# Inventory of molecular markers affecting biological characteristics of avian influenza A viruses

Annika Suttie, Yi-Mo Deng, Andrew R. Greenhill, Philippe Dussart, Paul F. Horwood, Erik A. Karlsson

► To cite this version:

Annika Suttie, Yi-Mo Deng, Andrew R. Greenhill, Philippe Dussart, Paul F. Horwood, et al.. Inventory of molecular markers affecting biological characteristics of avian influenza A viruses. Virus Genes, 2019, 55 (6), pp.739 - 768. 10.1007/s11262-019-01700-z. pasteur-03214753

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Distributed under a Creative Commons Attribution 4.0 International License
# Inventory of molecular markers affecting biological characteristics of avian influenza A viruses

Annika Suttie<fnref n="1" />,<fnref n="2" />,<fnref n="3" /> · Yi-Mo Deng<fnref n="3" /> · Andrew R. Greenhill<fnref n="2" /> · Philippe Dussart<fnref n="1" /> · Paul F. Horwood<fnref n="4" /> · Erik A. Karlsson<fnref n="1" /> ID

Received: 9 June 2019 / Accepted: 9 August 2019

© The Author(s) 2019

## Abstract

Avian influenza viruses (AIVs) circulate globally, spilling over into domestic poultry and causing zoonotic infections in humans. Fortunately, AIVs are not yet capable of causing sustained human-to-human infection; however, AIVs are still a high risk as future pandemic strains, especially if they acquire further mutations that facilitate human infection and/or increase pathogenesis. Molecular characterization of sequencing data for known genetic markers associated with AIV adaptation, transmission, and antiviral resistance allows for fast, efficient assessment of AIV risk. Here we summarize and update the current knowledge on experimentally verified molecular markers involved in AIV pathogenicity, receptor binding, replicative capacity, and transmission in both poultry and mammals with a broad focus to include data available on other AIV subtypes outside of A/H5N1 and A/H7N9.

**Keywords** Avian influenza · Influenza virus · Molecular marker · Mutation · Risk assessment · Molecular inventory

## Introduction

Avian influenza viruses (AIVs) (Family: *Orthomyxoviridae*, genus: *Influenzavirus A*) are negative sense, single-stranded RNA viruses found globally in their natural reservoir hosts, wild waterfowl, and other aquatic birds (mainly of the orders *Anseriformes* and *Charadriiformes*). AIV genomes are composed of eight genomic RNA segments encoding at least twelve viral proteins. Viral nomenclature is based on combinations of the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). To date, sixteen HA

(H1–H16) and nine NA (N1–N9) subtypes circulate in wild aquatic birds [1], and two novel HA subtypes (H17 and H18) and two novel NA subtypes (N10 and N11) have recently been identified in bats [2–6]. AIVs are sporadically transmitted from waterfowl to domestic avian species, resulting in a number of stable AIV lineages in domestic poultry. These domestic lineages typically circulate in poultry flocks as low pathogenic (LPAIV) variants, causing little to no apparent illness; however, some subtypes (namely A/H5 and A/H7) have the potential to mutate to form highly pathogenic (HPAIV) variants capable of causing high mortality rates in domestic avian species. These HPAIV lineages then spread back to wild bird species, potentiating global spread [1].

Occasionally, AIVs spillover into mammalian species such as humans, swine, marine mammals, and various members of the Equidae, Felidae, and Canidae families [7–10], generally through close contact with domestic poultry, the primary intermediate host [11]. These spillovers sometimes result in stable, mammalian-adapted lineages as have occurred with subtype A/H3N8 AIVs in equines and canines [9]. A number of zoonotic infections with different AIV subtypes have occurred in humans, including: A/H5 (H5N1, H5N6), A/H6N1, A/H7 (H7N2, H7N3, H7N4, H7N7, H7N9), A/H9N2, and A/H10 (H10N7, H10N8) [12]. Clinical symptoms vary in severity from asymptomatic infections, to conjunctivitis or influenza-like-illness, to

Edited by William Dundon.

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Erik A. Karlsson
ekarlsson@pasteur-kh.org

<footnote n="1">¹ Virology Unit, Institut Pasteur du Cambodge, Institut Pasteur International Network, 5 Monivong Blvd, PO Box #983, Phnom Penh, Cambodia</footnote>

<footnote n="2">² School of Health and Life Sciences, Federation University, Churchill, Australia</footnote>

<footnote n="3">³ World Health Organization Collaborating Centre for Reference and Research on Influenza, Peter Doherty Institute for Infection and Immunity, Melbourne, Australia</footnote>

<footnote n="4">⁴ College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, QLD, Australia</footnote>
severe acute respiratory illness. Infections with particularly virulent viral subtypes, such as A/H5 and A/H7, or infections in immunocompromised, high-risk hosts can cause high morbidity, respiratory distress and/or multiple-organ failure, and, in some cases, mortality [13]. Fortunately, minimal human-to-human transmission occurs in human AIV cases; however, each zoonotic event in mammals represents a risk for AIVs to adapt replicative and transmissible properties in the mammalian host. Indeed, several studies using ferret models have shown that these AIVs have the potential to mutate into airborne transmissible forms, potentially with some mutations already present in nature [14, 15]. However, while these observations are very important, point mutations associated with airborne transmission between ferrets are not universal, as they may not confer similar changes in biological characteristics of these viruses in other mammalian species, including humans.

Given the continual circulation of AIVs in wild birds and domestic poultry, the potential for human spillover, and the mutable nature of AIV itself, it is paramount to understand the potential risk of any emerging AIV. Indeed, risk assessment tools are vital in pandemic preparedness planning [16]. Genetic variation resulting in changes in viral properties such as receptor binding, replicative capacity, and transmission is a critical component of risk assessment. Historically, assessing these factors occurs in vivo; however, the ability to evaluate these properties in silico from sequence data allows for faster, more efficacious assessment of novel, emerging strains. Numerous studies identify molecular markers for AIV risk, and several previous papers compile markers of interest. Here, we summarize and update the current knowledge on experimentally verified molecular markers affecting biological characteristics of avian influenza viruses important for risk assessment and broaden the scope outside of the A/H5N1 and A/H7N9 subtypes.

# Methodology

## Data collection

All available information on AIV molecular markers/mutations was collected from: the CDC H5N1 Genetic changes inventory [17], the WHO Working Group on Surveillance of Influenza Antiviral Susceptibility (WHO-AVWG) [18], and publications summarizing AIV mutations and molecular markers affecting biological characteristics and potential risk [19–21]. Journal articles were sourced for each specific subtype using PubMed searches with MeSH Terms, Boolean operators and wild cards. For example, terms used to search for mutations in H6 AIVs: (influenza A virus[MeSH Terms]) AND (mutation OR mutagenesis OR

virulence[MeSH Terms]) AND (H6[Title/Abstract] OR H6N*[Title/Abstract]).

## Inclusion/exclusion

All mutations/molecular markers from influenza viruses of avian origin were included in the initial screening and tabulation, including viruses isolated from humans AIV cases. However, we do not consider this inventory as an exhaustive list. Several studies computationally predict molecular markers of viral adaptation to humans [22–26]. While this work is extremely valuable, experimental validation of the majority of the markers described in these studies is not available and, therefore, necessitated exclusion from this data summary. In addition, since this inventory focuses on AIV and zoonotic infection, genome mutations in human seasonal influenza viruses were also excluded. As several publications observe the same mutations causing similar biological characteristics that could indicate risk markers, we excluded duplicate information from the tables.

## Numbering

For all data presented, HA mutations are numbered according to the H3 subtype to maintain consistency with available literature; however, H5 numbering was also included in the table. N2 numbering was used for NA as this is most commonly used in the current literature. Internal proteins (including deletions) and deletions in NA are numbered according to the full length of A/Goose/Guangdong/1/1996 genome segments.

# Surface proteins

## Hemagglutinin (HA; Table 1)

Hemagglutinin is a homotrimeric transmembrane protein and is the most abundant protein present on the surface of influenza virions. For virions to successfully enter and replicate in host cells, host proteases cleave the HA0 precursor into two subunits, HA1 and HA2. The main role of the HA1 subunit is to initiate infection by recognizing and binding receptors on the host cell surface. After internalization and entrance into the endosomal pathway, the HA2 subunit fuses the viral and endosomal membranes, creating a pore for viral RNA entry into the host cell, and initiation of transcription and translation of viral products [27, 28]. Several mutations in HA are associated with changes in viral fitness and transmissibility, as they affect viral receptor binding avidity/ specificity or viral membrane fusion activities (Table 1).
<table><caption>**Table 1** Molecular markers/motifs in the hemagglutinin (HA; segment 4) gene of influenza virus experimentally verified molecular markers involved in AIV pathogenicity, receptor binding, replicative capacity, and transmission in both poultry and mammals</caption><thead><tr><th>Mutation/motif<br>H3 numbering<sup>a</sup></th><th>H5 numbering<sup>b</sup></th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td>D101N</td><td>D94N</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[200]</td></tr><tr><td>S126N</td><td>S121N</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[201]</td></tr><tr><td>S137A</td><td>S133A</td><td>Increased pseudovirus binding to α2-6</td><td>H5N1</td><td>[44]</td></tr><tr><td>A138V</td><td>A134V</td><td>Increased infectivity in SIAT Cells</td><td>H5N1</td><td>[202, 203]</td></tr><tr><td>G143R</td><td>G139R</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>S158N</td><td>S154N</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[201]</td></tr><tr><td>N158D</td><td>N154D</td><td>Decreased virulence in mice</td><td>H9N2</td><td>[205]</td></tr><tr><td>S159N</td><td>S155N</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[201]</td></tr><tr><td>T160A</td><td>T156A</td><td>Increased virus binding to α2-6, increased transmission in guinea pigs</td><td>H5N1</td><td>[201, 206]</td></tr><tr><td>G186V</td><td>G182V</td><td>Increased virus binding to α2-3</td><td>H7N9</td><td>[207]</td></tr><tr><td>N186K/D</td><td>N182K/D</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[201, 203, 204, 206]</td></tr><tr><td>V186N</td><td>V182N</td><td>Increased binding to α2-6, decreased binding to α2-3</td><td>H13N6</td><td>[208]</td></tr><tr><td>P186L</td><td>P182L</td><td>Decreased binding to α2-3</td><td>H6N1</td><td>[209]</td></tr><tr><td>D187G</td><td>D183G</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[47]</td></tr><tr><td>E190G</td><td>E186G</td><td>Increased virus binding to α2-6, maintained α2-3 binding, decreased virulence in mice</td><td>H5N1</td><td>[47, 210]</td></tr><tr><td>E190V</td><td>E186V</td><td>Decreased binding to α2-3 and α2-6</td><td>H6N2</td><td>[211]</td></tr><tr><td>T190V</td><td>T186V</td><td>Enhances binding affinity to mammalian cells and replication in mammalian cells</td><td>H9N2</td><td>[212]</td></tr><tr><td>T192I</td><td>T188I</td><td>Increased pseudovirus binding to α2-6</td><td>H5N1</td><td>[44]</td></tr><tr><td>K193R/T</td><td>K189R/T</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[201, 213]</td></tr><tr><td>Q196R/H</td><td>Q192R/H</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[47, 204, 214]</td></tr><tr><td>N197K</td><td>N193K</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>V214I</td><td>V210I</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[214]</td></tr><tr><td>G225D</td><td>G221D</td><td>Increased virus binding to α2,6</td><td>H6N1</td><td>[43]</td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th colspan="2">Mutation/motif</th><th rowspan="2">Phenotype</th><th rowspan="2">Subtypes tested</th><th rowspan="2">References</th></tr><tr><th>H3 numbering<sup>a</sup></th><th>H5 numbering<sup>b</sup></th></tr></thead><tbody><tr><td rowspan="6">Q226L</td><td rowspan="6">Q222L</td><td>Increased virus binding to α2-6</td><td>H4N6</td><td>[50]</td></tr><tr><td>Increased virus binding to α2-6</td><td>H6N2</td><td>[211]</td></tr><tr><td>Increased virus binding to α2-6, decreased binding to α2-3</td><td>H5N1</td><td>[46]</td></tr><tr><td>Increased virus binding to α2-3, decreased binding to α2-6</td><td>H7N9</td><td>[52, 215]</td></tr><tr><td>Increased virus binding to α2-6, enhanced replication in mammalian cells and ferrets, enhanced contact transmission in ferrets</td><td>H9N2</td><td>[49, 54]</td></tr><tr><td>Loss of binding to α2-3</td><td>H10N8 (human isolate)</td><td>[53, 216, 217]</td></tr><tr><td>L226I</td><td>L222I</td><td>Decreased binding to α2-3</td><td>H7N9</td><td>[52]</td></tr><tr><td>S227N</td><td>S223N</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[46, 47, 203, 218, 219]</td></tr><tr><td>G228A/S</td><td>G224A/S</td><td>Increased binding to α2-6, dual receptor specificity</td><td>H4N6</td><td>[50]</td></tr><tr><td rowspan="6">G228S</td><td rowspan="6">G224S</td><td>Increased viral replication in mammalian cells and virulence in mice</td><td>H1N2</td><td>[220]</td></tr><tr><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[45, 46, 201, 209]</td></tr><tr><td>Decreased virus binding to α2-3</td><td>H6N2</td><td>[211]</td></tr><tr><td>Decreased binding to α2-3 and α2-6 receptors</td><td>H7N9</td><td>[51]</td></tr><tr><td>Decreased binding to α2-3, no binding to α2-6</td><td>H10N8 (human isolate)</td><td>[53, 217]</td></tr><tr><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[214]</td></tr><tr><td>E255K</td><td>E251K</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[47]</td></tr><tr><td rowspan="2">326 to 329</td><td rowspan="2">323 to 330 (R-X-R, K-R)</td><td>Polybasic cleavage motif sequence required for high pathogenicity avian influenza viruses</td><td>H5Nx</td><td>[221-228]</td></tr><tr><td>Decreased pH of fusion, increased HA stability, increased replication efficiency and virulence in mice</td><td>H7Nx</td><td>[229-231]</td></tr><tr><td>K387I</td><td>K388I</td><td>Decreased pH of fusion, increased HA stability, increased replication efficiency and virulence in mice</td><td>H5N1</td><td>[130, 232, 233]</td></tr><tr><td>K393E</td><td>K394E</td><td>Increased pH of fusion, decreased HA stability, decreased virulence in mice</td><td>H7N9</td><td>[234]</td></tr><tr><td>E83K, S128P</td><td>E75K, S123P</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>E83K, S128P, R496K</td><td>E75K, S123P, R497K</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr></tbody></table>

<figure><img src="image_3.png" /></figure>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th>Mutation/motif</th><th>H3 numbering<sup>a</sup></th><th>H5 numbering<sup>b</sup></th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td>E83K, S128P, N197K, R496K</td><td>E75K, S123P, N193K, R497K</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>E83K, N197K</td><td>E75K, N193K</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>E83K, N197K, R496K</td><td>E75K, N193K, R497K</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>E83K, R496K</td><td>E75K, R497K</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>H110Y, T160A, Q226L, G228S</td><td>H103Y, T156A, Q222L, G224S (with PB2: E627K; PB1: H99Y)</td><td></td><td>Airborne transmissible in ferrets</td><td>H5N1</td><td>[15, 58]</td></tr><tr><td>S114R, T115I</td><td>S107R, T108I</td><td></td><td>Increased virulence in chickens and mice, increased pH of fusion</td><td>H5N1</td><td>[235]</td></tr><tr><td>S128P, N197K</td><td>S123P, N193K</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>S128P, N197K, R496K</td><td>S123P, N193K, R497K</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>S128P, R496K</td><td>S123P, R497K</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>Δ<sup>c</sup>, A138V</td><td>L129V, A134V</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[236]</td></tr><tr><td>Δ, I155T</td><td>L129del, I151T</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[214, 236]</td></tr><tr><td>S137A, T192I</td><td>S133A, T188I</td><td></td><td>Increased pseudovirus binding to α2-6</td><td>H5N1</td><td>[44]</td></tr><tr><td>G143R, N186K</td><td>G139R, N182K</td><td></td><td>Decreased binding to α2-3, increased virus binding to α2-6</td><td>H5N1</td><td>[46, 204]</td></tr><tr><td>N158D, N224K, Q226L, T318I</td><td>N154D, N220K, Q222L, T315I</td><td></td><td>Transmissible among ferrets</td><td>H5N1</td><td>[14]</td></tr><tr><td>N158S, Q226L</td><td>N154S, Q222L</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[237]</td></tr><tr><td>N158S, Q226L, N248D</td><td>N154S, Q222L, N244D</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[237]</td></tr><tr><td>S159N, T160A</td><td>S155N, T156A</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[201, 209]</td></tr><tr><td>S159N, T160A, S227N</td><td>S155N, T156A, S223N</td><td></td><td>Increased virus binding to α2-6, reduced lethality and systemic spread in mice</td><td>H5N1</td><td>[238]</td></tr><tr><td>T160A, K193T, N224K, Q226L</td><td>T156A, K189T, N220K, Q222L</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[213]</td></tr><tr><td>T160A, Q226L</td><td>T156A, Q222L</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[201, 209]</td></tr><tr><td>T160A, Q226L, G228S</td><td>T156A, Q222L, G224S</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[201, 209, 239]</td></tr><tr><td>T160A, S227N</td><td>T156A, S223N</td><td></td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[201, 209]</td></tr><tr><td>V186N, N228K</td><td>V182N, N224K</td><td></td><td>Increased virus binding to α2-6</td><td>H7N9</td><td>[51]</td></tr><tr><td>V186K/G, K193T, G228S</td><td>V182 K/G, K189T, G224S</td><td></td><td>Increased virus binding to α2-6</td><td>H7N9</td><td>[51]</td></tr><tr><td>V186N, N224K, G228S</td><td>V182N, N220K, G224S</td><td></td><td>Increased virus binding to α2-6</td><td>H7N9</td><td>[51]</td></tr></tbody></table>
<table><caption>**Table 1** (continued)</caption><thead><tr><th>Mutation/motif<br>H3 numbering<sup>a</sup></th><th>H5 numbering<sup>b</sup></th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td>N186K, Q196R, Q226L, S227N, G228S</td><td>N182K, Q192R, Q222L, S223N, G224S</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[46]</td></tr><tr><td>N186K, Q226L, S227N, G228S</td><td>N182K, Q222L, S223N, G224S</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[46]</td></tr><tr><td>N186K, Q226L, G228S</td><td>N182K, Q222L, G224S</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[46]</td></tr><tr><td>E187G, E190D, K193S, Q226L, G228S</td><td>E183G, E186D, K189S, Q222L, G224S</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[240]</td></tr><tr><td>E187G, Q226L, G228S</td><td>E183G, Q222L, G224S</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[47]</td></tr><tr><td>D187G, S227N</td><td>D183G, S223N</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[47]</td></tr><tr><td>T189A, G192R</td><td>T185A, G188R</td><td>Enhanced replication in ferrets, transmitted via aerosols among ferrets</td><td>H9N2 (with human H3N2 backbone)</td><td>[241]</td></tr><tr><td>E190G, Q226E, G228S</td><td>E186G, Q222E, G224S</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[47]</td></tr><tr><td>K193T, G228S</td><td>K189T, G224S</td><td>Dual α2-3 and α2-6 binding</td><td>H7N9</td><td>[51]</td></tr><tr><td>K193R, Q226L, G228S</td><td>K189R, Q222L, G224S</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[239, 240]</td></tr><tr><td>Q196R, Q226L, S227N, G228S</td><td>Q192R, Q222L, S223N, G224S</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[46]</td></tr><tr><td>Q196R, Q226L, G228S</td><td>Q192R, Q222L, G224S</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[46, 47]</td></tr><tr><td>Q196R, S227N</td><td>Q192R, S223N</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[46, 47]</td></tr><tr><td>N197K, R496K</td><td>N193K, R497K</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[204]</td></tr><tr><td>K222Q, S227R</td><td>K218Q, S223R</td><td>Increased virus binding to α2-3 and α2-6</td><td>H5N1</td><td>[242]</td></tr><tr><td>N224K, G228S</td><td>N220K, G224S</td><td>Increased virus binding to α2-6</td><td>H7N9</td><td>[51]</td></tr><tr><td>Q226L, S227N, G228S</td><td>Q222L, S223N, G224S</td><td>Increased virus binding to α2-6</td><td>H5N1</td><td>[46]</td></tr><tr><td>Q226L, G228S</td><td>Q222L, G224S</td><td>Increased virus binding to α2-6</td><td>H4N6</td><td>[50]</td></tr><tr><td></td><td></td><td>Increased virus binding to α2-6; decreased antiviral response in host; reduced tissue tropism in guinea pigs</td><td>H5N1</td><td>[45-47, 201, 206, 209, 215, 237, 239, 240, 243, 244]</td></tr><tr><td></td><td></td><td>Increased virus binding to α2-6</td><td>H7N7 (human isolate)</td><td>[245]</td></tr><tr><td></td><td></td><td>Loss of binding to α2-3, no gain of binding to α2-6</td><td>H10N8 (human isolate)</td><td>[53, 216, 217]</td></tr></tbody></table>

Single mutations are presented first followed by motifs involving multiple mutations. Individual markers and motifs are listed in numerical order for ease of identifying mutation of interest

<footnote><sup>a</sup>Mutation/location H3 numbering relative to A/Aichi/2/1968 (H3N2)</footnote>

<footnote><sup>b</sup>Mutation/location H5 numbering relative to A/Vietnam/1203/2004 (H5N1)</footnote>

<footnote><sup>c</sup>Δ indicates amino acids present in A/H5 but are deleted compared to A/H3</footnote>
## Receptor binding specificity

The HA protein initiates influenza virus infections by recognizing and binding to sialylated glycans on the surface of host cells. Distribution and type of sialic acid residues in the host respiratory tract and the receptor binding preference of AIVs are major determinants of viral host range and transmissibility [29–32]. AIVs typically bind to sialic acids in α2,3 linkage to galactose, whereas human adapted viruses bind sialic acids in α2,6 linkage. While the respiratory tract of humans contains both linkages of sialic acids, α2,6 linkages are more abundant on the surface of epithelial cells lining the upper respiratory tract, allowing for the spread of human adapted viruses through the production of aerosols by sneezing and coughing [33, 34]. α2,3 linkages are mainly found in the lower respiratory tract of humans, permitting infection with AIVs but restricting transmission [35]. In contrast, avian species have both α2,6 and α2,3 linked sialic acids in their respiratory and intestinal tracts, though abundance and distribution differs between species [36–39]. Generally, epithelial cells with α2,3 linkages are more abundant in the avian intestinal tract, facilitating transmission via the fecal–oral route.

