From 78 patients under the surveillance of Smallpox Eradication Program in Bihar State, India, 113 virological specimens were collected by the authors in February and March, 1975. Specimens were sent through W.H.O. to the authors' laboratory in Tokyo, Japan, for the assay of virus contents under rigid freezing in liquid nitrogen.
1) Specimens from 55 cases gave positive virus isolation on chick chorioallantoic membranes (CAM) ; all cases were smallpox-positive in immunofluorescence (IF) diagnosis in the field study.
2) Isolated viruses were identified with variola virus by the hyperplastic focus formation on HeLa cell monolayers.
3) Average virus titers of positive specimens expressed in terms of log10 PFU (pock-forming units) /specimen were 2.87, 4.61, and 4.81 in throat swab (TS, 7 cases), vesicular-pustular fluid (VP, 20 cases) and scab (SB, 41 cases), respectively. Highest titers were 4.01 (TS), 6.06 (VP), and 6.78 (SB) . There was significant correlation between the virus contents of TS and VP specimens and the days of illness at the collection, whereas no significant decline in virus contents of SB specimens was observed up to the 30th day of illness.
4) Scabs from one case were kept at room temperature under dry conditions. No significant loss in virus contents was shown during the first 4 weeks and a loss of only 0.7 log10 at the end of 16 weeks' storage, suggesting an important role of scabs in the genesis of remote outbreaks.
5) Typing of isolated virus strains by temperature-sensitivity (Ts) of hyperplastic focus formation on HeLa cell monolayers were attempted by a semi-micro culture system. Of 55 isolates, 51 were variola major type, while the remaining four strains demonstrated Ts between V. major and minor, suggesting the presence of an intermediate type in Indian smallpox. Epidemiological situations among cases of intermediate isolates are discussed.
