Previous studies (9, 10) have indicated that HA titers of tissue-culture fluids can be used to estimate the concentration of total viral particles, there being approximately a 1 : 1 ratio between physical particles and erythrocytes at the endpoint of an HA titration. The number of particles determined in this manner must be regarded as a minimum estimate, and dependent on the efficiency of removal of hemagglutination inhibitors from the test material.

Since the infectivity titrations on experiments 1 and 2 were not done with sufficient precision to justify calculation of particle-infectivity ratios from the data in tables 1 and 2, it is worthwhile to present data from another experiment carried out under similar conditions in which significant comparisons could be made.

Newborn Swiss mice were inoculated subcutaneously with 0.05 ml. of undiluted infectious tissue-culture fluid, representing approximately 10^6 ID50 per mouse. Seven days later, 1 mouse from each of 5 litters was sacrificed and the kidneys harvested individually; the remaining mice were sacrificed, and the kidneys removed and pooled. The individual and pooled kidneys were made into 10 percent suspensions in

Eagle's medium, clarified, and titrated for infectivity by the MAP procedure; 5 mice per titration dilution were used. Two titration tests were done: the different kidneys of the individual mice being tested in separate tests and the pooled kidney suspension being titrated both times. HA titers were determined on the suspensions after RDE treatment, with 10^7 erythrocytes per HA tube; the estimate of particle number was taken as 10^7 times the HA titer. The results are given in table 6. The ratios obtained varied from 10^1.6 to 10^3.2 particles per ID50, with a mean of 10^2.15. This ratio is comparable to that generally found with polyoma-infected tissue-culture fluids (10).

Table 6. Relationship Of Infectivity And Ha Titers Of Mouse Kidney Suspensions

Infectivity titer*

HA titer*

Ratio

HA X 10^7 ID50

Right

Left

Right

Left

Right

Left

Mouse No.

kidney

kidney

kidney

kidney

kidney

kidney

1

10^7.8

10^8.5

800

1600

10^2.1

10^1.7

2

10^7.6

10^8.0

1600

1600

10^2.6

10^2.2

3

10^7.4

10^7.8

400

400

10^2.2

10^1.8

4

10^7.4

10^6.8

100

100

10^1.6

10^2.2

5

10^5.8

10^7.0

100

100

10^3.2

10^2.0

Pool

10^8.0

10^7.9

800,

800

10^2.0

*Titers expressed per 0.2 ml. of 10 percent suspension.

Development Of Hemagglutinin In Mouse Tissues After Infection With Low Virus Dosage

Since the results described in the preceding sections were obtained by inoculation with massive doses of virus, it appeared desirable to determine if a similar growth pattern was induced by low-dose inoculums. Newborn general purpose mice were inoculated subcutaneously with 0.05 ml. of a 10^-6 dilution of fluid from mouse embryo tissue cultures infected

Table 7. Ha Titers Of Salivary-Gland And Kidney Suspensions At Intervals After Inoculation Of Newborn Swiss Mice With A Low Dose Of Polyoma Virus

Time (days)

10

Group No. (4 mice each)

1

HA titer*

Salivary gland

<10

Kidney 1280

10

2

<10

1280

12

3

<10

80

12

4

<10

5120

15

5

<10

<10

15

6

<10

80

18

7

<10

<10

18

8

<10

<10

21

9

<10

<10

21

10

<10

<10

*Titers expressed per 0.2 ml. of 10 percent suspension.

with a strain of polyoma virus recovered from the viscera of a normal antibody-positive, AKR mouse from the colony of Dr. Ludwik Gross. This fluid had an infectivity titer of 10^7.5 TC ID50 per 0.2 ml. when assayed in mouse embryo tissue culture, and 10^8.2 mouse ID50 per 0.2 ml. when titrated by the MAP procedure; thus, each newborn mouse received approximately 10 to 40 infectious doses of virus. At intervals 2 groups of 4 mice each were sacrificed, and HA titers were determined on the pooled salivary glands and on the pooled kidneys (table 7). No hemagglutinin was detected in the salivary-gland extracts, but the kidney extracts gave titers comparable to those obtained with large inoculums, and the time course of hemagglutinin development and disappearance was similar to that in the previous groups.