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Recent studies indicate that respiratory syncytial virus (RSV), like influenza, causes significant morbidity and mortality among elderly persons. There are currently no animal models to study the effects of aging on RSV disease and immunity. This manuscript provides an initial description of such a model. Aged and young BALB/c mice (22-24 and 2-4 months, respectively) were infected with 104TCID50of RSV A2. RSV was detected by culture in lung and nose wash specimens obtained 4-6 days following infection at a slightly higher titer in old mice in comparison with young mice. RT-PCR assay detected RSV in the lungs and nose washes of all mice on 4, 8, and 21 days postinoculation, with only a slightly less frequency in young mice. Splenic lymphocytes from old mice exhibited significantly lower RSV-specif ic MHC class I-restricted CD8+CTL responses (P < 0.01-0001), and reduced IFN-production (P < 0.03) than young mice. Conversely, IL-4 production was somewhat elevated in old mice. These results demonstrate diminished RSV virus-specific CD8+CTL responses and IFN-γ production in old mice in comparison with young. It is speculated that the deficient RSV-specific CTL responses may account for the Increased morbidity and mortality from RSV Infections in elderly persons. Although detailed hlstopathological, virological, and immunological analyses are incomplete at present, the old BALB/c RSV infection model described provides an opportunity to evaluate the role of CD8+ CTL and cytokines in RSV disease in aging.

Background : Monophosphoryl lipid A (MPL), QS-21 and QS-7 were evaluated in mice for their ability to increase the immunogenicity and protective efficacy of formalin-inactivated (FI) influenza A/Texas/91 virus vaccine. Freund's incomplete adjuvant (FIA) was used as a positive control. Methods : Mice were inoculated twice, 28 days apart, either intramuscularly (I.M.) with vaccine mixed with phosphate buffered saline, FIA, MPL or QS21, or intranasally (I.N.) with vaccine containing QS-21 or QS-7. The mice were bled on days 0, 28 and 49 and challenged I.N. on this last day with live virus. Four days later, the lungs from each animal were assessed for influenza virus. All sera were tested for virus-specific neutralizing (Nt), hemagglutination inhibiting (HI) and ELISA antibodies. Studies to account for the mechanism(s) of adjuvant activity have been initiated. Results : FIA, MPL and QS-21 all enhanced the production of virus-specific antibodies and increased protection from pulmonary virus infection following I.M. administration. Maximal adjuvanticity occurred in groups inoculated with "low" doses of vaccine and in groups administered vaccine mixed with QS-21. Both QS adjuvants exhibited significant adjuvant activity following I.N. inoculation. Protection correlated best with levels of virus-specific serum Nt and HI antibodies. Conclusions : The present studies support continued development of adjuvants for inactivated influenza virus vaccines.

ABSTRACTNo practical animal models for the testing of chemotherapeutic or biologic agents identified in cell culture assays as being active against measles virus (MV) are currently available. Cotton rats may serve this purpose. To evaluate this possibility, 5-ethynyl-1-β-d-ribofuranosylimidazole-4-carboxamide (EICAR) and poly(acrylamidomethyl propanesulfonate) (PAMPS), two compounds that have been reported to inhibit MV in vitro, and ribavirin, an established antiviral drug with MV-inhibitory activity, were evaluated for their antiviral activities against MV and respiratory syncytial virus (RSV) in tissue culture and in hispid cotton rats. A single administration of PAMPS markedly inhibited pulmonary RSV or MV replication (>3 log10reduction in pulmonary titer compared to that for controls), but only if this compound was administered intranasally at about the time of virus inoculation. Both EICAR and ribavirin exhibited therapeutic activity against RSV and MV in cotton rats when they were administered parenterally. However, both of these compounds were less effective against MV. On the basis of the pulmonary virus titers on day 4 after virus inoculation, the minimal efficacious dose of EICAR against MV (120 mg/kg of body weight/day when delivered intraperitoneally twice daily) appeared to be three times lower against this virus than that of ribavirin delivered at a similar dose (i.e., 360 mg/kg/day). These findings correlated with those obtained in vitro. The data obtained suggest that cotton rats may indeed be useful for the initial evaluation of the activities of antiviral agents against MV.

