Detection and Genome Sequence Analysis of Avian Metapneumovirus Subtype A Viruses Circulating in Commercial Chicken Flocks in Mexico

SIMPLE SUMMARY: The use of next-generation sequencing to identify pathogens in clinical samples is continually improving, providing genomic data that can be used to: (1) identify what viruses are present in particular samples, (2) determine the viral genotype, pathotype, or lineage, (3) predict which viruses are likely to cause severe disease, and (4) help guide what vaccines to use for control efforts. As part of a project to improve NGS for diagnostics, samples were collected from commercial chicken farms in Mexico and transported on FTA cards. One of the commonly identified viruses was avian metapneumovirus, an important upper respiratory avian pathogen consisting of subtypes A-D. Complete genomes of seven subtype-A isolates were identified, which are distinct from other previously reported subtype-A strains. Although subtype-A viruses were previously reported in Mexico, this is the first comprehensive complete genome sequence analysis of Mexican subtype-A viruses. These data provide a much clearer picture of viruses circulating in Mexico and demonstrate the value of next-generation sequencing to identify pathogens where surveillance is not routinely performed. Because subtype-A is not found in the U.S., a modified real-time reverse-transcriptase polymerase chain reaction test was evaluated that can quickly identify the virus in poultry samples. ABSTRACT: Avian metapneumoviruses (aMPV subtypes A-D) are respiratory and reproductive pathogens of poultry. Since aMPV-A was initially reported in Mexico in 2014, there have been no additional reports of its detection in the country. Using nontargeted next-generation sequencing (NGS) of FTA card-spotted respiratory samples from commercial chickens in Mexico, seven full genome sequences of aMPV-A (lengths of 13,288–13,381 nucleotides) were de novo assembled. Additionally, complete coding sequences of genes N (n = 2), P and M (n = 7 each), F and L (n = 1 each), M2 (n = 6), SH (n = 5) and G (n = 2) were reference-based assembled from another seven samples. The Mexican isolates phylogenetically group with, but in a distinct clade separate from, other aMPV-A strains. The genome and G-gene nt sequences of the Mexican aMPVs are closest to strain UK/8544/06 (97.22–97.47% and 95.07–95.83%, respectively). Various amino acid variations distinguish the Mexican isolates from each other, and other aMPV-A strains, most of which are in the G (n = 38), F (n = 12), and L (n = 19) proteins. Using our sequence data and publicly available aMPV-A data, we revised a previously published rRT-PCR test, which resulted in different cycling and amplification conditions for aMPV-A to make it more compatible with other commonly used rRT-PCR diagnostic cycling conditions. This is the first comprehensive sequence analysis of aMPVs in Mexico and demonstrates the value of nontargeted NGS to identify pathogens where targeted virus surveillance is likely not routinely performed.