A switch in binding preference from α2,3 “avian-type” receptors to α2,6 “human-type” receptors is considered a key factor for pandemic potential of AIVs. There are numerous HA mutations that, individually or in combination, affect viral receptor binding preference, pathogenicity and transmissibility [40]. Two mutations, E190D and G225D, increase the preference for “human-type” receptors in both the 1918, “Spanish” A/H1N1 pandemic virus and the 2009 A/H1N1 “Swine Flu” pandemic virus [41, 42]. However, the impact of these mutations on other AIVs appears to be subtype specific [43–45]. A single G225D mutation alters the receptor preference of an A/H6N1 virus isolated from a human in Taiwan to bind α2,6 human-type receptors [43]. In contrast, while the same single substitution in A/H5 viruses does not increase affinity for α2,6 glycans [44–46], E190G produces a dual α2,3/α2,6 receptor binding phenotype [47]. Double E190D/G225D mutants have minimal binding to either α2,3 or α2,6 glycans in both A/H5 and A/H6 subtypes [43–45].

Unlike the 1918 and “Swine Flu” pandemic viruses, the 1957 “Asian” pandemic A/H2N2 virus and the 1968 “Hong Kong” A/H3N2 pandemic virus gained dual Q226L/G228S mutations [31, 48]. Both Q226L and G228S mutations have a profound effect on the HA receptor specificity for a large range of AIV subtypes. Individually, Q226L decreases, or completely negates, HA affinity for α2,3 “avian-type” receptors in several subtypes, namely A/H4, A/H5, A/H7, A/H9, and A/H10 [46, 49–53]. However, the effect of Q226L on α2,6 binding preference varies. In A/H5, A/H7, and A/H10 subtype AIVs, Q226L either mildly enhances, or does not

influence, α2,6 binding [45, 46, 52, 53]. Conversely, Q226L substantially increases HA preference for α2,6 “human-type” receptors in A/H4 and A/H9 subtype AIVs [49, 50, 54]. In contrast to Q226L, a singular G228S mutation in A/H5 subtype isolates produces a dual binding phenotype, increasing α2,6 binding and maintaining α2,3. Therefore, the combination of Q226L/G228S dual mutations decreases, or even ablates, α2,3 binding while simultaneously increasing affinity for α2,6. However, this elegant switch does not hold true for all subtypes. For instance, single Q226L or dual Q226L/G228S mutations in a human A/H10N8 isolate significantly decrease α2,3 binding with only a minimal increase in α2,6 affinity [33, 34]. In addition, a number of other HA mutations, individually or in combination, affect viral receptor binding preference, pathogenicity and transmissibility, and more work is necessary to understand the risk of these mutations in all subtypes of concern.

Overall, the variable effect of HA mutations on AIV receptor binding preference between different AIV subtypes likely occurs due to differences in the HA receptor binding site (RBS). Four key structural elements are present in the HA RBS of all AIV subtypes: the 130-loop, 150-loop, 190-helix, and 220-loop. Conformation and amino acid composition of these structures is a primary determinant of HA receptor specify. Therefore, the variability of RBS loop length and amino acid composition between AIV subtypes could account for the variable effects observed with the same mutations in the RBS [40].

## pH of fusion and HA stability

HA stability, or pH of fusion, refers to the pH required to trigger an irreversible conformational change in the HA1/HA2 trimer that activates the HA2 fusion peptide to mediate the fusion of the viral and endosomal membranes. This fusion creates pores through which viral ribonucleoproteins (RNPs) can exit the endosome, initiating AIV infection in the cytoplasm of the host cell. Following internalization, the endosome progressively becomes more acidic until the contents are destroyed in the host lysosome [55]. Therefore, pH of fusion dictates the efficient timing of the release of viral RNPs. If released too early, host cell recognition of viral products heightens host antiviral response, attenuating viral infection [56, 57]. If released too late, endosomal contents are destroyed, preventing release of viral products. The optimal pH of fusion varies substantially. In avian species, optimal pH of fusion can differ dramatically; however, a pH of fusion above 5.5 is believed to enhance AIV replication and transmission. In humans and ferrets, increased stability with a pH of fusion of less than 5.5 favors replication [28].

Understanding mutations affecting HA stability is vitally important since pH of fusion may partly contribute to the ability of AIVs to transmit by aerosol droplet in the ferret
model [14, 15]. In subtype A/H5, a H103Y mutation stabilizes the HA, possibly increasing transmissibility [58]. A separate mutation, K387I, decreases the pH of fusion, increasing viral replication efficiency and virulence in mice while attenuating virulence in ducks [59]. A comprehensive list of mutations affecting HA stability can be found in publications by Russell [60] and Mair et al. [61]; however, the overall biological significance of pH of fusion requires further investigation. Therefore, mutations that affect pH stability without experimentally verified effects on viral replication efficiency, transmissibility, or pathogenicity have not been included in the inventory.

## Neuraminidase (NA; Table 2)

### Viral infectivity and release from host cells

Neuraminidase (NA) is the second major transmembrane protein present on the influenza virus surface [62]. NA is a sialidase, cleaving sialic acid from glycoproteins, which enables virions to move through the mucus lining epithelial cells to initiate viral infections [63, 64], as well as mediating the release of progeny virions from the surface of infected cells [65]. HA and NA need to operate in equilibrium for efficient viral replication (reviewed in [66]) and perturbations can result in decreased viral infectivity and replication. For instance, a reduction in NA expression on seasonal human influenza viruses by experimentally introduced mutations in the viral promoter decreases virulence in mice [67]. Additionally, deletions in the NA stalk domain, the area between the enzymatically active head and the hydrophobic, envelope region, inhibit sialidase activity of NA, and alter HA binding and viral infectivity [68–70]. NA stalk length varies considerably between AIVs and a shortening of this domain is associated with adaptation of AIVs from wild birds to domestic poultry [71, 72]. A nineteen amino acid NA stalk deletion is commonly observed in highly pathogenic A/H5N1 viruses, and this deletion associates with enhanced replication capacity in mice [53, 54]. Similarly, experimentally introduced NA stalk deletions in A/H1N1, A/H7N1, A/H9N2 and A/H7N9 subtypes increase viral pathogenicity in mice and/or chickens [71, 73–78].

### Antiviral susceptibility and resistance

The function of NA is essential for productive AIV infection, as exemplified by conserved NA catalytic sites across influenza virus strains. Developed as antivirals in the 1990s, neuraminidase inhibitors (NAIs) bind to this active site of NA and prevent the release of new viruses from the surface of the infected host cell. While these antiviral compounds have been used successfully for several decades, the prevalence of NAI resistance is increasing [79]. Environmental

contamination with NAIs is also a recent concern, increasing the possibility of NAI resistance in AIVs from wild birds and domestic poultry [80]. The majority of NA mutations in this updated inventory are markers associated with resistance to the major NAIs currently in use globally: oseltamivir, zanamivir, laninamivir, and peramivir. The WHO-AVWG periodically releases a comprehensive table of mutations shown to affect NAI susceptibility in seasonal human influenza viruses (including both A and B genera viruses) as well as in subtype A/H5N1 and A/H7N9 AIVs [18]. The NA table (Table 2) provides a summary of WHO-AVWG NAI susceptibility markers for A/H5N1 and A/H7N9 viruses.

## Proteins of the ribonucleoprotein complex: PB2, PB1, PA, and NP

Inside the viral envelope, the eight genomic viral RNA (vRNA) segments of influenza A viruses form part of the viral ribonucleoprotein complex (vRNP). The vRNP consists of vRNA associated with multiple copies of the nucleoprotein (NP), and an RNA-dependent RNA polymerase sub-complex (RdRP), formed by polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), and the polymerase acidic protein (PA). Cryo-EM and crystal structures of the RdRP show that PB1 forms the core of the structure, associating with PA via its N-terminal and PB2 via its C-terminal [81–83]. Subunits of the RdRP associate with the 5' and 3' ends of viral RNA and with NP. The RdRP is crucial for viral transcription and replication, producing vRNA, complementary RNA and viral messenger RNA (mRNA) (reviewed in [84, 85]). The synthesis of viral mRNA is dependent on RdRP cap snatching. Whereby, PB2 binds the 5' cap of host RNA polymerase II transcripts [86–89], 10–13 nucleotides are cleaved by the endonuclease site of PA, and PB1 uses the cleaved fragment as a primer to initiate transcription [90]. Cap snatching not only facilitates the translation of viral mRNA, it also inhibits the production of host mRNA, referred to as host shutoff [91, 92]. Mutations that hinder AIV cap snatching ability affinity of vRNP proteins can affect viral replicative capacity and, consequently, AIV virulence.

Mutations that increase the polymerase activity of AIVs are important for the adaptation of AIVs to mammalian hosts [84]. In a number of studies, the polymerase activity of AIVs is impaired in mammalian cell lines [93]. This reduction in polymerase activity limits the transcription of viral RNA resulting in less viral material available to be packaged into progeny viruses. Additionally, limited replication capacity reduces viral genomic mutation, hindering the ability of AIVs to create progeny with beneficial mutations. A number of mutations in proteins of the polymerase complex enhance the replicative capacity of AIVs in mammalian
<table><caption>**Table 2** Experimentally verified molecular markers/motifs in the neuraminidase (NA; segment 6) gene of avian influenza viruses associated with enhanced virulence and antiviral resistance</caption><thead><tr><th>Mutation/motif (N2 numbering<sup>a</sup>)</th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td rowspan="2">49–68 deletion<sup>b</sup></td><td>Enhanced virulence in mice</td><td>H5N1 (with H1N1 backbone)</td><td>[73]</td></tr><tr><td>Enhanced virulence in mice</td><td>H7N9 (human isolate)</td><td>[74]</td></tr><tr><td rowspan="4">54–72 deletion</td><td>Enhanced virulence in mice but not chickens</td><td>H5N1</td><td>[75]</td></tr><tr><td>Enhanced virulence in mice</td><td>H7N9 (human isolate)</td><td>[75]</td></tr><tr><td>Enhanced virulence in mice and chickens</td><td>H1N1 (avian)</td><td>[71]</td></tr><tr><td>Enhanced virulence in mice</td><td>H7N9 (human isolate)</td><td>[75]</td></tr><tr><td>54–73 deletion</td><td>Enhanced virulence in mice</td><td>H7N1</td><td>[76]</td></tr><tr><td>54–75 deletion</td><td>Enhanced virulence in chickens but not ducks</td><td>H2N2</td><td>[246]</td></tr><tr><td>54–81 deletion</td><td>Enhanced replication in chicken, but not duck, cell line. Enhanced replication in respiratory tract of chickens.</td><td>H7N9 (human isolate)</td><td>[77]</td></tr><tr><td>57–65 deletion</td><td>Enhanced virulence in mice</td><td>H5N1</td><td>[247, 248]</td></tr><tr><td>V116A</td><td>Reduced susceptibility to oseltamivir and zanamivir</td><td>H5N1</td><td>[249]</td></tr><tr><td>I117T</td><td>Reduced susceptibility to oseltamivir and zanamivir</td><td>H5N1</td><td>[250]</td></tr><tr><td rowspan="2">E119A</td><td>Reduced susceptibility to zanamivir</td><td>H4N2</td><td>[251, 252]</td></tr><tr><td>Reduced susceptibility to oseltamivir, zanamivir, peramivir, and laninamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td rowspan="4">E119D</td><td>Reduced susceptibility to zanamivir</td><td>H4N2</td><td>[250]</td></tr><tr><td>Reduced susceptibility to oseltamivir, zanamivir, and peramivir</td><td>H5N1</td><td>[251]</td></tr><tr><td>Reduced susceptibility to oseltamivir, zanamivir, peramivir, and laninamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td>Reduced susceptibility to zanamivir</td><td>H9N2</td><td>[254]</td></tr><tr><td rowspan="4">E119G</td><td>Reduced susceptibility to zanamivir</td><td>H4N2</td><td>[250]</td></tr><tr><td>Reduced susceptibility to zanamivir and peramivir</td><td>H5N1</td><td>[251]</td></tr><tr><td>Reduced susceptibility to zanamivir, peramivir, and laninamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td>Reduced susceptibility to oseltamivir and zanamivir</td><td>H5N1</td><td>[255]</td></tr><tr><td rowspan="2">Q136L</td><td>Reduced susceptibility to zanamivir, peramivir, and laninamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td>Reduced susceptibility to oseltamivir and zanamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td>R152K</td><td>Reduced susceptibility to laninamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td>D198G</td><td>Reduced susceptibility to oseltamivir and zanamivir</td><td>H5N1</td><td>[256]</td></tr><tr><td>I222M</td><td>Reduced susceptibility to oseltamivir</td><td>H5N1</td><td>[256]</td></tr><tr><td>I222K</td><td>Reduced susceptibility to oseltamivir, zanamivir, peramivir, and laninamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td>I222R</td><td>Reduced susceptibility to oseltamivir and laninamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td>S246N</td><td>Reduced susceptibility to oseltamivir</td><td>H5N1</td><td>[248]</td></tr><tr><td>T247P</td><td>Reduced susceptibility to oseltamivir and zanamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td rowspan="2">H274Y</td><td>Reduced susceptibility to oseltamivir and peramivir</td><td>H5N1</td><td>[251–253, 256–258]</td></tr><tr><td>Reduced susceptibility to oseltamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td>E276D</td><td>Reduced susceptibility to oseltamivir, zanamivir, peramivir, and laninamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td>E277Q</td><td>Reduced susceptibility to oseltamivir</td><td>H5N1</td><td>[259]</td></tr><tr><td rowspan="4">R292K</td><td>Reduced susceptibility to zanamivir</td><td>H4N2</td><td>[250]</td></tr><tr><td>Reduced susceptibility to oseltamivir</td><td>H6N2</td><td>[260]</td></tr><tr><td>Reduced susceptibility to oseltamivir, zanamivir, peramivir, and laninamivir</td><td>H7N9</td><td>[261, 262]</td></tr><tr><td>Reduced susceptibility to oseltamivir</td><td>H9N2</td><td>[254]</td></tr><tr><td rowspan="2">N294S</td><td>Reduced susceptibility to oseltamivir, zanamivir, and peramivir</td><td>H5N1</td><td>[251, 252, 257, 263]</td></tr><tr><td>Reduced susceptibility to zanamivir</td><td>H7N9</td><td>[253]</td></tr></tbody></table>
<table><caption>**Table 2** (continued)</caption><thead><tr><th>Mutation/motif (N2 numbering<sup>a</sup>)</th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td>R371K</td><td>Reduced susceptibility to oseltamivir, zanamivir, peramivir, and laninamivir</td><td>H7N9</td><td>[253]</td></tr><tr><td>A401T</td><td>Increased virus binding to α2-3</td><td>H7N9</td><td>[264]</td></tr><tr><td>K432T</td><td>Reduced susceptibility to zanamivir</td><td>H5N1</td><td>[265]</td></tr><tr><td>I117V, I314V</td><td>Reduced susceptibility to oseltamivir</td><td>H5N1</td><td>[247]</td></tr><tr><td>E119V, E222V</td><td>Reduced susceptibility to oseltamivir</td><td>H7N9</td><td>[266]</td></tr><tr><td>E119A/D/G, H274Y</td><td>Reduced susceptibility to oseltamivir, zanamivir, and peramivir</td><td>H5N1</td><td>[251]</td></tr><tr><td>I222L, S246N</td><td>Reduced susceptibility to oseltamivir</td><td>H5N1</td><td>[265]</td></tr><tr><td>I222M/V, H274Y</td><td>Reduced susceptibility to oseltamivir and peramivir</td><td>H5N1</td><td>[256]</td></tr><tr><td>K150N, I222L, S246N</td><td>Reduced susceptibility to oseltamivir</td><td>H5N1</td><td>[248]</td></tr></tbody></table>

Single mutations are presented first followed by motifs involving multiple mutations. Individual markers and motifs are listed in numerical order for ease of identifying mutation of interest

However, these increases in polymerase activity and/or replicative capacity do not always correlate with an increase in viral pathogenicity [94].

## Polymerase basic protein 2 (PB2; Table 3)

Mutations in the PB2 protein, namely one mutation—the substitution of glutamate with lysine at position 627 (E627K)—are, by far, the best-known mutations in the polymerase complex proteins to be associated with increases in viral fitness [95]. Avian viruses typically have glutamate at this position and the substitution with lysine arises during adaptation to replication in mammalian species and supports transmission [96, 97]. E627K increases viral polymerase activity and replication in mammalian cell lines at temperatures similar to those of the upper respiratory tract of humans (~33 °C), a trait that increases transmissibility of AIVs in mammals [98–100]. This mutation increases virulence of A/H5N1, A/H6N1, A/H7N7, A/H7N9, and A/H9N2 AIVs in mammalian models of disease [101–105]. In contrast, E627K decreases, or has no effect, on AIV virulence in chickens [106–108]. Interestingly, position 627 is situated on the surface of PB2 [84, 95, 109], and, therefore, mutations at this position influence the association of PB2 with interacting partners e.g., NP, importin α, and ANP32A [110–112]. However, the exact mechanism behind the influence of position 627 on AIV host range remains unknown. Indeed, other mutations in PB2 aside from E627K include, but are not limited to: T271A, K526R, A588V, Q591K, E627V, D701N, D701V, and S714R. All of these mutations increase viral polymerase activity in mammalian cell lines for multiple AIV subtypes. Combinations of these mutations, such as E627K/D701N/S714R, can also further enhance viral polymerase activity, replication and virulence [102].

PB2 also potentially antagonizes the host interferon (IFN) response. A subset of AIVs contain PB2 proteins with an N-terminal mitochondrial targeting signal (MTS) that facilitates import of PB2 into the mitochondrial matrix [66, 67]. Mitochondrial PB2 then antagonizes host IFN production by interfering with the action of mitochondrial antiviral signaling proteins (MAVS) [113, 114]. Disruption of the MTS through a single amino acid substitution at position 9 prevents mitochondrial localization of PB2, heightening host IFN response and attenuating viral virulence [115, 116]. Asparagine (N9) or threonine (T9) at position 9 facilitates import of PB2 into the mitochondrial matrix. N9 is typically present in human seasonal viruses (A/H1N1 prior to 2009, A/H2N2, and A/H3N2). In contrast, AIVs typically have aspartic acid at position 9 (D9), and AIV PB2s are non-mitochondrial and predominantly found in the nucleus. Experimentally mutating seasonal human influenza viruses to contain the non-mitochondrial D9 heightens production of IFN-β and attenuates pathogenicity in mice [115]. Conversely, mutating avian A/H5N1 to contain N9 increases pathogenicity in mice, though specific cellular localization was not investigated [116].