Between December 1996 and September 1998, 13 patients with advanced recurrent malignant brain tumors (9 with glioblastoma multiforme, 1 with gliosarcoma, and 3 with anaplastic astrocytoma) were treated with a single intratumoral injection of 2 × 109, 2 × 1010, 2 × 1011, or 2 × 1012vector particles (VP) of a replication-defective adenoviral vector bearing the herpes simplex virus thymidine kinase gene driven by the Rous sarcoma virus promoter (Adv.RSVtk), followed by ganciclovir (GCV) treatment. The VP to infectious unit ratio was 20:1. Our primary objective was to determine the safety of this treatment. Injection of Adv.RSVtk in doses ≤2 × 1011VP, followed by GCV, was safely tolerated. Patients treated with the highest dose, 2 × 1012VP, exhibited central nervous system toxicity with confusion, hyponatremia, and seizures. One patient is living and stable 29.2 months after treatment. Two patients survived >25 months before succumbing to tumor progression. Ten patients died within 10 months of treatment, 9 from tumor progression and 1 with sepsis and endocarditis. Neuropathologic examination of postmortem tissue demonstrated cavitation at the injection site, intratumoral foci of coagulative necrosis, and variable infiltration of the residual tumor with macrophages and lymphocytes.Molecular Therapy (2000) 1, 195–203; doi: 10.1006/mthe.2000.0030

An alternative model to nonhuman primates to study measles virus (MV) pathogenesis, to evaluate potential MV vaccines, or to screen for potential antivirals effective against this virus is highly desirable. The laboratory-adapted Edmonston strain of MV has been reported to replicate in the lungs of hispid cotton rats following intranasal inoculation, immunosuppress infected animals, and disseminate widely from the lungs, making these animals a candidate model. However, clinical MV strains have generally not been found to grow in these animals, limiting the utility and acceptance of this model. In the present studies we demonstrate reproducible replication of several clinical MV strains in hispid cotton rats. As with the Edmonston strain, leukocytes appear to be the primary target cells of these viruses following intranasal inoculation, and extrapulmonary dissemination is common. It is also demonstrated that prior MV infection or immunization of test animals with MV vaccine prevents pulmonary tract infection. These findings should make the MV-cotton rat model more acceptable.

Billions of people are infected with respiratory viruses annually. Infants and young children, the elderly, immunocompromised individuals and those debilitated by other diseases or nutritional deficiencies are most at risk for serious disease. There are few vaccines available for use against these viruses, and even where there are (influenza, measles and adenovirus), infections remain common. The continued prevalence of respiratory virus infections has lead to renewed efforts to find safe agents effective against the most medically important respiratory viruses: influenza, respiratory syncytial, parainfluenza, measles, rhino- and adenovirus. Copyright 1999 Harcourt Publishers Ltd

We have produced a genetically-engineered chimeric protein composed of the external domains of the respiratory syncytial virus (RSV) fusion (F) protein and the parainfluenza virus type 3 (PIV-3) hemagglutinin-neuraminidase (HN) protein in insect cells using the baculovirus expression system. The yield of the soluble chimeric FRSV-HNPIV-3protein could be increased approximately 2-fold by using Trichoplasia ni(High Five) insect cells in place of Spodoptera frugiperda(Sf9) for expression. The chimeric protein, purified from the supernatant of baculovirus-infected High Five cells by immunoaffinity chromatography was correctly processed at the F2-F1 proteolytic cleavage site. Immunochemical analysis of the chimera with a panel of anti-F and anti-HN monoclonal antibodies suggested that the antigenicity of the major F and HN neutralization epitopes of the chimeric protein was preserved. Immunization of cotton rats with two 1 or 10 μg doses of the chimeric protein adsorbed to aluminum phosphate elicited strong PIV-3 specific HAI responses as well as PIV-3 and RSV specific neutralizing antibodies, and at either dose completely protected against challenge with live RSV and PIV-3.

Recombinant (r) human (hu) manganese (Mn) and copper-zinc (CuZn) superoxide dismutase (SOD) were evaluated for their cytotoxicity and antiviral activity against respiratory syncytial virus (RSV) in tissue culture and in cotton rats. No apparent cytotoxicity or inhibition of RSV was observed in the tissue culture studies (both compounds had IC50and EC50values ⩾ 1000 μg/ml and a selective index = 1). However, significant reductions in mean pulmonary RSV titers (ranging between 0.5 and 1.9 log10/g of lung compared with the mean pulmonary viral titers detected in similarly inoculated, placebo-treated control animals) were seen in most of the experiments, in which experimentally infected cotton rats were exposed to continuous small-particle aerosols (reservoir concentrations ⩾ 20 mg/ml) containing either rhuMnSOD or rhuCuZnSOD. This protective effect was dose dependent and not observed when either rSOD compound was administered parenterally (intraperitoneally) or intranasally. No toxic effects were noted in any of the cotton rats exposed to aerosols of either rhuMn or CuZnSOD: nor was any evidence of drug-induced histopathology observed in sections of lung prepared from these animals.

