Emergence of a highly pathogenic avian influenza virus from a low-pathogenic progenitor

Abstract

Avian influenza (AI) viruses of the H7 subtype have the potential to evolve into highly pathogenic (HP) viruses that represent a major economic problem for the poultry industry and a threat to global health. However, the emergence of HPAI viruses from low-pathogenic (LPAI) progenitor viruses currently is poorly understood. To investigate the origin and evolution of one of the most important avian influenza epidemics described in Europe, we investigated the evolutionary and spatial dynamics of the entire genome of 109 H7N1 (46 LPAI and 63 HPAI) viruses collected during Italian H7N1 outbreaks between March 1999 and February 2001. Phylogenetic analysis revealed that the LPAI and HPAI epidemics shared a single ancestor, that the HPAI strains evolved from the LPAI viruses in the absence of reassortment, and that there was a parallel emergence of mutations among HPAI and later LPAI lineages. Notably, an ultradeep-sequencing analysis demonstrated that some of the amino acid changes characterizing the HPAI virus cluster were already present with low frequency within several individual viral populations from the beginning of the LPAI H7N1 epidemic. A Bayesian phylogeographic analysis revealed stronger spatial structure during the LPAI outbreak, reflecting the more rapid spread of the virus following the emergence of HPAI. The data generated in this study provide the most complete evolutionary and phylogeographic analysis of epidemiologically intertwined high- and low-pathogenicity viruses undertaken to date and highlight the importance of implementing prompt eradication measures against LPAI to prevent the appearance of viruses with fitness advantages and unpredictable pathogenic properties.

Importance: The Italian H7 AI epidemic of 1999 to 2001 was one of the most important AI outbreaks described in Europe. H7 viruses have the ability to evolve into HP forms from LP precursors, although the mechanisms underlying this evolutionary transition are only poorly understood. We combined epidemiological information, whole-genome sequence data, and ultradeep sequencing approaches to provide the most complete characterization of the evolution of HPAI from LPAI viruses undertaken to date. Our analysis revealed that the LPAI viruses were the direct ancestors of the HPAI strains and identified low-frequency minority variants with HPAI mutations that were present in the LPAI samples. Spatial analysis provided key information for the design of effective control strategies for AI at both local and global scales. Overall, this work highlights the importance of implementing rapid eradication measures to prevent the emergence of novel influenza viruses with severe pathogenic properties.

FIG 1

Map of the discrete geographical locations used in the spatial analysis. Locations are colored gray for Verona; red for Brescia; light blue for Mantua; yellow for Ferrara, Ravenna, Rovigo, and Bologna; orange for Venice, Padua, and Vicenza; violet for Pordenone and Udine; blue for Bergamo and Como; and green for Milan and Lodi.

FIG 2

ML tree of the HA gene segment of H7N1 avian influenza virus. Viruses are colored blue for LPAI viruses collected from March 1999 to January 2000, pink for LPAI viruses sampled between August 2000 and February 2001, and yellow for HPAI viruses (December 1999 to March 2000). The numbers at nodes represent bootstrap values (>70%), while branch lengths are scaled according to the numbers of nucleotide substitutions per site. The number highlighted in red represents the number of amino acid changes identified along the main branches of the tree. Sequences obtained using the NGS platform are marked with a black square. The closest relative of the LPAI-1 group is identified with a black arrow. The tree is midpoint rooted for clarity only.

FIG 3

MCC tree inferred for HA gene sequences of the Italian H7N1 viruses. Sequences are colored according to the province of origin. Light blue, Mantua; gray, Verona; red, Brescia; yellow, Ferrara-Ravenna-Bologna-Rovigo; orange, Venice-Padua-Vicenza; blue, Bergamo-Como; green, Milan-Lodi; violet, Pordenone-Udine. The numbers at branch points represent the probability of spatial states. The location state posterior probability distributions for the root and for the internal branch of the HPAI group are shown in the two graphs to the upper right of the phylogenetic tree.