## Polymerase basic protein 1 (PB1; Table 4) and polymerase protein (PA; Table 5)

Mutations in PB1 and PA affect viral replicative capacity and polymerase activity by altering the affinity between components of the RdRP [86–92]. A number of single amino acid substitutions increase AIV virulence in mice, PB1: K577E, D622G; PA: V63I, T97I, K142N/E, K356R, S421I, R443K, and K615N. Of these mutations PB1: N105S, K577E and PA: T97I, K356R also increase AIV polymerase activity in mammalian cell lines at 33 °C (a

<footnote><sup>a</sup>Mutation/location N2 numbering relative to A/Aichi/2/1968 (H3N2)</footnote>

<footnote><sup>b</sup>Deletions are numbered according to alignments with A/Goose/Guangdong/1/1996 (H5N1)</footnote>
<table><caption>**Table 3** Experimentally verified molecular markers/motifs in the polymerase basic protein 2 (PB2; segment 1) gene of avian influenza virus associated with polymerase activity, virulence, and transmissibility</caption><thead><tr><th>Mutation/motif</th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td>D9N<sup>a</sup></td><td>Increased virulence in mice</td><td>H5N1</td><td>[115, 116]</td></tr><tr><td>V25A</td><td>Increased virulence in mice</td><td>H5N1 backbone with H1N1 NS</td><td>[267]</td></tr><tr><td>I63T</td><td>Decreased pathogenicity in mice</td><td>H5N1</td><td>[72]</td></tr><tr><td rowspan="2">E158G</td><td rowspan="2">Increased polymerase activity in mammalian cell line, increased virulence in mice</td><td>H5N2</td><td>[268]</td></tr><tr><td>H5N9</td><td>[268]</td></tr><tr><td>E158K</td><td>Increased polymerase activity and replication in mammalian cell line, increased virulence in mice</td><td>H4N6</td><td>[269]</td></tr><tr><td>E192K</td><td>Increased polymerase activity in mammalian and avian cell line, increased virulence in mice</td><td>H5N1</td><td>[117]</td></tr><tr><td>A199S</td><td>Increased virulence in mice</td><td>H5N1</td><td>[116]</td></tr><tr><td>D253N</td><td>Increased polymerase activity in mammalian cell line</td><td>H9N2</td><td>[270]</td></tr><tr><td>D256G</td><td>Increased polymerase activity in mammalian cell line</td><td>H5N1 backbone with pH1N1 PB2</td><td>[271]</td></tr><tr><td rowspan="3">T271A</td><td rowspan="3">Increase polymerase activity in avian and mammalian cell line</td><td>H3N2 (avian)</td><td>[272]</td></tr><tr><td>H5N1</td><td>[273]</td></tr><tr><td>H7N9</td><td>[274]</td></tr><tr><td rowspan="2">I292V</td><td rowspan="2">Increased polymerase activity in mammalian cell line, increased virulence in mice</td><td>H9N2</td><td>[275]</td></tr><tr><td>H10N8</td><td>[276]</td></tr><tr><td>E358V</td><td>Decreased virulence in mice</td><td>H7N3</td><td>[277]</td></tr><tr><td>K389R</td><td>Increased polymerase activity and replication in mammalian cell line</td><td>H7N9</td><td>[278]</td></tr><tr><td>L339T</td><td>Decreased polymerase activity and decreased virulence in mice</td><td>H5N1</td><td>[279]</td></tr><tr><td>K482R</td><td>Increased polymerase activity in mammalian cell line</td><td>H7N9</td><td>[280, 281]</td></tr><tr><td rowspan="2">K526R</td><td rowspan="2">Increased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[282]</td></tr><tr><td>H7N9</td><td>[282]</td></tr><tr><td>M535L</td><td>Increased polymerase activity in mammalian cell line</td><td>H7N9</td><td>[25]</td></tr><tr><td rowspan="3">A588V</td><td rowspan="3">Increased polymerase activity and replication in mammalian and avian cell lines, increased virulence in mice</td><td>H7N9</td><td>[276]</td></tr><tr><td>H9N2</td><td>[276]</td></tr><tr><td>H10N8</td><td>[276]</td></tr><tr><td rowspan="4">Q591K</td><td rowspan="4">Increased polymerase activity in mammalian and avian cell line, increased replication in mammalian cell line, increased virulence in mice</td><td>H5N1</td><td>[117, 283]</td></tr><tr><td>H7N9</td><td>[25, 274]</td></tr><tr><td>Increased polymerase activity in mammalian and avian cell lines</td><td>H9N2</td><td>[284]</td></tr><tr><td>Increased polymerase activity and replication in mammalian cell line, increased virulence in mice</td><td>H7N9</td><td>[278]</td></tr><tr><td>V598T/I</td><td>Increased polymerase activity and replication in mammalian cells, increased virulence in mice</td><td>H7N9</td><td>[278]</td></tr></tbody></table>
<table><caption>**Table 3 (continued)**</caption><thead><tr><th>Mutation/motif</th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td rowspan="7">E627K</td><td>Increased polymerase activity and replication in mammalian cell line, increased virulence in mice</td><td>H4N6</td><td>[269]</td></tr><tr><td>Enhanced polymerase activity, increased virulence in mice, contributes to airborne pathogenicity of IAVs in ferrets and contact transmission in guinea pigs. Decreases polymerase activity and replication in avian cell lines. Decreases virulence in chickens.</td><td>H5N1</td><td>[15, 100, 101, 106–108, 116, 257, 271, 285–289]</td></tr><tr><td>Increased polymerase activity in mammalian cell line, increased virulence in mice</td><td>H6N1</td><td>[103]</td></tr><tr><td>Increased polymerase activity in mammalian cell lines, increased virulence in mice</td><td>H7N7</td><td>[102, 104]</td></tr><tr><td>Increased polymerase activity and replication in mammalian cell lines, increased virulence in mice</td><td>H7N9</td><td>[102, 105]</td></tr><tr><td>Increased polymerase activity in mammalian cell line, increased virulence in mice</td><td>H9N2</td><td>[102, 290]</td></tr><tr><td>Increased polymerase activity and replication in mammalian cell lines, increased virulence in mice</td><td>H5N1</td><td>[117]</td></tr><tr><td>K627E</td><td>Increased virulence in chickens</td><td>H5N1</td><td>[291]</td></tr><tr><td rowspan="2">D701N</td><td>Increased viral replication in mammalian cells and virulence in mice</td><td>H1N2</td><td>[220]</td></tr><tr><td>Increased polymerase activity, enhanced replication efficiency, increased virulence and contact transmission in guinea pigs, increased virulence in mice</td><td>H5N1</td><td>[96, 117, 206, 292, 293]</td></tr><tr><td rowspan="3">E627V</td><td>Increased polymerase activity in mammalian cell line</td><td>H7N9</td><td>[25, 274]</td></tr><tr><td>Increased polymerase activity in mammalian cell line</td><td>H9N2</td><td>[102]</td></tr><tr><td>Increased polymerase activity and replication in mammalian cell lines, increased virulence in mice</td><td>H5N1</td><td>[117]</td></tr><tr><td>S714R</td><td>Increased polymerase activity and replication in mammalian cell line</td><td>H7N7</td><td>[94, 102, 124]</td></tr><tr><td>S715N</td><td>Decreased virulence in mice</td><td>H5N1</td><td>[294]</td></tr><tr><td rowspan="2">M28I, A274T, K526R, I553V, L607V</td><td>Decreased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[295]</td></tr><tr><td>Increased polymerase activity in mammalian cell line and increased virulence in mice</td><td>H5N1</td><td>[296]</td></tr><tr><td>L89V, G309D</td><td>Increased polymerase activity in mammalian cell line and increased virulence in mice</td><td>H5N1</td><td>[296]</td></tr><tr><td>L89V, G309D, T339K, R477G, I495V, K627E, A676T</td><td>Increased polymerase activity in mammalian cell line and increased virulence in mice</td><td>H5N1</td><td>[296]</td></tr><tr><td>M147L, E627K</td><td>Increased polymerase activity in mammalian cell line and virulence in mice</td><td>H9N2</td><td>[297]</td></tr><tr><td>I147T, K339T, A588T</td><td>Increased polymerase activity in mammalian cell line and virulence in mice</td><td>H5N1</td><td>[298]</td></tr></tbody></table>

<figure><img src="image_4.png" /></figure>
<table><caption>**Table 3** (continued)</caption><thead><tr><th>Mutation/motif</th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td rowspan="2">K526R, E627K</td><td rowspan="2">Increased polymerase activity and viral replication in mammalian cell lines, increased virulence in mice</td><td>H5N1</td><td>[282]</td></tr><tr><td>H7N9</td><td>[282]</td></tr><tr><td>E627K, D701N</td><td>Increased polymerase activity in mammalian cell line</td><td>H7N9</td><td>[299]</td></tr><tr><td rowspan="3">E627K, S714R</td><td rowspan="3">Increased polymerase activity in mammalian cell lines</td><td>H7N7</td><td>[102]</td></tr><tr><td>H7N9</td><td>[102]</td></tr><tr><td>H9N2</td><td>[102]</td></tr><tr><td>E627K, D701N, S714R</td><td>Increased polymerase activity in mammalian cells, increased virulence in mice</td><td>H9N2</td><td>[102]</td></tr><tr><td rowspan="5">D701N, S714R</td><td>Increased polymerase activity in mammalian cell line, increased virulence in mice</td><td>H5N1</td><td>[300]</td></tr><tr><td>Increased polymerase activity in mammalian cell lines</td><td>H7N7</td><td>[102]</td></tr><tr><td>Increased polymerase activity in mammalian cell lines</td><td>H7N9</td><td>[102]</td></tr><tr><td>Increased polymerase activity in mammalian cell lines</td><td>H9N2</td><td>[102]</td></tr><tr><td>Enable airborne transmissibility between ferrets and contact transmission between guinea pigs</td><td>H5N1</td><td>[15, 58]</td></tr></tbody></table>

Single mutations are presented first followed by motifs involving multiple mutations. Individual markers and motifs are listed in numerical order for ease of identifying mutation of interest

number of mutations were only tested at 37 °C) [117–119]. The mechanism behind changes in viral replication and virulence caused by these substitutions is not always clear. PA substitutions at positions 63, 97 and 142 could hinder RdRP cap snatching by reducing cleavage of host mRNA caps. Interestingly, four substitutions in PA at positions 142, 147, 171 and 182 that are associated with low polymerase activity of AIVs can drive the emergence of compensatory mutations in other RdRP proteins, as demonstrated for the emergence of PB2 E627K in A(H7N9) viruses [120].

Interestingly, a combination of two mutations in PA, S224P and N383D, increase polymerase activity of a A/ H5N1 subtype virus and enhance viral replication in both mammalian and avian cell lines [121, 122]. This dual mutation also increases A/H5N1 AIV virulence in both mice and ducks. The mutations act in synergy, N383D increases AIV polymerase activity alone while the combination of S224P and N383D significantly increases AIV virulence. This combination differs from previously mentioned mutations as single point mutations in polymerase proteins typically affect viral virulence in mammalian species, with minimal changes in the polymerase activity, replication or virulence in avian species. Some mutations reportedly increase polymerase activity in avian and

mammalian cell lines (PB2: A588V, Q591K; PB1: D3V, S678N). However, in these reports, only murine models were utilized to test AIV virulence.

## Nucleoprotein (NP; Table 6)

The NP protein encapsulates viral genomic RNA and mediates import into the nucleus to initiate viral replication through nuclear localization signals and the importin-α/ importin-β nuclear import pathway. Mutations that affect the functions of NP have not been extensively investigated; however, substitutions I41V, R91K, R198K, E210D, K227R, K229R, N319K, E434K, K470R enhance polymerase activity in mammalian cell lines [53, 94, 111, 123]. These mutations do not always increase AIV virulence and the mechanism behind the increase in viral replication is not always clear. One particular mutation, N319K, improves A/H7N7 viral replication in mammalian cells by enhancing the interaction between NP and importin-α isoforms [94, 111, 124]. M105V, I109T, and A184K enhance viral replication and increase AIV virulence in chickens [125–127]. Interestingly, the M105V mutation may be involved in spillover adaption from ducks to chickens as M105V affects viral replication in embryo fibroblasts from chickens but not from ducks [127].

<footnote>ªMutations/motifs are numbered according to alignments with A/Goose/Guangdong/1/1996 (H5N1)</footnote>
<table><caption>**Table 4** Experimentally verified molecular markers/motifs in the polymerase basic protein 1 (PB1; segment 2) gene of influenza virus associated with polymerase activity, replication, virulence, and transmissibility</caption><thead><tr><th>Protein</th><th>Mutation/motif</th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td>PB1</td><td>D3V<sup>a</sup></td><td>Increased polymerase activity and viral replication in avian and mammalian cell lines</td><td>H5N1</td><td>[301]</td></tr><tr><td></td><td>N105S</td><td>Increase polymerase activity and replication in mammalian cell line, increased virulence in mice</td><td>H5N1</td><td>[117]</td></tr><tr><td></td><td>K207R</td><td>Decreased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[302]</td></tr><tr><td></td><td>Y436H</td><td>Decreased polymerase activity in mammalian cell line; decreased virulence in ducks, ferrets and mice</td><td>H5N1</td><td>[302]</td></tr><tr><td></td><td>V473L</td><td>Decreased polymerase activity and replication efficiency in mammalian cells</td><td>H1N1 with PB2, PB1, PA NP from H5N1</td><td>[303]</td></tr><tr><td></td><td>K577E</td><td>Increased polymerase activity and virulence in mice</td><td>H9N2</td><td>[118]</td></tr><tr><td></td><td>V598P</td><td>Decreased polymerase activity and replication efficiency in mammalian cells</td><td>H1N1 with PB2, PB1, PA NP from H5N1</td><td>[303]</td></tr><tr><td></td><td>D622G</td><td>Increased polymerase activity and virulence in mice</td><td>H5N1</td><td>[304]</td></tr><tr><td></td><td>T677M</td><td>Increased polymerase activity in mammalian cell line, decreased replication efficiency, decreased virulence in mice</td><td>H5N1</td><td>[72]</td></tr><tr><td></td><td>S678N</td><td>Increased replication in avian and mammalian cell lines</td><td>H7N7</td><td>[124]</td></tr><tr><td></td><td>V3A, N328K, N375S</td><td>Decreased replication efficiency and virulence in ferrets</td><td>H5N1</td><td>[93]</td></tr><tr><td></td><td>V473L, P598L</td><td>Decreased polymerase activity and replication in mammalian cells</td><td>H1N1 with PB2, PB1, PA NP from H5N1</td><td>[303]</td></tr><tr><td></td><td>H99Y (with HA: H110Y, T160A, G226L, G228S; PB2: E627K)</td><td>Airborne transmissible in ferrets</td><td>H5N1</td><td>[15, 58]</td></tr><tr><td>PB1-F2</td><td>N66S</td><td>Enhanced replication, virulence and antiviral response in mice</td><td>H5N1</td><td>[171, 305]</td></tr><tr><td></td><td>T51M, V56A, E87G</td><td>Decrease polymerase activity, replication and virulence in ducks</td><td>H5N1</td><td>[173]</td></tr></tbody></table>

Single mutations are presented first followed by motifs involving multiple mutations. Individual markers and motifs are listed in numerical order for ease of identifying mutation of interest

## Non-structural proteins 1 and 2 (NS1/NS2; Table 7)

Influenza virus genomic segment 8 encodes for three viral proteins: NS1, NS2 (Nuclear Export Protein; NEP), and NS3. The NS3 protein was only recently identified and its function remains largely unknown, therefore, mutations in this protein are not included in the inventory [128]. The NS1 protein performs multiple functions that affect AIV replication and virulence (reviewed in [129]). Typically 230 amino acids in length, NS1 ranges between 202 and 238 amino acids in length as the C-terminus is frequently truncated or, occasionally, elongated [130]. Structurally, NS1 proteins contain an RNA-binding domain (RBD) at the N-terminus with a flexible linker region connecting the

RBD to a C-terminal effector domain (ED). The RBD and ED are functional domains that mediate the associations between NS1 and interacting partners. NS1 interacts with the vRNP complex, specifically NP and PA, as well as a number of proteins involved in cellular signaling pathways, the host antiviral response, and nuclear/cytoplasmic trafficking or translation of mRNA. Overall, NS1 is the major viral antagonist of the host IFN response and plays a role in host cell shutoff and viral replication [129].

Single amino acid substitutions, deletions, and C-terminal truncations in NS1 affect AIV replicative capacity and pathogenicity. Point mutations in NS1 decreasing the host antiviral response (in chickens, ferrets or mice), include: P42S, F89Y, V149A, N200S (with NS2: T47A—see below) and

<figure><img src="image_5.png" /></figure>

<footnote><sup>a</sup>Mutations/motifs in both PB1 and PB1-F2 proteins are numbered according to alignments with A/Goose/Guangdong/1/1996 (H5N1)</footnote>
<table><caption>**Table 5** Experimentally verified molecular markers/motifs in the polymerase (PA; segment 3) gene of avian influenza viruses associated with polymerase activity, replication, virulence, and host inflammatory response</caption><thead><tr><th>Protein</th><th>Mutation/motif</th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td rowspan="15">PA</td><td>S37A<sup>a</sup></td><td>Increased polymerase activity in mammalian cell line</td><td>H7N9</td><td>[280]</td></tr><tr><td>A37S</td><td>Decreased polymerase activity in mammalian cell line</td><td>H7N7</td><td>[306]</td></tr><tr><td>V63I</td><td>Increase polymerase activity and enhanced replication in mammalian cell line, increased virulence in mice</td><td>H7N7</td><td>[306, 307]</td></tr><tr><td rowspan="5">T97I</td><td>Increased polymerase activity and replication in mammalian cell line, increased virulence in mice</td><td>H5N1</td><td>[117]</td></tr><tr><td>Increased polymerase activity in mammalian cell line and enhanced replication in mice</td><td>H5N2</td><td>[308]</td></tr><tr><td>Increased polymerase activity in mammalian cells</td><td>H6N1</td><td>[103]</td></tr><tr><td>Increased virulence in mice</td><td>H5N1</td><td>[116]</td></tr><tr><td>Increased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[301]</td></tr><tr><td>K142N/E</td><td>Decreased virulence in mice</td><td>H7N3</td><td>[277]</td></tr><tr><td>K158R</td><td>Increase polymerase activity and enhanced replication in mammalian cell line, increased virulence in mice</td><td>H9N2</td><td>[119]</td></tr><tr><td>P190S</td><td>Increased polymerase activity in mammalian and avian cell lines</td><td>H5N1</td><td>[121, 122]</td></tr><tr><td>K356R</td><td>Decreased virulence in mice</td><td>H7N3</td><td>[277]</td></tr><tr><td>N383D</td><td>Increase polymerase activity and replication in mammalian cell line</td><td>H7N9</td><td>[280]</td></tr><tr><td>Q400P</td><td>Increased polymerase activity and replication in mammalian cell line</td><td>H5N1</td><td>[116]</td></tr><tr><td>N409S</td><td>Increased virulence in mice</td><td>H5N1</td><td>[267]</td></tr><tr><td>S421I</td><td>Increased polymerase activity in mammalian cell line</td><td>H7N9 (human isolate)</td><td>[281]</td></tr><tr><td>R443K</td><td>Decreased polymerase activity in mammalian cell line, decreased virulence in ducks</td><td>H5N1</td><td>[302]</td></tr><tr><td>K497R</td><td>Increased polymerase activity in mammalian cell line and increased virulence in mice</td><td>H7N7</td><td>[94, 124]</td></tr><tr><td>T515A</td><td>Increased polymerase activity in mammalian cell line and increased virulence in mice</td><td>H5N1</td><td>[309]</td></tr><tr><td>K615N</td><td>Increased polymerase activity in mammalian cell line, increased virulence in mice</td><td>H7N1</td><td>[310]</td></tr><tr><td>A343S, D347E</td><td>Decreased polymerase activity replication in mammalian cell line, decreased virulence in mice</td><td>H7N7</td><td>[310]</td></tr><tr><td>P103H, S659L</td><td>Increased polymerase activity and enhanced viral replication in duck and mouse cell lines, increased virulence in mice and ducks</td><td>H5N1</td><td>[121, 122]</td></tr><tr><td>S224P, N383D</td><td>Increased polymerase activity in mammalian cell line</td><td>H7N9</td><td>[120]</td></tr><tr><td>K142R, I147V, I171V, M182L</td><td>Increased replication in mammalian cell line virulence in mice</td><td>H5N1</td><td>[311]</td></tr><tr><td>V44I, V127A, C241Y, A343T, I573V</td><td>Increased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[295]</td></tr><tr><td>S149P, H266R, I357K, S515T</td><td>Increase polymerase activity, enhanced replication capacity in mammalian cell line, increased virulence in mice</td><td>H9N2</td><td>[119]</td></tr><tr><td>K356R (with PB2 E627K)</td><td>Increased viral replication in mammalian and avian cell lines; increased inflammatory response in mice; increased virulence in mice, chickens, and ducks</td><td>H5N1</td><td>[121, 177, 178, 182, 183]</td></tr><tr><td rowspan="2">PA-X</td><td rowspan="2">Truncations resulting in loss of PA-X expression</td><td>Decreased virulence in mice, inhibited host inflammatory response</td><td>H9N2</td><td>[183]</td></tr><tr><td></td><td></td><td></td></tr></tbody></table>

Single mutations are presented first followed by motifs involving multiple mutations. Individual markers and motifs are listed in numerical order for ease of identifying mutation of interest

G205R (with NS2: M51I—see below) [131–133]. Deletions and C-terminal truncations of NS1 occur commonly. Contemporary A/H5N1 subtype viruses have five amino acids deleted from position 80 to 84 (relative to A/goose/Guangdong/1996) associated with an increase in virulence in mice and chickens [134]. C-terminal truncations result in deletion

of the NS1 PDZ domain, a four amino acid motif that modulates protein-protein interactions with PDZ proteins important for cellular signaling pathways [135]. Human influenza viruses typically contain the PDZ motifs RSKV or RSEV whereas avian viruses have ESEV or EPEV [136] and the influence of PDZ motifs on viral phenotype is host and strain

<footnote><sup>a</sup>Mutations/motifs in both PA and PA-X proteins are numbered according to alignments with A/Goose/Guangdong/1/1996 (H5N1)</footnote>
<table><caption>**Table 6** Experimentally verified molecular markers/motifs in the nucleoprotein (NP; segment 5) gene of influenza virus associated with polymerase activity, virulence, and transmissibility</caption><thead><tr><th>Mutation/motif</th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td>I41V<sup>a</sup></td><td>Increased polymerase activity in mammalian cell line</td><td>H7N9</td><td>[299]</td></tr><tr><td>K91R</td><td>Decreased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[123]</td></tr><tr><td>M105V</td><td>Increased virulence in chickens</td><td>H5N1</td><td>[126, 127]</td></tr><tr><td>I109T</td><td>Increased polymerase activity and viral replication in chickens (but not ducks), increased virulence in chickens</td><td>H5N1</td><td>[126, 127]</td></tr><tr><td>A184K</td><td>Increased replication in avian cells and virulence in chickens, enhanced IFN response</td><td>H5N1</td><td>[125]</td></tr><tr><td>K198R</td><td>Decreased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[123]</td></tr><tr><td>E210D</td><td>Increased polymerase activity in mammalian cell line</td><td>H7N9</td><td>[299]</td></tr><tr><td>K227R</td><td>Increased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[123]</td></tr><tr><td>K229R</td><td>Increased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[123]</td></tr><tr><td>N319K</td><td>Increased polymerase activity and replication in mammalian cell line</td><td>H7N7</td><td>[94, 111]</td></tr><tr><td>E434K</td><td>Increased polymerase activity in mammalian cell line</td><td>H9N2</td><td>[53]</td></tr><tr><td>K470R</td><td>Increased polymerase activity and replication in mammalian cell line, increased virulence in mice</td><td>H5N1</td><td>[123]</td></tr><tr><td>Q357L (with PB2: E627K)</td><td>Increased virulence in mice</td><td>H5N1</td><td>[116]</td></tr><tr><td>E434K (with HA: Q227P, D375E)</td><td>Enhanced contact transmission in guinea pigs</td><td>H9N2</td><td>[53]</td></tr><tr><td>E434K (with HA: Q227P, PB2: D195N)</td><td>Enhanced contact transmission in guinea pigs</td><td>H9N2</td><td>[53]</td></tr><tr><td>R99K, S345N (with HA: H110Y, T160A, Q226L, G228S; PB2: E627K; PB1: H99Y, I368V)</td><td>Airborne transmissible in ferrets</td><td>H5N1</td><td>[15]</td></tr></tbody></table>

Single mutations are presented first followed by motifs involving multiple mutations. Individual markers and motifs are listed in numerical order for ease of identifying mutation of interest

specific. The “avian” ESEV motif decreases viral replication in human and duck cell lines compared to the “human” RSKV motif, whereas these substitutions do not affect virulence in chickens [137, 138]. Overall, the PDZ domain does not appear to be a major determinant of AIV pathogenicity. Indeed, PDZ deletions in the 2009 pandemic A/H1N1 virus as well as in avian A/H5N1 and A/H7N1 subtype AIVs do not significantly affect viral virulence [139, 140].

