Isolation and Molecular Characterization of Avian Metapneumovirus Subgroup B Virus Isolated from a Chicken Farm in...

Avian metapneumovirus (aMPV) is a contagious respiratory disease affecting mainly turkeys and chickens, causing respiratory problems along with reproductive disorders such as egg drop. In the United States, the recent introduction of the aMPV subgroup B initially and later subgroup A in poultry farms resulted in significant economic losses. To isolate aMPV-B, 22 clinical samples from chicken and turkey farms that tested strongly positive by real-time RT-PCR were inoculated in four different cell lines, including turkey tracheal cells (TT cells), the Japanese quail fibrosarcoma cell line (QT35), chicken embryo fibroblasts, and Vero E6 cells. After five blind passages, successful isolation was achieved in one tracheal sample pool inoculated on TT cells. Real-time RT-PCR, indirect immunofluorescence assay, and next-generation sequencing confirmed the successful isolation. After primary isolation, successful adaptation and propagation in Vero E6 cells was achieved. The whole-genome sequence-based molecular characterization of this isolate (TT P6) revealed 100% identity with the field strain sequence of the source sample (ADRDL-6). The TT P6 isolate and all newly detected U.S. aMPV-B isolates showed >99% identity and clustered separately from previously reported Asian, European, and South American sequences, based on whole-genome and individual-gene analyses. The highest heterogeneity was detected in the G protein, which exhibited unique amino acid substitutions, and the SH protein. Interestingly, TT P6 possessed unique threonine157isoleucine (Thr157Ile) and isoleucine322threonine (Ile322Thr) substitutions compared to all other U.S. aMPV-Bs. However, turkey origin isolate NC 23734-GA showed maximum identity with TT P6 isolate (chicken origin), including a unique Ile322Thr mutation. Complete Vero cell adaptation and in-depth molecular characterization of TT P6 isolate make it a suitable candidate for pathogenesis study and vaccine development. The distinct phylogenetic clustering and unique amino acid changes of U.S. aMPV-Bs, particularly in the G protein, suggest the possibility of new variant emergence in the United States, requiring further genetic and in vivo studies.