Approximately one fifth of the macrophages obtained from the lungs of mice infected 2 to 5 days with influenza A/HK virus were found to rosette well with either unmodified human, chicken, or guinea pig erythrocytes, but not with erythrocytes from hamsters, sheep, or mice. Rosette-forming macrophages were seldom seen in suspensions from uninfected mice (3±3%) or mice infected 24 h previously (3±3%). Rosette formation was not due to virus hemadsorption, as indicated by the failure of specific antiserum to influenza virus to block rosette formation; by the induction of comparable levels of rosette-forming macrophages in the lungs of mice infected with herpes simplex virus type 2, a nonhemadsorbing virus; and by the inhibition of rosette formation at 4°C. Instead, rosette formation appeared to be directly related to macrophage elicitation or activation since nonstimulated macrophage populations such as peripheral blood monocytes, macrophages from uninfected lungs, or noninduced peritoneal macrophages were not observed to rosette to any significant extent. Furthermore, peritoneal macrophages induced with filter-sterilized normal horse serum rosetted at levels comparable to that observed with cells from infected lungs. These results indicate that hemadsorption alone can not be used as a criterion of virus infection of macrophages. However, rosette formation may serve to identify macrophage subpopulations which are active in host defense against viral infections.

Hispid cotton rats were inoculated intranasally with either measles virus (MV) Edmonston, a multipassaged, tissue culture-adapted strain of MV, or with one of three clinical MV isolates that had limited passages (three to five times) in tissue culture cells. MV Edmonston was recovered from the lungs of every (n= 37) hispid cotton rat inoculated with this virus for at least 7 days after virus inoculation. Peak pulmonary titers occurred on Day +4 (3.3–4.4 log10/g lung). Scattered areas of inflammation were observed interstitially in lung sections from infected animals stained with hematoxylin and eosin, and a similar pattern of diffuse fluorescence was seen in cryostat sections stained with an indirect fluorescent antibody procedure specific for virus antigens. Fluorescent antibody and virus isolation studies on lung lavage cells both suggested that lung leukocytes were a primary target of the virus. In contrast to these findings, virus was isolated only sporadically from hispid cotton rats inoculated with any of the clinical measles virus isolates. Despite the restricted growth of MV in these animals, cotton rats may be useful for studying certain aspects of measles virus pathogenesis and for screening potential antiviral compounds in vivo.

We studied the effects of combined administration of human immunoglobulin (IVIG) and ribavirin aerosol on respiratory syncytial virus (RSV) infection in cotton rats (Sigmodon hispidus). Cotton rats assigned to receive combined therapy were administered Gamimune, a preparation of purified IVIG with a high titer of anti-RSV neutralizing activity, intraperitoneally 24 h prior to intranasal RSV challenge and then treated with ribavirin aerosol 3 days after challenge. Lung viral titers from these cotton rats (geometric mean titers [GMT] log100.15 ± 0.5) were lower than titers from untreated animals (GMT, log103.7 ± 0.6) and animals treated with either IVIG alone (GMT, log101.8 ± 0.9) or ribavirin alone (GMT, log101.9 ± 1.1). Only one of 12 cotton rats treated with both IVIG and ribavirin had a demonstrable titer of virus after RSV challenge. When IVIG administration was delayed until day 3 after virus challenge, lung viral titers were still lowest in animals receiving both IVIG and ribavirin. In comparison, there was no additive antiviral effect between IVIG and ribavirin against RSV infections of HEp-2 cells in vitro. Pathologic changes on histologic examination of pulmonary tissues from animals challenged with RSV were least prominent in animals treated with both IVIG and ribavirin. Despite the apparent absence of in vitro additive antiviral effect, combined use of IVIG and ribavirin was more efficacious against RSV infection in the cotton rat than use of either agent alone.

