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Veterinary Microbiology 167 (2013) 296–306
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merging multiple reassortant H5N5 avian influenza viruses ducks, China, 2008
un-Guo Liu a,b, Ming Liu a,*, Fei Liu c, Rang Lv a, Da-Fei Liu a, Lian-Dong Qu a,n Zhang a,**
tate Key Lab of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin
0001, China
ollege of Veterinary Medicine, Northeast of Agricultural University, No. 59 Mucai Street, Harbin 150030, China
anghai Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Shanghai 200241, China
Introduction
The highly pathogenic H5N1 avian influenza (HPAI)ruses were first discovered in southern China in 1996.
Despite numerous strategic efforts to control H5N1 avianinfluenza virus spread, these emerging viruses havecontinued to survive and evolve. So far, the H5N1 avianinfluenza viruses have evolved into over 32 distinct cladesbased on their hemagglutinin (HA) genes (WHO/OIE/FAO,2008). The H5N1 HPAI viruses have been persistentlyendemic in poultry and have the potential ability to crossthe species barrier to transmit to humans (Li et al., 2004). In2005, an outbreak of H5N1 HPAI occurred in wild birds atQinghai Lake in western China (Chen et al., 2005). Subse-quently, the Qinghai-like H5N1 HPAI virus (clade 2.2) hasbeen detected in many other countries (Lipatov et al., 2007;
R T I C L E I N F O
icle history:
ceived 8 July 2013
ceived in revised form 3 September 2013
cepted 5 September 2013
ywords:
uenza virus
N5
lecular and phylogenetic analysis
thogenicity
A B S T R A C T
Three highly pathogenic H5N5 avian influenza viruses (HPAI), A/duck/Guangdong/wy11/
2008 (WY11), A/duck/Guangdong/wy19/2008 (WY19), and A/duck/Guangdong/wy24/
2008 (WY24) were isolated from ducks in southern China in April 2008. Here, we
characterized these viruses by performing sequencing and phylogenetic analyses of their
viral genes, assessing their virulence in ducks and mice, and performing cross-protection
experiments in chickens. Sequence analysis revealed that the HA genes of these H5N5
viruses showed 97.1–97.8% homology to A/wild duck/Hunan/211/2005 (H5N1) influenza
virus and that their NA genes showed 96.4–96.8% nucleotide identity to the NA gene of A/
duck/Hunan/5613/2003 (H6N5) influenza virus, which belongs to the Eurasian lineage.
Genotypic analysis indicated that these H5N5 viruses were multiple reassortants among
H5N1, H5N2, H6N2, and H6N5 viruses. The analysis of HA clade showed that these H5N5
viruses are clustering into clade 2.3.4. In animal experiments, these H5N5 viruses caused
50% mortality in ducks and 100% mortality in chickens. In cross-protection experiments,
the clade 2.3.2 avian influenza vaccine could provide only 75% protection with chickens
against H5N5 virus challenge. Moreover, the H5N5 virus replicated efficiently in the lungs
of mice, which suggested that the H5N5 viruses have the potential to infect mammalian
hosts. Since ducks have served as reassortant vessels, playing pivotal roles in the
generation of new subtypes of influenza viruses, it is important to monitor the emergence
of this novel subtype of influenza viruses in waterfowl to understand their ecology and
evolution and to control the spread of new viruses.
� 2013 Elsevier B.V. All rights reserved.
Corresponding author at: Harbin Veterinary Research Institute,
ngang District, No. 427 Maduan Street, Harbin 150001, China. Tel.:
6 45182765628; fax: +86 45182733132.
Corresponding author at: Harbin Veterinary Research Institute,
ngang District, No. 427 Maduan Street, Harbin 150001, China.
E-mail addresses: [email protected] (M. Liu),
[email protected] (Y. Zhang).
