THE JOURNAL OF INFECTIOUS DISEASES • VOL. 133, NO.1· © 1976 by the University of Chicago. All rights reserved.
JANUARY 1976
Influenza Vims Infection in Nude Mice John L. Sullivan, * Ronald E. Mayner, David W. Barry, and Francis A. Ennis
From the Division of Virology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland
from the Rodent and Rabbit Production Section of the NIH. All mice were six to eight weeks of age at the time of the study. Virus and mode of infection. Groups of 1850 nude and normal Balb/c mice were inoculated with 4 ml of twofold dilutions of a PR-8 influenza virus-infected allantoic fluid. This inoculum contained 100 50% mouse lethal doses (MLD5o)/ml, as determined by administration in an aerosol chamber for 15 min to Balb/ c mice [4]. Both strains of mice receiving the same dilution of virus were exposed in the same chamber and at the same time. Control mice of both strains were exposed to an aerosol of phosphate-buffered saline in a similar manner. Serologic testing and virus isolation. Animals were exsanguinated and the lungs and spleens were removed at appropriate intervals after exposure. Individual sera were collected, treated with Vibrio cholerae neuraminidase, heated at 56 C for 30 min, and incubated with chick erythrocytes to remove nonspecific inhibitors [5] before being tested for HAl antibodies. Sera were tested by the HAl procedure as modified for microtitration plates [6]. Seroconversion is defined as a change in titer of HAl antibodies from < 1:8 to ~ 1:8, because our experience has shown that sera of uninfected nude, Balb /c, and outbred mice in our colonies do not contain antibodies to influenza virus when tested undiluted in neutralizing antibody assays or when diluted 1: 8 (the lowest dilution possible in the assay for HAl antibody). Specimens of lung and spleen were prepared for isolation of virus by homogenization in 2 ml of beef heart infusion broth containing gentamicin
The role of the T-lymphocyte (T-cell) component of the immune response in influenza virus infection has not been well characterized. Recent studies in mice have shown that (1) thymectomized, lethally irradiated, bone marrow-reconstituted mice do not form antibody to purified influenza hemagglutinin [1]; (2) nude (nu/nu) mice that are deficient in T-lymphocytes do not form antibody to hemagglutinin after exposure to live PR-8 influenza virus [2]; and (3) treatment of normal mice with antilymphocyte serum prior to and during infection with a live A2 influenza virus results in a decreased mortality rate [3]. To delineate the relation between antibody , T-cell function, and the pathogenesis of fatal influenza virus infection, we have performed a series of experiments in which we have observed the effect of influenza virus infection in nude mice. Materials and Methods
Mice. Nude mice bred on a Balb/c background (seventh backcross, obtained from the Mammalian Genetics and Animal Production Section of the National Cancer Institute, National Institutes of Health [NIH], Bethesda, Md.) were housed in filter-top cages in a room isolated from other animals. Normal Balb/c mice were obtained Received for publication June 6, 1975, and in revised form September 10, 1975. Please address requests for reprints to Dr. Francis A. Ennis, Division of Virology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20014, * Present address: Department of Pediatrics RD20, School of Medicine, University of Washington, Seattle, Washington 98195.
91
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 30, 2015
The role of T-cell function in influenza virus infection was studied by aerosol infection of nude mice with an influenza A virus (PR-8 strain). Nude mice died somewhat later than normal mice, and the antibody response of nude mice to the virus was minimal. Furthermore, nude mice did not eliminate the virus, which persisted for relatively long periods (two to three weeks).
Sullivan et al.
92
(50 ug/rnl) and amphotericin B (0.3 ug/rnl) with a Potter tissue homogenizer. Suspensions were then inoculated into nine-day-old embryonated hens' eggs. Allantoic fluids, collected 48 hr later, were titrated for hemagglutinating virus in microtitration plates. Viral isolates were characterized as PR-8 by HAl tests in which specific PR-8 antisera were used.
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JANUARY 1976
Influenza Vims Infection in Nude Mice John L. Sullivan, * Ronald E. Mayner, David W. Barry, and Francis A. Ennis
From the Division of Virology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland
from the Rodent and Rabbit Production Section of the NIH. All mice were six to eight weeks of age at the time of the study. Virus and mode of infection. Groups of 1850 nude and normal Balb/c mice were inoculated with 4 ml of twofold dilutions of a PR-8 influenza virus-infected allantoic fluid. This inoculum contained 100 50% mouse lethal doses (MLD5o)/ml, as determined by administration in an aerosol chamber for 15 min to Balb/ c mice [4]. Both strains of mice receiving the same dilution of virus were exposed in the same chamber and at the same time. Control mice of both strains were exposed to an aerosol of phosphate-buffered saline in a similar manner. Serologic testing and virus isolation. Animals were exsanguinated and the lungs and spleens were removed at appropriate intervals after exposure. Individual sera were collected, treated with Vibrio cholerae neuraminidase, heated at 56 C for 30 min, and incubated with chick erythrocytes to remove nonspecific inhibitors [5] before being tested for HAl antibodies. Sera were tested by the HAl procedure as modified for microtitration plates [6]. Seroconversion is defined as a change in titer of HAl antibodies from < 1:8 to ~ 1:8, because our experience has shown that sera of uninfected nude, Balb /c, and outbred mice in our colonies do not contain antibodies to influenza virus when tested undiluted in neutralizing antibody assays or when diluted 1: 8 (the lowest dilution possible in the assay for HAl antibody). Specimens of lung and spleen were prepared for isolation of virus by homogenization in 2 ml of beef heart infusion broth containing gentamicin
The role of the T-lymphocyte (T-cell) component of the immune response in influenza virus infection has not been well characterized. Recent studies in mice have shown that (1) thymectomized, lethally irradiated, bone marrow-reconstituted mice do not form antibody to purified influenza hemagglutinin [1]; (2) nude (nu/nu) mice that are deficient in T-lymphocytes do not form antibody to hemagglutinin after exposure to live PR-8 influenza virus [2]; and (3) treatment of normal mice with antilymphocyte serum prior to and during infection with a live A2 influenza virus results in a decreased mortality rate [3]. To delineate the relation between antibody , T-cell function, and the pathogenesis of fatal influenza virus infection, we have performed a series of experiments in which we have observed the effect of influenza virus infection in nude mice. Materials and Methods
Mice. Nude mice bred on a Balb/c background (seventh backcross, obtained from the Mammalian Genetics and Animal Production Section of the National Cancer Institute, National Institutes of Health [NIH], Bethesda, Md.) were housed in filter-top cages in a room isolated from other animals. Normal Balb/c mice were obtained Received for publication June 6, 1975, and in revised form September 10, 1975. Please address requests for reprints to Dr. Francis A. Ennis, Division of Virology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20014, * Present address: Department of Pediatrics RD20, School of Medicine, University of Washington, Seattle, Washington 98195.
91
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on May 30, 2015
The role of T-cell function in influenza virus infection was studied by aerosol infection of nude mice with an influenza A virus (PR-8 strain). Nude mice died somewhat later than normal mice, and the antibody response of nude mice to the virus was minimal. Furthermore, nude mice did not eliminate the virus, which persisted for relatively long periods (two to three weeks).
Sullivan et al.
92
(50 ug/rnl) and amphotericin B (0.3 ug/rnl) with a Potter tissue homogenizer. Suspensions were then inoculated into nine-day-old embryonated hens' eggs. Allantoic fluids, collected 48 hr later, were titrated for hemagglutinating virus in microtitration plates. Viral isolates were characterized as PR-8 by HAl tests in which specific PR-8 antisera were used.
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