Characterization of mutations associated with the adaptation of a low-pathogenic H5N1 avian influenza virus to chicken embryos

G Model

VETMIC-5970; No. of Pages 8 Veterinary Microbiology xxx (2012) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

Characterization of mutations associated with the adaptation of a low-pathogenic H5N1 avian influenza virus to chicken embryos Il-Hwan Kim a,d, Hyuk-Joon Kwon b,**,1, Jun-Gu Choi c, Hyun-Mi Kang c, Youn-Jeong Lee c, Jae-Hong Kim a,d,1,* a

Laboratory of Avian Diseases, Seoul National University, Seoul 151-742, Republic of Korea Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea Avian Disease Division, Animal, Plant and Fisheries Quarantine and Inspection Agency, 175 Anyangro, Anyangsi, Gyeonggido 430-757, Republic of Korea d College of Veterinary Medicine and BK21 for Veterinary Science, Seoul National University, Seoul 151-742, Republic of Korea b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 September 2012 Received in revised form 21 October 2012 Accepted 25 October 2012

Migratory waterfowls are the most common reservoir for avian influenza virus (AIV), thus viral adaptation is required for efficient replication in land fowls. To date, low pathogenic (LP) H5 subtype AIVs have been isolated from migratory waterfowls, and the adaptation of these viruses to land fowls might lead to the generation of highly pathogenic AIVs. Thus, A/ wild duck/Korea/50-5/2009 (H5N1) LPAIV was passaged 20 times through embryonated chicken eggs (ECEs), and the resulting genetic and phenotypic changes were investigated. The pathogenicities of the early (50-5-E2) and final passage (50-5-E20) strains to chicken embryos were similarly high, but the 50-5-E20 titer was 100 times higher than that of 505-E2. 50-5-E20 showed 8 amino acid changes in PA (1), HA (4), NA (1), M1 (1) and M2 (1), with different frequencies among influenza A viruses (0–99.6%). The relevance of these changes, except H103Y in HA, to viral replication remains unknown. To investigate the roles of internal genes and mutations in HA and NA in viral replication, four recombinant viruses possessing combinations of HA and NA genes of 50-5-E2 and 50-5-E20 with 6 internal genes of PR8 were generated through reverse genetics. The embryo pathogenicities of the H5N1 recombinant viruses carrying internal PR8 genes were reduced, and the titers of the recombinant viruses with 50-5-E20 HA were higher than those with 50-5-E2 HA. Therefore, the identified mutations might be useful as chicken adaptation markers for the generation of high growth H5N1 recombinant viruses in ECEs. ß 2012 Elsevier B.V. All rights reserved.

Keywords: H5N1 Low pathogenic avian influenza virus Chicken embryo-adapted Reverse genetics Replication efficiency

1. Introduction

* Corresponding author at: Laboratory of Avian Diseases, Seoul National University, Gwanakro 599, Daehak-Dong, Gwanak-Gu, Seoul, Republic of Korea. Tel.: +82 2 880 1250; fax: +82 2 885 6614. ** Corresponding author at: Research Institute for Veterinary Science, Seoul National University, Gwanakro 599, Daehak-Dong, Gwanak-Gu, Seoul 151-742, Republic of Korea. Tel.: +82 2 880 1288; fax: +82 2 880 1233. E-mail addresses: [email protected] (H.-J. Kwon), [email protected] (J.-H. Kim). 1 Hyuk-Joon Kwon and Jae-Hong Kim contributed equally to this study.

Avian influenza viruses (AIVs) are type A influenza viruses that possess 8 segmented, single-stranded, negative sense RNA genomes, which encode 11 proteins: PB1, PB1-F2, PB2, PA, HA, NP, NA, M1, M2, NS1 and NEP (Chen et al., 2001; O’Neill et al., 1998; Webster et al., 1992). The pathogenicity, host adaptation and viral replication efficiency of AIVs are multigenic traits, and various mutations in the viral genes have been reported. PB1, PB2 and PA form a polymerase complex, and PB1 acts as the polymerase. PB1-F2 is encoded from an alternate (+1) open reading frame in the PB1 gene and is regarded as

0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.10.034

Please cite this article in press as: Kim, I.-H., et al., Characterization of mutations associated with the adaptation of a lowpathogenic H5N1 avian influenza virus to chicken embryos. Vet. Microbiol. (2012), http://dx.doi.org/10.1016/ j.vetmic.2012.10.034

