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

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.

Unique Variants of Avian Coronaviruses from Indigenous Chickens in Kenya

“…Raw NGS data were preprocessed and analyzed using a nontargeted taxonomic classification and assembly pipeline developed by BASE₂BIO LLC (Oshkosh, WI, USA), as recently described [ 35 ]. The pipeline utilized megahit v 1.2.9 for de novo assembly using default parameters [ 36 ].…”

Comparable outcomes from long and short read random sequencing of total RNA for detection of pathogens in chicken respiratory samples

“…More recently, the next generation sequencing (NGS) platforms have changed this paradigm by providing the possibility for simultaneous diagnostic testing and sequencing of novel and re-emerging pathogens directly from a clinical sample ( 23 ). We have recently optimized conditions for efficient detection or multiple respiratory pathogens in poultry by directly sequencing clinical samples with the Illumina short read sequences ( 24 – 26 ). The widespread application of these sequencing platforms for routine diagnostics is still limited due to the associated longer processing time, complex bioinformatics expertise of random sequencing data analysis, and higher cost, hence need for the improving recent alternative diagnostic methods to counter these challenges.…”