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.

In case of APMV-5, antibody titers were measured by NAI assay. For the NAI assay, APMV-5 strain budgerigar/Kunitachi/ 74 was used as the source of NA. The NA activity of APMV-5 was measured by a modified fluorometric assay [49] . Briefly, serial twofold dilutions of serum samples were prepared in 20 ml volumes of enzyme buffer (33 mM 2-N-morpholino ethanesulfonic acid [MES], pH 6.5, and 4 mM calcium chloride) in a 96-well plate. 20 ml APMV-5, diluted in enzyme buffer to a constant NA amount (an optical density at 450 nm [OD 450 ] of 100,000), was added as source of NA, and incubated for 1 h at room temperature. Ten microliters of 12.5% (vol/vol) dimethyl sulfoxide was added to each well of a fluorometric assay plate (black 96-well plates; Microfluor, Franklin, MA). Ten microliters of each serum and virus mixture was transferred in duplicate to the assay plate. 10 ml of diluted virus or enzyme buffer alone were used as positive and negative controls, respectively. The reaction was initiated by the addition of 30 ml of substrate mix [1 volume of 330 mM MES, pH 6.4; 3 volumes of 10 mM calcium chloride; and 2 volumes of 0.5 mM 29-(4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (MUN) (Sigma)] to give a final concentration of 100 mM MUN in the assay. The reaction mixture was incubated at 37uC for 15 min with shaking, and the reaction was terminated by the addition of 150 ml of termination buffer (0.014 M sodium hydroxide in 83% [vol/vol] ethanol). The extent of the reaction was quantified by fluorometry at an excitation wavelength of 360 nm and an emission wavelength of 450 nm using the Victor3 multilabel plate reader (PerkinElmer). Readings from the substrate blanks were subtracted from the virus sample readings. The average background-corrected NA activity was calculated from 12 independent wells. The neuraminidase inhibition (NAI) titer for each sample was reported as the reciprocal log 2 of the highest serum dilution resulting in a 50% or greater reduction in input NA activity.