G Model

VETMIC-5970; No. of Pages 8 2

I.-H. Kim et al. / Veterinary Microbiology xxx (2012) xxx–xxx

virulence factor (Chen et al., 2001). PB2 and PA participate in ‘cap snatching’ and play important roles in viral replication and in overcoming host barriers (Boivin et al., 2010; Plotch et al., 1981). HA binds to sialic acid on the surface of host cells, and the binding affinity of HA is affected by the amino acid sequence of the receptor binding site (RBS) and the type of sialic acid linkage to galactose (a2,3-, avian and a2,6-, mammalian receptors) (Matrosovich et al., 1997). Neuraminidase (NA) is important for viral budding (Seto and Rott, 1966). M1 binds to ribonucleoprotein (RNP) and NEP for cytoplasmic transport from the nucleus and forms a layer beneath the viral envelope (Akarsu et al., 2003). M2 is translated from spliced M1 mRNAs and functions as an ion channel, which plays a role in the fusion between the viral envelope and the endosome for viral entry (Sugrue and Hay, 1991). NS1 is a nonstructural protein involved in the inhibition of antiviral host responses, pathogenicity and viral replication efficiency (Hale et al., 2008). Migratory waterfowls serve as reservoirs for all known subtypes of AIVs. The majority of AIVs are nonpathogenic in domestic poultry, such as chicken and ducks, but H5N1 highly pathogenic avian influenza viruses (HPAIVs) have caused enormous economic losses in the poultry industry and have become one of the most important threats to public health (Webster et al., 1992; Claas et al., 1998). Currently, H5 subtype low pathogenic avian influenza viruses (LPAIVs) have been reported in migratory waterfowls and swine; however, H5N1 LPAIVs in land fowls might evolve into HPAIVs through adaptations (Baek et al., 2010; Kim et al., 2011; Ping et al., 2012). During passages through land fowls, AIVs might acquire multiple mutations for the efficient replication and inhibition of host antiviral mechanisms. Amino acid deletions in the stalk of NA and N-linked glycan changes in HA are well-known mutations for balancing the neuraminidase activity and receptor binding affinity of HA, which improves viral replication (Matrosovich et al., 1999; Mitnaul et al., 2000). However, mutations in H5N1 LPAIV during passages through embryonated chickens eggs (ECEs) have rarely been reported, and an H5N1 LPAIV has been evaluated for the vaccine strain against H5N1 HPAIV (Isoda et al., 2008). Therefore, in the present study we adapted a H5N1 LPAIV from migratory wild ducks to chicken embryos via passaging through ECEs and compared the genetic and phenotypic characteristics of early passage strains with those of the last passage strain. We observed a significant increase in viral titer and detected 8 cumulative amino acid changes in PA, HA, NA, M1 and M2. With the exception of one mutation in HA, Most of the mutations were novel and were present among influenza A viruses (IAVs) at different frequencies. A reverse genetics study revealed that the mutations in HA and NA of the last passage virus were important for increasing viral replication efficiency. The substitution of 6 internal genes of H5N1 LPAIV with those of PR8 decreased the pathogenicity of recombinant viruses in chicken embryos. Therefore, the mutations identified in the present study might be useful as molecular markers for the adaptation of AIVs to generate high growth recombinant vaccine viruses in ECEs.

2. Materials and methods 2.1. Viruses, cells and plasmids A/wild duck/Korea/SNU50-5/2011 (H5N1) (50-5) was isolated from migratory wild duck feces. H5N1 (50-5) was passaged 20 times through 10-day-old (10-d-o) SPF ECEs (VALO BioMedia, Adel, IA), and the viruses passaged 2 and 20 times were referred to as 50-5-E2 and 50-5-E20, respectively. The Hoffmann vector system was used to generate the rPR8 virus, which was passaged three times in 10-d-o SPF ECEs and used for the experiment (Hoffmann et al., 2002). The influenza viruses were inoculated in 10-do ECEs via the allantoic cavity and incubated for 24–120 h. After incubating at 4 8C overnight, the allantoic fluids were harvested and stored at 70 8C until further use. Chicken embryo kidney (CEK) cells were cultured in Eagle’s minimum essential medium (MEM; Life Technologies Co., NY, Grand Island, USA) supplemented with 5% fetal bovine serum (FBS; Life Technologies Co., NY, USA). 293T and MDCK cells were purchased from the American Type Culture Collection (ATCC, VA) and maintained in DMEM (Life Technologies Co.) supplemented with 5% FBS. The 293T cells were used for the generation of recombinant viruses through reverse genetics. 2.2. Virus isolation, chicken embryo adaptation and virus titration The 10% feces suspension in antibiotics-treated PBS was centrifuged at 3,000xg for 30 min, and the supernatant was inoculated into three 10-d-o SPF ECEs for virus isolation. The presence of AIV in the allantoic fluid was confirmed using a hemagglutination (HA) test with 1% (v/v) chicken red blood cells (RBC) according to the WHO Manual on Animal Influenza Diagnosis and Surveillance. This procedure was repeated 20 times to adapt the H5N1 50-5 virus to chicken embryos. To measure the virus titer, the individual samples were serially diluted 10-fold from 101 to 109, and each dilution (106 to 109) was inoculated into five 10-d-o SPF ECEs, CEK or MDCK cells. The 50% chicken embryo infection dose (EID50/ml) and 50% tissue culture infection dose (TCID50/ml) were calculated according to the SpearmanKarber method (Hamilton et al., 1977). 2.3. RT-PCR, sequencing and sequence analysis Total RNA was extracted from 150 ml of allantoic fluid using the Viral Gene Spin kit (iNtRON Biotechnology Co., Korea). Reverse transcription was performed using the RT&GOTM Mastermix (MP Biomedicals Inc., OH, USA), and cDNAs were amplified using TaKaRa Ex TaqTM (TaKaRa, Japan) with the universal primer set for influenza A viruses (Hoffmann et al., 2001). To amplify the noncoding 30 - and 50 -ends of viral genomes, genomic RNA was ligated using T4 RNA ligase (NEB Co., MA, USA), and one-step RT-PCR was conducted using the Qiagen OneStep RT-PCR kit (Qiagen GmbH, Hilden, Germany) with segment-specific genes (Table 1) and universal primer sets, as previously described (de Wit et al., 2007; Wang and Lee, 2009). The nucleotide sequences of the purified PCR amplicons were

Please cite this article in press as: Kim, I.-H., et al., Characterization of mutations associated with the adaptation of a lowpathogenic H5N1 avian influenza virus to chicken embryos. Vet. Microbiol. (2012), http://dx.doi.org/10.1016/ j.vetmic.2012.10.034

G Model

VETMIC-5970; No. of Pages 8 I.-H. Kim et al. / Veterinary Microbiology xxx (2012) xxx–xxx

3

Table 1 Primers used in the present study. Primer

Sequence (50 –30 )

Usage

cmv-SF bGH-SR PB2-338R PB2-1819F PB1-321R PB1-2074F PA-289R PA-1970F H5-308R H5-1416F NP-232R NP-1223F N1-445R N1-985F M-251R M-763F NS-385R NS-617F

