood. The local augmentation of NK activity in the lung correlated with an increase in interferon (IFN) titer in the lung wash and lung homogenate of PR8 inoculated mice. The virus failed to induce IFN or augment NK activity when it was inoculated systemically. The observed local augmentation of NK activity and local induction of interferon production following it inoculation suggests that the NK population in the lung is capable of responding to locally derived regulatory factors.     

The Role of Interferon in Influenza Virus Tissue Tropism

We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1−/− animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1−/− mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT−/− mice, and mice lacking gamma interferon (IFNγ−/− mice) or the IFN-α receptor (IFNαR−/− mice). Influenza A/WSN/33 virus replicates to high titers in STAT1−/− or IFNαR−/− fibroblasts, while cells derived from WT or IFNγ−/− animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro.     

Protection of OK-432, a Streptococcus pyogenes preparation, against lethal infection of mice with herpes simplex virus

We have studied the protective effect of OK-432, a biological response modifier (BRM) of Streptococcus pyogenes origin, on the lethal infection of mice with herpes simplex virus (HSV)-1. A single intraperitoneal (i.p.) injection of more than 10 micrograms of OK-432, when given at least two days before the infection, gave a marked effect yielding nearly 100% protection against ordinarily lethal infection. The protection was independent of the amount of infected virus inoculated. When given after the infection, the agent even at the maximal dose (100 micrograms), produced only a marginal effect. A single i.p. administration of OK-432 augmented the natural killer (NK) activity of peritoneal exudate cells and spleen mononuclear cells in mice 2 to 3 days after injection of OK-432, coinciding with the times when it induced a survival effect on HSV-infection. Treating OK-432-treated mice with a combination of an anti-macrophage agent, silica, and an anti-NK cell agent, anti-asialo GM1 serum, before infection diminished the antiviral effect of OK-432. The OK-432 protection against HSV infection was also markedly diminished in athymic nude mice. Thus, the protective effect of OK-432 on lethal HSV infection seems to be based on the activation of NK cells, macrophages, and T lymphocytes.     

Stress-induced neuroinvasiveness of a neurovirulent noninvasive Sindbis virus in cold or isolation subjected mice.

Effects of cold or isolation stress on brain penetration by the neurovirulent noninvasive Sindbis virus strain (SVN) were studied in mice. SVN injected intracerebrally (i.c.) causes acute encephalitis and kills adult mice but is unable to invade the brain and kill when injected intraperitoneally (i.p.). Mice inoculated i.p. with SVN were exposed to cold stress or were singly housed. Both stress patterns induced SVN encephalitis and death in 42% (cold) and 37% (isolation) of the tested mice. No death was observed in the control injected mice. Brain virus levels were found to be more than 10(6) PFU in all dying mice. No virus was detected in the control group brains. The virus that was isolated from the brains of moribund mice demonstrated no changes in neuroinvasive and neurovirulent properties. We suggest a stress induced blood-brain-barrier opening with subsequent virus entrance as the mechanism of stress induced SVN encephalitis.     

Trypsin action on the growth of Sendai virus in tissue culture cells. V. An activating enzyme for Sendai virus in the chorioallantoic fluid of the embryonated chicken egg

A trypsin-like protease which is responsible for activation of Sendai virus was found in the chorioallantoic fluid (CAF) of embryonated chicken eggs. Treatment of the inactive form of Sendai virus, grown in LLC-MK2 cells, with CAF enhanced both hemolytic activity and infectivity for the cells. Soybean trypsin inhibitor restrained the enhancing activity of CAF. These results indicate that CAF contains a trypsin-like protease which activates the inactive form of Sendai virus. The activation was strongly inhibited by phenylmethylsulfonylfluoride, ethylenediaminetetraacetate, antipain, and leupeptin but not by tosyllysylchloromethylketone, suggesting that the activating enzyme in CAF is a protease similar to but not identical with trypsin. The inactive form of the virion was produced in ovo when the seed virus was inoculated along with antipain or leupeptin. In deembryonated chicken eggs in which CAF was substituted for a culture medium, multiple cycle growth occurred, but not when soybean trypsin inhibitor was present. These observations indicate that some activating enzyme, possibly the same one as found in CAF, was secreted from the chorioallantoic membrane.