Contents lists available at ScienceDirect
Veterinary Microbiology
jou r nal h o mep ag e: w ww .e ls evier . co m/lo c ate /vetm i c
78-1135/$ – see front matter � 2013 Elsevier B.V. All rights reserved.
p://dx.doi.org/10.1016/j.vetmic.2013.09.004
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C.-G. Liu et al. / Veterinary Microbiology 167 (2013) 296–306 297
ingst et al., 2006). In addition, reassortment amongfluenza viruses is a common event that gives rise to
ifferent subtypes of influenza virus and can also causefluenza outbreaks (Bragstad et al., 2006; Liu et al., 2009;
rown, 2000; Zell et al., 2008; Nicoll and Danielsson,013; Lee et al., 2012). The HPAI H5N1 virus in 1997 inong Kong was a triple reassortant among H5N1, H6N1,nd H9N2 viruses (Hoffmann et al., 2000; Guan et al.,999). Aquatic birds are well known as a stock reservoirool for 16 subtypes of HA and 9 subtypes of neurami-idase (NA) influenza virus. It is believed that all of theseubtypes are widespread in naturally infected Aquaticirds and that reassortment among them occurs with eachther in high frequency (Chen et al., 2005; Lipatov et al.,007; Brown et al., 2006).
Among the nine NA subtypes of influenza virus, the N5ubtype is rare. To date, there are only three documentedeports of H5N5-subtype influenza virus infection (Zhaot al., 2013; Zou et al., 2012; Gu et al., 2011). In this study,ree influenza viruses were isolated from fecal samples of
ucks in southern China, in April 2008. Hemagglutinationnd neuraminidase inhibition assays with mono-specificntiserum confirmed that these three viruses belong to5N5 subtype. Here, we defined and analyzed the fullenome sequences of these three isolates. We alsoxamined their pathogenicity in ducks and mice andxamined cross-protection with a heterogeneous virus inhickens.
. Materials and methods
.1. Viruses and animals
During our routine surveillance of avian influenzairuses at a live bird market in Guangdong province ofhina in April 2008, three H5N5 viruses were isolated frompparently healthy ducks from a local farm, which haveeen vaccinated with an inactivated influenza vaccine
5N1 subtype). The three isolates were designated as A/uck/Guangdong/wy11/2008 (WY11), A/duck/Guang-ong/wy19/2008 (WY19), and A/duck/Guangdong/wy24/008 (WY24), respectively. These H5N5 viruses wereurified by dilution end point method in 10-day-oldpecific pathogen free (SPF) embryonated chicken eggs andere verified by hemagglutination and neuraminidasehibition assays. The currently prevalent influenza virus
/muscovy duck/Jilin/MHK513/2011 (H5N1) of clade.3.2, was propagated in 10-day-old SPF embryonatedhicken eggs and then stored at �70 8C until use. The 10-ay-old SPF embryonated chicken eggs, 4-week-old SPFhite Leghorn chickens, 4-week-old SPF ducks, and 6-eek-old Balb/c mice were supplied by the experimental
nimal center of HVRI. All birds were housed in isolationabinets that were ventilated under negative pressure withEPA-filtered air; mice were housed in individuallyentilated cages. Food and water were available ad libitum.ll animals were maintained in the animal facility atarbin Veterinary Research Institute under standard
onditions prescribed by the Institutional Guidelines.he study protocol was approved by the Institutionalnimal Care and Use Committee.
2.2. Sequencing and phylogenetic analyses of viral genes
Viral RNA was extracted from allantoic fluids by using aviral RNA extraction kit (Qiagen, Shanghai, China). Virus-specific cDNAs were obtained by using influenza universalreverse transcription primer uni-12: 50-AGCAAAAGCAGG-30 with the AMV reverse transcriptase (TaKaRa, Dalian,China). The PB2, PB1, PA, HA, NP, NA, M, and NS genes wereamplified by using their respective gene-specific primers.All gene segments were cloned into the pMD18-T vector(TaKaRa, Dalian, China) and then sequenced by using anABI PRISM 3700 DNA Analyzer (Applied Biosystems,Shanghai Invitrogen, China). All gene sequence data forthe three H5N5 viruses were deposited in GenBank: WY11:CY091624-CY091631; WY19: CY091632-CY091639; andWY24: CY091640-CY091647. LASERGENE 7.1 (DNASTAR,Madison, WI) was used for sequences analysis. Phyloge-netic analysis and tree construction were based on theopen reading frame of each gene’s nucleotide sequence.Phylogenetic analysis of viral genes was conducted byusing the MEGA 4.0 program (Tamura et al., 2007);phylogenetic trees were generated by means of theNeighbor-Joining method in the MEGA 4.0 software usingp-distance; 1.000 NJ bootstrap replicates were obtained toestimate the phylogenies.
