Field Monitoring of Avian Influenza Viruses: Whole-Genome Sequencing and Tracking of Neuraminidase Evolution Using 454 Pyrosequencing

Abstract

Adaptation of avian influenza viruses (AIVs) from waterfowl to domestic poultry with a deletion in the neuraminidase (NA) stalk has already been reported. The way the virus undergoes this evolution, however, is thus far unclear. We address this question using pyrosequencing of duck and turkey low-pathogenicity AIVs. Ducks and turkeys were sampled at the very beginning of an H6N1 outbreak, and turkeys were swabbed again 8 days later. NA stalk deletions were evidenced in turkeys by Sanger sequencing. To further investigate viral evolution, 454 pyrosequencing was performed: for each set of samples, up to 41,500 reads of ca. 400 bp were generated and aligned. Genetic polymorphisms between duck and turkey viruses were tracked on the whole genome. NA deletion was detected in less than 2% of reads in duck feces but in 100% of reads in turkey tracheal specimens collected at the same time. Further variations in length were observed in NA from turkeys 8 days later. Similarly, minority mutants emerged on the hemagglutinin (HA) gene, with substitutions mostly in the receptor binding site on the globular head. These critical changes suggest a strong evolutionary pressure in turkeys. The increasing performances of next-generation sequencing technologies should enable us to monitor the genomic diversity of avian influenza viruses and early emergence of potentially pathogenic variants within bird flocks. The present study, based on 454 pyrosequencing, suggests that NA deletion, an example of AIV adaptation from waterfowl to domestic poultry, occurs by selection rather than de novo emergence of viral mutants.

Deep sequencing: Becoming a critical tool in clinical virology

“…Given the importance of transmission and adaptation of avian influenza viruses, and more recently swine strains for epidemics and pandemics in humans, an important number of studies based on deep sequencing techniques have described avian [77–81] and porcine [82–85] influenza virus evolution. For example, the selection of minority variants with a deletion in the neuraminidase (NA) gene that allows the adaptation of avian influenza viruses from waterfowl to domestic poultry [77], suggests a high frequency of mixed infections and genetic reassortment within these viruses [78, 79, 85].…”

“…For example, the selection of minority variants with a deletion in the neuraminidase (NA) gene that allows the adaptation of avian influenza viruses from waterfowl to domestic poultry [77], suggests a high frequency of mixed infections and genetic reassortment within these viruses [78, 79, 85]. Other studies described the prevalence and spread patterns of different human influenza viruses in specific geographic areas, based not only on the hemagglutinin (HA) and NA genes but by analyzing all viral segments [86–89].…”

Emergence of a Highly Pathogenic Avian Influenza Virus from a Low-Pathogenic Progenitor

“…In most cases, the number of LPAI isolates collected prior to the emergence of the HPAI strain was insufficient to determine the genesis of the virulent phenotype, including whether this occurs via a stepwise evolutionary process (13-15, 17, 18, 31). Importantly, improved deep-sequencing technologies allow for greater insight into the emergence of new variants of influenza type A viruses (32,33), potentially including HPAI strains.…”

A low-pathogenic avian influenza H6N1 outbreak in a turkey flock in France: a comprehensive case report

“…A few reports indicated infections of commercial turkeys by LPAI virus of migratory waterfowl origin: in the USA in the mid-1960s (Halvorson et al, 1985), as well as more recently in Italy (Campitelli et al, 2004). To date the transmission of AI viruses from commercial ducks to domestic turkeys has never been formally demonstrated in the field, but is suggested by recent observations (Croville et al, 2012). However, the literature provides several reports of transmission of AIVs from commercial swine to turkeys, including H3N2 (Yassine et al, 2007) and, recently, pandemic H1N1 subtypes (Pantin-Jackwood et al, 2010.…”