The NS2 protein, also referred to as the nuclear export protein (NEP) [141], regulates several factors, including: transcription and translation of viral products [142]; nuclear export of vRNP complexes [143]; and viral budding from host cells [144, 145]. NS2 regulates viral polymerase activity, enhancing the synthesis of viral cRNA, vRNA, and, in some cases, mRNA [142, 146]. Very few NS2 mutations that affect viral fitness and host adaptation are characterized in AIVs. Adaptive mutations described in a human A/H5N1 isolate include M16I that, individually or in combination with Y41C and E75G, increase viral polymerase activity in mammalian cell lines [146, 147]. Additionally, two combinations of NS1 and NS2 mutations, T47A with NS1 N200S and M51I with NS1 G205R, decrease the host antiviral response in ferrets [133]. However, the effect of

these mutations on AIV pathogenicity is either negligible or unclear [146, 147].

### Matrix protein (MP; Table 8)

Influenza virus genomic segment 7 (MP) encodes for the matrix 1 and 2 proteins (M1 and M2, respectively). M1 is located beneath the viral envelope where it associates with the lipid membrane and viral RNPs. This interaction needs to be ablated for viral RNP to enter the host cell cytoplasm [148, 149]. In the nucleus, M1 associates with vRNPs and mediates export into the cytoplasm [149]. M1 is also involved in viral assembly and budding [150]. M2 is a trans-membrane protein present on the surface of influenza virions that acts as an ion channel, acidifying endosomes and contributing to the release of viral RNP into the cytoplasm [151]. The cytoplasmic domain of M2 is also involved in viral genome packaging for progeny viruses [152, 153]. Three more M proteins have also recently been identified: M3, M4 and M42 [154]; however, the functional roles of these proteins are not well established and currently no mutations have been shown to affect viral fitness so they are not included in this inventory.

<footnote><sup>a</sup>Mutations/motifs are numbered according to alignments with A/Goose/Guangdong/1/1996 (H5N1)</footnote>
<table><caption>**Table 7** Experimentally verified molecular markers/motifs in the non-structural protein (NS; segment 8) gene of influenza virus associated with replication, virulence, pathogenicity, and antiviral response</caption><thead><tr><th>Protein</th><th>Mutation/motif</th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td rowspan="12">NS1</td><td>P42S<sup>a</sup></td><td>Increased virulence and decreased antiviral response in mice</td><td>H5N1</td><td>[131]</td></tr><tr><td>D74N</td><td>Enhanced replication in mammalian cells and pathogenicity in mice</td><td>H7N1 backbone with H5N1 NS</td><td>[312]</td></tr><tr><td rowspan="3">80–84 deletion</td><td>Increased virulence in chickens</td><td>H1N1 (avian)</td><td>[313]</td></tr><tr><td>Increased virulence in chickens and mice</td><td>H1N1 backbone with H5N1 HA, NA and NS</td><td>[314]</td></tr><tr><td>Increased virulence in swine</td><td>H1N1 backbone with H5N1 NS</td><td>[314]</td></tr><tr><td>Y84F</td><td>Decreases replication in mammalian cells and enhances interferon response</td><td>H1N1 with H5N1 NS</td><td>[132]</td></tr><tr><td rowspan="2">D92E [D87E]<sup>b</sup></td><td>Increased virulence in swine and mice</td><td>H1N1 backbone with H5N1 NS</td><td>[314, 315]</td></tr><tr><td>Increased virulence in chickens and mice</td><td>H1N1 backbone with H5N1 HA, NA and NS</td><td>[314]</td></tr><tr><td>I106M [I101M]</td><td>Increased viral replication in mammalian cells virulence in mice</td><td>H1N1 with all internal genes from H7N9</td><td>[316]</td></tr><tr><td>C138F</td><td>Increased replication in mammalian cells, decreased interferon response</td><td>H5N1</td><td>[317]</td></tr><tr><td>V149A</td><td>Increased virulence and decreased interferon response in chickens</td><td>H5N1</td><td>[318]</td></tr><tr><td>L103F, I106M [L98F, I101M]</td><td>Increased virulence in mice</td><td>H5N1</td><td>[319, 320]</td></tr><tr><td>N205S (with NS2: T47A)</td><td>Decreased antiviral response in ferrets</td><td>H5N1</td><td>[133]</td></tr><tr><td>G210R (with: NS2 M51I)</td><td>Decreased antiviral response in ferrets</td><td>H5N1</td><td>[133]</td></tr><tr><td>P3S, R41K, D74N</td><td>Enhanced replication in mammalian cells and pathogenicity in mice</td><td>H7N1 backbone with H5N1 NS</td><td>[312]</td></tr><tr><td>R38A, K41A</td><td>Decreased replication in mammalian and avian cell line</td><td>H7N1</td><td>[321]</td></tr><tr><td>K55E, K66E, C138F</td><td>Enhanced replication in mammalian cells, decrease IF response</td><td>H5N1</td><td>[317]</td></tr><tr><td>222–230 deletion</td><td>Increased replication in mammalian and avian cell lines</td><td>H5N1</td><td>[138]</td></tr><tr><td rowspan="2">225–230 deletion</td><td>Increased viral replication in avian cell line</td><td>H7N1</td><td>[130, 322]</td></tr><tr><td>No impact on viral replication in avian cell lines</td><td>H7N1</td><td>[140]</td></tr><tr><td rowspan="3"><sup>227</sup>ESEV<sup>230</sup> (PDZ domain)</td><td>Increased virulence in mice</td><td>H1N1 pdm09 virus with ‘avian’ PDZ motif</td><td>[323]</td></tr><tr><td>Decreased viral replication in mammalian and avian cell lines</td><td>H5N1</td><td>[138]</td></tr><tr><td>Increased viral replication and virulence in mice, decreased viral replication in human and duck cell lines</td><td>H7N1</td><td>[137]</td></tr><tr><td><sup>227</sup>RSKV<sup>230</sup> (PDZ domain)</td><td>Increased viral replication in human and duck cell lines but no effect in murine cells</td><td>H7N1</td><td>[137]</td></tr><tr><td>230–237 elongation</td><td>Increased replication and inflammatory cytokine production in chickens</td><td>H9N2</td><td>[324]</td></tr><tr><td rowspan="4">NS2/NEP</td><td>M16I</td><td>Increased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[325]</td></tr><tr><td>M16I, Y41C, E75G</td><td>Increased polymerase activity in mammalian cell line</td><td>H5N1</td><td>[325]</td></tr><tr><td>T47A (with NS1: N205S)</td><td>Decreased antiviral response in ferrets</td><td>H5N1</td><td>[133]</td></tr><tr><td>M51I (with NS1: G2010R)</td><td>Decreased antiviral response in ferrets</td><td>H5N1</td><td>[133]</td></tr></tbody></table>

Single mutations are presented first followed by motifs involving multiple mutations. Individual markers and motifs are listed in numerical order for ease of identifying mutation of interest

Only a small number of mutations in segment 7 are associated with host adaptation. All occur in the region encoding the M1 protein. Four mutations, namely N30D, I43M,

T139A and T215A, increase the virulence of A/H5N1 sub-type AIVs in mice [155–157]. I43M also increases virulence in chickens and ducks; however, the underlying mechanisms

<footnote><sup>a</sup>Mutations/motifs in NS1 and NEP are presented with numbering relative to A/goose/Guangdong/1/1996 (H5N1)</footnote>

<footnote><sup>b</sup>The CDC molecular inventory numbers NS1 mutations relative to A/Vietnam/1203/2014 (H5N1) which contains an NS1 deletion. For entries from the CDC inventory that uses alternate numbering, the numbering according to A/Vietnam/1203/2014 (H5N1) is included in brackets</footnote>
<table><caption>**Table 8** Experimentally verified molecular markers/motifs in the matrix (M; segment 7) gene of influenza virus associated with virulence and antiviral resistance</caption><thead><tr><th>Protein</th><th>Mutation/motif</th><th>Phenotype</th><th>Subtypes tested</th><th>References</th></tr></thead><tbody><tr><td rowspan="3">M1</td><td>N30D<sup>a</sup></td><td>Increased virulence in mice</td><td>H5N1</td><td>[156]</td></tr><tr><td>I43M</td><td>Increased virulence in mice, chickens and ducks</td><td>H5N1</td><td>[157]</td></tr><tr><td>T215A</td><td>Increased virulence in mice</td><td>H5N1</td><td>[156]</td></tr><tr><td rowspan="6">M2</td><td>L26F</td><td>Increased resistance to amantadine and rimantadine</td><td>H5N1</td><td>[159, 160, 326]</td></tr><tr><td rowspan="2">I/V27A/T/S</td><td>Increased resistance to amantadine and rimantadine</td><td>H5N1<br>H5N2</td><td>[159–161, 326, 327]</td></tr><tr><td>Increased resistance to amantadine and rimantadine</td><td>H5N1<br>H5N2<br>H7N2</td><td>[161, 326, 327]</td></tr><tr><td>A30V/T/S</td><td>Increased resistance to amantadine and rimantadine</td><td>H5N1<br>H5N2<br>H7N2</td><td>[160, 161, 326–330]</td></tr><tr><td>S31N/G</td><td>Increased resistance to amantadine and rimantadine</td><td>H5N1<br>H5N2<br>H9N2</td><td>[160, 161, 326–330]</td></tr><tr><td>G34E</td><td>Increased resistance to amantadine and rimantadine</td><td>H5N1</td><td>[159]</td></tr></tbody></table>

Single mutations are presented first followed by motifs involving multiple mutations. Individual markers and motifs are listed in numerical order for ease of identifying mutation of interest

remain unclear [157]. The M2 protein is an important target of antiviral compounds and mutations in this region contribute to antiviral resistance phenotypes. Adamantanes (amantadine and rimantadine) block the M2 ion channel and inhibit early stages of virus replication. This class of drugs is no longer recommended against seasonal human influenza as these viruses display a high degree of adamantine resistance. In addition, adamantine resistance is increasing in AIVs globally, including subtypes of major concern, such as A/H5 and A/H7 [158]. Well-known mutations associated with adamantane resistance in M2 include: L26F, V27A, A30V/T/S, S31N/G, and G34E [159–161].

## Auxiliary proteins

### PB1-F2 (Table 4)

PB1-F2 is an auxiliary protein expressed in a majority of influenza A viruses and produced from a +1 alternate reading frame of PB1 [162]. Full-length PB1-F2 is approximately 90 amino acids in length, with frequent variation by truncation. Overall expression and length of PB1-F2 affect influenza virus pathogenicity in both a host and strain dependent manner [163]. PB1-F2 induces host cell apoptosis, antagonizes the host antiviral innate immunity, enhances the production of pro-inflammatory cytokines, and affects viral polymerase activity (reviewed in [164]). Pro-apoptotic activity of PB1-F2 has been reported in human seasonal A/H1N1 influenza viruses but not in A/H5N1 subtype AIVs [165]. The association between PB1-F2, mitochondrial-associated proteins, and cellular factors

also inhibits antiviral responses and enhances production of pro-inflammatory cytokines. Interaction of PB1-F2 with MAVS, TBK1 and IRF3. PB1-F2 inhibits the action of TBK1 and IRF3, downregulating host production of type 1 interferon [166, 167]. Additionally, interaction between PB1-F2 and MAVS enhances TRAF6-mediated NF-kB activation, promoting the production of pro-inflammatory cytokines [168, 169]. In A/H5N1 subtype AIVs and human pandemic influenza viruses from 1918, 1957 and 1968, expression of full-length PB1-F2 heightens the inflammatory response to infection in mice, rendering them more susceptible to secondary bacterial pneumonia [170].

Only a few PB1-F2 mutations in AIVs subtypes affect viral pathogenicity. Truncation of the A/H5N1 PB1-F2 protein increases pathogenicity in mice [165, 171], but complete deletion of A/H5N1 PB1-F2 does not significantly alter viral virulence [163, 171]. Perhaps the best-known PB1-F2 mutation, N66S, inhibits host interferon production, increasing pro-inflammatory cytokine responses. However, these effects remain strain and host specific. For example, N66S in A/H5N1 subtype AIV enhances viral replication and pathogenicity in mice but not ducks [171]. Indeed, the few reports that analyze the effect of PB1-F2 expression in avian species show viral attenuation. PB1-F2 expression in A/H5N1 and A/H9N2 subtype AIVs decreases viral pathogenicity in chickens, possibly through inducing the host immune response earlier in chickens compared to mice [172]. Additionally, a combination of mutations T51M/V56A/E87G in PB1-F2 decrease viral polymerase activity, replication, and virulence in mallard ducks [173].

<footnote><sup>a</sup>Mutations/motifs in M1 and M2 proteins are presented with numbering relative to A/goose/Guangdong/1/1996 (H5N1)</footnote>
## PA-X (Table 5)

PA-X is a fusion protein comprised of 191N-terminal amino acids (including the endonuclease domain) and 61 amino acids from the C-terminus from segment 3 (PA) formed as the result of ribosomal frameshifting [174]. PA-X has plays a role in host shutoff [174–176], modulating the host immune response [174, 177–179], viral polymerase activity, viral replication, viral induced apoptosis, and virulence (reviewed in [180]). Approximately 75% of all influenza virus isolates possess a full-length PA-X sequence, with the remaining 25% expressing PA-X truncations. These truncations of up to 41 amino acids in length most commonly occur in the PA-X C-terminus and are most commonly found in the 2009 pandemic A/H1N1, canine A/H3N2 and A/H3N8, equine A/H7N7, and bat influenza viruses. However, they also occur in some A/H5N1 and A/H9N2 subtype AIVs [181]. Overall, the function of PA-X appears to be subtype specific. In A/H5N1 subtype AIV, loss of PA-X expression increases viral replication, host inflammatory response, and virulence in mice, chickens, and ducks [177, 178, 182]. In contrast, loss of PA-X in the A/H9N2 subtype limits the host inflammatory response and decreases virulence in mice [183].

## Limitations and usage considerations of this molecular inventory

As stated in the methodology, this inventory was not intended as an exhaustive list so several caveats and limitations need to be considered when utilizing these tables. First, the inventory only includes experimentally validated (in vitro or in vivo) markers/mutations. As stated previously, several studies utilize in silico approaches to predict molecular markers of viral adaptation to humans [22–26]. This work is extremely valuable and findings from these computational studies guide future research. Indeed, novel machine learning approaches have helped to identify novel markers and amino acid positions in human and avian influenza strains associated with pandemic potential [184, 185]. Further work on these novel mutations should be conducted in vitro and in vivo. Second, as stated in the text, changes in biological characteristics for specific point mutations and motifs are sometimes only associated with specific viral subtypes or hosts, so caution should be made when generalizing findings for novel strains or hosts. Finally, consideration must be made for viral sequences themselves. Influenza viruses, like many other RNA viruses, replicate as a quasispecies, allowing for rapid changes in diversity and viral adaptation in response to environmental pressures [186]. The majority of molecular markers/motifs described in these studies come from consensus sequences which may not adequately reflect the entire population of viruses in a given sample. With the

continual advancement of Next Generation Sequencing (NGS) and bioinformatics we are better able to understand how minority variants could contribute to pandemic potential of influenza strains. Indeed, recent work by Welkers et al. using NGS on human respiratory A/H5N1 samples identifies multiple single amino acid variants in all three polymerase subunits. In vitro analysis of these markers shows substantial increases in polymerase activity [187]. Therefore, we must move towards consideration of the influenza viral quasispecies as a whole when considering the dynamic evolutionary and adaptive pathways/processes and the meaning and predictive power for zoonotic and/or pandemic potential.

## Discussion/conclusions

Since the discovery of the Goose/Guangdong-lineage A/H5 viruses in China in 1996, HPAIV of the A/H5N1 subtype have spread globally, causing numerous outbreaks in wild birds and poultry. Overall, 861 confirmed human A/H5N1 infections have been reported with a case fatality rate of 52.8% [188]. In addition, newly emerged and emerging AIVs with zoonotic potential continue to appear, including subtypes A/H7N9, A/H7N4, A/H5Nx, A/H9N2, A/H10N7, and A/H10N8 [189–193]. As of June 2019, there have been 1,568 human A/H7N9 cases reported with 616 deaths (CFR: 39%) [194]. There have been 23 human cases of A/H5N6 infection reported from 10 different provinces across mainland China, of which 15 were fatal (CFR 65.2%) [195]. A single human non-fatal infection with an A/H7N4 subtype virus occurred in an elderly woman in Jiangsu, China in December 2017 [192, 196]. This virus is antigenically distinct from formerly circulating A/H7 strains, and, concerningly, appears to be spreading across Southeast Asia, continually reassorting with other viruses in the region [197, 198]. To date, none of the novel A/H7N4 viruses contains known amino acid mutations that confer adaptation of AIV to humans (e.g., PB2 627/701 or HA 186/226/228) or anti-viral resistance. However, A/H7N4 isolated from Cambodia does contain the M gene amino acid mutations N30D and T215A that increase pathogenicity of A/H5N1 virus in mice [198]. Due to the antigenic differences between the A/H7N4 viruses and other H7 lineages, including the A/Anhui/1/2013-like A/H7N9 lineage, the continual spread, and risk for human infection, this newly detected A/H7N4 lineage is now in preparation as a candidate vaccine virus for pandemic preparedness [199]. To date, no avian origin influenza viruses can transmit efficiently from human-to-human via aerosols. Given the ongoing spillover of avian influenza viruses into domestic poultry, as well as the risk of human infection and potential for adaptation, continual, vigilant surveillance and risk assessment is vital to combat endemic and emerging AIVs. This inventory provides a list
of the currently known, experimentally verified mutations/
molecular markers affecting host adaptation in AIVs and can
be utilized for molecular characterization and risk assess-
ment of novel AIV strains.

**Acknowledgements** The authors would like to thank all of the mem-
bers of the Influenza Team in the Virology Unit at the Institute Pasteur
in Cambodia, as well as at the WHO Collaborating Center and Peter
Doherty Institute at the University of Melbourne for their insightful
discussions and input.

**Funding** This publication is the result, in part, of work conducted under a cooperative agreement with the Office of the Assistant Secretary for Preparedness and Response in the U.S. Department of Health and Human Services (HHS), grant number IDSEP140020-01-00 awarded to Dr. Philippe Dussart and Dr. Erik Karlsson. Its contents and conclusions are solely the responsibility of the authors and do not represent the official views of HHS. Annika Suttie is funded by an Australian Government Research Training Program Scholarship and a Faculty of Science and Technology Research Scholarship from Federation University. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health.

Compliance with Ethical Standards

**Conflict of Interest** All authors declare that they have no conflicts of interest.

**Ethical approval** This article does not contain any studies with human participants or animals performed by any of the authors.

