Accepted Manuscript Title: Effects of different NS genes of avian influenza viruses and amino acid changes on pathogenicity of recombinant A/Puerto Rico/8/34 viruses Author: Il-Hwan Kim Hyuk-Joon Kwon Su-Hyung Lee Dae-Yong Kim Jae-Hong Kim PII: DOI: Reference:

S0378-1135(14)00522-7 http://dx.doi.org/doi:10.1016/j.vetmic.2014.11.010 VETMIC 6814

To appear in:

VETMIC

Received date: Revised date: Accepted date:

6-8-2014 4-11-2014 7-11-2014

Please cite this article as: Kim, I.-H., Kwon, H.-J., Lee, S.-H., Kim, D.-Y., Kim, J.H.,Effects of different NS genes of avian influenza viruses and amino acid changes on pathogenicity of recombinant A/Puerto Rico/8/34 viruses, Veterinary Microbiology (2014), http://dx.doi.org/10.1016/j.vetmic.2014.11.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights A mice nonpathogenic NS gene originated from an attenuated H9N2 virus was identified. Mice pathogenic NS genes among LPAIVs were identified.

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Novel amino acid changes related to mice pathogenicity were identified.

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A method to generate nonpathogenic PR8-based recombinant virus was suggested.

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Effects of different NS genes of avian influenza viruses and amino acid changes on

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pathogenicity of recombinant A/Puerto Rico/8/34 viruses

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Seoul, Republic of Korea b

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Laboratory of Avian Diseases, College of Veterinary Medicine, Seoul National University,

Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National

University, Seoul, Republic of Korea c

Laboratory of Poultry Clinical Science, College of Veterinary Medicine, Seoul National

University, Pyeongchang, Gangwon-do, Republic of Korea d

Laboratory of Pathology, College of Veterinary Medicine, Seoul National University, Seoul,

Republic of Korea

(Running title: Characterization of a novel NS gene) 1

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Il-Hwan Kima,b, Hyuk-Joon Kwonb, c, 1, Su-Hyung Leed, Dae-Yong Kimb,d, Jae-Hong Kima,b,1

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Hyuk-Joon Kwon and Jae-Hong Kim contributed equally to the study.

†Corresponding Authors

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Jae-Hong Kim

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Laboratory of Avian Diseases, Seoul National University, Gwanakro 599, Daehak-Dong,

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Gwanak-Gu, Seoul, Republic of Korea

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TEL: 82-2-880-1250; FAX: 82-2-885-6614; E-mail: [email protected]

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Hyuk-Joon Kwon

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Laboratory of Poultry Clinical Science, Seoul National University, Pyeongchangdaero 1597,

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Pyeongchang, Gangwon-do, 232-916, Korea; TEL: 82-33-339-6111; FAX: 82-33-339-6199;

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E-mail: [email protected]

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Abstract To examine the effects of the NS1 and NEP genes of avian influenza viruses (AIVs)

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on pathogenicity in mice, we generated recombinant PR8 viruses containing 3 different NS

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genes of AIVs. In contrast to the reverse genetics-generated PR8 (rPR8) strain and other

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recombinant viruses, the recombinant virus rPR8-NS(0028), which contained the NS gene of

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A/chicken/KBNP-0028/2000 (H9N2) (0028), was non-pathogenic to mice. The novel single

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mutations of 0028 NS1 to corresponding amino acid of PR8 NS1, G139D and S151T

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increased the pathogenicity of rPR8-NS(0028). The replacement of the PL motifs (EPEV or

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RSEV) of pathogenic recombinant viruses with that of 0028 (GSEV) did not reduce the

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pathogenicity of the viruses. However, a recombinant virus with an EPEV-grafted 0028 NS

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gene was more pathogenic than rPR8-NS(0028) but less than rPR8. The lower pathogenicity

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of rPR8-NS(0028) might be associated with the lower virus titer and IFN-β level in the lungs

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of infected mice, and be attributed to G139, S151 and GSEV-PL motif of NS1 gene of 0028.

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In conclusion we defined new amino acid residues of NS1 related to mice pathogenicity and

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the presence of pathogenic NS genes among low pathogenic AIVs may encourage continuous

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monitoring of their mammalian pathogenicity.

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Key words: avian influenza virus, NS gene, reverse genetics, pathogenicity, PL motif

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1. Introduction

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The nonstructural protein (NS) genome segment of the influenza A virus (IAV) encodes

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NS1 and nuclear export protein (NEP) (Lamb and Lai, 1980; O'Neill et al., 1998).

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Multifunctional NS1 inhibits the activation of antiviral mechanisms and regulates viral RNA

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transcription (Hale et al., 2008; Kuo and Krug, 2009; Min et al., 2007; Nemeroff et al., 1998).

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NEP plays a role in the export of viral ribonucleoproteins and the matrix 1 protein complex

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and in the regulation of viral transcription and replication (Akarsu et al., 2003; Robb et al.,

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2009). As a result, the NS genes affect the pathogenicity and replication efficiency of IAVs.

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To date, various NS1 gene mutations associated with replication efficiency and viral

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pathogenicity have been reported (Hale et al., 2008). Among them, PDZ domain ligand (PL)

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motifs in the C-terminal of NS1 are clearly different between avian (ESEV) and mammalian

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(RSKV) IAVs (Hale et al., 2008; Obenauer et al., 2006). The amino acid sequences of PL

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motifs are variable, and these motifs interact differently with different PDZ domains of host

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proteins (Obenauer et al., 2006). However, same PL motif showed different pathogenicity

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and replication efficiency in different genetic background of virus and host. Thus, these

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motifs were regarded as strain- or host-specific (Hale et al., 2008; Hale et al., 2010a; Jackson

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et al., 2008; Soubies et al., 2010; Zielecki et al., 2010). In addition, the constellation of an NS

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gene with certain viral polymerase genes affects replication efficiency and viral pathogenicity

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(Shelton et al., 2012). Although AIVs are constantly isolated from poultry in farms, live birds

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in markets and migratory birds, the potential virulence of NS genes from low pathogenic

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AIVs has not been studied sufficiently (Kim et al., 2011; Lee et al., 2010; Park et al., 2011).