TAA GCA GAG CTC TCT GGC TA TGG TGG CGT TTT TGG GGA CA GGY CCA TTC CTR TTC CAC CA ACA YTA TTC CAR CAR ATG CG GGA TTC TTC AAG GAA AGC CA CTT GAG GAT GAA CAG ATG TA TCC AGG CCA TYG TTC GGT C TAT ATG CAT CTY CAC AAC T CTT CTC YAC TAT GTA AGA CCA YTC CGA CTA CAG CTT ARG GAY AAT GC CCA TTC TCT CTA TTG TTA TGC TG CAR CAG AGR GCA TCT GCA GG CCA TTR GAA TGC TTG TCA TTC AA AAT GAT GGR ACA GGC AGT TG TGC AGT CCT CGC TCA CTG GTG CAR ATG CAG CGA TTC AA TCC AYT ATT GCY TGG TCC AT TMT ACA GAG ATT CRC TTG G

Sequencing of pHW2000 plasmid insert Amplification and sequencing of the non-coding 30 - and 50 -ends of viral genomes

determined using an ABI3711 automatic sequencer (Macrogen Co. Seoul, Korea), and the resulting sequences were deposited in the GenBank database. The nucleotide and deduced amino acid sequences were aligned and compared using the BioEdit program (McMaster University, Hamilton, Canada). The frequencies of the identified amino acid changes were determined through comparisons with the amino acid sequences in the GenBank and EMBL databases using a BLAST search. The locations of amino acid residues were numbered from the first amino acid residue after the HA5 signal peptide, except the amino acid residues in RBS and the 158N-glycan, which followed H3-numbering. The following IAV genes were used in the comparisons: A/wild duck/Korea/CSM4-12/2009 (H5N1) (Wd/Kr/CSM4-12/09, JF510040- JF510047), A/ Mallard duck/Hokkaido/24/2009 (H5N1) (Md/Hok/24/09, AB530989-AB530996), A/Swine/Korea/C13/2008 (H5N2) (Sw/Kr/C13/08, FJ461593, FJ461595, FJ461597, FJ461599, FJ461601, FJ461603, FJ461605 and FJ461607), A/Indonesia/5/2005 (H5N1) (parents virus; CY116643–CY116650, ferret-adapted virus; CY116651–CY116658), A/duck/ Hong Kong/702/1979 (H9N2) (chicken-adapted virus, CY031264-CY031271; quail-adapted virus, CY031272CY031279), A/Viet Nam/1203/2004 (H5N1) [VN/1203/ 04(H5N1), HM006756- HM006773], and A/Puerto Rico/ 8/1934 (H1N1) (PR8, EF467817- EF467824). The mutated amino acid residues of HA and NA were identified on the 3D structure of HA (3s11.pdb) and NA (2hty.pdb) proteins using SWISS-PdbViewer 4.04 (http:// www.expasy.org/spdbv/). 2.4. Cloning of HA and NA genes and rescue of recombinant viruses The bi-directional transcription vector pHW2000 and 8 plasmid vectors containing 8 genome segments of PR8 were previously constructed (Hoffmann et al., 2002). The HA and NA amplicons of 50-5-E2 and 50-5-E20 were cloned into pHW2000, as previously reported, and the

nucleotide sequences of the inserts were confirmed through sequencing using the primers in Table 1. To understand the effects of HA and NA genes on the virus replication efficiency in ECEs and the effect of internal genes on viral pathogenicity in chicken embryos, we transfected Hoffmann’s eight reverse genetics plasmids to generate 4 PR8-derived recombinant viruses with different combinations of HA and NA genes of 50-5-E2 and 50-5-E20, namely (rPR8-HN(E2), rPR8-HN(E20), rPR8-H(E2)N(E20) and rPR8-H(E20)N(E2) and parent PR8 (rPR8), as previously described with some modifications (Hoffmann et al., 2002). Briefly, 293T cells were cultured (5  105 cells/well in 6-well plates) and transfected with 300 ng of each plasmid using Lipofectamine and Plus reagent (Invitrogen Co., CA, USA) in a final volume of 1 ml of Opti-MEM (Invitrogen). After a 3-h incubation, 1 ml of fresh medium was added. The cells were incubated for an additional 20 h, and 0.5 ng/ml of trypsin (Invitrogen Co.) was subsequently added. After 12 h, the culture medium was harvested, and 200 ml was injected into 10-day-old SPF ECEs via the allantoic cavity. After incubating for 2–3 days, the allantoic fluid was harvested and subjected to the HA test. The genetic markers of each recombinant virus were confirmed using RT-PCR and sequencing. 2.5. The mean death time (MDT) of H5N1 virus The mean death times (MDTs) of the tested H5N1 viruses were calculated to compare the pathogenicity to chicken embryos as previously described (Isoda et al., 2011). The tested H5N1 viruses were inoculated into the allantoic cavity of ten 10-d-o SPF ECEs with 102 EID50/0.1 ml and incubated at 37 8C. Embryonic death was observed every twelve hours until 120 h post inoculation (hpi), and the MDT of each virus was calculated as the mean hours of embryo death. The MDT of the surviving chicken embryos until 120 hpi was read as 120 h.

Please cite this article in press as: Kim, I.-H., et al., Characterization of mutations associated with the adaptation of a lowpathogenic H5N1 avian influenza virus to chicken embryos. Vet. Microbiol. (2012), http://dx.doi.org/10.1016/ j.vetmic.2012.10.034

G Model

VETMIC-5970; No. of Pages 8 I.-H. Kim et al. / Veterinary Microbiology xxx (2012) xxx–xxx

4

2.6. Nucleotide sequence accession numbers The 8 complete genome sequences of 50-5-E2 and 505-E20 were deposited in the GenBank database under the accession numbers: JX497765-JX497780. 2.7. Statistical analyses The significance of the viral titer and MDT were assessed using one-way analysis-of-variance (ANOVA) (95% confidence intervals).

