Experimental studies for a model of inapparent influenzal infection
It is generally agreed that a large proportion of influenzal infections in man is inapparent, and this has been proven serologically (Keogh et al. 1958, Broun et al. 1960). In human influenza epidemics the clinical manifestation of the infection is well characterized by sudden onset of fever, generalized muscular pains and rapid recovery in uncomplicated cases. Significant pneumonic complications among cases from the general population in usual epidemics scarcely exceed one per cent of the
total (Francis and Maassab 1965).
Following the isolation of influenza virus from the patients (Smith et al. 1933, Francis 1934), and the successful serial passages of the isolates in mouse and ferret, the mouse has been used extensively in studies of the pathogenesis, pathology and immunology of influential infection (An-drewes et al. 1935, Francis and Magill 1937, Burnet and Stone 1945, Anderson and Burnet 1947, Sugg 1949, Jones 1950, Smith et al. 1951, Ledinko and Perry 1955, Ishida et al. 1959, Dolowy and Muldoon 1964, Schulman and Kilbourne 1965). In the mouse the main criteria of
infection have been death and pulmonary consolidation.
Ferrets are known to be one of the most sensitive animal to experimental influenzal infection, and have also been used widely in the studies of influenza (Shope 1934, Francis 1934, Taylor and Dreguss 1941, Hull and Loosli 1951, Sugg and Nagaki 1955, Liu 1955, Basarab and Smith 1965). In ferret influenza, there is an
abrupt onset of fever with lassitude, sneezing, yawning, nasal discharge and loss of appetite after an incubation of 48 hours. Generally animal recovers rapidly and is immune to reinfection at that time (Francis and Maassab 1965).
However, most of the studies on influenzas infections in experimental animals have been made with severe apparent infection that resulted from inoculation of a large amount of the virus into the animals.
In efforts of establishing a model of inapparent influenzal infection in experimental animals, studies were made 1) with the temperature response of mice to infections with influenza virus to determine whether the mousse could serve as an experimental model for the less severe infections that usually occur in man, and 2) on the occurrence of overt and inapparent influenzal infections in ferrets in relation to the size of infecting doses of the virus.
Materials and Methods
A. The temperature response of mice to infection with influenza virus.
Mice: Female mice of an inbred strain, BALB/C, 4 weeks old were used. The room temperature in which mice were kept was maintained at 24.9 C to 26.6 C (75 to 80 F). For virus inoculation a mouse was anesthetized lightly wish ether and 0.05 ml of the inoculum was given intranasally.
Virus: Two sublines of the PR 8 strain of type A influenza virus, i,e., egg-line virus and mousepassaged virus, were rosed. The allantois-on-shell technique (Fazekas de St Groth and White 1958) was applied for virus titration and the titers were expressed in terms of 60% infections dose (Membrane Piece Infectious Dose: MPID^^50).
Scoring of lung lesion: Gross lung lesions were scored by the extent of the pulmonary consolidation seen on the lung surface in relation to total lung surface area, i.e., 4+, 3+, 2+ aud 1+. The total lung-lesion score of each group of mice was obtained by calculating a percentage of the total possible score (Cohen 1960).
Measurement of rectal temperature: Rectal temperature was measured daily once between 2 and 3 PM with a Thermistemp Telethermometer Model TK (Yellow Springs Instrument Co., Yellow Springs, Ohio, U.S.A.).
B. Comparison of overt and inapparent influenzal infection in ferrets.
Criteria of infection: Overt infection is the one in which the ferret bad fever during the acute phase of the infection and produced a high level of antibody (HI) in its serum which could be detected during the convalescent stage. Inapparent infection is that indicated by antibody prpduction as in the case of overt
infection but without fever during the course of the infection.
Ferrets: Twelve to 18-month old female ferrets, weighing from 700 to 1,000 gm, were purohased from Gilman Marshall (N.Y., U.S.A.). For virus inoculation the ferret was anesthetized by infraperitoneal injection of Sodium Nembutal and 0.5 ml of the inoculum was given intranasally.
Rectal temperature was measured twice daily at 10 AM and 2 PM with a Thermistemp Telethermometer Model TK. A sharp rise in rectal temperature to 38.9 C (102 F) or more after virus inoculation was considered as fever. Room temperature of normal
animals and isolation room was maintained at 23.3C to 25.6C (74 to 78 F).
Virus: A mouse-passaged subline of the PR 8 sprain of type A influenza virus used in the mouse experiment was further passaged 8 time in BALB/C mice with 3-day intervals. A pooled infected allantoic fluid obtained by inoculation of lung homogenate of the 15th passage mouse was used as inoculum.
Virus titration was made by egg infectivity titration and the titers were expressed in terms of EID^^50.
Hemagglutination-inhibition test of sera and nasal washings: Hl antibody titer was determined by Salk's method (Salk 1944).
Interferon-like substance in nasal and lung homogenates: Interference activities of nasal homogenates (and a few lung homogenates) in which viral growth was demonstrated were determined by the method of Isaacs and Hitchcock (1960).
Techniques required for Plaque formation by encephalomyocarditis virus in L cell culture were the same as described by Takemoto and Liebhaber (1961).
1. Hypothermia rather than fever was the characteristic thermal response of mice to intranasal inoculation of the PR 8 strain of type A influenza virus.
2. Mice showed marked hypothermia wish fatal termination in response to inoculation of large amounts, e.g., 1,000 or more infectious doses (ID^^50), of 2 sublines of the virus, one adapted to embryonated eggs and the other to mice.
3. Inoculation of mice with a small amount of virus, e.g., one to two ID^^50, did not cause hypothermia. In mice inoculated with about 2 ID^^50 of egg-line virus, the maximum level of virus in the lungs was lower than in mice inoculated with larger amounts of virus, and pulmonary lesions were present but failed to reach the level of 50% total lung-lesion score.
4. In mice inoculated with a moderate amount of egg-line virus, e.g., about 200 ID^^50, the maximum virus level in the lungs was reached 3 to 4 days before the occurrence of hypothermia. Time of occurrence and degree of hypothermia appeared to be closely related to the extent of pulmonary lesions.
5. The mouse-passaged virus caused more rapid development of hypothermia than the egg-line virus.
6. When ferrets were inoculated with a highly diluted (10**-7) mouse-passaged virus, a small proportion of them experienced inapparent infections and the rest of them escaped the infection.
7 With the increased sine of viral inocula, there was a good correlation between the size of infecting doses and the frequency of overt infections in ferrets.
8. Nasal tissues were the main locus of viral multiplication in ferrets at 72 hours after virus inoculatien. Viral multiplication in nasal tissues was demonstrated only in a small proportion of ferrets which were inoculated with a 10**-7 dilution of virus; however, when the size of vinal inoculum was increased above this level, all ferrets had viral growth in their nasal tissues.
9. The involvement of pulmonary tissues, viral growth in those tissues and the development of gross lung lesions were significantly rare. There was no clear-cut relationship between the size of infecting doses and the frequency of such pulmonary involvements in ferrets.
It is concluded 1) that hypothermia, rather than fever, proved to be a striking feature of the thermal response of mice to infection with influenza virus and it is related to the extent of pulmonary lesion rather than to the extent of virus increase per so, and therefore, the mouse could not be used as an experimental
animal for studying the difference between symptomless infection and illness without pulmonary consolidation, and 2) that though an intensive attempt has not been made for the establishment of inapparent influential infection in a large number of ferrets, the inoculation of ferrets with a very small amount of virus such as 1 ferret infectious dose might result in talc production of satisfactory model of inapparent influenzal infection with certain regularity.