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Thus, studies on the effects of different NS genes and single amino acid mutation on viral

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pathogenicity in different genetic backgrounds from previous studies may be valuable. Therefore, in the present study we exchanged the NS gene of PR8 with 3 intact NS genes

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of low pathogenic AIVs (LPAIVs) by reverse genetics to investigate the effects of different

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NS genes on pathogenicity in mice. We found pathogenic NS genes among LPAIVs from

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poultry and wild birds and defined new amino acid residues involved in mammalian

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pathogenicity of recombinant PR8 virus.

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2. Materials and methods

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2.1. Viruses, cells and plasmids.

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A/chicken/Korea/01310/2001 (H9N2) (01310) is the parent strain of a commercial

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inactivated H9N2 oil emulsion vaccine in Korea (Choi et al., 2008). The 01310 (CE3) strain

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was passaged through 10-day-old (10-d-o) SPF embryonated chicken eggs (ECEs) (VALO

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BioMedia, Adel, IA) three times. This strain was obtained from the Laboratory of Influenza

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Viruses at the Animal, Plant and Fisheries Quarantine and Inspection Agency (Choi et al.,

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2008). A/chicken/Korea/KBNP-0028/2000 (H9N2) (0028) was an attenuated and highly

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productive strain that was established by passaging the virus 19 times at a high titer and

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without embryonic death for 3 days of incubation in 10-d-o SPF ECEs (Kwon, 2009).

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A/chicken/Korea/SNU8011/2008 (H9N2) (8011) and A/chicken/Korea/SNU9037/2008

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(H9N2) (9037) were isolated from commercial chicken farms, Low pathogenic

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A/duck/Korea/J31/2009 (H7N2) (J31-D) was isolated from a chicken in a live bird market,

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and Low pathogenic A/wild duck/Korea/SNU50-5/2011 (H5N1) (50-5) was isolated from

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migratory bird feces during a nationwide surveillance program supported by the Korean

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veterinary authority, QIA. A/canine/Korea/SNU9046/2009 (H3N2) (9046) was isolated from

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a dog suffering from respiratory symptoms and consigned for diagnosis from Seoul National

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University Veterinary Medical Teaching Hospital. The rPR8 virus generated by the

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Hoffmann vector system was passaged three times in 10-d-o ECEs and then used in

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experiments (Hoffmann et al., 2002). All of the influenza viruses were inoculated in 10-d-o

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ECEs via the allantoic cavity route and incubated for 36-72 h. After chilling at 4°C overnight,

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the allantoic fluid was harvested and stored at -70°C until use.

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293T cells were purchased from the American Type Culture Collection (ATCC, VA)

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and maintained in DMEM (Invitrogen Co., CA, USA) supplemented with 10% fetal bovine

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serum (Invitrogen).

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2.2. Titration of viruses

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Each sample was inoculated into three 10-d-o SPF ECEs for virus isolation. The presence of

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AIV in the allantoic fluid was confirmed using the HA assay. To measure the virus titer, each

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individual sample was serially diluted from 10–1 to 10–9 in 10-fold increments, and each

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dilution (10–6 to 10–9) was inoculated into 5 10-d-o SPF ECEs. The 50% chicken embryo

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infectious dose (EID50 ml–1) was calculated by the Spearman-Karber method (Hamilton,

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1977).

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2.3. RT-PCR, sequencing and sequence analysis

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The viral genomic RNA samples of strains 01310, 0028, 8011, 9037, J31-D, 50-5 and 9046 6 Page 6 of 32

were extracted from 150 µl of allantoic fluid using the Viral Gene Spin kit (iNtRON

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Biotechnology Co., Korea). The NS amplicon of each virus was obtained by RT-PCR as

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described previously, and the nucleotide sequences were directly determined with PCR

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primers using an ABI3711 automatic sequencer (Macrogen Co. Seoul, Korea) (Hoffmann et

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al., 2001). The nucleotide and deduced amino acid sequences of the avian and canine

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influenza viruses used in the study and the reference strains in the GenBank database were

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aligned with the Clustal method in the MegAlign program (Windows version 3.12e:

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DNASTAR, Madison, WI), and the nucleotide sequences of the NS genes were compared

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with each other and to those of the IAVs in GenBank using the BLAST search method.

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Phylogenetic trees were constructed with the neighbor-joining method (Tamura-Nei distance,

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500 repeats of bootstrap, MEGA 5.05 version). The abbreviations and accession numbers of

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compared IAV genes are as follows: A/chicken/Korea/KBNP-0028/2000 (H9N2) (0028,

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EF620904), A/chicken/Korea/01310/2001 (H9N2) (01310, JX094858), A/chicken/Korea/

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SNU8011/2008 (H9N2) (8011, JX273031), A/chicken/Korea/SNU9037/ 2009 (H9N2) (9037,

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JX273030),

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duck/Korea/SNU50-5/2011 (H5N1) (50-5, JX273027) , A/canine/Korea/SNU9046/2009

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(H3N2)

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A/DK/England/1/1956 (H11N6) (DK/England/56, GU052206), A/WSN/1933 (H1N1)

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(WSN/33,

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A/mallard/New

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A/pintail/Alberta/119/1979 (H4N6) (Pin/Alb/119/79, M25374), A/Udorn/72 (H3N2)

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(Udorn/72,

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A/pintail/Alberta/358/1979

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(Pin/Alb/121/79, M25371), A/mallard/Alberta/827/1978 (H8N4) (Mal/alb/827/78, M25372),

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A/Brevig_Mission/1/18 (H1N1) (Brevig Mission/1/18, AF333238).

Korea/J31/2009

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(9046,

JX273029),

CY034136),

A/Puerto

A/pintail/

York/6750/1978

CY009640),

(H7N2)

(J31-D,

Rico/8/34

(H1N1)

Alberta/268/1978 (H2N2)

(PR8,

(Pin/Alb/268/78,

(Mal/NY/6750/78,

A/mallard/Alberta/88/1976 (Pin/Alb/358/79,

JX273028),

M25370),

(Mal/Alb/88/76,

A/wild

JX120148),

M25369), CY116839),

M25373),

A/pintail/Alberta/121/1979

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The variable amino acids of NS1 that were identified in the present study were

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localized on the 3D structure of the RNA-binding (2z0a.pdb) and effector (2RHK.pdb)

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domains of the NS1 protein using SWISS-PdbViewer 4.04 (http://www.expasy.org/spdbv/).