To initiate evaluation of the cell-mediated immunological response to influenza virus in a major site of disease, lung cells were obtained by transpleural lavage from lungs of uninfected mice and from those infected 3 or 6 days previously with 5 50% mouse infectious doses (MID50) of avirulent (P3) or virulent (P9) influenza A Hong Kong (H3N2) virus. The number of cells recovered by lavage was dependent on the dose, time after inoculation, and the type of virus used for inoculation. Although lavage pools were shown to contain peripheral blood leukocytes, this contamination was shown to be consistently less than 5% of the total leukocytes harvested. Among the ca. 0.75 × 106lavage cells obtained from each uninfected mouse, about 90% were macrophages or lymphocytes in approximately equal proportion. T, B, and null (lyphocytes lacking theta or surface immunoglobulin markers) lymphocytes averaged 23, 9, and 7% of cells in these suspensions, respectively. After infection with either P3 or P9 virus, increased numbers of activated macrophages and lymphoblasts were observed. The major change during P3 infection was an increase in absolute numbers of null lymphocytes. In contrast, during P9 infection, T and B lymphocytes and macrophages progressively increased in absolute numbers while null cells decreased. These data suggest that cell-mediated immunological responses to influenza virus occur in the lung during infection, but that the responses to virulent and avirulent variants may differ both qualitatively and quantitatively.

A human isolate of type A Hong Kong influenza virus (H3N2) was adapted to mice by serial passage. Lung homogenates from mice who received low passage levels contained about the same quantity of virus (106.2−6.9550% tissue culture infective doses/ml) as those from mice who received high passage levels (105.95−6.4550% tissue culture infective doses/ml); however, death occurred only in animals given high-passage virus. Passage 3 (P3) and passage 9 (P9) viruses were selected as representative of low-passage and high-passage viruses, respectively. Although minimal differences were detected in infectivity for rhesus monkey kidney tissue cultures and mice, P9 virus was at least 10,000 times more lethal for mice (mean lethal dose = 104.2). Infection with P3 virus was accompanied by minimal bronchitis and bronchiolitis only, whereas P9-infected animals exhibited marked bronchitis, bronchiolitis, and pneumonia. Striking thymic cortical atrophy was also demonstrable in the P9-infected animals and, although virus was more commonly recovered from thymuses from these animals, immunofluorescent studies revealed only a few cells containing influenza virus antigens. To further explore the participation of thymus-derived lymphocytes in influenza, athymic nude mice and furred immunocompetent littermates were given 500 50% mouse infectious doses of P9 virus. Nude mice exhibited an increased survival time and, in contrast to the extensive lung pathology seen in furred littermates, manifested minimal cellular infiltration and no tissue destruction in lungs. Brains from nude mice exhibited encephalomalacia with lymphocytic perivascular cuffing, which was not seen in furred animals. Virus was recovered from brains of 6 of 13 nude mice and 1 of 10 furred animals. The contrasting models suggest that thymus-dependent cells play a significant role in the inflammatory response to influenza virus infection and should prove useful for probing host-virus interactions which characterize influenza virus virulence.

Systemically administered CsA has not consistently suppressed the pulmonary immunoreactivity that leads to rejection in lung transplant patients. Pulmonary T cells from patients given CsA systemically still retain their immunoreactivity, which can be suppressed with added CsA. Direct application of CsA by aerosol to the respiratory epithelium should achieve high lung concentrations with minimum systemic effects. In the present study, CsA was most efficiently incorporated into liposomes composed of egg yolk phosphatidylcholine at a molar ratio of CsA to egg yolk phosphatidylcholine of 1:20. These CsA liposomes retained their biological activity and were as effective as free CsA in the suppression of anti-CD3-stimulated [3H]thymidine incorporation by mouse spleen cells. The generation of a small-particle aerosol of CsA liposomes had no effect on this biological activity. CsA liposome aerosol particles have a mass median aerodynamic diameter of 2 μm, which allows for distribution of drug throughout the respiratory tract. Quantitation of CsA in the lungs and blood of mice exposed to CsA liposome aerosols for 4 days showed that as little as 15 min daily (0.11 mg/kg/day) was sufficient to achieve an estimated concentration of CsA in respiratory secretions of 6 μg/ml without detectable blood levels. Thus, CsA liposomes can be produced and aerosolized that achieve pulmonary concentrations with sufficient immunosuppressive activity to be effective in the treatment of lung diseases.