2.3. Determination of intravenous pathogenicity index (IVPI)
To determine the IVPI, 30 chickens (10 for each virus)were intravenously inoculated with 0.1 mL of a 10�1
dilution of WY11, WY19, or WY24 virus, respectively. Allchickens were observed daily for 10 days for clinical signsand death.
2.4. Virulence in ducks
Sixteen 4-week-old SPF ducks were randomly dividedinto two groups, each containing eight ducks. The WY11virus was selected as representative of the three H5N5viruses in this experiment. Each duck of one group waschallenged with WY11 virus in 106 EID50 by means ofintranasal and eye drop administration; birds in anothergroup served as co-housed control were inoculated with0.3 mL of sterile phosphate-buffered saline (PBS) per duck.The ducks were monitored daily for signs of illness andtracheal and cloacal swabs were collected on days 3, 5, 8,and 14 post-challenge for virus detection by inoculating10-day-old SPF chicken eggs. Antibodies titers weredetermined by means of the HI test 14 days post-challenge.
2.5. Virulence in mice
To determine the pathogenicity of the H5N5 influenzaviruses in mice, the WY11 virus was again selected as arepresentative virus. Forty-eight 6-week-old female Balb/cmice were randomly divided into six groups. The mice ingroups 1 to 3 were inoculated intranasally with 106 EID50
of WY11 virus and the mice in groups 4–6 were given0.05 mL of PBS by the same route. The mice in groups 1 and4 were weighed and observed daily for 14 days for clinicalsigns; sera were collected for the HI antibody test on day
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C.-G. Liu et al. / Veterinary Microbiology 167 (2013) 296–306298
post-challenge. The mice in groups 2 and 5 were fed ine same cage; every two days two mice from each groupere sacrificed to assess virus replication in the lung,leen, liver, and kidney by inoculating 10-day-old SPFicken eggs. Two mice in groups 3 and 6 were sacrificedery two days to examine lung, spleen, liver, and kidneyr pathological lesions by using histochemistry hematox-in and eosin (HE) stains.
. Cross-protection test in chickens
To determine whether a clade 2.3.2 heterologous H5N1fluenza virus could provide effective protection againste H5N5 viruses in chickens, the clade 2.3.2 recentlylated influenza virus MHK513 was selected as vaccinetigen to vaccinate SPF chickens; the WY11 virus served
a representative virus for this challenge test. In brief, theHK513 virus was inactivated with 0.1% formalin for 24 hd then emulsified with ISA 70 VG adjuvant (SEPPIC) (thetio of antigen to adjuvant was 2.6:7.4) (Liu et al., 2011).teen 4-week-old SPF chickens (8 chickens in group 1d 3) were vaccinated with 0.3 mL of vaccine (containing
1 mg HA protein) subcutaneously in the neck, respec-ely. Another sixteen chickens (divided into groups 2 and
were injected with 0.3 mL of PBS per chicken. Theallenge test was conducted with the WY11 virus in dose
106 EID50 per chicken in groups 1 and 2; the virus wasministered intranasally and via eye drops 2 weeks post-ccination. Chickens in group 3 and 4 were challengedith MHK513 in dose of 106 EID50 per chicken. Theickens were monitored daily for signs of illness; tracheald cloacal swabs were collected on days 3, 5, 8, and 14st-challenge for virus detection. HI antibodies wereonitored by using the MHK513 and WY11 viruses astection antigen.
