Techniques 
Bird sera (fall 1966-spring 1968, many of them pooled by species), 
plasmas (spring 1968, again often pooled), and erythrocytes (fall 1968- 
fall 1969, not pooled) were received at YARU frozen and stored at -70®C 
or colder until inoculated intracerebrally (i*c.) into 1-4 day-old Swiss 
mice of YARU Charles River stock, 6 mice per sample. Brains of mice sick 
or dead during a 2-week observation period were sub-passaged as 10% sus¬ 
pension until a passage line was established. Reisolation was attempted 
in many cases. 
Characterization of infectious agents included serological comparison 
with mouse pathogens (mostly arboviruses) in the YARU-WHO Intemationl Re¬ 
ference Centre for Arboviruses collection, and in the cases of some of the 
new viruses, filtration through gradacol membranes, deoxycholate sensitivity, 
and infection of, and transmission by Aedes aegypti mosquitoes of the YARU 
colony. Details of the identification procedures as used at YARU are descri¬ 
bed by Shope (1970). 
Ticks were pooled, limiting the pools to individuals of the same tick 
species and host species, with 25 ticks or less per pool. They were tritur¬ 
ated in 2.0 ml. amounts of phosphate buffered, 0.75% bovine albumin contain¬ 
ing antibiotics, centrifuged and the supernatant inoculated into mice as above. 
Serological tests were carried out on bird sera and plasmas stored at 
-70®C until processing. For HI tests the techniques of Clarke and Casals 
(1958) were used with acetone extraction of sera and plasmas and a micro tech¬ 
nique in leucite plates. The sera were screened at a 1:10 dilution and those 
positive were titered. HI tests of the 1966 sera were done with antigens of 
group A ( Middelburg, Sindbis, Semliki Forest, Y-62-33, chikungunya, Ndumu), 
group B (Entebbe bat, yellow fever, Wesselsbron, Israel turkey meningo-enceph- 
alitis, Zika, Ntaya, louping ill, West Nile) and also Tahyna and Bunyamwera. 
67 