(Matrosovich et al., 1999). The representative mutations at 627 and 701 of PB2 and D92E and the deletion of 80–84 residues of NS1, which are important for mammalian pathogenicity, were not observed (Hatta et al., 2001; Li et al., 2005; Seo et al., 2002; Viseshakul et al., 2004). The PDZ-domain ligand (PL) motif was avian type, ESEV (Obenauer et al., 2006). The amino acid residues important for virus replication and pathogenicity are summarized in Table 3. 3.2. Comparison of phenotypic characteristics of 50-5-E2 and 50-5-E20

3. Results 3.1. Analyses of nucleotide and amino acid sequences of 50-5-E2 The entire genome sequence of 50-5-E2 was determined. The nucleotide and amino acid sequences were compared with the corresponding genes in the GenBank database and 2 similar H5N1 strains isolated from wild ducks in 2009 in Korea (Wd/Kr/CSM4-12/09) and Hokkaido in Japan (Md/Hok/24/09) were identified. Wd/Kr/ CSM4-12/09 showed high similarities (99.2–99.8%) in all of the genes, except PA (96.1%), and Md/Hok/24/09 also showed high similarities (98.9–99.8%) in all of the genes, except NP (92.6%), M (93.6%) and PA (95.9%). However, the amino acid similarities of Wd/Kr/CSM4-12/09 and Md/ Hok/24/09 were 98.9–100% and 97.9–100%, respectively (Table 2). 50-5-E2 contained the avian type amino acid residues 226Q and 228G in the RBS and a single basic amino acid (RETR) at the cleavage site of HA, which are characteristics of LPAIVs (Duan et al., 2007). The 158N-glycan and amino acid deletions in the NA stalk were not observed

The pathogenicity of the early (50-5-E2) and final passage (50-5-E20) strains were determined using the MDTs of chicken embryos. The MDTs of 50-5-E2 and 50-5E20 were 35.0 and 38.0 h, respectively, and these strains were pathogenic to chicken embryos (Table 3). The viral titers of 50-5-E20 in ECEs, CEK and MDCK were approximately 10- to 100-fold higher than those of 50-5-E2. The viral titers of 50-5-E2 and 50-5-E20 in MDCK were significantly lower than those of the ECEs (P < 0.05). The optimal viral replication temperature of 50-5-E2 and 50-5E20 was 37 8C, but the viral titer of 50-5-E2 at 42 8C was reduced in stiff compared with 50-5-E20 (Table 4). 3.3. Comparison of genetic characteristics of 50-5-E2 and 505-E20 Ten nucleotide mutations were observed in PB2 (G1149A), PA (T632C and C1794T), HA (C355T, A529G, T998C and G1178A), NA (G1106A), M1 (G691A) and M2 (T116C); these mutations were nonsynonymous, except for G1149A and C1794T, which were synonymous. The 8

Table 2 Comparison of nucleotide and amino acid sequences of A/wild duck/Korea/50-5-E2/2009 (H5N1) with similar isolates in the GenBank database. Genome segment

Protein

Identity (%)

Compared nucleotide sequence

Wd/Kr/CSM4-12/09a PB2 PB1 PA HA NP NA M NS a b c

PB2 PB1/PB1-F2 PA HA NP NA M1/M2 NS1/NEP

99.5 99.8 96.1 99.5 99.2 99.7 99.8 99.7

Md/Hok/24/09b

(99.5)c (99.8/100.0) (99.5) (99.4) (100.0) (99.5) (100.0/98.9) (99.5/99.1)

99.4 99.6 95.9 98.9 92.6 99.6 93.6 99.8

37–2280b 1–2274 34–2151 46–1680 1–1386 16–1395 1–982 1–838

(99.4) (99.8/99.8) (99.4) (98.7) (100.0) (99.5) (100.0/97.9) (99.5/99.1)

A/wild duck/Korea/CSM4-12/2009 (H5N1). A/Mallard duck/Hokkaido/24/2009 (H5N1). % Identity of nucleotide sequence (% identity of amino acid sequence).

Table 3 Comparison of important amino acid residues associated with viral pathogenicity and host range determination. Strain

Wd/Kr/50-5/09 (H5N1) VN/1203/04 (H5N1) PR8 (H1N1) a b

HAa

NA

PB2

Q226L

G228S

158N-Glycan

Cleavage site

Stalk del.

E627K

D701N

NS1 D92E

Deletion of 80-84 aa

PL motifb

Q Q Q

G G G

– + –

RETR RERRRKKR IQSR

– + +

E K K

D D D

D E D

– + –

ESEV – RESV

H3 numbering. PDZ domain ligand motif: ESEV (avian type) and RSEV (mammalian type) (Obenauer et al., 2006).