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2.4. Cloning and mutagenesis of NS genes

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The bi-directional transcription vector pHW2000 and 8 plasmid vectors with 8 genome

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segments of PR8 were provided by Hoffmann et al. (Hoffmann et al., 2002). The NS

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amplicons of 01310, 0028 and 50-5 were cloned into pHW2000, as described previously, and

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the nucleotide sequences of the inserts were confirmed by sequencing with the primers listed

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in Table 1. To understand the effects of the single amino acid mutation and different PL

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motifs of NS1, we mutated 0028 NS1 (F70K, T127N, G139D, S151T, N189D and Q220R),

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PR8 NS1 (T151S) and the nucleotide sequences encoding the PL motif of each virus by site-

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directed mutagenesis (iNtRON Biotechnology; Enzynomics Co., Korea). The mutagenesis

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primer sets are listed in Table 1. The PL motifs of the rPR8 (RSEV) and 01310 (EPEV) NS1

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genes were converted to GSEV using the PR8-GSEV-F/PR8-GSEV-R and 01310-GSEV-

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F/01310-GSEV-R primer sets, and the PL motif (GSEV) of 0028 was changed into EPEV

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and RSEV using the 0028-EPEV-F/0028-EPEV-R and 0028-RSEV-F/0028-RSEV-R primer

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sets, respectively.

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2.5. Rescue of recombinant viruses

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The recombinant PR8 (rPR8) was generated by transfecting Hoffmann’s eight reverse

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genetics plasmids as described previously, with some modifications. The NS gene 8 Page 8 of 32

recombinant viruses, including rPR8-NS(01310), rPR8-NS(0028) and rPR8-NS(50-5), were

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generated by replacing the PR8 NS gene plasmid with each of the 8 altered viral NS gene

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plasmids. The single amino acid and PL motif mutant recombinant viruses, rPR8-NS(0028)-

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F70K, rPR8-NS(0028)-T127N, rPR8-NS(0028)-G139D, rPR8-NS(0028)-S151T, rPR8-

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NS(0028)-N189D, rPR8-NS(0028)-Q220R, rPR8-NS(PR8)-T151S, rPR8-NS(PR8)-GSEV,

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rPR8-NS(01310)-GSEV, rPR8-NS(0028)-EPEV and rPR8-NS(0028)-RSEV, were generated

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by replacing the PR8 NS gene plasmid with the 7 single amino acid mutant and 4 PL motif

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mutant NS gene plasmids. Briefly, 293T cells were cultured (1 × 106 cells/well in 6-well

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plates) and transfected with 300 ng of each plasmid using Lipofectamine 2000 and Plus

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reagents (Invitrogen) in a final volume of 1 ml of Opti-MEM (Invitrogen). After 3 h of

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incubation, 1 ml of fresh medium was added. The cells were incubated for an additional 36 h,

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and 0.5 mg/ml of trypsin (Invitrogen) was then added. After 12 h, the culture medium was

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harvested and 200 µl of the medium was injected into 10-day-old SPF ECEs via the allantoic

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cavity. After incubating for 2–3 days, the allantoic fluid was harvested and tested via the HA

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assay using 1% (v/v) chicken red blood cells (RBCs) according to the WHO Manual on

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Animal Influenza Diagnosis and Surveillance. The genetic markers of each recombinant

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viruses were confirmed by RT-PCR and sequencing.

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2.6. Animal experiments

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The five-week-old (w-o) female BALB/c mice were purchased from KOATEC (Pyeongtaek,

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Korea), and mouse pathogenicity tests were conducted by BioPOA Co. (Yongin, Korea).

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General animal care was provided under an Animal Use Protocol (BP-2012-0001-1, BP-

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2013-0006-1) approved by the Institutional Animal Care and Use Committee of BioPOA Co.

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(Yongin, Korea). The 50% lethal doses in mice (MLD50) for rPR8, rPR8-NS(01310), rPR8-

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NS(0028), rPR8-NS(50-5), rPR8-NS(0028)-F70K, rPR8-NS(0028)-T127N, rPR8-NS(0028)-

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N189D, rPR8-NS(0028)-Q220R, rPR8-NS(PR8)-GSEV and rPR8-NS(01310)-GSEV were

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measured as previously described, with some modifications (Matsuoka et al., 2009). Briefly

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each recombinant virus was diluted to 106, 105 and 104 EID50 (50 µl)–1, and 5 mice were

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assigned to each viral dilution. The mice were anesthetized with Zoletil (15 mg kg–1; Virbac

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S.A., France), and the mortality and weight loss were observed every day for 14 days. When

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the body weight of a mouse decreased more than 25%, the mouse was humanly euthanized by

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CO2 asphyxiation.

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The pathogenicities of rPR8, rPR8-NS(01310), rPR8-NS(0028), rPR8-NS(50-5),

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rPR8-NS(0028)-F70K, rPR8-NS(0028)-T127N, rPR8-NS(0028)-G139D, rPR8-NS(0028)-

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S151T, rPR8-NS(0028)-N189D, rPR8-NS(0028)-Q220R, rPR8-NS(PR8)-T151S, rPR8-NS-

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GSEV, rPR8-NS(01310)-GSEV, rPR8-NS(0028)-EPEV and rPR8-NS(0028)-RSEV were

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tested. Five mice were assigned to each virus or the mock virus condition. The anesthetized

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mice were challenged with 106 EID50 (50 µl)–1 of each virus as described above. The control

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(mock) mice were inoculated with same amount of sterilized PBS. The mortality and weight

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loss were observed as above.