**Open Access** This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

References

1. Bodewes R, Kuiken T (2018) Changing role of wild birds in the epidemiology of avian influenza A viruses. In: Kielian M, Mettenleiter TC, Roossinck MJ (eds) Advances in virus Resarach. Academic Press, Cambridge, pp 279–307

2. Tong S, Zhu X, Li Y, Shi M, Zhang J, Bourgeois M, Yang H, Chen X, Recuenco S, Gomez J, Chen LM, Johnson A, Tao Y, Dreyfus C, Yu W, McBride R, Carney PJ, Gilbert AT, Chang J, Guo Z, Davis CT, Paulson JC, Stevens J, Rupprecht CE, Holmes EC, Wilson IA, Donis RO (2013) New world bats harbor diverse influenza A viruses. PLoS Pathog 9:e1003657

3. Wu Y, Wu Y, Tefsen B, Shi Y, Gao GF (2014) Bat-derived influenza-like viruses H17N10 and H18N11. Trends Microbiol 22:183–191

4. Tong S, Li Y, Rivailler P, Conrardy C, Castillo DAA, Chen L-M, Recuenco S, Ellison JA, Davis CT, York IA, Turmelle AS, Moran D, Rogers S, Shi M, Tao Y, Weil MR, Tang K, Rowe LA, Sammons S, Xu X, Frace M, Lindblade KA, Cox NJ, Anderson LJ, Rupprecht CE, Donis RO (2012) A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci USA 109:4269–4274

5. Li Q, Sun X, Li Z, Liu Y, Vavricka CJ, Qi J, Gao GF (2012) Structural and functional characterization of neuraminidase-like molecule N10 derived from bat influenza A virus. Proc Natl Acad Sci USA 109:18897–18902

6. Zhu X, Yu W, McBride R, Li Y, Chen L-M, Donis RO, Tong S, Paulson JC, Wilson IA (2013) Hemagglutinin homologue from H17N10 bat influenza virus exhibits divergent receptor-binding and pH-dependent fusion activities. Proc Natl Acad Sci USA 110:1458–1463

7. Kaplan BS, Webby RJ (2013) The avian and mammalian host range of highly pathogenic avian H5N1 influenza. Virus Res 178:3–11

8. Bourret V (2018) Avian influenza viruses in pigs: an overview. Vet J 239:7–14

9. Chambers TM (2014) In: Spackman E (ed) Animal influenza virus. Springer, New York, pp 365–370

10. Karlsson EA, Ip HS, Hall JS, Yoon SW, Johnson J, Beck MA, Webby RJ, Schultz-Cherry S (2014) Respiratory transmission of an avian H3N8 influenza virus isolated from a harbour seal. Nat Commun 5:4791

11. Sikkema RS, Freidl GS, de Bruin E, Koopmans M (2016) Weigh-
ing serological evidence of human exposure to animal influenza
viruses—a literature review. Eurosurveillance 21:30388

12. Mostafa A, Abdelwhab EM, Mettenleiter TC, Pleschka S (2018) Zoonotic potential of influenza A viruses: a comprehensive overview. Viruses 10:497

13. Koutsakos M, Kedzierska K, Subbarao K (2019) Immune responses to avian influenza viruses. J Immunol 202:382–391

14. Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, Shinya K, Zhong G, Hanson A, Katsura H, Watanabe S, Li C, Kawakami E, Yamada S, Kiso M, Suzuki Y, Maher EA, Neumann G, Kawaoka Y (2012) Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 486:420–428

15. Herfst S, Schrauwen EJA, Linster M, Chutinimitkul S, de Wit E, Munster VJ, Sorrell EM, Bestebroer TM, Burke DF, Smith DJ, Rimmelzwaan GF, Osterhaus ADME, Fouchier RAM (2012) Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336:1534–1541

16. Burke SA, Trock SC (2018) Use of Influenza risk assessment tool for prepandemic preparedness. Emerg Infect Dis 24:471

17. Centers for Disease Control and Prevention (2012) H5N1 genetic changes inventory: a tool for influenza surveillance and preparedness. https://www.cdc.gov/flu/avianflu/h5n1-genetic-changes.htm

18. World Health Organisation (2018) Summary of neuraminidase amino acid substitutions associated with reduced inhibition by neuraminidase inhibitors (NAI). https://www.who.int/influenza/gisrs_laboratory/antiviral_susceptibility/NAI Reduced_Susceptibility_Marker_Table_WHO.pdf?ua=1

19. Mertens E, Dugan VG, Stockwell TB, Lindsay LL, Plancarte M, Boyce WM (2013) Evaluation of phenotypic markers in full genome sequences of avian influenza isolates from California. Comp Immunol Microbiol Infect Dis 36:521–536

20. Gabriel G, Czudai-Matwich V, Klenk H-D (2013) Adaptive mutations in the H5N1 polymerase complex. Virus Res 178:53–62

21. Lloren KKS, Lee T, Kwon JJ, Song MS (2017) Molecular markers for interspecies transmission of avian influenza viruses in mammalian hosts. Int J Mol Sci 18:2706

22. Wen L, Chu H, Wong BH-Y, Wang D, Li C, Zhao X, Chiu M-C, Yuan S, Fan Y, Chen H, Zhou J, Yuen K-Y (2018) Large-scale sequence analysis reveals novel human-adaptive markers in PB2 segment of seasonal influenza A viruses. Emerg Microb Infect 7:1–12
23. Finkelstein DB, Mukatira S, Mehta PK, Obenauer JC, Su X, Webster RG, Naeve CW (2007) Persistent host markers in pandemic and H5N1 influenza viruses. J Virol 81:10292-10299

24. Shaw M, Cooper L, Xu X, Thompson W, Krauss S, Guan Y, Zhou N, Klimov A, Cox N, Webster R, Lim W, Shortridge K, Subbarao K (2002) Molecular changes associated with the transmission of avian influenza a H5N1 and H9N2 viruses to humans. J Med Virol 66:107-114

25. Chen G-W, Kuo S-M, Yang S-L, Gong Y-N, Hsiao M-R, Liu Y-C, Shih S-R, Tsao K-C (2016) Genomic signatures for avian H7N9 viruses adapting to humans. PLoS ONE 11:e0148432

26. Gamblard F, Sami A, Hemmatzadeh F, Ebrahimie E (2019) Identifying mutation positions in all segments of influenza genome enables better differentiation between pandemic and seasonal strains. Gene 697:78-85

27. Gamblin SJ, Skehel JJ (2010) Influenza hemagglutinin and neuraminidase membrane glycoproteins. J Biol Chem 285:28403-28409

28. Russell CJ, Hu M, Okda FA (2018) Influenza hemagglutinin protein stability, activation, and pandemic risk. Trends Microbiol 26:841-853

29. Matrosovich MN, Gambaryan AS, Teneberg S, Piskarev VE, Yamnikova SS, Lvov DK, Robertson JS, Karlsson KA (1997) Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology 233:224-234

30. Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk H-D (2004) Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci USA 101:4620-4624

31. Connor RJ, Kawaoka Y, Webster RG, Paulson JC (1994) Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology 205:17-23

32. Suzuki Y, Ito T, Suzuki T, Holland RE, Chambers TM, Kiso M, Ishida H, Kawaoka Y (2000) Sialic acid species as a determinant of the host range of influenza A viruses. J Virol 74:11825-11831

33. Thompson CI, Barclay WS, Zambon MC, Pickles RJ (2006) Infection of human airway epithelium by human and avian strains of influenza A virus. J Virol 80:8060-8068

34. van Riel D, den Bakker MA, Leijten LME, Chutinimitkul S, Munster VJ, de Wit E, Rimmelzwaan GF, Fouchier RAM, Osterhaus ADME, Kuiken T (2010) Seasonal and pandemic human influenza viruses attach better to human upper respiratory tract epithelium than avian influenza viruses. Am J Pathol 176:1614-1618

35. Shinya K, Ebina M, Yamada S, Ono M, Kasai N, Kawaoka Y (2006) Influenza virus receptors in the human airway: avian flu. Nature 440:435-436

36. França M, Stallknecht DE, Howerth EW (2013) Expression and distribution of sialic acid influenza virus receptors in wild birds. Avian Pathol 42:60-71

37. Kimble B, Ramirez Nieto G, Perez DR (2010) Characterization of influenza virus sialic acid receptors in minor poultry species. Virol J 7:365

38. Costa T, Chaves AJ, Valle R, Darji A, van Riel D, Kuiken T, Majó N, Ramis A (2012) Distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species. Vet Res 43:28

39. Pillai SPS, Lee CW (2010) Species and age related differences in the type and distribution of influenza virus receptors in different tissues of chickens, ducks and turkeys. Virol J 7(1):5

40. Laznowski M, Dawson WK, Szczepinska T, Plewczynski D (2018) The structural variability of the influenza A hemagglutinin receptor-binding site. Brief Funct Genom 17:415-427

41. Glaser L, Stevens J, Zamarin D, Wilson IA, Garcia-Sastre A, Tumpey TM, Basler CF, Taubenberger JK, Palese P (2005) A single amino acid substitution in 1918 Influenza virus hemagglutinin changes receptor binding specificity. J Virol 79:11533-11536

42. Matrosovich M, Tuzikov A, Bovin N, Gambaryan A, Klimov A, Castrucci MR, Donatelli I, Kawaoka Y (2000) Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J Virol 74:8502-8512

43. de Vries RP, Tzarum N, Peng W, Thompson AJ, Ambepitiya Wickramasinghe IN, de la Pena ATT, van Breemen MJ, Bouwman KM, Zhu X, McBride R, Yu W, Sanders RW, Verheije MH, Wilson IA, Paulson JC (2017) A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors. EMBO Mol Med 9:1314-1325

44. Yang Z-Y, Wei C-J, Kong W-P, Wu L, Xu L, Smith DF, Nabel GJ (2007) Immunization by avian H5 influenza hemagglutinin mutants with altered receptor binding specificity. Science 317:825-828

45. Stevens J, Blixt O, Tumpey TM, Taubenberger JK, Paulson JC, Wilson IA (2006) Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312:404-410

46. Chutinimitkul S, van Riel D, Munster VJ, van den Brand JMA, Rimmelzwaan GF, Kuiken T, Osterhaus ADME, Fouchier RAM, de Wit E (2010) In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity. J Virol 84:6825-6833

47. Chen L-M, Blixt O, Stevens J, Lipatov AS, Davis CT, Collins BE, Cox NJ, Paulson JC, Donis RO (2012) In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity. Virology 422:105-113

48. Rogers GN, Paulson JC, Daniels RS, Skehel JJ, Wilson IA, Wiley DC (1983) Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature 304:76

49. Wan H, Perez DR (2007) Amino acid 226 in the hemagglutinin of H9N2 influenza viruses determines cell tropism and replication in human airway epithelial cells. J Virol 81:5181-5191

50. Song H, Qi J, Xiao H, Bi Y, Zhang W, Xu Y, Wang F, Shi Y, Gao GF (2017) Avian-to-human receptor-binding adaptation by influenza a virus hemagglutinin H4. Cell Rep 20:1201-1214

51. de Vries RP, Peng W, Grant OC, Thompson AJ, Zhu X, Bouwman KM, de la Pena ATT, van Breemen MJ, Ambepitiya Wickramasinghe IN, de Haan CAM, Yu W, McBride R, Sanders RW, Woods RJ, Verheije MH, Wilson IA, Paulson JC (2017) Three mutations switch H7N9 influenza to human-type receptor specificity. PLoS Path 13:e1006390

52. Xu R, de Vries RP, Zhu X, Nycholat CM, McBride R, Yu W, Paulson JC, Wilson IA (2013) Preferential recognition of avian-like receptors in human influenza A H7N9 viruses. Science 342:1230-1235

53. Sang X, Wang A, Ding J, Kong H, Gao X, Li L, Chai T, Li Y, Zhang K, Wang C, Wan Z, Huang G, Wang T, Feng N, Zheng X, Wang H, Zhao Y, Yang S, Qian J, Hu G, Gao Y, Xia X (2015) Adaptation of H9N2 AIV in guinea pigs enables efficient transmission by direct contact and inefficient transmission by respiratory droplets. Sci Rep 5:15928

54. Wan H, Sorrell EM, Song H, Hossain MJ, Ramirez-Nieto G, Monne I, Stevens J, Cattoli G, Capua I, Chen L-M, Donis RO, Busch J, Paulson JC, Brockwell C, Webby R, Blanco J, Al-Natour MQ, Perez DR (2008) Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential. PLoS ONE 3:e2923

55. Mellman I, Fuchs R, Helenius A (1986) Acidification of the endocytic and exocytic pathways. Annu Rev Biochem 55:663-700
56. Gerlach T, Hensen L, Matrosovich T, Bergmann J, Winkler M, Peteranderl C, Klenk H-D, Weber F, Herold S, Pohlmann S, Matrosovich M (2017) pH optimum of hemagglutinin-mediated membrane fusion determines sensitivity of influenza A viruses to the interferon-induced antiviral state and IFITMs. J Virol 91:e00246-17

57. Marvin SA, Russier M, Huerta CT, Russell CJ, Schultz-Cherry S (2017) Influenza virus overcomes cellular blocks to productively replicate, impacting macrophage function. J Virol 91:e014717-16

58. Linster M, van Boheemen S, de Graaf M, Schrauwen Eefje JA, Lexmond P, Mänz B, Bestebroer Theo M, Baumann J, van Riel D, Rimmelzwaan Guus F, Osterhaus Albert DME, Matrosovich M, Fouchier Ron AM, Herfst S (2014) Identification, characterization, and natural selection of mutations driving airborne transmission of A/H5N1 virus. Cell 157:329-339

59. Zaraket H, Bridges OA, Russell CJ (2013) The pH of activation of the hemagglutinin protein regulates H5N1 influenza virus replication and pathogenesis in mice. J Virol 87:4826-4834

60. Russell CJ (2014) Acid-induced membrane fusion by the hemagglutinin protein and its role in influenza virus biology. Curr Top Microbiol Immunol 385:93-116

61. Mair CM, Ludwig K, Herrmann A, Sieben C (1838) Receptor binding and pH stability—how influenza A virus hemagglutinin affects host-specific virus infection. Biochim Biophys Acta 1153-1168:2014

62. McAuley JL, Gilbertson BP, Trifkovic S, Brown LE, McKimm-Breschkin JL (2019) Influenza virus neuraminidase structure and functions. Front Microbiol 10:39. https://doi.org/10.3389/fmicb.2019.00039

63. Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD (2004) Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J Virol 78:12665-12667

64. Cohen M, Zhang X-Q, Senaati HP, Chen H-W, Varki NM, Schooley RT, Gagneux P (2013) Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase. Virol J 10:321

65. Palese P, Tobita K, Ueda M, Compans RW (1974) Characterization of temperature sensitive influenza virus mutants defective in neuraminidase. Virology 61:397-410

66. Byrd-Leotis L, Cummings RD, Steinhauer DA (2017) The interplay between the host receptor and influenza virus hemagglutinin and neuraminidase. Int J Mol Sci 18:1541

67. Solorzano A, Zheng H, Fodor E, Brownlee GG, Palese P, García-Sastre A (2000) Reduced levels of neuraminidase of influenza A viruses correlate with attenuated phenotypes in mice. J Gen Virol 81:737-742

68. Mitnaul LJ, Matrosovich MN, Castrucci MR, Tuzikov AB, Bovin NV, Kobasa D, Kawaoka Y (2000) Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus. J Virol 74:6015-6020

69. Wagner R, Matrosovich M, Klenk H-D (2002) Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Rev Med Virol 12:159-166

70. Baigent SJ, McCauley JW (2001) Glycosylation of haemagglutinin and stalk-length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture. Virus Res 79:177-185

71. Munier S, Larcher T, Cormier-Aline F, Soubieux D, Su B, Guigand L, Labrosse B, Cherel Y, Quere P, Marc D, Naffakh N (2010) A genetically engineered waterfowl influenza virus with a deletion in the stalk of the neuraminidase has increased virulence for chickens. J Virol 84:940-952

72. Li J, Dohna ZH, Cardona CJ, Miller J, Carpenter TE (2011) Emergence and genetic variation of neuraminidase stalk deletions in avian influenza viruses. PLoS ONE 6:e14722

73. Matsuoka Y, Swayne DE, Thomas C, Rameix-Welti M-A, Naffakh N, Warnes C, Altholtz M, Donis R, Subbarao K (2009) Neuraminidase stalk length and additional glycosylation of the hemagglutinin influence the virulence of influenza H5N1 viruses for mice. J Virol 83:4704-4708

74. Zhou H, Yu Z, Hu Y, Tu J, Zou W, Peng Y, Zhu J, Li Y, Zhang A, Yu Z, Ye Z, Chen H, Jin M (2009) The special neuraminidase stalk-motif responsible for increased virulence and pathogenesis of H5N1 influenza A virus. PLoS ONE 4:e6277

75. Bi Y, Xiao H, Chen Q, Wu Y, Fu L, Quan C, Wong G, Liu J, Haywood J, Liu Y, Zhou B, Yan J, Liu W, Gao GF (2016) Changes in the length of the neuraminidase stalk region impact H7N9 virulence in mice. J Virol 90:2142-2149

76. Hoffmann TW, Munier S, Larcher T, Soubieux D, Ledevin M, Esnault E, Tourdes A, Croville G, Guerin JL, Quere P, Volmer R, Naffakh N, Marc D (2012) Length variations in the NA stalk of an H7N1 influenza virus have opposite effects on viral excretion in chickens and ducks. J Virol 86:584-588

77. Park S, Il Kim J, Lee I, Bae J-Y, Yoo K, Nam M, Kim J, Sook Park M, Song K-J, Song J-W, Kee S-H, Park M-S (2017) Adaptive mutations of neuraminidase stalk truncation and glycosylation confer enhanced pathogenicity of influenza A viruses. Sci Rep 7:10928

78. Sun Y, Tan Y, Wei K, Sun H, Shi Y, Pu J, Yang H, Gao GF, Yin Y, Feng W, Perez DR, Liu J (2013) Amino acid 316 of hemagglutinin and the neuraminidase stalk length influence virulence of H9N2 influenza virus in chickens and mice. J Virol 87:2963-2968

79. Davidson S (2018) Treating influenza infection, from now and into the future. Front Immunol 9:1946

80. Jarhult JD (2018) Environmental resistance development to influenza antivirals: a case exemplifying the need for a multidisciplinary One Health approach including physicians. Acta Vet Scand 60:6

81. Chang S, Sun D, Liang H, Wang J, Li J, Guo L, Wang X, Guan C, Boruah Bhargavi M, Yuan L, Feng F, Yang M, Wang L, Wang Y, Wojdyla J, Li L, Wang J, Wang M, Cheng G, Wang H-W, Liu Y (2015) Cryo-EM structure of influenza virus RNA polymerase complex at 4.3 Å resolution. Mol Cell 57:925-935

82. Pflug A, Guilligay D, Reich S, Cusack S (2014) Structure of influenza A polymerase bound to the viral RNA promoter. Nature 516:355

83. Reich S, Guilligay D, Pflug A, Malet H, Berger I, Crépin T, Hart D, Lunardi T, Nanao M, Ruigrok RWH, Cusack S (2014) Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516:361

84. Stevaert A, Naesens L (2016) The influenza virus polymerase complex: an update on its structure, functions, and significance for antiviral drug design. Med Res Rev 36:1127-1173

85. Lo C-Y, Tang Y-S, Shaw P-C (2018) Structure and function of influenza virus ribonucleoprotein. In: Harris JR, Bhella D (eds) Virus protein and nucleoprotein complexes. Springer, Singapore, pp 95-128

86. Fechter P, Mingay L, Sharps J, Chambers A, Fodor E, Brownlee GG (2003) Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding. J Biol Chem 278:20381-20388

87. Li M-L, Rao P, Krug RM (2001) The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits. EMBO J 9:2078-2086

88. Guilligay D, Tarendeau F, Resa-Infante P, Coloma R, Crepin T, Sehr P, Lewis J, Ruigrok RWH, Ortin J, Hart DJ, Cusack S (2008) The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol 15:500-506

89. Plotch SJ, Bouloy M, Ulmanen I, Krug RM (1981) A unique cap(m7G pppXm)-dependent influenza virion endonuclease
cleaves capped RNAs to generate the primers that initiate viral
RNA transcription. Cell 23:847-858

90. Dias A, Bouvier D, Crépin T, McCarthy AA, Hart DJ, Baudin F, Cusack S, Ruigrok RWH (2009) The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458:914-918

91. Rodriguez A, Perez-Gonzalez A, Nieto A (2007) Influenza virus infection causes specific degradation of the largest subunit of cellular RNA polymerase II. J Virol 81:5315-5324

92. Bercovich-Kinori A, Tai J, Gelbart IA, Shitrit A, Ben-Moshe S, Drori Y, Itzkovitz S, Mandelboim M, Stern-Ginossar N (2016) A systematic view on influenza induced host shutoff. eLife 5:e18311

93. Salomon R, Franks J, Govorkova EA, Ilyushina NA, Yen H-L, Hulse-Post DJ, Humberd J, Trichet M, Rehg JE, Webby RJ, Webster RG, Hoffmann E (2006) The polymerase complex genes contribute to the high virulence of the human H5N1 influenza virus isolate A/Vietnam/1203/04. J Exp Med 203:689-697