Reshaped human MAb RSHZ19, which is specific for the surface fusion protein of respiratory syncytial virus (RSV) is in clinical development for the prevention and treatment of RSV-induced disease in human infants. The current studies profile lung virus clearance and evaluate lung histopathology in MAb-treated, RSV-infected cotton rats, a well characterized model of RSV infection. The highest dose of this MAb (10 mg/kg) administered parenterally 24 h before infection decreased subgroup A or B RSV lung titers to below detectable levels (=2.3 log10reduction), and significantly reduced lung virus titers (=2.0 log10reduction) when administered 96 h postinfection. Prophylactic administration of 10 mg/kg RSHZ19 was significantly more protective than 1000 mg/kg conventional human immune serum globulin (HSIg), and protective serum-neutralizing titers in MAb-treated animals (1:32, which correlated with approximately 40 µg/ml determined by anti-idiotype ELISA) were significantly lower than those reported previously for HSIg or for convalescent human serum (1:200-1:400). MAb concentration in lung lavages was determined by ELISA to be approximately 1% of the serum MAb concentration, but was not detectable by neutralization assay. The degree of lung histopathology in MAb-treated cotton rats was proportional to lung virus titer, and inversely proportional to the RSHZ19 dose administered. There was no evidence of exacerbated disease in the lungs of MAb-treated animals. These studies thus support the potential clinical utility of RSHZ19 MAb in the prevention and treatment of RSV-induced disease in humans.

ABSTRACTStreptococcal pyrogenic exotoxin B (SpeB), a conserved cysteine protease expressed by virtually all Streptococcus pyogenesstrains, has recently been shown to be an important virulence factor (S. Lukomski, S. Sreevatsan, C. Amberg, W. Reichardt, M. Woischnik, A. Podbielski, and J. M. Musser, J. Clin. Invest. 99:2574–2580, 1997). Genetic inactivation of SpeB significantly decreased the lethality of a serotype M49 strain for mice and abolished the lethality of a serotype M3 strain after intraperitoneal (i.p.) injection. In the present study, a wild-type M3 isolate and an M3 speBmutant derivative were used to investigate the mechanism responsible for altered virulence. Following i.p. injection, the mutant and wild-type strains induced virtually identical cellular inflammatory responses, characterized largely by an influx of polymorphonuclear leukocytes (PMNs). In addition, the mutant and wild-type strains rapidly entered the blood and were recovered from all organs examined. However, significantly fewer (P< 0.05) CFUs of the isogenic mutant derivative than of the wild-type parent strain were recovered from blood and organs. PMNs effectively cleared the M3 speBmutant from the peritoneum by 22 h, thereby sparing the host. In contrast, the wild-type M3 strain continued to replicate intraperitoneally and had the ability to kill phagocytes. This process allowed the wild-type strain to continuously disseminate, resulting in host death. Our results indicate that genetic inactivation of the cysteine protease decreased the resistance of the mutant to phagocytosis and impaired its subsequent dissemination to organs. These results provide insight into the detrimental effect of SpeB inactivation on virulence.

ABSTRACT: We studied the effects of combined administration of human immunoglobulin (IVIG) and ribavirin aerosol on respiratory syncytial virus (RSV) infection in cotton rats (Sigmodon hispidus). Cotton rats assigned to receive combined therapy were administered Gamimune, a preparation of purified IVIG with a high titer of anti-RSV neutralizing activity, intraperitoneally 24 h prior to intranasal RSV challenge and then treated with ribavirin aerosol 3 days after challenge. Lung viral titers from these cotton rats (geometric mean titers [GMT] log10= 0.15 ± 0.5) were lower than titers from untreated animals (GMT, log10= 3.7 ± 0.6) and animals treated with either IVIG alone (GMT, log10= 1.8 ± 0.9) or ribavirin alone (GMT, log10= 1.9 ± 1.1). Only one of 12 cotton rats treated with both IVIG and ribavirin had a demonstrable titer of virus after RSV challenge. When IVIG administration was delayed until day 3 after virus challenge, lung viral titers were still lowest in animals receiving both IVIG and ribavirin. In comparison, there was no additive antiviral effect between IVIG and ribavirin against RSV infections of HEp-2 cells in vitro. Pathologic changes on histologic examination of pulmonary tissues from animals challenged with RSV were least prominent in animals treated with both IVIG and ribavirin. Despite the apparent absence of in vitro additive antiviral effect, combined use of IVIG and ribavirin was more efficacious against RSV infection in the cotton rat than use of either agent alone.