Results
. Homology analysis
To expand the genetic information available regardingN5 viruses and to examine the degree of sequenceilarity between the H5N5 viruses and other viruses, the
ll-length fragments of each gene of the three H5N5ruses were completely sequenced and pairwisequences comparisons were made (Table S1). The PB2nes of the H5N5 viruses were closely related to that ofe H5N2 virus A/duck/Korea/A14/2008, with 96.5–96.8%
ilarity at the nucleotide level (Table S1). Nucleotidequence comparisons also revealed that the PB1 genes ofe three H5N5 viruses were highly homologous to that ofduck/Mongolia/54/2001 (H5N2) and A/duck/Hokkaido/c-1/04 (H5N1), with 97.1–97.2% homology. The PA ande NS genes showed 97.9–98.1% and 97.2–98.4% similar-, respectively, to A/wild duck/Hunan/021/2005 (H5N1).e HA genes showed the highest homology to the HA of A/
ild duck/Hunan/211/2005 (H5N1), with 97.1–97.8%mology. The NP genes of the three H5N5 showed.3–97.7% homology to the NP gene of A/duck/Shantou/4/2002 (H6N2). The NA genes were closely related toat of A/duck/Hunan/5613/2003 (H6N5), with 96.4–96.8%
nucleotide identity. The M genes shared 98.5–98.6%similarities to that of the H5N1 influenza virus A/China/GD01/2006, which is of human origin.
Supplementary material related to this article can befound, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2013.09.004.
3.2. Phylogenetic analysis
To understand the evolutionary relationship betweenthe three H5N5 viruses and other influenza viruses inGenBank, phylogenetic trees for each gene were con-structed by using MEGA 4. The clade of the HA genes of thethree H5N5 influenza viruses was identified according tothe unified nomenclature system (WHO/OIE/FAO, 2008).Phylogenetic analysis of the HA gene revealed that thethree H5N5 viruses belong to clade 2.3.4 (Fig. 1). The HAgenes of the three H5N5 viruses in this study and otherH5N5 viruses (EC031 and EC008) (Gu et al., 2011) isolatedin eastern China in 2008 fell onto the same branch.
Phylogenetic analysis of the N5 genes indicated that allof the N5 genes have evolved into two distinct lineages, theNorth American lineage and the Eurasian lineage. LikeEC031 and EC008, the N5 genes of the three H5N5 virusesbelonged to the Eurasian lineage and clustered with theH6N5 virus A/duck/Hunan/5613/2003 (Fig. 2).
Phylogenetic analysis also suggested that the PB1 genesof the H5N5 viruses, as well as those of the EC031 andEC008 viruses, were located in the same cluster with A/duck/Hokkaido/Vac-1/04 (H5N1) and A/duck/Mongolia/54/2001 (H5N2) viruses, but formed a single branchdistinct from other recent isolates (Fig. S1). The PB2 genesof the H5N5 viruses isolated in China clustered withviruses represented by A/duck/Korea/A14/2008 (H5N2),but formed a single branch that was distinct from otherrecent isolates (Fig. S2). The PA genes of the H5N5 virusesand that of EC031 fell into the same cluster with the A/wildduck/Hunan/021/2005 (H5N1) virus, but EC008 did notcluster with these viruses; rather it was located outside ofthe branch (Fig. S3). Phylogenetic analysis of the NP genesrevealed that the H5N5 viruses, EC031, and EC008 allclustered with the NP genes of H6N2 influenza viruses,represented by A/duck/Shantou/494/2002(H6N2) (Fig. S4).The trees for the M and NS genes revealed that the H5N5viruses, EC031, and EC008 were grouped with the M andNS genes of H5N1 human and avian influenza virusesisolated between 2005 and 2008, represented by A/China/GD01/2006(H5N1) and A/wild duck/Hunan/021/2005(H5N1) (Figs. S5 and S6), respectively.
Supplementary material related to this article can befound, in the online version, at http://dx.doi.org/10.1016/j.vetmic.2013.09.004.