Please cite this article in press as: Kim, I.-H., et al., Characterization of mutations associated with the adaptation of a lowpathogenic H5N1 avian influenza virus to chicken embryos. Vet. Microbiol. (2012), http://dx.doi.org/10.1016/ j.vetmic.2012.10.034

G Model

VETMIC-5970; No. of Pages 8 I.-H. Kim et al. / Veterinary Microbiology xxx (2012) xxx–xxx

5

Table 4 Comparison of pathogenicity and replication efficiency of early embryo-passaged (50-5-E2), the last embryo-passaged (50-5-E20) and PR8-derived recombinant (rPR8 series) H5N1 influenza viruses. MDTa (hpi)

Virus

EID50/mlb (log10)

TCID50/ml (log10)c CEKd

35.0  4.0 38.0  4.2 104.0  31.7 120  0 NTe NT NT

50-5-E2 50-5-E20 rPR8-HN(E2) rPR8-HN(E20) rPR8-H(E2)N(E20) rPR8-H(E20)N(E2) rPR8 a b c d e

MDCKd

37 8C

32 8C

37 8C

42 8C

32 8C

37 8C

42 8C

7.9  0.3 10.0  0.1 7.6  0.3 9.2  0.3 7.8  0.6 8.6  0.3 9.4  0.3

6.4  0.1 7.3  0.3 6.6  0.3 7.3  0.3 NT NT 8.3  0.1

6.8  0.5 7.8  0.3 7.0  0.3 7.9  0.6 6.3  0.3 7.3  0.4 8.6  0.3

3.8  0.1 5.5  0.1 2.8  0.0 3.8  0.0 NT NT 4.3  0.0

5.4  0.4 6.0  0.2 5.7  0.2 6.0  0.2 NT NT 7.9  0.3

6.7  0.1 7.2  0.1 6.8  0.1 7.8  0.3 NT NT 8.7  0.2

3.9  0.0 5.3  0.0 < 2.25 3.8  0.0 NT NT 3.8  0.0

Mean death time of 10-day-old SPF chicken embryos with standard deviation; hpi: hours post inoculation. 50% of chicken embryo infection dose, EID50/ml, geometric mean log10 titer with standard deviation. 50% of tissue cell infection dose, TCID50/ml, geometric mean log10 titer with standard deviation. CEK: chicken embryo kidney cell; MDCK: Mardin-Darby canine kidney cell line. NT: not tested.

amino acid changes of 50-5-E2, PA (M211T), HA1 (H103Y, K161E, and L317P), HA2 (R51K), NA (S369N), M1 (D231N) and M2 (I39T), which occurred during embryonic adaptation, are summarized in Table 4. The amino acid changes were cumulative. 50-5-E10 showed P317L of HA1 and R51K of HA2 mutations but had subpopulations of viruses with H103Y of HA1 and D231N of M1 mutation, which were represented as overlapping peaks in the electropherograms of the sequencing results. 50-5-E20 showed 8 complete amino acid changes. The frequency of each amino acid change was calculated on the basis of the BLAST search results (Table 5). M211T of PA was rare, but R51K and S369N showed 99.6 and 46.5% frequencies, respectively. However, H103Y, K161E, L317P, D231N and I39T were infrequent (0.2–8.7%). Interestingly, a swine H5N1 LPAIV isolate (Sw/Kr/C13/08) reported in Korea in 2008 (Lee et al., 2009) contained 3 identical amino acid changes, including H103Y, L317P and R51K, and an HPAIV isolate [A/ Indonesia/5/2005 (H5N1)] containing R51K acquired H103Y after ferret-adaptation (Table 5) (Herfst et al.,

2012). The PR8 strain exhibited the same amino acid changes in M1 and M2, and the M2 mutation was observed in a chicken- or quail-adapted H9N2 virus (A/duck/Hong Kong/702/1979 (H9N2)). The HA and NA mutations were located in the 3D structure of each protein, and H103Y, K161E and R51K mutations of HA were represented in the 3D structure (Fig. 1). The 103 and 161 amino acid residues of HA1 were located far from the RBS of HA, and the 103-residue side chain was located near the 79-residue central helix of neighboring HA2 (Fig. 1A). Approximately 161 residues of HA1 were located near 219 residues of another neighboring HA1 (Fig. 1B). Thus, these proteins might participate in intermolecular interactions. The biological role of K161E was not reported, but H103Y increases the affinity of HA to avian receptors (Herfst et al., 2012). The 317 and 51 residues of HA1 and HA2, respectively, were located far from the RBS and were predicted to participate in intramolecular interactions. The 317 amino acid (HA1) residue was located near the proteolytic cleavage site of HA and the

Table 5 The amino acid changes acquired during passages of A/wild duck/Korea/SNU50-5/2009 (H5N1) (50-5) through 10-day-old (10-d-o) SPF embryonated chicken eggs (ECEs) and the presence of similar amino acid changes in swine and adapted influenza A viruses. Protein

Positiona

Frequency (%)g

Amino acid 50-5

PA HA1

HA2 NA M1 M2

211 103 161 317 51 369 231 39

E2b

E5

E10

E15

E18

E20

M H K L R S D I

M H K L R S D I

M H/Yc K P K S D/N I

T Y E P K N N I

T Y E P K N N I

T Y E P K N N T

Sw/Kr/C13/08 (H5N2)

Ferret-adapted virus (H5N1)e

Ck or quail-adapted virus (H9N2)f

PR8 (H1N1)

M Y K P K –d D I

M Y K L K S D I

M – – – – – D T

M – – – – D N T

0 0.5 0.3 2.2 99.6 46.5 8.7 0.2

a

The location of the amino acid residue in the protein. 50-5 virus passaged two times through 10-d-o SPF ECEs. Presence of mixed sequences representing mixed populations of the virus. d Not compared due to different subtypes of NA or HA. e A ferret-adapted virus through serial lung passages of A/Indonesia/5/2005 (H5N1). f The chicken or quail-adapted viruses obtained through serial lung passages of A/duck/Hong Kong/702/1979 (H9N2). g The frequency of each amino acid change acquired during the passages of 50-5. All sequences were analyzed using a BLAST search and are available in the influenza virus resource database (http://www.ncbi.nlm.nih.gov/genomes/FLU/). b c

