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 this study, we chose to evaluate nine different APMV strains. Six of these were wt viruses representing six different serotypes, specifically biologically-derived wt APMV -2, -3, -5, -7, and -9, as well as a recombinant version of wt APMV-4. We chose wt APMV-2, -5, -7 and wt rAPMV-4 because we previously showed that replication of these viruses in cell culture does not require, and is not enhanced by, exogenous protease supplementation [8, 11, 13, 19] . Since this property is known to correlate with pantropic replication and virulence by NDV in chickens, it was possible that it might confer increased replication of these non-NDV APMVs in the rhesus macaques, although we note that this NDV paradigm had not been consistent for these non-NDV APMVs in chickens [16, 18, 19] . Wt APMV-3 was selected because this virus replicates to high titer in cell culture and a reverse genetics system has been developed for this serotype [9, 17] . Wt APMV-9 was selected because this virus replicates well in nasal turbinate and lungs of mice and hamsters [4, 5] . The other three viruses evaluated in this study were recombinant versions of APMV-2, -4, and -7 in which the naturally-occurring F protein cleavage sites were replaced by multi-basic cleavage sites containing the optimal furin motif. Specifically, rAPMV-2 (type 1 Africa) contained the multi-basic cleavage site RRRRRQF that is present in a virulent African NDV strain; rAPMV-4/Fc-BC contained the cleavage site RRQKRQF derived from the mesogenic NDV strain Baudette C; and rAPMV-7/Fcs-5B contained the cleavage site RRKKRQF derived from the velogenic NDV strain Nigeria/95. As noted, in NDV, multi-basic cleavage sites generally are associated with greater virulence in chickens. Rhesus macaques infected with APMV-2, -3, -4, -5, -7 and -9 showed no signs of any disease. APMV-2, -3, -7, and -9 replicated to low titers in upper respiratory tract as evidenced by low level of shedding of these viruses in nasal washes of animals from day 1 to 7. APMV-4 and -5 were found negative for shedding from upper respiratory tract. Similarly, we previously showed that rhesus macaques or African green monkeys that were infected by the combined intranasal and intratracheal routes with NDV strain BC were mostly negative for viral shedding [29] . Together, these results indicate that APMVs are highly restricted in replication in the upper respiratory tract of monkeys. However, analysis of BAL samples in the present study revealed replication of all these APMVs except wt APMV-5 in lower respiratory tract until day 6 post infection, although at different levels. The magnitude of shedding of wt APMV-2, rAPMV-2 (type 1 Africa), wt APMV-3, wt APMV-7, and rAPMV-7/Fcs-5B was moderate, while in case of wt APMV-9, wt rAPMV-4, and rAPMV-4/Fc-BC it was low. In contrast, a very low level of shedding (as determined by plaque assay of tracheal lavages or samples of lung tissues) was detected from lower respiratory tract of AGMs infected with NDV in earlier studies [29] . This indicates that, compared to NDV, wt and recombinant APMV-2 and 7, and wt APMV-3 replicate more efficiently in the lower respiratory tract in non-human primates. This apparent increased replication may provide an advantageous increase in immunogenicity for an expressed foreign antigen. Despite the somewhat greater replication of certain APMVs in the respiratory tract, analysis of fecal samples of rhesus macaques infected with all these APMVs revealed no shedding, su