Evaluation of the Replication, Pathogenicity, and Immunogenicity of Avian Paramyxovirus (APMV) Serotypes 2, 3, 4, 5, 7, and 9 in Rhesus Macaques

Khattar, Sunil K.; Nayak, Baibaswata; Kim, Shin-Hee; Xiao, Sa; Samal, Sweety; Paldurai, Anandan; Buchholz, Ursula J.; Collins, Peter L.; Samal, Siba K.

The serum antibody levels to the specific APMV serotype used for immunization were evaluated pre-and post-immunization by hemagglutination inhibition (HI) assay, virus neutralization (VN) assay, and Western blot analysis, except for the serum antibody response to APMV-5, which was evaluated by VN and by neuraminidase inhibition (NAI) assay. For the HI assay, 25 ml of each serum sample was first treated with 50 ml of receptor destroying enzyme II (catalog number YCC 340-122; Accurate Chemical and Scientific, Westbury NY) at a 1:3 ratio (vol/vol) at 37uC overnight. Then, 25 ml of 5% sodium citrate was added and incubation was continued at 56uC for 30 min. Each serum sample was allowed to cool to room temperature and 100 ml of packed chicken RBCs were added. After incubation at 4uC for 30 min, samples were centrifuged at 1000 6 g for 10 min. Supernatants were used for HI assay. For the HI assay, twofold serial dilutions of treated sera (50 ml) were prepared, and each dilution was combined with 4 HA units of a particular live and homologous APMV serotype. Following 1 h of incubation, 50 ml of 1% chicken RBC was added and incubated for 30 min at room temperature, and HA was scored as the mean reciprocal log 2 (6 standard errors of the mean) of the highest serum dilution causing complete inhibition of four HA units of the indicated APMV.