Please cite this article in press as: Kim, I.-H., et al., Characterization of mutations associated with the adaptation of a lowpathogenic H5N1 avian influenza virus to chicken embryos. Vet. Microbiol. (2012), http://dx.doi.org/10.1016/ j.vetmic.2012.10.034

G Model

VETMIC-5970; No. of Pages 8 6

I.-H. Kim et al. / Veterinary Microbiology xxx (2012) xxx–xxx

Fig. 1. Locations of identified mutations (H103Y, K161E and R51K) and predictions of their interactions with neighboring amino acid residues in the 3D structures of a HA5 trimer (3s11.pdb) using SWISS-PdbViewer 4.04 (http://www.expasy.org/spdbv/). The 130-residue loop (pear blue), 190-residue helix (yellow) and 220-residue loop (purple), which form the receptor-binding pocket of HA, are represented. (A) The location of 103H (green) and 103Y (green) and their interactions with 79N (red) in the central helices of neighboring HA2 are represented. (B) The location of 161K (green) and 161E (green) and their interactions with 219T (red) in HA1 of the neighboring monomer are represented. (C) The location of 51K (green) and 51R (green) in the helices of HA2 and their interactions with 107T (red) in central helices of neighboring HA2 are represented. The putative hydrogen bonding between 51R and 107T is represented as a broken line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

51 amino acid residue in helix A of HA2 was predicted to form hydrogen bonds with 107T in the central helix of HA2 (Fig. 1C). The correlation of L317P and R51K mutations to viral replication efficiency in ECEs has never been reported. The 369 residue of NA was also located far from the neuraminidase binding site of NA, and the function of the S369N mutation has not been reported. In addition, the roles of other mutations, such as M211T, D231N and I39T, in viral replication in ECEs have also not been reported. 3.4. The effects of the internal genes of PR8 and the HA and NA genes of 50-5-E2 and 50-5-E20 on the pathogenicity to chicken embryos and the replication efficiency in ECEs, CEK and MDCK The four H5N1 recombinant viruses rPR8-HN(E2), rPR8HN(E20), rPR8-H(E2)N(E20) and rPR8-H(E20)N(E2), and

rPR8 were generated using reverse genetics. The embryo pathogenicities of rPR8-HN(E2) and rPR8-HN(E20) were significantly reduced compared with the parent strains (P < 0.05) (Table 4), indicating roles for the internal genes of 50-5-E2 and 50-5-E20 in chicken embryo pathogenicity. The viral titers of rPR8-HN(E20) and rPR8H(E20)N(E2) were higher than those of rPR8-HN(E2) and rPR8-H(E2)N(E20) in ECEs and CEK cells (Table 4). The viral titer of rPR8-HN (E20) was similar to that of 505-E20 in CEK cells at 32 8C and 37 8C but significantly lower than that of 50-5-E20 in ECEs (P < 0.05). However, the titer of rPR8-HN (E20) was comparable with that of rPR8. The viral titer of rPR8-HN(E20) was higher than that of rPR8-HN(E2) in MDCK at 37 8C but significantly lower than that of rPR8 in MDCK at 32 8C and 37 8C (P < 0.05). The optimal viral replication temperature of rPR8-HN (E2) and rPR8-HN (E20) was 37 8C, and the viral titers at

Please cite this article in press as: Kim, I.-H., et al., Characterization of mutations associated with the adaptation of a lowpathogenic H5N1 avian influenza virus to chicken embryos. Vet. Microbiol. (2012), http://dx.doi.org/10.1016/ j.vetmic.2012.10.034

G Model

VETMIC-5970; No. of Pages 8 I.-H. Kim et al. / Veterinary Microbiology xxx (2012) xxx–xxx

42 8C also decreased in stiff compared with 50-5-E20 (Table 4). 4. Discussion H5N1 LPAIVs can serve as precursors of HPAIVs through the acquisition of mutations for efficient replication in land fowls. Recently, H5 subtype LPAIVs have been reported in wild ducks and swine in Korea, and these viruses are considered as dormant threats to the poultry industry and public health. During the passages, only 10 nucleotide changes and 8 nonsynonymous mutations were observed. The nonsynonymous mutations were cumulative and acquired in step-by-step manner, which involved at least four steps. L317P and R51K were acquired first, followed by H103Y and D231N; M211T, K161E and S369N; and finally I39T. The occurrence of the first and most frequent mutation in HA might reflect the importance of HA in host adaptation. Recently, H103Y was identified in a ferretpassaged H5N1 mutant virus that acquired airborne transmission capacity (Herfst et al., 2012). Together with T156A, which does not contain the 154N-glycan (158Nglycan according to H3-numbering), H103Y increases the receptor affinities of HA proteins to avian or mammalian RBSs (Herfst et al., 2012). The 50-5-E2 had no potential 158N-glycan and was maintained in 50-5-E20 through chicken embryo passages. Thus, the higher replication efficiency of 50-5-E20 might reflect an increased receptor affinity. For efficient viral replication, a balance between HA affinity to the receptor and neuraminidase activity is essential. The effect of the S369N mutation in NA remains unknown, and the location of this residue is not directly associated with the sialic acid binding site. However, we can speculate that the S369N mutation might occur later than H103Y to modulate neuraminidase activity to balance the increased receptor affinity of HA. To date, H5N1 or H9N2 viruses with a 158N-glycan acquire stalk deletions of NA to balance reduced HA affinity to the receptor through reduction in neuraminidase activity during adaptation in chickens (Matrosovich et al., 1997; Mitnaul et al., 2000). Thus, the H103Y mutation in HA, which does not possess a 158N-glycan, and the S369N mutation in NA might reflect novel balancing mechanisms for increasing both HA and NA activities. However, further studies to estimate the affinities of different HA/NA combinations will be required to confirm these potential mechanisms. Although L317P affects the binding affinity of a human monoclonal scFv, CR6261, to target an epitope of the HA protein of a H5N1 virus, other mutations have unknown biological functions in viral replication in ECEs (Throsby et al., 2008). The existence of similar mutations in other mammalian and land fowl-adapted viruses might provide insight into the functions of these mutations. It has been reported that a swine H5N2 virus (Sw/Kr/C13/08) derived from AIVs was excreted for a longer period with higher titers in the nasal discharges of swine and ferrets than in Sw/Kr/C12/08 (Lee et al., 2009). Sw/Kr/C13/08 contains the same H103Y and P317L and R51K mutations in HA1 and HA2, respectively, as those of 50-5-E20. However, it is unclear whether these mutations were acquired during replication in land fowls or in swine. However, future