3.3. Molecular characterization
All of the H5N5 viruses in this study maintained themultiple basic amino acid motif -PLREKRRKRG- (WY11 orWY19) and -PLREKRRRKRG- (WY24) at the HA cleavagesite (Table 1), which is characteristic of HPAI. The receptorbinding site of the HA protein retained its avian cell-surface receptor characteristics, in that it contains the
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C.-G. Liu et al. / Veterinary Microbiology 167 (2013) 296–306 299
mino acid residues 222Q and 224G, which preferentiallyind to 2, 3-NeuAcGal linkages (Matrosovich et al., 1999).ompared with clade 2.3.4 viruses, all of the China H5N5iruses lost the N-link glycosylation site 154NNT156,hich was mutated to 154NDA156. All other amino acid
residues known to be relevant to antigenic sites orvirulence were identical to those of clade 2.3.4 virusesrepresented by A/China/GD01/2006 (H5N1), A/wild duck/Hunan/211/2005 (H5N1), and A/wild duck/Hunan/021/2005 (H5N1), with the exception of positions 263 and 156,
ig. 1. Phylogenetic analysis of the HA genes of the H5N5 WY influenza viruses isolated in China. The scale bar represents the nucleotide substitutions per
te. The numbers above and below the branches indicate bootstrap values. Bootstrap support values >70 are shown (1000 replicates). The blue font
dicated the three viruses characterized in this study. (For interpretation of the references to color in this figure legend, the reader is referred to the web
ersion of the article.)
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C.-G. Liu et al. / Veterinary Microbiology 167 (2013) 296–306300
here an A to T or a T to A mutation, respectively, wasserved in the H5N5 viruses isolated in China (Table 1).e antigenic sites 83 and 86 were also different betweene three H5N5 viruses and EC031/EC008.The deduced amino acid sequences of the NA protein of
e three H5N5 viruses compare with that of A/duck/assachusetts/Sg-00440/2005 (H5N5) and A/mallard/N/105/2000(H5N5), one amino acid deletion at NA stalksition 41 was observed. Compared with the NA gene ofgoose/Guangdong/1/96 (H5N1), the N5 genes of theree H5N5 viruses had lost four amino acids at positions–57, whereas the NA genes of clade 2.3.4 viruses had a-amino acid deletion at positions 49–68 (Table 2).For the drug-resistance genes, the NA protein of all of
e H5N5 viruses contained 119E, 274H, and 292R,ggesting that they remained sensitive to neuraminidasehibitors (Table 2). The amino acid residue at positions 26,
27, and 30 in the M2 protein were L, V, and A, respectively,indicating that the H5N5 viruses retained their sensitivityto amantadine and rimantadine (Table 2). However, the Sto N resistance substitution at position 31 in the M2protein of the H5N5 viruses was observed. The NS1 proteinof all of the China H5N5 viruses contained a 5-amino aciddeletion at positions 80–84 but retained its virulencecharacteristics, with 92D and the PDZ-binding motif ofEPEV. The H5N5 viruses also have the conserved E atposition 627 and D at position 701 in PB2 (Table 2).
3.4. Genotyping
To determine the genotypes of the H5N5 viruses, all ofthe gene fragments were put into the influenza A virusgenotype tool (Lu et al., 2007). The results showed that thegenotypes of the PB2, PB1, PA, HA, NP, NA, M, and NS
. 2. Phylogenetic analysis of the NA genes of the H5N5 WY influenza viruses isolated in China. The numbers above and below the branches indicate
otstrap values. Bootstrap support values >70 are shown (1000 replicates). The scale bar represents the nucleotide substitutions per site. The blue font
icates the viruses characterized in this study. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
article.)
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C.-G. Liu et al. / Veterinary Microbiology 167 (2013) 296–306 301
egments of the H5N5 viruses were defined as K, G, D, 5J, F,B, F, and 1E, respectively (Fig. 3). Fig. 3 shows that the5N5 viruses originated from multiple recombinants ofifferent virus subtypes, including H5N1, H5N2, H6N5, and6N2. The HA and NA genes of the H5N5 viruses appear toe from A/wild duck/Hunan/211/2005 (H5N1) and A/duck/unan/5613/2003 (H6N5), respectively. The NP gene mayave been provided by the H6N2 virus A/duck/Shantou/94/2002. The PA and NS genes originated from theommon ancestor virus A/wild duck/Hunan/021/2005
5N1). The M gene was contributed by human H5N1irus:A/China/GD01/2006 (H5N1). The PB2 and PB1 genes
of the H5N5 viruses may have been donated by the H5N2viruses A/duck/Korea/A14/2008 and A/duck/Mongolia/54/2001, respectively, or the PB1 gene maybe also be rooted inthe H5N1 virus A/duck/Hokkaido/Vac-1/04.