94. Gabriel G, Dauber B, Wolff T, Planz O, Klenk HD, Stech J (2005) The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc Natl Acad Sci USA 102:18590-18595

95. Nilsson BE, Te Velthuis AJW, Fodor E (2017) Role of the PB2 627 domain in influenza A virus polymerase function. J Virol 91:e02467-02416

96. Steel J, Lowen AC, Mubareka S, Palese P (2009) Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627 K or 627E/701N. PLoS Pathog 5:e1000252

97. Van Hoeven N, Pappas C, Belser JA, Maines TR, Zeng H, Garcia-Sastre A, Sasisekharan R, Katz JM, Tumpey TM (2009) Human HA and polymerase subunit PB2 proteins confer transmission of an avian influenza virus through the air. Proc Natl Acad Sci USA 106:3366-3371

98. Subbarao KE, Murphy BR, London W (1993) A single amino acid in the PB2 gene of influenza a virus is a determinant of host range. J Virol 4:1761-1764

99. Massin P, van der Werf S, Naffakh N (2001) Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses. J Virol 75:5398-5404

100. Hatta M, Hatta Y, Kim JH, Watanabe S, Shinya K, Nguyen T, Lien PS, Le QM, Kawaoka Y (2007) Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice. PLoS Pathog 3:1374-1379

101. Hatta M, Gao P, Halfmann P, Kawaoka Y (2001) Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293:1840-1842

102. Sediri H, Thiele S, Schwalm F, Gabriel G, Klenk H-D (2016) PB2 subunit of avian influenza virus subtype H9N2: a pandemic risk factor. J Gen Virol 97:39-48

103. Cheng K, Yu Z, Chai H, Sun W, Xin Y, Zhang Q, Huang J, Zhang K, Li X, Yang S, Wang T, Zheng X, Wang H, Qin C, Qian J, Chen H, Hua Y, Gao Y, Xia X (2014) PB2-E627K and PA-T97I substitutions enhance polymerase activity and confer a virulent phenotype to an H6N1 avian influenza virus in mice. Virology 468-470:207-213

104. de Jong RM, Stockhofe-Zurwieden N, Verheij ES, de Boer-Luijtze EA, Ruiter SJ, de Leeuw OS, Cornelissen LA (2013) Rapid emergence of a virulent PB2 E627K variant during adaptation of highly pathogenic avian influenza H7N7 virus to mice. Virol J 10:276

105. Zhang H, Li X, Guo J, Li L, Chang C, Li Y, Bian C, Xu K, Chen H, Sun B (2014) The PB2 E627K mutation contributes to the high polymerase activity and enhanced replication of H7N9 influenza virus. J Gen Virol 95:779-786

106. Bogs J, Kalthoff D, Veits J, Pavlova S, Schwemmle M, Mänz B, Mettenleiter TC, Stech J (2011) Reversion of PB2-627E to -627 K during replication of an H5N1 Clade 2.2 virus in mammalian hosts depends on the origin of the nucleoprotein. J Virol 85:10691-10698

107. Long JS, Howard WA, Núñez A, Moncorgé O, Lycett S, Banks J, Barclay WS (2013) The effect of the PB2 mutation 627 K on highly pathogenic H5N1 avian influenza virus is dependent on the virus lineage. J Virol 87:9983-9996

108. Richard M, Herfst S, van den Brand JMA, de Meulder D, Lexmond P, Bestebroer TM, Fouchier RAM (2017) Mutations driving airborne transmission of A/H5N1 Virus in mammals cause substantial attenuation in chickens only when combined. Sci Rep 7:7187

109. Tarendeau F, Crepin T, Guilligay D, Ruigrok RWH, Cusack S, Hart DJ (2008) Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit. PLoS Pathog 4:e1000136

110. Long JS, Giotis ES, Moncorgé O, Frise R, Mistry B, James J, Morisson M, Iqbal M, Vignal A, Skinner MA, Barclay WS (2016) Species difference in ANP32A underlies influenza A virus polymerase host restriction. Nature 529:101

111. Gabriel G, Herwig A, Klenk H-D (2008) Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of influenza A virus. PLoS Pathog 4:e11

112. Labadie K, Dos Santos Afonso E, Rameix-Welti M-A, van der Werf S, Naffakh N (2007) Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells. Virology 362:271-282

113. Long JCD, Fodor E (2016) The PB2 subunit of the influenza A virus RNA polymerase is imported into the mitochondrial matrix. J Virol 90:8729-8738

114. Carr SM, Carnero E, Garcia-Sastre A, Brownlee GG, Fodor E (2006) Characterization of a mitochondrial-targeting signal in the PB2 protein of influenza viruses. Virology 344:492-508

115. Graef KM, Vreede FT, Lau Y-F, McCall AW, Carr SM, Subbarao K, Fodor E (2010) The PB2 subunit of the influenza virus RNA polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon. J Virol 84:8433-8445

116. Kim JH, Hatta M, Watanabe S, Neumann G, Watanabe T, Kawaoka Y (2010) Role of host-specific amino acids in the pathogenicity of avian H5N1 influenza viruses in mice. J Gen Virol 91:1284-1289

117. Taft AS, Ozawa M, Fitch A, Depasse JV, Halfmann PJ, Hill-Batorski L, Hatta M, Friedrich TC, Lopes TJS, Maher EA, Ghedin E, Macken CA, Neumann G, Kawaoka Y (2015) Identification of mammalian-adapting mutations in the polymerase complex of an avian H5N1 influenza virus. Nat Commun 6:7491

118. Kamiki H, Matsugo H, Kobayashi T, Ishida H, Takenaka-Uema A, Murakami S, Horimoto T (2018) A PB1-K577E mutation in H9N2 influenza virus increases polymerase activity and pathogenicity in mice. Viruses 10:653

119. Xu G, Zhang X, Gao W, Wang C, Wang J, Sun H, Sun Y, Guo L, Zhang R, Chang K-C, Liu J, Pu J (2016) Prevailing PA mutation K356R in avian influenza H9N2 virus increases mammalian replication and pathogenicity. J Virol 90:8105-8114

120. Liang L, Jiang L, Li J, Zhao Q, Wang J, He X, Huang S, Wang Q, Zhao Y, Wang G, Sun N, Deng G, Shi J, Tian G, Zeng X, Jiang Y, Liu L, Liu J, Chen P, Bu Z, Kawaoka Y, Chen H, Li C (2019) Low polymerase activity attributed to PA drives the acquisition of the PB2 E627K mutation of H7N9 avian influenza virus in mammals. MBio 10:e01162-01119

121. Song J, Xu J, Shi J, Li Y, Chen H (2015) Synergistic effect of S224P and N383D substitutions in the PA of H5N1 avian
influenza virus contributes to mammalian adaptation. *Sci Rep*
5:10510

122. Song J, Feng H, Xu J, Zhao D, Shi J, Li Y, Deng G, Jiang Y, Li X, Zhu P, Guan Y, Bu Z, Kawaoka Y, Chen H (2011) The PA protein directly contributes to the virulence of H5N1 avian influenza viruses in domestic ducks. J Virol 85:2180–2188

123. Chen L, Wang C, Luo J, Li M, Liu H, Zhao N, Huang J, Zhu X, Ma G, Yuan G, He H (2017) Amino acid substitution K470R in the nucleoprotein increases the virulence of H5N1 influenza A virus in mammals. Front Microbiol 8:1308

124. Gabriel G, Abram M, Keiner B, Wagner R, Klenk HD, Stech J (2007) Differential polymerase activity in avian and mammalian cells determines host range of influenza virus. J Virol 81:9601–9604

125. Wasilenko JL, Sarmento L, Pantin-Jackwood MJ (2009) A single substitution in amino acid 184 of the NP protein alters the replication and pathogenicity of H5N1 avian influenza viruses in chickens. Arch Virol 154:969–979

126. Tada T, Suzuki K, Sakurai Y, Kubo M, Okada H, Itoh T, Tsukamoto K (2011) NP body domain and PB2 contribute to increased virulence of H5N1 highly pathogenic avian influenza viruses in chickens. J Virol 85:1834–1846

127. Tada T, Suzuki K, Sakurai Y, Kubo M, Okada H, Itoh T, Tsukamoto K (2011) Emergence of avian influenza viruses with enhanced transcription activity by a single amino acid substitution in the nucleoprotein during replication in chicken brains. J Virol 85:10354–10363

128. Selman M, Dankar SK, Forbes NE, Jia J-J, Brown EG (2012) Adaptive mutation in influenza A virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing. Emerg Microb Infect 1:1–10

129. Marc D (2014) Influenza virus non-structural protein NS1: interferon antagonism and beyond. J Gen Virol 95:2594–2611

130. Abdelwhab E-SM, Veits J, Breithaupt A, Gohrbandt S, Ziller M, Teifke JP, Stech J, Mettenleiter TC (2016) Prevalence of the C-terminal truncations of NS1 in avian influenza A viruses and effect on virulence and replication of a highly pathogenic H7N1 virus in chickens. Virulence 7:546–557

131. Jiao P, Tian G, Li Y, Deng G, Jiang Y, Liu C, Liu W, Bu Z, Kawaoka Y, Chen H (2008) A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice. J Virol 82:1146–1154

132. Wang BX, Wei L, Kotra LP, Brown EG, Fish EN (2017) A conserved residue, tyrosine (Y) 84, in H5N1 influenza A virus NS1 regulates IFN Signaling responses to enhance viral infection. Viruses 9:107

133. Imai H, Shinya K, Takano R, Kiso M, Muramoto Y, Sakabe S, Murakami S, Ito M, Yamada S, Le MTQ, Nidom CA, Sakai-Tagawa Y, Takahashi K, Omori Y, Noda T, Shimojima M, Kaku-gawa S, Goto H, Iwatsuki-Horimoto K, Horimoto T, Kawaoka Y (2010) The HA and NS genes of human H5N1 influenza A virus contribute to high virulence in ferrets. PLoS Pathog 6:e1001106

134. Long J-X, Peng D-X, Liu Y-L, Wu Y-T, Liu X-F (2008) Virulence of H5N1 avian influenza virus enhanced by a 15-nucleotide deletion in the viral nonstructural gene. Virus Genes 36:471–478

135. Lee H-J, Zheng JJ (2010) PDZ domains and their binding partners: structure, specificity, and modification. Cell Commun Signal 8:8

136. Obenauer JC (2006) Large-scale sequence analysis of avian influenza isolates. Science 311:1576–1580

137. Soubies SM, Volmer C, Croville G, Loupias J, Peralta B, Costes P, Lacroux C, Guerin J-L, Volmer R (2010) Species-specific contribution of the four C-terminal amino acids of influenza A virus NS1 protein to virulence. J Virol 84:6733–6747

138. Zielecki F, Semmler I, Kalthoff D, Voss D, Mael S, Gruber AD, Beer M, Wolff T (2010) Virulence determinants of avian H5N1 influenza A virus in mammalian and avian hosts: role of the C-terminal ESEV motif in the viral NS1 protein. J Virol 84:10708–10718

139. Dundon WG, Capua I (2009) A closer look at the NS1 of influenza virus. Viruses 1:1057–1072

140. Soubies SM, Hoffmann TW, Croville G, Larcher T, Ledevin M, Soubieux D, Quéré P, Guérin J-L, Marc D, Volmer R (2013) Deletion of the C-terminal ESEV domain of NS1 does not affect the replication of a low-pathogenic avian influenza virus H7N1 in ducks and chickens. J Gen Virol 94:50–58

141. O'Neill RE, Talon J, Palese P (1998) The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17:288–296

142. Robb NC, Vreede FT, Smith M, Fodor E (2009) NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome. J Gen Virol 90:1398–1407

143. Shimizu T, Takizawa N, Watanabe K, Nagata K, Kobayashi N (2011) Crucial role of the influenza virus NS2 (NEP) C-terminal domain in M1 binding and nuclear export of vRNP. FEBS Lett 585:41–46

144. Gorai T, Goto H, Noda T, Watanabe T, Kozuka-Hata H, Oyama M, Takano R, Neumann G, Watanabe S, Kawaoka Y (2012) F1Fo-ATPase, F-type proton-translocating ATPase, at the plasma membrane is critical for efficient influenza virus budding. Proc Natl Acad Sci USA 109:4615–4620

145. Paterson D, Fodor E (2012) Emerging roles for the influenza A virus nuclear export protein (NEP). PLoS Pathog 8:e1003019

146. Mänz B, Brunotte L, Reuther P, Schwemmler M (2012) Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells. Nat Commun 3:802

147. Mänz B, Schwemmler M, Brunotte L (2013) Adaptation of avian influenza A virus polymerase in mammals to overcome the host species barrier. J Virol 87:7200–7209

148. Bui M, Whittaker G, Helenius A (1996) Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins. J Virol 70:8391–8401

149. Martin K, Helenius A (1991) Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell 67:117–130

150. Gomez-Puertas P, Albo C, Perez-Pastrana E, Vivo A, Portela A (2000) Influenza virus matrix protein is the major driving force in virus budding. J Virol 74:11538–11547

151. Pinto LH, Holsinger LJ, Lamb RA (1992) Influenza virus M2 protein has ion channel activity. Cell 69:517–528

152. Chen BJ, Leser GP, Jackson D, Lamb RA (2008) The influenza virus M2 protein cytoplasmic tail interacts with the M1 protein and influences virus assembly at the site of virus budding. J Virol 82:10059–10070

153. McCown MF, Pekosz A (2005) The influenza A virus M2 cytoplasmic tail is required for infectious virus production and efficient genome packaging. J Virol 79:3595–3605

154. Wise HM, Hutchinson EC, Jagger BW, Stuart AD, Kang ZH, Robb N, Schwartzman LM, Kash JC, Fodor E, Firth AE, Gog JR, Taubenberger JK, Digard P (2012) Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain. PLoS Pathog 8:e1002998

155. Smeenk CA, Wright KE, Burns BF, Thaker AJ, Brown EG (1996) Mutations in the hemagglutinin and matrix genes of a virulent influenza virus variant, A/FM/1/47-MA, control different stages in pathogenesis. Virus Res 44:79–95

156. Fan S, Deng G, Song J, Tian G, Suo Y, Jiang Y, Guan Y, Bu Z, Kawaoka Y, Chen H (2009) Two amino acid residues in the
matrix protein M1 contribute to the virulence difference of H5N1
avian influenza viruses in mice. Virology 384:28-32

157. Nao N, Kajihara M, Manzoor R, Maruyama J, Yoshida R, Muramatsu M, Miyamoto H, Igarashi M, Eguchi N, Sato M, Kondoh T, Okamatsu M, Sakoda Y, Kida H, Takada A (2015) A single amino acid in the M1 protein responsible for the different pathogenic potentials of H5N1 highly pathogenic avian influenza virus strains. PLoS ONE 10:e0137989

158. Dong G, Peng C, Luo J, Wang C, Han L, Wu B, Ji G, He H (2015) Adamantane-resistant influenza a viruses in the world (1902–2013): frequency and distribution of M2 gene mutations. PLoS ONE 10:e0119115–e0119115

159. Abed Y, Goyette N, Boivin G (2005) Generation and characterization of recombinant influenza A (H1N1) viruses harboring amantadine resistance mutations. Antimicrob Agents Chemother 49:556–559

160. Lan Y, Zhang Y, Dong L, Wang D, Huang W, Xin L, Yang L, Zhao X, Li Z, Wang W, Li X, Xu C, Yang L, Guo J, Wang M, Peng Y, Gao Y, Guo Y, Wen L, Jiang T, Shu Y (2010) A comprehensive surveillance of adamantane resistance among human influenza A virus isolated from mainland China between 1956 and 2009. Antivir Ther 15:853–859

161. Cheung C-L, Rayner JM, Smith GJD, Wang P, Naipospos TSP, Zhang J, Yuen K-Y, Webster RG, Peiris JSM, Guan Y, Chen H (2006) Distribution of amantadine-resistant H5N1 avian influenza variants in Asia. J Infect Dis 193:1626–1629

162. Chen W, Calvo PA, Malide D, Gibbs J, Schubert U, Bacik I, Basta S, O'Neill R, Schickli J, Palese P, Henklein P, Bennink JR, Yewdell JW (2001) A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 7:1306–1312

163. Chen CJ, Chen GW, Wang CH, Huang CH, Wang YC, Shih SR (2010) Differential localization and function of PB1-F2 derived from different strains of influenza A virus. J Virol 84:10051–10062

164. Kamal R, Alymova I, York I (2017) Evolution and virulence of influenza A virus protein PB1-F2. Int J Molec Sci 19:96

165. Kamal RP, Kumar A, Davis CT, Tzeng W-P, Nguyen T, Donis RO, Katz JM, York IA (2015) Emergence of highly pathogenic avian influenza A(H5N1) virus PB1-F2 variants and their virulence in BALB/c mice. J Virol 89:5835–5846

166. Dudek SE, Wixler L, Nordhoff C, Nordmann A, Anhlan D, Wixler V, Ludwig S (2011) The influenza virus PB1-F2 protein has interferon antagonistic activity. Biol Chem 392(12):1135–1144

167. Leymarie O, Meyer L, Tafforeau L, Lotteau V, Costa BD, Delmas B, Chevalier C, Le Goffic R (2017) Influenza virus protein PB1-F2 interacts with CALCOCO2 (NDP52) to modulate innate immune response. J Gen Virol 98:1196–1208

168. Reis AL, McCauley JW (2013) The influenza virus protein PB1-F2 interacts with IKKβ and modulates NF-κB signalling. PLoS ONE 8:e63852–e63852

169. Le Goffic R, Bouguyon E, Chevalier C, Vidic J, Da Costa B, Leymarie O, Bourdieu C, Decamps L, Dhorne-Pollet S, Delmas B (2010) Influenza A virus protein PB1-F2 exacerbates IFN-β expression of human respiratory epithelial cells. J Immunol 185:4812–4823

170. McAuley JL, Chipuk JE, Boyd KL, Van De Velde N, Green DR, McCullers JA (2010) PB1-F2 proteins from H5N1 and 20 century pandemic influenza viruses cause immunopathology. PLoS Pathog 6:e1001014

171. Schmolke M, Manicassamy B, Pena L, Sutton T, Hai R, Varga ZT, Hale BG, Steel J, Perez DR, Garcia-Sastre A (2011) Differential contribution of PB1-F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species. PLoS Pathog 7:e1002186

172. Leymarie O, Embury-Hyatt C, Chevalier C, Jouneau L, Moroldo M, Da Costa B, Berhane Y, Delmas B, Weingartl HM, Le Goffic R (2014) PB1-F2 attenuates virulence of highly pathogenic avian H5N1 influenza virus in chickens. PLoS ONE 9:e100679

173. Marjuki H, Scholtissek C, Franks J, Negovetich NJ, Aldridge JR, Salomon R, Finkelstein D, Webster RG (2010) Three amino acid changes in PB1-F2 of highly pathogenic H5N1 avian influenza virus affect pathogenicity in mallard ducks. Arch Virol 155:925–934

174. Jagger BW, Wise HM, Kash JC, Walters KA, Wills NM, Xiao YL, Dunfee RL, Schwartzman LM, Ozinsky A, Bell GL, Dalton RM, Lo A, Efstathiou S, Atkins JF, Firth AE, Taubenberger JK, Digard P (2012) An overlapping protein-coding region in influenza A virus segment 3 modulates the host response. Science 337:199–204

175. Desmet EA, Bussey KA, Stone R, Takimoto T (2013) Identification of the N-terminal domain of the influenza virus PA responsible for the suppression of host protein synthesis. J Virol 87:3108–3118

176. Khaperskyy DA, Schmaling S, Larkins-Ford J, McCormick C, Gaglia MM (2016) Selective degradation of host RNA polymerase II transcripts by influenza A virus PA-X host shutoff protein. PLoS Pathog 12:e1005427

177. Hu J, Mo Y, Wang X, Gu M, Hu Z, Zhong L, Wu Q, Hao X, Hu S, Liu W, Liu H, Liu X, Liu X (2015) PA-X decreases the pathogenicity of highly pathogenic H5N1 influenza A virus in avian species by inhibiting virus replication and host response. J Virol 89:4126–4142

178. Gao H, Sun Y, Hu J, Qi L, Wang J, Xiong X, Wang Y, He Q, Lin Y, Kong W, Seng L-G, Sun H, Pu J, Chang K-C, Liu X, Liu J (2009) The contribution of PA-X to the virulence of pandemic, H1N1 and highly pathogenic H5N1 avian influenza viruses. Sci Rep 5:2015

179. Hayashi T, MacDonald LA, Takimoto T (2015) Influenza A virus protein PA-X contributes to viral growth and suppression of the host antiviral and immune responses. J Virol 89:6442–6452

180. Hu J, Ma C, Liu X (2018) PA-X: a key regulator of influenza A virus pathogenicity and host immune responses. Med Microbiol Immunol 207:255–269

181. Shi M, Jagger BW, Wise HM, Digard P, Holmes EC, Taubenberger JK (2012) Evolutionary conservation of the PA-X open reading frame in segment 3 of influenza A virus. J Virol 86:12411–12413

182. Gao H, Sun H, Hu J, Qi L, Wang J, Xiong X, Wang Y, He Q, Lin Y, Kong W, Seng L-G, Pu J, Chang K-C, Liu X, Liu J, Sun Y (2015) Twenty amino acids at the C-terminus of PA-X are associated with increased influenza A virus replication and pathogenicity. J Gen Virol 96:2036–2049