7

studies concerning the roles of these mutations in swine pathogenicity might be valuable. The I39T mutation in M2 was observed in chicken and quail-adapted viruses, and D231N and I39T were observed in the PR8 strain, which replicate to high titers in ECEs. The M211T mutation in PA was not detected in any viruses using a BLAST search. Thus, reverse genetics studies might also be valuable in characterizing the roles of these mutations. In the present study, reverse genetics revealed that both HA and NA proteins of 50-5-E20 are important for efficient virus replication in ECEs. However, the viral titer of rPR8HN(E20) was significantly lower than that of 50-5-E20 (P < 0.05). Although the viral titers of rPR8-HN(E20) in CEK at 32 8C and 37 8C were similar, the viral titer at 42 8C was significantly less than that of 50-5-E20 (P < 0.05). D231N and I39T mutations have previously been shown in PR8 M1 and M2, respectively. Hence, further study might also be valuable to determine the function of M211T. The viral titers of H5N1 parent and recombinant viruses at 32 8C were lower than those at 37 8C; thus, 37 8C might be the optimal temperature for HA and NA activities. The pathogenicities of rPR8-HN(E2) and rPR8-HN(E20) were significantly less than those of their parent strains, 50-5-E2 and 50-5-E20, and the virus titers of 50-5-E20 in CEK and MDCK at 42 8C were significantly higher than those of 505-E2 and rPR8-HN(E20) (P < 0.05). Therefore, internal genes other than HA and NA genes might be associated with the chicken embryo pathogenicity of AIVs and viral replication at 42 8C. In conclusion, the mutations identified in this study may be useful as chicken adaptation markers for the generation of H5N1 recombinant viruses with high growth efficiency in ECEs. In addition, a functional study of the newly identified mutations might provide insight into the mechanism of chicken embryo adaptation of H5N1 LPAIVs. Acknowledgements This work was supported through a Top Brand Project grant from the Korean Research Council of Fundamental Science & Technology and the KRIBB Initiative program (KGM3110912), a grant from the Korean Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No. A103001) and a grant (ZAD15-2010-11-02) from the Animal, Plant & Fisheries Quarantine and Inspection Agency (QIA), Ministry of Food, Agriculture, Forestry and Fisheries, Republic of Korea from 2010 to 2011. References Akarsu, H., Burmeister, W.P., Petosa, C., Petit, I., Muller, C.W., Ruigrok, R.W., Baudin, F., 2003. Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2). EMBO J. 22, 4646–4655. Baek, Y.H., Pascua, P.N., Song, M.S., Park, K.J., Kwon, H.I., Lee, J.H., Kim, S.Y., Moon, H.J., Kim, C.J., Choi, Y.K., 2010. Surveillance and characterization of low pathogenic H5 avian influenza viruses isolated from wild migratory birds in Korea. Virus Res. 150, 119–128. Boivin, S., Cusack, S., Ruigrok, R.W., Hart, D.J., 2010. Influenza A virus polymerase: structural insights into replication and host adaptation mechanisms. J. Biol. Chem. 285, 28411–28417. Chen, W., Calvo, P.A., Malide, D., Gibbs, J., Schubert, U., Bacik, I., Basta, S., O’Neill, R., Schickli, J., Palese, P., Henklein, P., Bennink, J.R., Yewdell,

Please cite this article in press as: Kim, I.-H., et al., Characterization of mutations associated with the adaptation of a lowpathogenic H5N1 avian influenza virus to chicken embryos. Vet. Microbiol. (2012), http://dx.doi.org/10.1016/ j.vetmic.2012.10.034