3.5. Pathogenicity in chickens
Chickens intravenously inoculated with H5N5 virusesshowed neural symptoms and signs of depression. The 10chickens in each virus-infected group were all dead within60 h of infection. The IVPI values of WY11, WY19, and WY24 were calculated to be 2.9, 2.9, and 2.8, respectively.
able 1
olecular analysis of the HA protein of the WY11, WY19, and WY24 viruses with reference strains.
HA
Clade Cleavage site Receptor
binding
site
N-link Glycosylation
site
Antigenic site E Antigenic
site A
Amino acid site associ-
ates with virulence
(H5N5) 321-331 222 224 154-156 83 86 138 140 141 124 212 263 156
WY11 (H5N5) 2.3.5 PPREKRR-KRG Q G NDA A A Q T P D K T A
WY19 (H5N5) 2.3.5 PLREKRR-KRG Q G NDA A A Q T P D K T A
WY24 (H5N5) 2.3.5 PLREKRRRKRG Q G NNA A A Q T P D K T A
EC008 (H5N5) 2.3.5 PLREKRR-KRG Q G NNA T A Q T P D K T A
EC031 (H5N5) 2.3.5 PLREKRR-KRG Q G NNA T A Q T P D K T A
MN105 (H5N5) PQRE———TRG Q G DNA N V N R S N E A A
MHK513 (H5N1) 2.3.2 PQRERRR-KRG Q G DNA A A Q N S D K T A
HN021 (H5N1) 2.3.4 PLRERRR-KRG Q G NNT A A Q T P D K A T
HN211 (H5N1) 2.3.4 PLRERRR-KRG Q G NNT A A Q T P D K A T
GD01 (H5N1) 2.3.4 PLRERRR-KRG Q G NNT A A Q T P D K A T
AH1 (H5N1) 2.3.4 PLRERRR-KRG Q G NNT A A Q T P D K A T
SX2 (H5N1) 7 PQRERRRKKRG Q G NNT A A L E P N K A T
BGH05 (H5N1) 2.2 PQGERRRKKRG Q G DNA I A Q R S D K T A
GD196 (H5N1) 0 PQRERRRKKRG Q G NSA A A H R S N E A A
Vac-1 (H5N2) PQRE———TRG Q G NNA D I N R S D E A A
Mon54 (H5N2) PQRE———TRG Q G NNA D I N R S D E A A
A14 (H5N2) PQRE———TRG Q G NNA D T N R S N E A A
A protein amino acids numbered according to that of GD196. -: amino acid deletion.
able 2
olecular analysis of the NA, M2, NS1, and PB2 sequences of the WY11, WY19, and WY24 viruses with reference strains.