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2.7. Measurement of IFN-β level, virus titer in the infected lungs of mice and

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histopathology

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Three mice from each group inoculated intranasally with PBS (mock) and 106 EID50 (50 µl)–1

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of rPR8-NS(0028), rPR8-NS(0028)-S151T, rPR8-NS(0028)-EPEV and rPR8 were euthanized

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at 3 and 6 days post-inoculation (DPI) and their lungs were collected. The lungs were ground 10 Page 10 of 32

with a mortar and pestle, and 10% suspensions were prepared with PBS. After centrifugation

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at 2,000 × g for 15 min the supernatants were stored at -70°C until use. The IFN-β levels

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were measured with a mouse interferon-beta ELISA kit according to the manufacturer’s

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instructions (PBL Biomedical Lab., Piscataway, NJ), and the viral titers of the pooled lung

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samples were measured as above. To compare the effects of different NS genes and the PL

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motifs of AIVs on pathogenicity, 3 mice of each group were euthanized on 5 DPI and the

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lungs were collected for histopathological analyses. After fixation in 10% phosphate-buffered

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neutral formalin, one section from each lung lobe per mouse was pulled, processed,

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embedded in paraffin, and stained with hematoxylin and eosin (H&E) for histopathological

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analyses of the pulmonary lesions.

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2.8. Statistical analyses

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The body weight change, viral and IFN-β levels were evaluated for statistical significance

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using a one-way analysis-of-variance (ANOVA). The mortality difference in the

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pathogenicity test was assessed using Fisher’s exact test (95% confidence intervals).

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3. Results

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3.1. Comparison of nucleotide and amino acid sequences and phylogenetic

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analysis of NS genes

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The nucleotide and amino acid sequences of the 0028, 01310, 8011, 9037, J31-D, 50-5

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and 9046 NS genes were determined (accession nos., JX273027-JX273031) and compared 11 Page 11 of 32

with those of the human and avian NS genes. The nucleotide and amino acid sequences of

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0028, 01310, 8011, 9037, J31-D and 9046 showed higher identity for the A alleles (nt 83.3-

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96.9% and aa 80.1-99.5%) than for the B alleles of NS genes (nt 69.1-70.9% and aa 66.9-

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70.0%) (Treanor et al., 1989). The 50-5 NS gene showed higher identity to the B alleles (nt

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90.4-96.1% and aa 97.8-98.6%) than to the A alleles (nt 69.2-71.2% and aa 66.2-69.5%). The

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phylogenetic analysis revealed that the 0028, 01310, 8011, 9037, J31-D and 9046 NS genes

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formed a cluster with the A alleles and 50-5 formed a cluster with the B alleles of the NS

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genes (data not shown).

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3.2. The effects of NS genes of AIVs on the pathogenicity of recombinant PR8

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viruses

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To investigate the effects of the NS genes on pathogenicity in mice and virus replication

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in ECEs, the NS genes of 0028, 01310 and 50-5 were selected as the representatives of the A

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and B alleles. The recombinant PR8 viruses containing different NS genes [rPR8-NS(0028),

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rPR8-NS(01310) and rPR8-NS(50-5)] were generated by reverse genetics, and their viral

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titers were measured in ECEs. To test the pathogenicity of the recombinant viruses, the 50%

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of the mouse lethal dose (MLD50), the mortality, the mean day of death and the body-weight

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loss were measured using BALB/c mice. The MLD50, mortality and day of death for rPR8

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and the recombinant NS viruses are summarized in Table 2. The rPR8-NS(01310), rPR8-

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NS(50-5) and rPR8 were pathogenic to mice and showed 80-100% mortality within an

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average of 7 days when mice were challenged with 106 EID50 of virus. In contrast, rPR8-

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NS(0028) was nonpathogenic to mice, and the mortality and mean day of death were

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significantly different from those of rPR8, rPR8-NS(01310) and rPR8-NS(50-5). The MLD50

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of rPR8-NS(0028) was higher than 6.0 (log10), and this virus caused no mortality for 14 days.

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The body weights of the mice inoculated with rPR8-NS(01310), rPR8-NS(50-5) and rPR8

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decreased significantly from 3 DPI to 7 DPI compared to those of the mice in the control

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(mock) and rPR8-NS(0028) groups (P < 0.05) (Fig. 1a). The body weight loss of the rPR8-

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NS(0028) group was not significantly different from that of the control (mock) mice.

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The NS1 of 0028 proteins had 12 different amino acid residues (A/E60G, E/K70F,

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E/S71K, S/P87T, M119I, N/R127T, I/L137T, D/N139G, T151S, D189N, R220Q and

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E/R227G) and NEP had 2 (M31I and G/E65R) compared to the pathogenic PR8, 01310 and

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50-5 (Table 3). The variable amino acid residues were located on the 3D structure of the NS1

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protein dimers (2z0a.pdb and 2RHK.pdb). The G60, F70 and K71 residues were located on

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the opposite side of the RNA-binding tract relative to the RNA-binding domain, and the F70

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and K71 residues of one monomer were located close to G60 of the other monomer (Fig. 2a)

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(Chien et al., 2004; Yin et al., 2007). T87 and T137 were located close to G139 in the 3D

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structure of the effector domain of the NS1 protein (Fig. 2b). The nuclear export signal (NES)

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of NS1 is composed of residues 136, 139, 144 and 146, and high- and low-pathogenicity NS1

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proteins have different amino acids (I and V at 136, and N and D at 139, respectively). Strain

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0028 NS1 has residue G139, of which the function is unknown. Amino acid residues 123-127

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are important for the binding of NS1 to PKR, which plays a key role in the induction of the

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antiviral mechanisms of a host cell (Min et al., 2007). Interestingly, S151 and N189 are

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located close to amino acid residues 126 and 125, respectively (Fig. 2c). The PL motif of

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0028 NS1 (GSEV) was different from the major mammalian and avian PL motifs.

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3.3. The effect of single mutation of 0028 NS1 on the pathogenicity of

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recombinant PR8 virus

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Seven recombinant viruses possessing single amino acid changes in 0028 and PR8 NS1

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were generated and their titers and pathogenicity in mice were investigated (Table 2). The

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virus titers of rPR8-NS(0028)-F70K, rPR8-NS(0028)-T127N, rPR8-NS(0028)-G139D, rPR8-

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NS(0028)-S151T, rPR8-NS(0028)-N189D and rPR8-NS(PR8)-T151S were not significantly

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different from rPR8. According to the mouse virulence test the mortality of rPR8-NS(0028)-

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F70K, rPR8-NS(0028)-T127N, rPR8-NS(0028)-N189D and rPR8-NS(0028)-Q220R was 0%

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and the day of death and MLD50 were higher than 14 and 6.0 (log10), respectively. The

296

mortality of rPR8-NS(0028)-G139D, rPR8-NS(0028)-S151T and rPR8-NS(PR8)-T151S was

297

20, 80 and 100% within 8 days after inoculation, respectively. The body weights of the mice

298

inoculated with rPR8-NS(0028)-S151T and rPR8-NS(PR8)-T151S were significantly

299

different from that of the control (mock) during 3-6 DPI. The body weight of rPR8-

300

NS(0028)-G139D decreased during 7-9 DPI but was not significantly different from that of

301

the control (mock) (Fig. 1b).