G Model

VETMIC-5970; No. of Pages 8 8

I.-H. Kim et al. / Veterinary Microbiology xxx (2012) xxx–xxx

J.W., 2001. A novel influenza A virus mitochondrial protein that induces cell death. Nat. Med. 7, 1306–1312. Claas, E.C., Osterhaus, A.D., van Beek, R., De Jong, J.C., Rimmelzwaan, G.F., Senne, D.A., Krauss, S., Shortridge, K.F., Webster, R.G., 1998. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472–477. de Wit, E., Bestebroer, T.M., Spronken, M.I., Rimmelzwaan, G.F., Osterhaus, A.D., Fouchier, R.A., 2007. Rapid sequencing of the non-coding regions of influenza A virus. J. Virol. Methods 139, 85–89. Duan, L., Campitelli, L., Fan, X.H., Leung, Y.H., Vijaykrishna, D., Zhang, J.X., Donatelli, I., Delogu, M., Li, K.S., Foni, E., Chiapponi, C., Wu, W.L., Kai, H., Webster, R.G., Shortridge, K.F., Peiris, J.S., Smith, G.J., Chen, H., Guan, Y., 2007. Characterization of low-pathogenic H5 subtype influenza viruses from Eurasia: implications for the origin of highly pathogenic H5N1 viruses. J. Virol. 81, 7529–7539. Hale, B.G., Randall, R.E., Ortin, J., Jackson, D., 2008. The multifunctional NS1 protein of influenza A viruses. J. Gen. Virol. 89, 2359–2376. Hamilton, M.A., Russo, R.C., Thurston, R.V., 1977. Trimmed SpearmanKarber method for estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol. 11, 714–719. Hatta, M., Gao, P., Halfmann, P., Kawaoka, Y., 2001. Molecular basis for high virulenceof Hong Kong H5N1 influenza A viruses. Science 293,1840–1842. Herfst, S., Schrauwen, E.J., Linster, M., Chutinimitkul, S., de Wit, E., Munster, V.J., Sorrell, E.M., Bestebroer, T.M., Burke, D.F., Smith, D.J., Rimmelzwaan, G.F., Osterhaus, A.D., Fouchier, R.A., 2012. Airborne transmission of influenza A/H5N1 virus between ferrets. Science 336, 1534–1541. Hoffmann, E., Krauss, S., Perez, D., Webby, R., Webster, R.G., 2002. Eightplasmid system for rapid generation of influenza virus vaccines. Vaccine 20, 3165–3170. Hoffmann, E., Stech, J., Guan, Y., Webster, R.G., Perez, D.R., 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146, 2275–2289. Isoda, N., Sakoda, Y., Kishida, N., Soda, K., Sakabe, S., Sakamoto, R., Imamura, T., Sakaguchi, M., Sasaki, T., Kokumai, N., Ohgitani, T., Saijo, K., Sawata, A., Hagiwara, J., Lin, Z., Kida, H., 2008. Potency of an inactivated avian influenza vaccine prepared from a non-pathogenic H5N1 reassortant virus generated between isolates from migratory ducks in Asia. Arch. Virol. 153, 1685–1692. Isoda, N., Sakoda, Y., Okamatsu, M., Tsuda, Y., Kida, H., 2011. Improvement of the H5N1 influenza virus vaccine strain to decrease the pathogenicity in chicken embryos. Arch. Virol. 156, 557–563. Kim, B.S., Kang, H.M., Choi, J.G., Kim, M.C., Kim, H.R., Paek, M.R., Kwon, J.H., Lee, Y.J., 2011. Characterization of the low-pathogenic H5N1 avian influenza virus in South Korea. Poult. Sci. 90, 1449–1461. Lee, J.H., Pascua, P.N., Song, M.S., Baek, Y.H., Kim, C.J., Choi, H.W., Sung, M.H., Webby, R.J., Webster, R.G., Poo, H., Choi, Y.K., 2009. Isolation and genetic characterization of H5N2 influenza viruses from pigs in Korea. J. Virol. 83, 4205–4215. Li, Z., Chen, H., Jiao, P., Deng, G., Tian, G., Li, Y., Hoffmann, E., Webster, R.G., Matsuoka, Y., Yu, K., 2005. Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model. J. Virol. 79, 12058–12064.

Matrosovich, M., Zhou, N., Kawaoka, Y., Webster, R., 1999. The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J. Virol. 73, 1146–1155. Matrosovich, M.N., Gambaryan, A.S., Teneberg, S., Piskarev, V.E., Yamnikova, S.S., Lvov, D.K., Robertson, J.S., Karlsson, K.A., 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. Mitnaul, L.J., Matrosovich, M.N., Castrucci, M.R., Tuzikov, A.B., Bovin, N.V., Kobasa, D., Kawaoka, Y., 2000. Balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza A virus. J. Virol. 74, 6015–6020. 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. Ping, J., Selman, M., Tyler, S., Forbes, N., Keleta, L., Brown, E.G., 2012. Lowpathogenic avian influenza virus A/turkey/Ontario/6213/1966 (H5N1) is the progenitor of highly pathogenic A/turkey/Ontario/ 7732/1966 (H5N9). J. Gen. Virol. 93, 1649–1657. Plotch, S.J., Bouloy, M., Ulmanen, I., Krug, R.M., 1981. A unique cap (m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription. Cell 23, 847–858. Seo, S.H., Hoffmann, E., Webster, R.G., 2002. Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nat. Med. 8, 950–954. Seto, J.T., Rott, R., 1966. Functional significance of sialidose during influenza virus multiplication. Virology 30, 731–737. Sugrue, R.J., Hay, A.J., 1991. Structural characteristics of the M2 protein of influenza A viruses: evidence that it forms a tetrameric channel. Virology 180, 617–624. Throsby, M., van den Brink, E., Jongeneelen, M., Poon, L.L., Alard, P., Cornelissen, L., Bakker, A., Cox, F., van Deventer, E., Guan, Y., Cinatl, J., ter Meulen, J., Lasters, I., Carsetti, R., Peiris, M., de Kruif, J., Goudsmit, J., 2008. Heterosubtypic neutralizing monoclonal antibodies crossprotective against H5N1 and H1N1 recovered from human IgM + memory B cells. PLoS ONE 3, e3942. Viseshakul, N., Thanawongnuwech, R., Amonsin, A., Suradhat, S., Payungporn, S., Keawchareon, J., Oraveerakul, K., Wongyanin, P., Plitkul, S., Theamboonlers, A., Poovorawan, Y., 2004. The genome sequence analysis of H5N1 avian influenza A virus isolated from the outbreak among poultry populations in Thailand. Virology 328, 169–176. Wang, L., Lee, C.W., 2009. Sequencing and mutational analysis of the noncoding regions of influenza A virus. Vet. Microbiol. 135, 239–247. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56, 152–179.

Please cite this article in press as: Kim, I.-H., et al., Characterization of mutations associated with the adaptation of a lowpathogenic H5N1 avian influenza virus to chicken embryos. Vet. Microbiol. (2012), http://dx.doi.org/10.1016/ j.vetmic.2012.10.034