183. Gao H, Xu G, Sun Y, Qi L, Wang J, Kong W, Sun H, Pu J, Chang K-C, Liu J (2015) PA-X is a virulence factor in avian H9N2 influenza virus. J Gen Virol 96:2587–2594

184. Kargarfard F, Sami A, Mohammadi-Dehcheshmeh M, Ebrahimie E (2016) Novel approach for identification of influenza virus host range and zoonotic transmissible sequences by determination of host-related associative positions in viral genome segments. BMC Genom 17:925

185. Qiang X, Kou Z (2019) Scoring amino acid mutation to predict pandemic risk of avian influenza virus. BMC Bioinform 20:288

186. Lauring AS, Andino R (2010) Quasispecies theory and the behavior of RNA viruses. PLoS Pathog 6:e1001005–e1001005

187. Welkers MRA, Pawestri HA, Fonville JM, Sampurno OD, Pater M, Holwerda M, Han AX, Russell CA, Jeeninga RE, Setiawaty V, de Jong MD, Eggink D (2019) Genetic diversity and host adaptation of avian H5N1 influenza viruses during human infection. Emerg Microbe Infect 8:262–271
188. World Health Organization (2019) Cumulative number of confirmed human cases for avian influenza A(H5N1) reported to WHO, 2003–2019. https://www.who.int/influenza/human_anima l_interface/2019_06_24_tableH5N1.pdf?ua=1

189. Qi W, Zhou X, Shi W, Huang L, Xia W, Liu D, Li H, Chen S, Lei F, Cao L, Wu J, He F, Song W, Li Q, Li H, Liao M, Liu M (2014) Genesis of the novel human-infecting influenza A(H10N8) virus and potential genetic diversity of the virus in poultry, China. Eurosurveillance 19:20841

190. Arzey GG, Kirkland PD, Arzey KE, Frost M, Maywood P, Conaty S, Hurt AC, Deng YM, Iannello P, Barr I, Dwyer DE, Ratnamohan M, McPhie K, Selleck P (2012) Influenza virus A (H10N7) in chickens and poultry abattoir workers, Australia. Emerg Infect Dis 18:814–816

191. Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, Chen J, Jie Z, Qiu H, Xu K, Xu X, Lu H, Zhu W, Gao Z, Xiang N, Shen Y, He Z, Gu Y, Zhang Z, Yang Y, Zhao X, Zhou L, Li X, Zou S, Zhang Y, Li X, Yang L, Guo J, Dong J, Li Q, Dong L, Zhu Y, Bai T, Wang S, Hao P, Yang W, Zhang Y, Han J, Yu H, Li D, Gao GF, Wu G, Wang Y, Yuan Z, Shu Y (2013) Human infection with a novel avian-origin influenza A (H7N9) virus. N Engl J Med 368:1888–1897

192. Tong XC, Weng SS, Xue F, Wu X, Xu TM, Zhang WH (2018) First human infection by a novel avian influenza A(H7N4) virus. J Infect 77:249–257

193. Huang Y, Li X, Zhang H, Chen B, Jiang Y, Yang L, Zhu W, Hu S, Zhou S, Tang Y, Xiang X, Li F, Li W, Gao L (2015) Human infection with an avian influenza A (H9N2) virus in the middle region of China. J Med Virol 87:1641–1648

194. Food and Agriculture Organization of the United Nations (2019) H7N9 situation update. http://www.fao.org/ag/againfo/progr ammes/en/empres/h7n9/situation_update.html

195. World Health Organization (2019) Influenza at the human-animal interface. https://www.who.int/influenza/human_animal_inter face/Influenza_SummaryIRA_HA_interface_09_04_2019.pdf

196. Gao P, Du H, Fan L, Chen L, Liao M, Xu C, Xiang B, Ren T (2018) Human infection with an avian-origin influenza A (H7N4) virus in Jiangsu: a potential threat to China. J Infect 77:249–257

197. World Health Organisation (2018) Human infection with avian influenza A(H7N4) virus—China. Disease Outbreak News (DONs). https://www.who.int/csr/don/22-february-2018-ah7n4 -china/en/

198. Vijaykrishna D, Deng Y-M, Grau ML, Kay M, Suttie A, Hor-wood PF, Kalpravidh W, Claes F, Osbjer K, Dussart P, Barr IG, Karlsson EA (2019) Emergence of influenza A(H7N4) virus, Cambodia. Emerg Infect Dis. https://doi.org/10.3201/eid25 10.190506

199. World Health Organisation (2019) Antigenic and genetic charac- teristics of zoonotic influenza viruses and development of candi- date vaccine viruses for pandemic preparedness. Geneva. https:// www.who.int/influenza/vaccines/virus/201902_zoonotic_vacci nevirusupdate.pdf

200. Su Y, Yang H-Y, Zhang B-J, Jia H-L, Tien P (2008) Analysis of a point mutation in H5N1 avian influenza virus hemagglutinin in relation to virus entry into live mammalian cells. Arch Virol 153:2253–2261

201. Wang W, Lu B, Zhou H, Suguitan AL Jr, Cheng X, Subbarao K, Kemble G, Jin H (2010) Glycosylation at 158 N of the hemag- glutinin protein and receptor binding specificity synergistically affect the antigenicity and immunogenicity of a live attenuated H5N1 A/Vietnam/1203/2004 vaccine virus in ferrets. J Virol 84:6570–6577

202. Naughtin M, Dyason JC, Mardy S, Sorn S, von Itzstein M, Buchy P (2011) Neuraminidase inhibitor sensitivity and receptor-bind- ing specificity of Cambodian clade 1 highly pathogenic H5N1 influenza virus. Antimicrob Agents Chemother 55:2004–2010

203. Kongchanagul A, Suptawiwat O, Kanrai P, Uiprasertkul M, Puthavathana P, Auewarakul P (2008) Positive selection at the receptor-binding site of haemagglutinin H5 in viral sequences derived from human tissues. J Gen Virol 89:1805–1810

204. Yamada S, Suzuki Y, Suzuki T, Le MQ, Nidom CA, Sakai-Tagawa Y, Muramoto Y, Ito M, Kiso M, Horimoto T, Shinya K, Sawada T, Kiso M, Usui T, Murata T, Lin Y, Hay A, Haire LF, Stevens DJ, Russell RJ, Gamblin SJ, Skehel JJ, Kawaoka Y (2006) Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature 444:378–382

205. Jin F, Dong X, Wan Z, Ren D, Liu M, Geng T, Zhang J, Gao W, Shao H, Qin A, Ye J (2019) A single mutation N166D in hemagglutinin affects antigenicity and pathogenesis of H9N2 avian influenza virus. Viruses 11:709

206. Gao Y, Zhang Y, Shinya K, Deng G, Jiang Y, Li Z, Guan Y, Tian G, Li Y, Shi J, Liu L, Zeng X, Bu Z, Xia X, Kawaoka Y, Chen H (2009) Identification of amino acids in HA and PB2 critical for the transmission of H5N1 avian influenza viruses in a mammalian host. PLoS Pathog 5:e1000709

207. Dortmans JCFM, Dekkers J, Wickramasinghe INA, Verheije MH, Rottier PJM, van Kuppeveld FJM, de Vries E, de Haan CAM (2013) Adaptation of novel H7N9 influenza A virus to human receptors. Sci Rep 3:3058

208. Lu X, Qi J, Shi Y, Wang M, Smith DF, Heimburg-Molinaro J, Zhang Y, Paulson JC, Xiao H, Gao GF (2013) Structure and receptor binding specificity of hemagglutinin H13 from avian influenza A virus H13N6. J Virol 87:9077–9085

209. Wang F, Qi J, Bi Y, Zhang W, Wang M, Zhang B, Wang M, Liu J, Yan J, Shi Y, Gao GF (2015) Adaptation of avian influenza A (H6N1) virus from avian to human receptor-binding prefer- ence. EMBO J 34:1661–1673

210. Han P, Hu Y, Sun W, Zhang S, Li Y, Wu X, Yang Y, Zhu Q, Jiang T, Li J, Qin C (2015) Mouse lung-adapted mutation of E190G in hemagglutinin from H5N1 influenza virus contrib- utes to attenuation in mice. J Med Virol 87:1816–1822

211. Qu Z, Ma S, Kong H, Deng G, Shi J, Liu L, Suzuki Y, Chen H (2017) Identification of a key amino acid in hemagglutinin that increases human-type receptor binding and transmission of an H6N2 avian influenza virus. Microbe Infect 19:655–660

212. Teng Q, Xu D, Shen W, Liu Q, Rong G, Li X, Yan L, Yang J, Chen H, Yu H, Ma W, Li Z (2016) A single mutation at position 190 in hemagglutinin enhances binding affinity for human type sialic acid receptor and replication of H9N2 avian influenza virus in mice. J Virol 90:9806–9825

213. Peng W, Bouwman KM, McBride R, Grant OC, Woods RJ, Verheije MH, Paulson JC, de Vries RP (2018) Enhanced human-type receptor binding by ferret-transmissible H5N1 with a K193T mutation. J Virol 92:e02016-17

214. Watanabe Y, Ibrahim MS, Ellakany HF, Kawashita N, Mizuike R, Hiramatsu H, Sriwilaijaroen N, Takagi T, Suzuki Y, Ikuta K (2011) Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emer- gence in birds in Egypt. PLoS Pathog 7:e1002068

215. Ramos I, Krammer F, Hai R, Aguilera D, Bernal-Rubio D, Steel J, Garcia-Sastre A, Fernandez-Sesma A (2013) H7N9 influenza viruses interact preferentially with alpha2,3-linked sialic acids and bind weakly to alpha2,6-linked sialic acids. J Gen Virol 94:2417–2423

216. Tzarum N, de Vries RP, Peng W, Thompson AJ, Bouwman KM, McBride R, Yu W, Zhu X, Verheije MH, Paulson JC, Wilson IA (2017) The 150-loop restricts the host specificity of human H10N8 influenza virus. Cell Rep 19:235–245

217. Zhang H, de Vries RP, Tzarum N, Zhu X, Yu W, McBride R, Paulson JC, Wilson IA (2015) A human-infecting H10N8
influenza virus retains a strong preference for avian-type receptors. Cell Host Microbe 17:377-384

218. Gambaryan A, Tuzikov A, Pazynina G, Bovin N, Balish A, Klimov A (2006) Evolution of the receptor binding phenotype of influenza A (H5) viruses. Virology 344:432-438

219. Shinya K, Makino A, Hatta M, Watanabe S, Kim JH, Kawaka Y (2010) A mutation in H5 haemagglutinin that conferred human receptor recognition is not maintained stably during duck passage. J Gen Virol 91:1461-1463

220. Yu Z, Ren Z, Zhao Y, Cheng K, Sun W, Zhang X, Wu J, He H, Xia X, Gao Y (2019) PB2 and hemagglutinin mutations confer a virulent phenotype on an H1N2 avian influenza virus in mice. Arch Virol 164:2023-2029

221. Bosch FX, Garten W, Klenk HD, Rott R (1981) Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of Avian influenza viruses. Virology 113:725-735

222. Perdue ML, Garcia M, Senne D, Fraire M (1997) Virulence-associated sequence duplication at the hemagglutinin cleavage site of avian influenza viruses. Virus Res 49:173-186

223. Webster RG, Rott R (1987) Influenza virus A pathogenicity: the pivotal role of hemagglutinin. Cell 50:665-666

224. Subbarao K, Klimov A, Katz J, Regnery H, Lim W, Hall H, Perdue M, Swayne D, Bender C, Huang J, Hemphill M, Rowe T, Shaw M, Xu X, Fukuda K, Cox N (1998) Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279:393-396

225. Horimoto T, Kawaoka Y (1994) Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus. J Virol 68:3120-3128

226. Schrauwen EJA, Herfst S, Leijten LM, van Run P, Besteboer TM, Linster M, Bodewes R, Kreijtz JHCM, Rimmelzwaan GF, Osterhaus ADME, Fouchier RAM, Kuiken T, van Riel D (2012) The multibasic cleavage site in H5N1 virus is critical for systemic spread along the olfactory and hematogenous routes in ferrets. J Virol 86:3975-3984

227. Suguitan AL Jr, Matsuoka Y, Lau Y-F, Santos CP, Vogel L, Cheng LI, Orandle M, Subbarao K (2012) The multibasic cleavage site of the hemagglutinin of highly pathogenic A/ Vietnam/1203/2004 (H5N1) avian influenza virus acts as a virulence factor in a host-specific manner in mammals. J Virol 86:2706-2714

228. Zhang Y, Sun Y, Sun H, Pu J, Bi Y, Shi Y, Lu X, Li J, Zhu Q, Gao GF, Yang H, Liu J (2012) A single amino acid at the hemagglutinin cleavage site contributes to the pathogenicity and neurovirulence of H5N1 influenza virus in mice. J Virol 86:6924-6931

229. Zhu W, Zhou J, Li Z, Yang L, Li X, Huang W, Zou S, Chen W, Wei H, Tang J, Liu L, Dong J, Wang D, Shu Y (2017) Biological characterisation of the emerged highly pathogenic avian influenza (HPAI) A(H7N9) viruses in humans, in mainland China, 2016 to 2017. Eurosurveillance 22:30533

230. Sun X, Belser JA, Pulit-Penaloza JA, Zeng H, Lewis A, Shieh W-J, Tumpey TM, Maines TR (2016) Pathogenesis and transmission assessments of two H7N8 influenza A viruses recently isolated from Turkey farms in Indiana using mouse and ferret models. J Virol 90:10936-10944

231. Abdelwhab EM, Veits J, Ulrich R, Kasbohm E, Teifke JP, Mettenleiter TC (2016) Composition of the hemagglutinin polybasic proteolytic cleavage motif mediates variable virulence of H7N7 avian influenza viruses. Sci Rep 6:39505

232. Reed ML, Yen H-L, DuBois RM, Bridges OA, Salomon R, Webster RG, Russell CJ (2009) Amino acid residues in the fusion peptide pocket regulate the pH of activation of the H5N1 influenza virus hemagglutinin protein. J Virol 83:3568-3580

233. Krenn BM, Egorov A, Romanovskaya-Romanko E, Wolschek M, Nakowitsch S, Ruthsatz T, Kiefmann B, Morokutti A, Humer J, Geiler J, Cinatl J, Michaelis M, Wressnigg N, Sturlan S, Ferko B, Batishchev OV, Indenbom AV, Zhu R, Kastner M, Hinterdorfer P, Kiselev O, Muster T, Romanova J (2011) Single HA2 mutation increases the infectivity and immunogenicity of a live attenuated H5N1 intranasal influenza vaccine candidate lacking NS1. PLoS ONE 6:e18577-e18577

234. Sun X, Belser JA, Yang H, Pulit-Penaloza JA, Pappas C, Brock N, Zeng H, Creager HM, Stevens J, Maines TR (2019) Identification of key hemagglutinin residues responsible for cleavage, acid stability, and virulence of fifth-wave highly pathogenic avian influenza A(H7N9) viruses. Virology 535:232-240

235. Wessels U, Abdelwhab EM, Veits J, Hoffmann D, Mamerow S, Stech O, Hellert J, Beer M, Mettenleiter TC, Stech J (2018) A dual motif in the hemagglutinin of H5N1 goose/guangdong-like highly pathogenic avian influenza virus strains is conserved from their early evolution and increases both membrane fusion pH and virulence. J Virol 92:e00778-00718

236. Auewarakul P, Suptawiwat O, Kongchanagul A, Sangma C, Suzuki Y, Ungchusak K, Louisirirotchanakul S, Lerdsamran H, Pooruk P, Thitithanyanont A, Pittayawonganon C, Guo C-T, Hiramatsu H, Jampangern W, Chunsutthiwat S, Puthavathana P (2007) An avian influenza H5N1 virus that binds to a human-type receptor. J Virol 81:9950-9955

237. Ilyushina NA, Govorkova EA, Gray TE, Bovin NV, Webster RG (2008) Human-like receptor specificity does not affect the neuraminidase-inhibitor susceptibility of H5N1 influenza viruses. PLoS Pathog 4:e1000043

238. Yen H-L, Aldridge JR, Boon ACM, Ilyushina NA, Salomon R, Hulse-Post DJ, Marjuki H, Franks J, Boltz DA, Bush D, Lipatov AS, Webby RJ, Rehg JE, Webster RG (2009) Changes in H5N1 influenza virus hemagglutinin receptor binding domain affect systemic spread. Proc Natl Acad Sci USA 106:286-291

239. Stevens J, Blixt O, Chen L-M, Donis RO, Paulson JC, Wilson IA (2008) Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J Mol Biol 381:1382-1394

240. Maines TR, Chen L-M, Van Hoeven N, Tumpey TM, Blixt O, Belser JA, Gustin KM, Pearce MB, Pappas C, Stevens J, Cox NJ, Paulson JC, Raman R, Sasisekharan R, Katz JM, Donis RO (2011) Effect of receptor binding domain mutations on receptor binding and transmissibility of avian influenza H5N1 viruses. Virology 413:139-147

241. Sorrell EM, Wan H, Araya Y, Song H, Perez DR (2009) Minimal molecular constraints for respiratory droplet transmission of an avian-human H9N2 influenza A virus. Proc Natl Acad Sci USA 106:7565-7570

242. Guo H, de Vries E, McBride R, Dekkers J, Peng W, Bouwman KM, Nycholat C, Verheije MH, Paulson JC, van Kuppeveld FJM, de Haan CAM (2017) Highly pathogenic influenza A(H5Nx) viruses with altered H5 receptor-binding specificity. Emerg Infect Dis 23:220-231

243. Harvey R, Martin ACR, Zambon M, Barclay WS (2004) Restrictions to the adaptation of influenza A virus H5 hemagglutinin to the human host. J Virol 78:502-507

244. Ayora-Talavera G, Shelton H, Scull MA, Ren J, Jones IM, Pickles RJ, Barclay WS (2009) Mutations in H5N1 influenza virus hemagglutinin that confer binding to human tracheal airway epithelium. PLoS ONE 4:e7836-e7836

245. Srinivasan K, Raman R, Jayaraman A, Viswanathan K, Sasisekharan R (2013) Quantitative description of glycan-receptor binding of influenza A virus H7 hemagglutinin. PLoS ONE 8:e49597

246. Sorrell EM, Song H, Pena L, Perez DR (2010) A 27-amino-acid deletion in the neuraminidase stalk supports replication of an
avian H2N2 influenza A virus in the respiratory tract of chickens.
J Virol 84:11831-11840

247. Hurt AC, Selleck P, Komadina N, Shaw R, Brown L, Barr IG (2007) Susceptibility of highly pathogenic A(H5N1) avian influenza viruses to the neuraminidase inhibitors and adamantanes. Antivir Res 73:228-231

248. Boltz DA, Douangngeun B, Phommachanh P, Sinthasak S, Mondry R, Obert C, Seiler P, Keating R, Suzuki Y, Hiramatsu H, Govorkova EA, Webster RG (2010) Emergence of H5N1 avian influenza viruses with reduced sensitivity to neuraminidase inhibitors and novel reassortants in Lao People's Democratic Republic. J Gen Virol 91:949-959

249. Kode SS, Pawar SD, Tare DS, Keng SS, Hurt AC, Mullick J (2019) A novel I117T substitution in neuraminidase of highly pathogenic avian influenza H5N1 virus conferring reduced susceptibility to oseltamivir and zanamivir. Vet Microbiol 235:21-24

250. Gubareva LV, Robinson MJ, Bethell RC, Webster RG (1997) Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en. J Virol 71:3385-3390

251. Baek YH, Song M-S, Lee E-Y, Kim Y-I, Kim E-H, Park S-J, Park KJ, Kwon H-I, Pascua PNQ, Lim G-J, Kim S, Yoon S-W, Kim MH, Webby RJ, Choi Y-K (2015) Profiling and characterization of influenza virus N1 strains potentially resistant to multiple neuraminidase inhibitors. J Virol 89:287-299

252. Ilyushina NA, Seiler JP, Rehg JE, Webster RG, Govorkova EA (2010) Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets. PLoS Pathog 6:e1000933

253. Gubareva LV, Sleeman K, Guo Z, Yang H, Hodges E, Davis CT, Baranovich T, Stevens J (2017) Drug susceptibility evaluation of an influenza A(H7N9) virus by analyzing recombinant neuraminidase proteins. J Infect Dis 216:S566-S574

254. Kode SS, Pawar SD, Cherian SS, Tare DS, Bhoye D, Keng SS, Mullick J (2019) Selection of avian influenza A (H9N2) virus with reduced susceptibility to neuraminidase inhibitors oseltamivir and zanamivir. Virus Res 265:122-126

255. Hurt AC, Lowther S, Middleton D, Barr IG (2010) Assessing the development of oseltamivir and zanamivir resistance in A(H5N1) influenza viruses using a ferret model. Antivir Res 87:361-366

256. Hurt AC, Holien JK, Barr IG (2009) In vitro generation of neuraminidase inhibitor resistance in A(H5N1) influenza viruses. Antimicrob Agents Chemother 53:4433-4440

257. Le QM, Kiso M, Someya K, Sakai YT, Nguyen TH, Nguyen KHL, Pham ND, Ngyen HH, Yamada S, Muramoto Y, Horimoto T, Takada A, Goto H, Suzuki T, Suzuki Y, Kawaoka Y (2005) Avian flu: isolation of drug-resistant H5N1 virus. Nature 437:1108