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Four recombinant viruses were generated by exchanging the PL motifs of PR8 (RSEV),

303

01310 (EPEV) and 0028 (GSEV), and the viral titers and pathogenicity of these viruses in

304

mice were then investigated. According to the mouse virulence test, the mortality of rPR8-

305

NS(PR8)-GSEV and rPR8-NS(01310)-GSEV was 100% until 6 DPI. rPR8-NS(0028)-EPEV

306

and rPR8-NS(0028)-RSEV caused no mortality for 14 days, as was also observed for rPR8-

307

NS(0028) (Table 2). The MLD50 of rPR8-NS(PR8)-GSEV was less than 4.0 (log10), and that

308

of rPR8-NS(01310)-GSEV was identical to that of rPR8-NS(01310). The body weights of the

309

mice inoculated with rPR8-NS(PR8)-GSEV and rPR8-NS(01310)-GSEV started to decrease

310

at 3 DPI, and only the body weight loss of rPR8-NS(0028)-EPEV at 7 and 8 DPI was

311

significantly different from that of the control (mock) (P < 0.05) (Fig. 1c).

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14 Page 14 of 32

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3.4. The comparison of histopathological lesions of the rPR8-, rPR8-NS(0028)-,

314

and rPR8-NS(0028)-EPEV-infected lungs of mice

315

The lungs of the mice in the rPR8, rPR8-NS(0028), rPR8-NS(0028)-EPEV and the

317

control (mock) groups were compared (Fig. 3). The pulmonary lesions of rPR8-infected mice

318

were characterized by acute necrotizing bronchiolitis and interstitial pneumonia, and the

319

lesions observed in these mice were more severe than those observed in other groups. The

320

rPR8-NS(0028)-EPEV-infected mice showed a mild to moderate degree of necrotizing

321

bronchiolitis compared to the rPR8-infected mice. However, the lesions of the mice infected

322

with rPR8-NS(0028) were less severe than those of rPR8 and were similar in severity to those

323

of the control (mock) group.

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3.5. IFN-β levels and viral titers in the infected lungs of mice

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The IFN-β levels in the lungs of the mice infected with rPR8, rPR8-NS(0028), rPR8-

328

NS(0028)-S151T and rPR8-NS(0028)-EPEV were compared. The IFN-β levels in the rPR8-

329

infected lungs were the highest, followed by those of rPR8-NS(0028)-EPEV and rPR8-

330

NS(0028)-S151T on 3 DPI. The IFN-β levels of rPR8-NS(0028)-S151T-, rPR8-NS(0028)-

331

EPEV- and rPR8-infected lungs on 3 DPI were significantly higher than that of the control

332

(mock) (Fig. 4). The viral titers [TCID50 (0.1 ml)–1 (log10)] of rPR8-NS(0028)-infected lungs

333

on 3 and 6 DPI (4.9 and 4.3) were lower than those of rPR8-NS(0028)-S151T (6.5 and 5.1),

334

rPR8-NS(0028)-EPEV (6.3 and 4.7) and rPR8 (6.7 and 4.1) (Fig. 4).

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4. Discussion 15 Page 15 of 32

337

The effects of the NS genes on the pathogenicity and replication efficiency of IAVs have

339

been demonstrated. The replacement of the NS gene of H7N1, a highly pathogenic avian

340

influenza virus (HPAIV), with that of H5N1 HPAIV increased viral replication in

341

mammalian cells and pathogenicity in mice, and the A allele of the NS gene of AIVs

342

maintained the pathogenicity of an H3N2 virus (A/Udorn/307/72) in squirrel monkeys (Ma et

343

al., 2010; Treanor et al., 1989). In contrast, the B allele of the NS gene of AIVs attenuated an

344

H3N2 virus (A/Udorn/307/72) in squirrel monkeys, and the swine-related NS gene of a 1918

345

pandemic virus also attenuated the A/WSN/33 (H1N1) strain (WSN33) in mice (Basler et al.,

346

2001; Treanor et al., 1989). Most NS genes (01310, 0028, 9037, 8011 and 9046) analyzed in

347

the present study were classified into the A allele group, with the exception of 50-5.

348

Interestingly, rPR8-NS(0028) caused no body weight loss, mortality or pathological lesions

349

for 14 days. Although the NS gene of 50-5 was classified into the B allele group, rPR8-

350

NS(50-5) was as pathogenic as rPR8. Thus, the simple classification of NS genes as either A

351

or B alleles is not sufficient to predict the pathogenicity of the NS genes and the presence of

352

virulent NS genes among LPAIVs may be potential risk factor of new virulent virus

353

appearance.

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NS1 has multiple functions that require binding of NS1 to target molecules. To date,

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various amino acid substitutions resulting in the loss- or gain-of-function of NS1 have been

356

reported (Hale et al., 2008). The nonpathogenic NS1 of 0028 contains 12 amino acid residues

357

that are different from those of other pathogenic NS1 proteins. Most of the identified variable

358

amino acid residues of 0028 NS1 are unknown, but the residue 189 was reported to affect the

359

binding of NS1 to two zinc finger regions of CPSF30 (Nemeroff et al., 1998; Twu et al.,

360

2006). The binding of NS1 to CPSF30 inhibits the 3’-end processing of host pre-mRNAs and

361

results in the retention of immature mRNA in the nucleus and the suppressed expression of 16 Page 16 of 32

antiviral genes, including IFN-β (Noah et al., 2003). The single mutation D189N in 0028 NS1

363

is shared by WSN33 NS1, which binds to CPSF30, and the 0028 NS1 may bind to CPSF30.