Hybrid recombinant human alpha interferon A/D (rIFN A/D) is unusual in that it has equivalent antiviral activity in in vitroassays using mouse or human tissue cells. This interferon was delivered to outbred Swiss mice in small particle aerosols before and/or after these mice were inoculated with virulent influenza A/HK/68 virus. Although rIFN A/D was effective in inhibiting influenza virus replication in vitroin primary mouse embryo cells, it had only a limited degree of effectiveness in the in vivotests; only animals exposed to rIFN A/D for 4 hr and inoculated with influenza virus 4 hr later exhibited a significant decrease in mortality (approximately 25% reduction in deaths; P< 0.025) compared with that of the untreated control animals. The limited effectiveness of rIFN A/D seen in these studies indicated that the use of this or similar recombinant alpha interferons alone to prevent or treat influenza virus infection in humans is not promising.

Transpleural lavage of lungs from uninfected C3H mice yielded an average of 300,000 leukocytes per mouse. This number increased eightfold within 6 days after intranasal inoculation with virulent influenza A/Hong Kong/68 (H3N2) virus. Macrophages and lymphocytes in approximately equal numbers comprised 90% or more of the leukocytes both before and during infection. B, T, and null lymphocytes comprised, respectively, 9, 21, and 18% of the leukocytes before infection and 7, 26, and 5% by day 6. In absolute numbers, macrophages and T lymphocytes provided the major increments during infection. Cytotoxic activity of mononuclear cells from lung lavages was compared in a chromium release assay using syngeneic L929 target cells with the activity of mediastinal lymph nodes, spleens, and peripheral blood of uninfected and infected C3H mice. Nonspecific cytotoxicity for target cells infected with H3hkNeq1 or B/Lee influenza virus was found with mononuclear cells from uninfected mice. This activity tended to be highest with lavage leukocytes and was associated with adherent cells, presumably macrophages. Increased virus-specific cytotoxicity was detected with lavage cells by day 6 and persisted through day 9, the period of maximal pneumonia. Similar cytotoxic activity also appeared in cells from the nodes and spleen at this same time but was not detected in peripheral blood cells. The virus-specific cytotoxicity of lavage cells was due largely to a nonadherent cell possessing Fc receptors and theta antigen but lacking C3 receptors; these properties are compatible with actively cytotoxic T lymphocytes. The cytological characteristics of the infiltrating leukocytes and the cytotoxicity data suggest that the local T cell response to influenza virus infection in the lung is a major contributor to the pneumonia observed in this mouse model.

The virostatic activity of sophocarpines and gancyclovir (GCV) was tested using HHV‐6 Z29 strain and Molt‐3 cells. The cytotoxic (IC50) and the antiviral (ED50) values were first experimentally determined and selective indices (SI) were then calculated. The SI values for sophocarpines 1 and 2 and GCV were in the order 184, 183, and 23, respectively. Though preliminary, these findings indicate that sophocarpines have the potential to inhibit HHV‐6 replication. Copyright © 2002 John Wiley & Sons, Ltd.

The importance and significance of amantadine- or rimantadine-resistant influenza viruses in immunocompromised patients was studied in a population of adult bone marrow transplant (BMT) recipients and patients with leukemia prospectively cultured for respiratory viruses. Influenza A viruses were isolated from 29 patients with acute respiratory illness (14 BMT recipients and 15 patients with leukemia). Fifteen patients (52%) received amantadine (n = 4) or rimantadine (n = 11) therapy. All influenza isolates recovered from six patients shedding virus for 3 days were screened for antiviral susceptibility; resistant isolates were further genetically characterized. Initial influenza isolates were susceptible to amantadine or rimantadine, but subsequent isolates from five of six patients were resistant. Influenza-associated mortality was similar among patients with and without documented antiviral resistance (2 of 5 vs. 5 of 24). We conclude that development of antiviral resistance in immunocompromised individuals should be considered when they have been treated with antivirals and have shed influenza virus for a prolonged period. Isolation procedures should be instituted for all immunocompromised patients with influenza, both during and after therapy with amantadine or rimantadine.