1 3 Evolutionary trajectories of two distinct avian influenza epidemics: 4 Parallelisms and divergences 5 6 7 Alice Fusaro a,⇑ , Luca Tassoni a , Joseph Hughes b , Adelaide Milani a , Annalisa Salviato a , Alessia Schivo a , 8 Pablo Murcia b , Lebana Bonfanti a , Giovanni Cattoli a , Isabella Monne a 9 a Istituto Zooprofilattico Sperimentale delle Venezie, viale dell’Università, 10, Legnaro (PD), Italy 10 b MRC-University of Glasgow Center for Virus Research, 464 Bearsden Road, Glasgow, United Kingdom 11 12 14 article info 15 Article history: 16 Received 13 March 2015 17 Received in revised form 5 May 2015 18 Accepted 19 May 2015 19 Available online xxxx 20 Keywords: 21 Avian influenza virus 22 H7 subtype 23 Parallel evolution 24 Deep sequencing 25 Evolutionary dynamics 26 Molecular analysis 27 28 abstract 29 Influenza A virus can quickly acquire genetic mutations that may be associated with increased virulence, 30 host switching or antigenic changes. To provide new insights into the evolutionary dynamics and the 31 adaptive strategies of distinct avian influenza lineages in response to environmental and host factors, 32 we compared two distinct avian influenza epidemics caused by the H7N1 and H7N3 subtypes that circu- 33 lated under similar epidemiological conditions, including the same domestic species reared in the same 34 densely populated poultry area for similar periods of time. 35 The two strains appear to have experienced largely divergent evolution: the H7N1 viruses evolved into 36 a highly pathogenic form, while the H7N3 did not. However, a more detailed molecular and evolutionary 37 analysis revealed several common features: (i) the independent acquisition of 32 identical mutations 38 throughout the entire genome; (ii) the evolution and persistence of two sole genetic groups with similar 39 genetic characteristics; (iii) a comparable pattern of amino acid variability of the HA proteins during the 40 low pathogenic epidemics; and (iv) similar rates of nucleotide substitutions. These findings suggest that 41 the evolutionary trajectories of viruses with the same virulence level circulating in analogous epidemio- 42 logical conditions may be similar. In addition, our deep sequencing analysis of 15 samples revealed that 43 17 of the 32 parallel mutations were already present at the beginning of the two epidemics, suggesting 44 that fixation of these mutations may occur with different mechanisms, which may depend on the fitness 45 gain provided by each mutation. This highlighted the difficulties in predicting the acquisition of muta- 46 tions that can be correlated to viral adaptation to specific epidemiological conditions or to changes in 47 virus virulence. 48 Ó 2015 Published by Elsevier B.V. 49 50 51 52 1. Introduction 53 Since the 90s, outbreaks caused by avian influenza virus of the 54 H7 subtype have been frequently reported in domestic poultry 55 throughout the world, causing not only important damage to the 56 poultry industry, but also a great concern for human health, as 57 demonstrated by the recent H7N9 epidemic in China (Chen et al., 58 2013). Once in poultry, this subtype can evolve into a highly patho- 59 genic form. While the low pathogenic avian influenza (LPAI) virus 60 causes only a mild, primarily respiratory disease in the infected 61 domestic fowl along with production drops, the highly pathogenic 62 avian influenza virus (HPAI) produces an extremely serious disease 63 that can devastate the poultry population. 64 As shown in previous studies (Campitelli et al., 2004; 65 Lebarbenchon and Stallknecht, 2011), the H7 viruses collected 66 from poultry are genetically related to the viruses from wild birds, 67 suggesting relative frequent interspecies transmissions. Similarly, 68 the distribution of the HPAI H7 strains throughout the phyloge- 69 netic trees indicates the evolution of multiple independent highly 70 pathogenic forms from the low pathogenic progenitors (Röhm 71 et al., 1995; Lebarbenchon and Stallknecht, 2011; Abdelwhab 72 et al., 2014). 73 Following the transmission from wild to domestic birds the 74 virus can experience an accelerated fixation of beneficial mutations 75 to adapt to new species and new environmental conditions. 76 Sequence adaptations to land-based avian species, such as the 77 acquisition of new additional glycosylation sites near the hemag- 78 glutinin (HA) receptor binding site (RBS), deletions at the http://dx.doi.org/10.1016/j.meegid.2015.05.020 1567-1348/Ó 2015 Published by Elsevier B.V. ⇑ Corresponding author at: Division of Comparative Biomedical Sciences, OIE and National Reference Laboratory for avian influenza & Newcastle disease, FAO Reference Centre for animal influenza and Newcastle disease, OIE Collaborating Centre for Diseases at the Human-Animal Interface, Istituto Zooprofilattico Sper- imentale delle Venezie, viale dell’Università, 10, 35020 Legnaro (PD), Italy. E-mail address: afusaro@izsvenezie.it (A. Fusaro). Infection, Genetics and Evolution xxx (2015) xxx–xxx Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid MEEGID 2351 No. of Pages 10, Model 5G 20 May 2015 Please cite this article in press as: Fusaro, A., et al. Evolutionary trajectories of two distinct avian influenza epidemics: Parallelisms and divergences. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.020