364

The CPSF30-binding activity of NS1 does not guarantee an increase in pathogenicity. In case

365

of A/California/04/09 (H1N1) NS1, the pathogenicity was decreased (Hale et al., 2010b). The

366

loss of CPSF30- binding activity of NS1 can be compensated by cognate PA and NP, and the

367

cooperation of NS1 with PA and NP is also important for viral pathogenicity (Kuo and Krug,

368

2009; Shelton et al., 2012; Twu et al., 2007). Considering that PR8 NS1 also does not bind to

369

CPSF30, the observed pathogenicity of the PR8 NS gene may be the result of cooperation

370

between PA and NP in PR8 (Kochs et al., 2007). Although the 1918 pandemic virus NS1 is

371

almost the same as that of the pathogenic 01310 in terms of the 12 variable amino acids of

372

0028 NS1, the 1918 pandemic viral NS gene was nonpathogenic in the genetic background of

373

WSN33 (Basler et al., 2001). Thus, the optimal constellation of an NS gene with other viral

374

genes may be important for viral pathogenicity. The single mutations of 0028 NS1 to

375

corresponding amino acids of PR8, F70K, T127N, N189D and Q220R did not increase the

376

pathogenicity of recombinant viruses, and they may not play role in the viral pathogenicity by

377

itself. The effect of G139D on the pathogenicity of recombinant virus can be supported by a

378

recent report on the importance of residue 138 comprising p85β-binding domain in systemic

379

infection of virus in mice (Fan et al., 2013). The S151T mutation significantly increased the

380

mouse pathogenicity of rPR8-NS(0028)-S151T and it was a novel mutation which had not

381

been reported previously. The close proximity of S151 with residue 126 of the PKR (123-

382

127) binding site in the 3D structure may reflect the importance of PKR interaction for viral

383

pathogenicity (Min et al., 2007). However, the T151S mutation of PR8 NS1 did not decrease

384

the pathogenicity of rPR8-NS(PR8)-T151S. Thus, other amino acid residues are also

385

important for complete expression of pathogenicity.

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The role of the PL motif in the replication efficiency and pathogenicity of IAVs has 17 Page 17 of 32

been reported, and the variable PL motifs are known to interact differently with different

388

PDZ domains of host proteins (Jackson et al., 2008; Obenauer et al., 2006; Soubies et al.,

389

2010; Zielecki et al., 2010). The PL motif of 0028 (GSEV) was observed in minor avian and

390

porcine IAVs, and its role in pathogenicity has only rarely been reported (Obenauer et al.,

391

2006; Wang et al., 2012). The recombinant PR8 viruses containing GSEV-grafted PR8 and

392

01310 NS genes showed increased and similar pathogenicity relative to their parent viruses

393

with intact NS genes, respectively. The increased pathogenicity of rPR8-NS(PR8)-GSEV was

394

unexpected, but a similar result was reported recently for a recombinant swine influenza virus

395

(Wang et al., 2012). The RSEV- and EPEV-grafted 0028 NS genes did not increase the

396

mortality of rPR8-NS(0028)-RSEV and rPR8-NS(0028)-EPEV. However, rPR8-NS(0028)-

397

EPEV caused more significant weight loss on 7 and 8 DPI and more histopathological lesions

398

than rPR8-NS(0028). The pathogenesis of the avian PL motif is still unclear, but the ESEV

399

PL motif is known to enhance viral replication by reducing apoptosis of host cell through

400

disruption of Scribble’s proapoptotic function (Liu et al., 2010). Thus, the amino acid context

401

of NS1, as well as the PL motif, may be important for any effects on the pathogenicity of

402

IAVs.

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NS1 inhibits expression of IFN-β by pre- and/or post-transcriptional mechanisms,

404

and the expression level of IFN-β differs from virus to virus (Kochs et al., 2007; Zhang et al.,

405

2011). In contrast to PR8 NS1 the 0028 NS1 may cause decreased post-transcriptional

406

expression of IFN-β by CPSF30-binding activity and reduce virus replication by decreased

407

interaction with viral polymerases and activated antiviral molecules (Shelton et al., 2012).

408

The increased IFN-β level in the PR8-infected lung tissues may have triggered more potent

409

immunopathological host response (‘cytokine storm’) which increased the virulence of the

410

PR8 virus (Yuen and Wong, 2005).

411

In conclusion, we defined new amino acid residues of NS gene which are important 18 Page 18 of 32

for virus pathogenicity in mice and the presence of pathogenic NS genes among LPAIVs may

413

reinforce continuous monitoring of mammalian pathogenicity of LPAIVs. Furthermore, the

414

nonpathogenic 0028 NS genome segment may be valuable to generate nonpathogenic

415

recombinant viruses which can be used to study pathogenicity and antigenicity of variable

416

IAVs.

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This work was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. A103001).