258. Nguyen HT, Nguyen T, Mishin VP, Sleeman K, Balish A, Jones J, Creanga A, Marjuki H, Uyeki TM, Nguyen DH, Nguyen DT, Do HT, Klimov AI, Davis CT, Gubareva LV (2013) Antiviral susceptibility of highly pathogenic avian influenza A(H5N1) viruses isolated from poultry, Vietnam, 2009-2011. Emerg Infect Dis 19:1963-1971

259. Govorkova EA, Ilyushina NA, Boltz DA, Douglas A, Yilmaz N, Webster RG (2007) Efficacy of oseltamivir therapy in ferrets inoculated with different clades of H5N1 influenza virus. Antimicrob Agents Chemother 51:1414-1424

260. Gillman A, Muradrasoli S, Mårdnäs A, Söderström H, Fedorova G, Löwenthal M, Wille M, Daggfeldt A, Järhult JD (2015) Oseltamivir resistance in influenza A(H6N2) caused by an R292K substitution in neuraminidase is not maintained in mammals without drug pressure. PLoS ONE 10:e0139415-e0139415

261. Hai R, Schmolke M, Leyva-Grado VH, Thangavel RR, Margine I, Jaffe EL, Krammer F, Solorzano A, Garcia-Sastre A, Palese P, Bouvier NM (2013) Influenza A(H7N9) virus gains neuraminidase inhibitor resistance without loss of in vivo virulence or transmissibility. Nat Commun 4:2854

262. Sleeman K, Guo Z, Barnes J, Shaw M, Stevens J, Gubareva LV (2013) R292K substitution and drug susceptibility of influenza A(H7N9) viruses. Emerg Infect Dis 19:1521-1524

263. Earhart KC, Elsayed NM, Saad MD, Gubareva LV, Nayel A, Deyde VM, Abdelsattar A, Abdelghani AS, Boynton BR, Mansour MM, Essmat HM, Klimov A, Shuck-Lee D, Monteville MR, Tjaden JA (2009) Oseltamivir resistance mutation N294S in human influenza A(H5N1) virus in Egypt. J Infect Public Health 2:74-80

264. Dai M, McBride R, Dortmans JCFM, Peng W, Bakkers MJG, de Groot RJ, van Kuppeveld FJM, Paulson JC, de Vries E, de Haan CAM (2017) Mutation of the second sialic acid-binding site, resulting in reduced neuraminidase activity, preceded the emergence of H7N9 influenza A virus. J Virol 91:e00049-00017

265. Creanga A, Hang NLK, Cuong VD, Nguyen HT, Phuong HVM, Thanh LT, Thach NC, Hien PT, Tung N, Jang Y, Balish A, Dang NH, Duong MT, Huong NT, Hoa DN, Tho ND, Klimov A, Kapella BK, Gubareva L, Kile JC, Hien NT, Mai LQ, Davis CT (2017) Highly pathogenic avian influenza A(H5N1) viruses at the animal-human interface in Vietnam, 2003-2010. J Infect Dis 216:S529-S538

266. Marjuki H, Mishin VP, Chesnokov AP, Jones J, De La Cruz JA, Sleeman K, Tamura D, Nguyen HT, Wu H-S, Chang F-Y, Liu M-T, Fry AM, Cox NJ, Villanueva JM, Davis CT, Gubareva LV (2015) Characterization of drug-resistant influenza A(H7N9) variants isolated from an oseltamivir-treated patient in Taiwan. J Infect Dis 211:249-257

267. Zhao D, Fukuyama S, Yamada S, Lopes TJS, Maemura T, Katsura H, Ozawa M, Watanabe S, Neumann G, Kawaoka Y (2015) Molecular determinants of virulence and stability of a reporter-expressing H5N1 influenza A virus. J Virol 89:11337-11346

268. Zhou B, Li Y, Halpin R, Hine E, Spiro DJ, Wentworth DE (2011) PB2 residue 158 is a pathogenic determinant of pandemic H1N1 and H5 influenza a viruses in mice. J Virol 85:357-365

269. Xu G, Wang F, Li Q, Bing G, Xie S, Sun S, Bian Z, Sun H, Feng Y, Peng X, Jiang H, Zhu L, Fan X, Qin Y, Ding J (2019) Mutations in PB2 and HA enhanced pathogenicity of H4N6 avian influenza virus in mice. J Gen Virol. https://doi.org/10.1099/jgv.0.001192

270. Zhang J, Su R, Jian X, An H, Jiang R, Mok CKP (2018) The D253N mutation in the polymerase basic 2 gene in avian influenza (H9N2) virus contributes to the pathogenesis of the virus in mammalian hosts. Virol Sin 33:531-537

271. Manzoor R, Sakoda Y, Nomura N, Tsuda Y, Ozaki H, Okamatsu M, Kida H (2009) PB2 protein of a highly pathogenic avian influenza virus strain A/chicken/Yamaguchi/7/2004 (H5N1) determines its replication potential in pigs. J Virol 83:1572-1578

272. Bussey KA, Bousse TL, Desmet EA, Kim B, Takimoto T (2010) PB2 residue 271 plays a key role in enhanced polymerase activity of influenza A viruses in mammalian host cells. J Virol 84:4395-4406

273. Foeglein A, Loucaides EM, Mura M, Wise HM, Barclay WS, Digard P (2011) Influence of PB2 host-range determinants on the intranuclear mobility of the influenza A virus polymerase. J Gen Virol 92:1650-1661

274. Mok CKP, Lee HHY, Lestra M, Nicholls JM, Chan MCW, Sia SF, Zhu H, Poon LLM, Guan Y, Peiris JSM (2014) Amino acid substitutions in polymerase basic protein 2 gene contribute to the pathogenicity of the novel A/H7N9 influenza virus in mammalian hosts. J Virol 88:3568-3576

275. Gao W, Zu Z, Liu J, Song J, Wang X, Wang C, Liu L, Tong Q, Wang M, Sun H, Sun Y, Liu J, Chang K-C, Pu J (2019)
Prevailing I292VPB2 mutation in avian influenza H9N2 virus increases viral polymerase function and attenuates IFN-β induction in human cells. J Gen Virol 20:19. https://doi.org/10.1099/jgv.0.001294

276. Xiao C, Ma W, Sun N, Huang L, Li Y, Zeng Z, Wen Y, Zhang Z, Li H, Li Q, Yu Y, Zheng Y, Liu S, Hu P, Zhang X, Ning Z, Qi W, Liao M (2016) PB2-588V promotes the mammalian adaptation of H10N8, H7N9 and H9N2 avian influenza viruses. Sci Rep 6:19474

277. DesRochers BL, Chen RE, Gounder AP, Pinto AK, Bricker T, Linton CN, Rogers CD, Williams GD, Webby RJ, Boon ACM (2016) Residues in the PB2 and PA genes contribute to the pathogenicity of avian H7N3 influenza A virus in DBA/2 mice. Virology 494:89-99

278. Hu M, Yuan S, Zhang K, Singh K, Ma Q, Zhou J, Chu H, Zheng B-J (2017) PB2 substitutions V598T/I increase the virulence of H7N9 influenza A virus in mammals. Virology 501:92-101

279. Liu Y, Qin K, Meng G, Zhang J, Zhou J, Zhao G, Luo M, Zheng X (2013) Structural and functional characterization of K339T substitution identified in the PB2 subunit cap-binding pocket of influenza A virus. J Biol Chem 288:11013-11023

280. Yamayoshi S, Yamada S, Fukuyama S, Murakami S, Zhao D, Uraki R, Watanabe T, Tomita Y, Macken C, Neumann G, Kawaoka Y (2014) Virulence-aflecting amino acid changes in the PA protein of H7N9 influenza A viruses. J Virol 88:3127-3134

281. Yamayoshi S, Kiso M, Yasuhara A, Ito M, Shu Y, Kawaoka Y (2018) Enhanced replication of highly pathogenic influenza A(H7N9) virus in humans. Emerg Infect Dis 24:746-750

282. Song W, Wang P, Mok BW-Y, Lau S-Y, Huang X, Wu W-L, Zheng M, Wen X, Yang S, Chen Y, Li L, Yuen K-Y, Chen H (2014) The K526R substitution in viral protein PB2 enhances the effects of E627K on influenza virus replication. Nat Commun 5:5509

283. Yamada S, Hatta M, Staker BL, Watanabe S, Imai M, Shinya K, Sakai-Tagawa Y, Ito M, Ozawa M, Watanabe T, Sakabe S, Li C, Kim JH, Myler PJ, Phan I, Raymond A, Smith E, Stacy R, Nidom CA, Lank SM, Wiseman RW, Bimber BN, O'Connor DH, Neumann G, Stewart LJ, Kawaoka Y (2010) Biological and structural characterization of a host-adapting amino acid in influenza virus. PLoS Pathog 6:e1001034

284. Wang C, Lee HHY, Yang ZF, Mok CKP, Zhang Z (2016) PB2-Q591K mutation determines the pathogenicity of avian H9N2 influenza viruses for mammalian species. PLoS ONE 11:e0162163

285. Chen H, Bright RA, Subbarao K, Smith C, Cox NJ, Katz JM, Matsuoka Y (2007) Polygenic virulence factors involved in pathogenesis of 1997 Hong Kong H5N1 influenza viruses in mice. Virus Res 128:159-163

286. Shinya K, Hamm S, Hatta M, Ito H, Ito T, Kawaoka Y (2004) PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5N1 influenza A viruses in mice. Virology 320:258-266

287. Fornek JL, Gillim-Ross L, Santos C, Carter V, Ward JM, Cheng LI, Proll S, Katze MG, Subbarao K (2009) A single-amino-acid substitution in a polymerase protein of an H5N1 influenza virus is associated with systemic infection and impaired T-cell activation in mice. J Virol 83:11102-11115

288. Mase M, Tanimura N, Imada T, Okamatsu M, Tsukamoto K, Yamaguchi S (2006) Recent H5N1 avian Influenza A virus increases rapidly in virulence to mice after a single passage in mice. J Gen Virol 87:3655-3659

289. Bortz E, Westera L, Maamary J, Steel J, Albrecht RA, Manicassamy B, Chase G, Martínez-Sobrido L, Schwemmle M, García-Sastre A (2011) Host- and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins. MBio 2:e00151-00111

290. Sang X, Wang A, Chai T, He X, Ding J, Gao X, Li Y, Zhang K, Ren Z, Li L, Yu Z, Wang T, Feng N, Zheng X, Wang H, Zhao Y, Yang S, Gao Y, Xia X (2015) Rapid emergence of a PB2-E627K substitution confers a virulent phenotype to an H9N2 avian influenza virus during adoption in mice. Arch Virol 160:1267-1277

291. Schat KA, Bingham J, Butler JM, Chen L-M, Lowther S, Crowley TM, Moore RJ, Donis RO, Lowenthal JW (2012) Role of position 627 of PB2 and the multibasic cleavage site of the hemagglutinin in the virulence of H5N1 avian influenza virus in chickens and ducks. PLoS ONE 7:e30960

292. Le QM, Sakai-Tagawa Y, Ozawa M, Ito M, Kawaoka Y (2009) Selection of H5N1 influenza virus PB2 during replication in humans. J Virol 83:5278-5281

293. Li Z, Chen H, Jiao P, Deng G, Tian G, Li Y, Hoffmann E, Webster RG, Matsuoka Y, Yu K (2005) Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model. J Virol 79:12058-12064

294. Sun H, Cui P, Song Y, Qi Y, Li X, Qi W, Xu C, Jiao P, Liao M (2015) PB2 segment promotes high-pathogenicity of H5N1 avian influenza viruses in mice. Front Microbiol 6:73

295. Leung BW, Chen H, Brownlee GG (2010) Correlation between polymerase activity and pathogenicity in two duck H5N1 influenza viruses suggests that the polymerase contributes to pathogenicity. Virology 401:96-106

296. Li J, Ishaq M, Prudence M, Xi X, Hu T, Liu Q, Guo D (2009) Single mutation at the amino acid position 627 of PB2 that leads to increased virulence of an H5N1 avian influenza virus during adaptation in mice can be compensated by multiple mutations at other sites of PB2. Virus Res 144:123-129

297. Wang J, Sun Y, Xu Q, Tan Y, Pu J, Yang H, Brown EG, Liu J (2012) Mouse-adapted H9N2 influenza A virus PB2 protein M147L and E627K mutations are critical for high virulence. PLoS ONE 7:e40752

298. Fan S, Hatta M, Kim JH, Halfmann P, Imai M, Macken CA, Le MQ, Nguyen T, Neumann G, Kawaoka Y (2014) Novel residues in avian influenza virus PB2 protein affect virulence in mammalian hosts. Nat Commun 5:5021

299. Zhu W, Zou X, Zhou J, Tang J, Shu Y (2015) Residues 41 V and/or 210D in the NP protein enhance polymerase activities and potential replication of novel influenza (H7N9) viruses at low temperature. Virol J 12:71

300. Czudai-Matwich V, Otte A, Matrosovich M, Gabriel G, Klenk H-D (2014) PB2 mutations D701N and S714R promote adaptation of an influenza H5N1 virus to a mammalian host. J Virol 88:8735-8742

301. Elgendy EM, Arai Y, Kawashita N, Daidoji T, Takagi T, Ibrahim MS, Nakaya T, Watanabe Y (2017) Identification of polymerase gene mutations that affect viral replication in H5N1 influenza viruses isolated from pigeons. J Gen Virol 98:6-17

302. Hulse-Post DJ, Franks J, Boyd K, Salomon R, Hoffmann E, Yen HL, Webby RJ, Walker D, Nguyen TD, Webster RG (2007) Molecular changes in the polymerase genes (PA and PB1) associated with high pathogenicity of H5N1 influenza virus in mallard ducks. J Virol 81:8515-8524

303. Xu C, Hu W-B, Xu K, He Y-X, Wang T-Y, Chen Z, Li T-X, Liu J-H, Buchy P, Sun B (2012) Amino acids 473 V and 598P of PB1 from an avian-origin influenza A virus contribute to polymerase activity, especially in mammalian cells. J Gen Virol 93:531-540

304. Feng X, Wang Z, Shi J, Deng G, Kong H, Tao S, Li C, Liu L, Guan Y, Chen H (2016) Glycine at position 622 in PB1 contributes to the virulence of H5N1 avian influenza virus in mice. J Virol 90:1872-1879
305. Conenello GM, Zamarin D, Perrone LA, Tumpey T, Palese P (2007) A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence. PLoS Pathog 3:1414–1421

306. Hu M, Yuan S, Ye Z-W, Singh K, Li C, Shuai H, Fai N, Chow BKC, Chu H, Zheng B-J (2017) PAN substitutions A37S, A37S/I61T and A37S/V63I attenuate the replication of H7N7 influenza A virus by impairing the polymerase and endonuclease activities. J Gen Virol 98:364–373

307. Hu M, Chu H, Zhang K, Singh K, Li C, Yuan S, Chow BKC, Song W, Zhou J, Zheng B-J (2016) Amino acid substitutions V63I or A37S/I61T/V63I/V100A in the PA N-terminal domain increase the virulence of H7N7 influenza A virus. Sci Rep 6:37800

308. Song M-S, Pascua PNQ, Lee JH, Baek YH, Lee O-J, Kim C-J, Kim H, Webby RJ, Webster RG, Choi YK (2009) The polymerase acidic protein gene of influenza a virus contributes to pathogenicity in a mouse model. J Virol 83:12325–12335

309. Zhong G, Le MQ, Lopes TJS, Halfmann P, Hatta M, Fan S, Neumann G, Kawaoka Y (2018) Mutations in the PA protein of avian H5N1 influenza viruses affect polymerase activity and mouse virulence. J Virol 92:e01557-01517

310. Zhao H, Chu H, Zhao X, Shuai H, Wong BH-Y, Wen L, Yuan S, Zheng B-J, Zhou J, Yuen K-Y (2016) Novel residues in the PA protein of avian influenza H7N7 virus affect virulence in mammalian hosts. Virology 498:1–8

311. Yamaji R, Yamada S, Le MQ, Ito M, Sakai-Tagawa Y, Kawaoka Y (2015) Mammalian adaptive mutations of the PA protein of highly pathogenic avian H5N1 influenza virus. J Virol 89:4117–4125

312. Kanrai P, Mostafa A, Madhugiri R, Lechner M, Wilk E, Schughart K, Ylosmaki L, Saksela K, Ziebuhr J, Pleschka S (2016) Identification of specific residues in avian influenza A virus NS1 that enhance viral replication and pathogenicity in mammalian systems. J Gen Virol 97:2135–2148

313. Trapp S, Soubieux D, Marty H, Esnault E, Hoffmann TW, Chan denier M, Lion A, Kut E, Quere P, Larcher T, Ledevin M, Munier S, Naffakh N, Marc D (2014) Shortening the unstructured, inter domain region of the non-structural protein NS1 of an avian H1N1 influenza virus increases its replication and pathogenicity in chickens. J Gen Virol 95:1233–1243

314. Seo SH, Hoffmann E, Webster RG (2002) Lethal H5N1 influ enza viruses escape host anti-viral cytokine responses. Nat Med 8:950–954

315. Lipatov AS, Andreansky S, Webby RJ, Hulse DJ, Rehg JE, Krauss S, Perez DR, Doherty PC, Webster RG, Sangster MY (2005) Pathogenesis of Hong Kong H5N1 influenza virus NS gene reassortants in mice: the role of cytokines and B- and T-cell responses. J Gen Virol 86:1121–1130

316. Ayllon J, Domingues P, Rajsbaum R, Miorin L, Schmolke M, Hale BG, García-Sastre A (2014) A single amino acid substitu- tion in the novel H7N9 influenza A virus NS1 protein increases CPSF30 binding and virulence. J Virol 88:12146–12151

317. Li J, Zhang K, Chen Q, Zhang X, Sun Y, Bi Y, Zhang S, Gu J, Li J, Liu D, Liu W, Zhou J (2018) Three amino acid substitutions in the NS1 protein change the virus replication of H5N1 influenza virus in human cells. Virology 519:64–73

318. Li Z, Jiang Y, Jiao P, Wang A, Zhao F, Tian G, Wang X, Yu K, Bu Z, Chen H (2006) The NS1 gene contributes to the virulence of H5N1 avian influenza viruses. J Virol 80:11115–11123

319. Kuo R-L, Krug RM (2009) Influenza a virus polymerase is an integral component of the CPSF30-NS1A protein complex in infected cells. J Virol 83:1611–1616

320. Spesock A, Malur M, Hossain MJ, Chen L-M, Njaa BL, Davis CT, Lipatov AS, York IA, Krug RM, Donis RO (2011) The viru lence of 1997 H5N1 influenza viruses in the mouse model is increased by correcting a defect in their NS1 proteins. J Virol 85:7048–7058

321. Trapp S, Soubieux D, Lidove A, Esnault E, Lion A, Guillory V, Wacquiez A, Kut E, Quéré P, Larcher T, Ledevin M, Nadan V, Camus-Bouclainville C, Marc D (2018) Major contribution of the RNA-binding domain of NS1 in the pathogenicity and rep- lication potential of an avian H7N1 influenza virus in chickens. Virol J 15:55

322. Keiner B, Maenz B, Wagner R, Cattoli G, Capua I, Klenk H-D (2010) Intracellular distribution of NS1 correlates with the infectivity and interferon antagonism of an avian influenza virus (H7N1). J Virol 84:11858–11865

323. Jackson D, Hossain MJ, Hickman D, Perez DR, Lamb RA (2008) A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity. Proc Natl Acad Sci USA 105:4381–4386

324. Kong W, Liu L, Wang Y, He Q, Wu S, Qin Z, Wang J, Sun H, Sun Y, Zhang R, Pu J, Liu J (2015) C-terminal elongation of NS1 of H9N2 influenza virus induces a high level of inflammatory cytokines and increases transmission. J Gen Virol 96:259–268

325. Reuther P, Giese S, Gotz V, Kilb N, Manz B, Brunotte L, Schwemmler M (2014) Adaptive mutations in the nuclear export protein of human-derived H5N1 strains facilitate a polymerase activity-enhancing conformation. J Virol 88:263–271

326. Bean WJ, Threlkeld SC, Webster RG (1989) Biologic potential of amantadine-resistant influenza A virus in an avian model. J Infect Dis 159:1050–1056

327. Ilyushina NA, Govorkova EA, Webster RG (2005) Detection of amantadine-resistant variants among avian influenza viruses iso- lated in North America and Asia. Virology 341:102–106

328. He G, Qiao J, Dong C, He C, Zhao L, Tian Y (2008) Amanta- dine-resistance among H5N1 avian influenza viruses isolated in Northern China. Antiviral Res 77:72–76

329. Puthavathana P, Auewarakul P, Charoenying PC, Sangsiriwut K, Pooruk P, Boonnak K, Khanyok R, Thawachsupa P, Kijphati R, Sawanpanyalert P (2005) Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand. J Gen Virol 86:423–433

330. Buranathai C, Amonsin A, Chaisigh A, Theamboonlers A, Pari- yothorn N, Poovorawan Y (2007) Surveillance activities and molecular analysis of H5N1 highly pathogenic avian influenza viruses from Thailand, 2004-2005. Avian Dis 51:194–200

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