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19 Page 19 of 32

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DNA coding for the two overlapping nonstructural proteins of influenza virus. Cell 21, 475-485. Lee, H.J., Kwon, J.S., Lee, D.H., Lee, Y.N., Youn, H.N., Lee, Y.J., Kim, M.C., Jeong, O.M., Kang, H.M., Kwon, J.H., Lee, J.B., Park, S.Y., Choi, I.S., Song, C.S., 2010. Continuing evolution and interspecies transmission of influenza viruses in live bird markets in Korea. Avian Dis 54, 738-748. Liu, H., Golebiewski, L., Dow, E.C., Krug, R.M., Javier, R.T., Rice, A.P., 2010. The ESEV PDZ-binding motif of the avian influenza A virus NS1 protein protects infected cells from apoptosis by directly targeting Scribble. J Virol 84, 11164-11174. Ma, W., Brenner, D., Wang, Z., Dauber, B., Ehrhardt, C., Hogner, K., Herold, S., Ludwig, S., Wolff, T., Yu, K., Richt, J.A., Planz, O., Pleschka, S., 2010. The NS segment of an H5N1 highly pathogenic avian influenza virus (HPAIV) is sufficient to alter replication efficiency, cell tropism, and host range of an H7N1 HPAIV. J Virol 84, 2122-2133. Matsuoka, Y., Lamirande, E.W., Subbarao, K., 2009. The mouse model for influenza. Curr Protoc Microbiol Chapter 15, Unit 15G 13. Min, J.Y., Li, S., Sen, G.C., Krug, R.M., 2007. A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology 363, 236-243. Nemeroff, M.E., Barabino, S.M., Li, Y., Keller, W., Krug, R.M., 1998. Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs. Mol Cell 1, 991-1000. Noah, D.L., Twu, K.Y., Krug, R.M., 2003. Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3' end processing of cellular pre-mRNAS. Virology 307, 386-395. O'Neill, R.E., Talon, J., Palese, P., 1998. The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17, 288-296. Obenauer, J.C., Denson, J., Mehta, P.K., Su, X., Mukatira, S., Finkelstein, D.B., Xu, X., Wang, J., Ma, J., Fan, Y., Rakestraw, K.M., Webster, R.G., Hoffmann, E., Krauss, S., Zheng, J., Zhang, Z., Naeve, C.W., 2006. Large-scale sequence analysis of avian influenza isolates. Science 311, 1576-1580. Park, K.J., Kwon, H.I., Song, M.S., Pascua, P.N., Baek, Y.H., Lee, J.H., Jang, H.L., Lim, J.Y., Mo, I.P., Moon, H.J., Kim, C.J., Choi, Y.K., 2011. Rapid evolution of low-pathogenic H9N2 avian influenza viruses following poultry vaccination programmes. J Gen Virol 92, 36-50. Robb, N.C., Smith, M., Vreede, F.T., Fodor, E., 2009. NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome. J Gen Virol 90, 1398-1407. Shelton, H., Smith, M., Hartgroves, L., Stilwell, P., Roberts, K., Johnson, B., Barclay, W., 2012. An influenza reassortant with polymerase of pH1N1 and NS gene of H3N2 influenza A virus is attenuated in vivo. J Gen Virol 93, 998-1006. Soubies, S.M., 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. Treanor, J.J., Snyder, M.H., London, W.T., Murphy, B.R., 1989. The B allele of the NS gene of avian influenza viruses, but not the A allele, attenuates a human influenza A virus for squirrel monkeys. Virology 171, 1-9. Twu, K.Y., Kuo, R.L., Marklund, J., Krug, R.M., 2007. The H5N1 influenza virus NS genes selected after 1998 enhance virus replication in mammalian cells. J Virol 81, 8112-8121. Twu, K.Y., Noah, D.L., Rao, P., Kuo, R.L., Krug, R.M., 2006. The CPSF30 binding site on the NS1A protein of influenza A virus is a potential antiviral target. J Virol 80, 3957-3965. Wang, J., Qi, X., Lu, C., 2012. Mutations in the C-terminal tail of NS1 protein facilitate the replication of classical swine H1N1 influenza A virus in mice. Folia Microbiol (Praha) 57, 169-175.

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Yin, C., Khan, J.A., Swapna, G.V., Ertekin, A., Krug, R.M., Tong, L., Montelione, G.T., 2007. Conserved surface features form the double-stranded RNA binding site of non-structural protein 1 (NS1) from influenza A and B viruses. J Biol Chem 282, 20584-20592. Yuen, K.Y., Wong, S.S., 2005. Human infection by avian influenza A H5N1. Hong Kong Med J 11, 189-199. Zhang, Z., Hu, S., Li, Z., Wang, X., Liu, M., Guo, Z., Li, S., Xiao, Y., Bi, D., Jin, H., 2011. Multiple amino acid substitutions involved in enhanced pathogenicity of LPAI H9N2 in mice. Infect Genet Evol 11, 1790-1797. Zielecki, F., Semmler, I., Kalthoff, D., Voss, D., Mauel, S., Gruber, A.D., 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.

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535

FIGURE LEGENDS

536

Fig. 1. Comparison of mouse virulence of recombinant PR8 viruses. Groups of 5 mice were

538

infected intranasally with 106 EID50 of recombinant PR8 viruses with different NS genes

539

from avian influenza viruses (a), artificial NS genes with single amino acid changes (b) and

540

modified PL motifs (c). Mice were monitored daily for 10 days. * represents a significant

541

difference between the control (mock) and other groups (P < 0.05).

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542

Fig. 2. The locations of the variable amino acids in the 0028 NS1 protein. A: The RNA-

544

binding domain of NS1; B and C: the effector domain of NS1. The variable amino acids of

545

0028 were located on the RNA-binding (2z0a.pdb) and effector (2RHK.pdb) domains of NS1

546

protein using SWISS-PdbViewer 4.04 (http://www.expasy.org/spdbv/).

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Fig. 3. Histopathology of the lung tissue of the BALB/c mice infected with recombinant PR8

549

viruses. The lung samples of 3 mice from each group inoculated intranasally with 106 EID50

550

of recombinant PR8 viruses or PBS were collected at 5 days post inoculation. The necrotizing

551

bronchiolitis and interstitial pneumonia were most severe in rPR8 [(b), (f)] and that of rPR8-

552

NS (0028)-EPEV [(c), (g)] was mild to moderate compared to rPR8. rPR8-NS(0028) [(a), (e)]

553

showed no significant histopathological lesions compared to the control (mock) [(d), (h)]

554

Tissue sections were stained with H&E (A, B, C, D = 100X; E, F, G, H = 400X).

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Fig. 4. Induction of IFN-β and the viral titers within the recombinant virus-infected lungs of

557

BALB/c mice. The lung samples of 3 mice from each group which had been inoculated

558

intranasally with PBS (mock) and 106 EID50 (50 µl)–1 of rPR8-NS(0028), rPR8-NS(0028)-

559

S151T, rPR8-NS(0028)-EPEV and rPR8 were collected at 3 and 6 days post-inoculation 23 Page 23 of 32

(DPI). The lungs were homogenized and 10% suspensions were prepared with PBS. The

561

IFN-β levels were measured with a mouse interferon-beta ELISA kit and the viral titers of the

562

pooled lung samples were estimated in 10-d-o SPF embryonated chicken eggs. * represents a

563

significant difference between the control (mock) and other groups (P < 0.05).

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24 Page 24 of 32

Table 1. Primers used in the present study. Sequence (5’ to 3’)

Usage

cmv-SF

TAAGCAGAGCTCTCTGGCTA

Sequencing

bGH-SR

TGGTGGCGTTTTTGGGGACA

Sequencing

PR8-GSEV-F

GAACAATTGGGTCAGAAGTTTGAAGAAATAAG

Mutagenesis

PR8-GSEV-R

CTTATTTCTTCAAACTTCTGACCCAATTGTTC

PR8-T151S-F

AAGAGGGAGCAATTGTTGGCG

PR8-T151S-R

CTGAGAAAGCCCTTAGCAATATTAG

01310-GSEV-F

GAACAATTGGGTCAGAAGTTTGAAGAAATAAG

Mutagenesis

01310-GSEV-R

CTTAT TTCTTCAAACTTCTGACCCAATTGTTC

Mutagenesis

0028-RSEV-F

TGGCGAGAACAATTAGGTCAGAAGT TTGAAGA

Mutagenesis

0028-RSEV-R

TCTTCAAACTTCTGACCTAATTGTTCTCGCCA

Mutagenesis

0028-EPEV-F

TGGCGAGAACAATTGAGCCAGAAGTTTGAAGA

Mutagenesis

0028-EPEV-R

TCTTCAAACTTCTGGCTCAATTGTTCTCGCCA

Mutagenesis

0028-F70K-F

AAAAAAGAATCCGATGAGGCAC

Mutagenesis

0028-F70K-R

AAGAATCCGCTCCACTATCTG

Mutagenesis

0028-T127N-F

ATTGAAAGCAAACTTCAGTGTGAC

Mutagenesis

0028-T127N-R

GTGATATTTTTATCCATTATTGCCTGG

Mutagenesis

0028-G139D-F

TTTGACCGGCTGGAAACCC

Mutagenesis

0028-G139D-R

AGTCACACTGAAGTTTGCTTTC

Mutagenesis

0028- S151T-F

AGGAAGGAGCAATTGTGGGAG

Mutagenesis

0028- S151T-R

CCGTGAAAGCTCTAAGTAGTATTAG

Mutagenesis

0028- N189D-F

AACACAGTTCGAGTCTCTGAAAC

Mutagenesis

0028-N189D-R

ATCATTCCATTCAAGTCCTCC

Mutagenesis

0028-Q220R-F

AATGGCGAGAACAATTGGGTC

Mutagenesis

0028-Q220R-R

TTTCGTTTCTGCTTTGGAGG

Mutagenesis

565

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Primer

Mutagenesis

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Mutagenesis

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Mutagenesis

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All primers were designed in this study.

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25 Page 25 of 32

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Titer ±SD [EID50 ml–1 (log10)]

MLD50 [EID50 (50 µl)–1 (log10)]

Mortality* (%)

Day of death* (mean ±SD)

rPR8

RSEV

9.0 ±0.4

4.7

5/5 (100)

5.2 ±0.4

rPR8-NS(01310)

EPEV

8.5 ±0.5

5.5

4/5 (80)

6.3 ±1.5

rPR8-NS(0028)

GSEV

9.0 ±0.4

>6.0

0/5 (0)

>14

rPR8-NS(50-5)

ESEV

8.5 ±0.0

4.7

4/5 (80)

5.8±1.0

rPR8-NS(0028)-F70K

GSEV

8.6 ±0.1

>6.0

0/5 (0)

>14

rPR8-NS(0028)-T127N

GSEV

9.4 ±0.2

>6.0

0/5 (0)

>14

GSEV

8.8 ±0.1

NT†

1/5 (20), 2/5(40)‡

7.0, 8.0 ±0.0‡

GSEV

8.8 ±0.3

NT

4/5 (80)

6.5 ±0.6

rPR8-NS(0028)-G139D

ed

ce

rPR8-NS(0028)-S151T

M

PL motif

Recombinant virus

rPR8-NS(0028)-N189D

GSEV

9.3 ±0.3

>6.0

0/5 (0)

>14

rPR8-NS(0028)-Q220R

GSEV

9.6±0.2

>6.0

0/5 (0)

>14

rPR8-NS(PR8)- T151S

RSEV

9.1±0.3

NT

5/5 (100)

4.8 ±0.4

rPR8-NS(PR8)-GSEV

GSEV

8.7±0.2

6.0

0/5 (0)

>14

rPR8-NS(0028)-RSEV

RSEV

9.0±0.3

0/5 (0)

>14

Ac

568 569 570 571

an

Table 2. The pathogenicity of the parent and recombinant NS viruses in BALB/c mice.

pt

567

>6.0 6

* The pathogenicity test was performed by inoculating intranasally 10 EID50 (50 µl) mice. The mortality and weight loss were observed every day for 14 days. † NT, not tested ‡ Repeated experiment.

–1

of a recombinant virus into five 5-week-old BALB/c

27

Page 26 of 32

Table 3. Comparison of the variable amino acids in NS1 and NEP of avian influenza viruses

573

and rPR8.

NEP

574

Function

Refrerence

G

60

A*/I/V/E

Unknown

F

70

E/K

Unknown

K

71

E/S/G

Unknown

T

87

S/P

Unknown

I

119

M

Unknown

T

127

N/R

(Min et 2007)

T

137

I/L

G

139

D/E/N

NES (I136/N139/L144/S146)

(Keiner et al., 2010)

S

151

T

Unknown

N

189

Q

220

G

227

R/E

I

31

M

Unknown

R

63

E

Unknown

us

cr

ip t

Location of Other amino acid viruses

PKR binding (123-127)

al.,

d

M

Unknown

D/G

te

Ac ce p

NS1

0028

an

572

R

CPSF30(G189)

(Hale et 2010)

al.,

NLS2(K219/R220)

(Melen et al., 2007)

PL motif(227-230)

(Jackson et al., 2008)

* The amino acid residues of rPR8 were represented in boldface.

575

28 Page 27 of 32

Ac

ce pt

ed

M

an

us

cr

ip t

Graphical Abstract (for review)

Page 28 of 32

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pt

ed

M

an

us

cr

i

Figure

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Ac

ce

pt

ed

M

an

us

cr

i

Figure

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Ac

ce

pt

ed

M

an

us

cr

i

Figure

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pt

ed

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cr

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Figure

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34 viruses.

To examine the effects of the NS1 and NEP genes of avian influenza viruses (AIVs) on pathogenicity in mice, we generated recombinant PR8 viruses conta...
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