NA M2 NS1 PB2
NA stalk dele-
tion
Oseltamivir-resistant
amino acid
Amantadine resistant
amino acids
5-aa deletion Virulence
determinant
Virulence
determinant
41 49–68 119 274 292 26 27 30 31 80–84 92 PBM 627 701
WY11 Yes 54–57 E H R L V A N Yes D EPEV E D
WY19 Yes 54–57 E H R L V A N Yes D EPEV E D
WY24 Yes 54–57 E H R L V A N Yes D EPEV E D
EC008 Yes 54–57 E H R L V A N Yes D EPEV E D
C031 Yes 54–57 E H R L V A N Yes D EPEV E D
MN105 S 54–57 E H R L V A S No D ESEV E D
SG05 S 54–57 E H R L V A S Yes D ESEV E A
HN5613 Yes 54–57 E H R L V A S No D ESEV E D
HN021 G Yes E H R L V A S Yes D ESEV E D
HN211 E Yes E H R L V A S Yes D ESEV E D
AH1 G Yes E H R L V A S Yes D ESEV E D
GD01 G Yes E H R L V A S Yes D ESEV E D
SX2 G Yes E H R L V A N Yes D ESEV E D
BGH05 G Yes E H R L V A S Yes D ESKV K D
Vac-1 G Yes E H R L V A S No D ESEV E D
GD196 G No E H R L V A S No D ESEV E D
Mon54 G 56 E H R L V A S No D ESEV E D
A14 E 56 E H R L V A S No D ESEV E D
ST494 E 56 E H R L I A S No D ESEV E D
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Fi
C.-G. Liu et al. / Veterinary Microbiology 167 (2013) 296–306302
. Virulence in ducks
The WY11 virus was partial lethal to ducks causing a% mortality rate in the challenge group, and a 25%
ortality rate in the co-housed group (Fig. 4). On day 7st-challenge, neural symptoms were observed in twok ducks. Virus titration of tracheal and cloacal swabs
dicated that viral shedding occurred from day 3 toy 8 in both the challenge group and the co-housed
group; however, no viral shedding was detected on day14 (Table 3). HI antibodies were detected in birds fromboth the challenge and the co-housed groups on day 14post-infection (6.5 log 2 and 5.3 log 2, respectively;Table 3).
3.7. Virulence in mice
The WY11 virus was able to replicate in the lungs ofmice from day 2 to day 8 post-challenge without prioradaptation (Table 4). Virus titers in the lungs ranged from3.8 � 0.7 to 6.5 � 1.2 log10 EID50/mL. There was nodetectable virus in any of the other organs tested, includingthe liver, spleen, and kidney. The WY11 virus caused about11% weight loss in the mice (data not shown). On day 10post-infection, the mice began to recover weight andultimately achieved the same level as that of the controlgroup on day 12 post-infection. On day 14 post-challenge,seven of the eight challenged mice seroconverted and meantiters of HI antibodies reached 1:18.75. Mice in the co-housed group showed no detectable HI antibody or weightloss. The challenged mice showed lung histopathologicallesions on days 2, 4, and 6 post-inoculation, including smallblood vessel passive congestion, fibrous pneumonia, andinterstitial pneumonia (Fig. 5). The lungs of the mice in thecontrol group showed no obvious histopathologicalchanges.
. 3. Genotypes of the novel H5N5 WY influenza viruses isolated in China. The eight gene segments are (horizontal bars starting from top to bottom) the
2, PB1, PA, HA, NP, NA, M, and NS genes. Each color represents a separate virus background. The simplified schematic illustration is based on full-length
cleotide-distance comparisons and phylogenetic analysis. The capital letters indicate the genotypes. (For interpretation of the references to color in this
ure legend, the reader is referred to the web version of the article.)
g. 4. Percent survival of ducks after challenge with the WY11 virus.
Table 3
Virus titers of tracheal and cloacal swabs from ducks and HI antibody titers post-infection.
DPI Tracheal swabsa
No. positive/no. sampled
Cloacal swabsa
No. positive/no. sampled
HI antibody titers (log2)
Challenged Co-housed Challenged Co-housed Challenged Co-housed
3 7/7 (5.5) 8/8 (2.2) 7/7(5.7) 8/8(3.3) – –
5 6/6 (6.7) 6/6(5.5) 6/6(6.2) 6/6(6.0) – –
8 2/5 (2.4) 2/6(1.8) 1/5(2.5) 2/6(2.4) – –
14 0/4 0/6 0/4 0/6 6.5 5.3
DPI: days post-infection; –, not done.a Virus titers post-challenge log10(EID50/mL).
Table 4
Replication of the H5N5 WY11 virus in mice.
Organs Virus titers on the indicated days post-challenge log10(EID50/mL)a
2 4 6 8
Lung 2/2 (5.2 � 0.8) 2/2 (6.5 � 1.2) 2/2 (6.1 � 0.5) 1/2 (3.8 � 0.7)
Liver 0/2 0/2 0/2 0/2
Spleen 0/2 0/2 0/2 0/2
Kidney 0/2 0/2 0/2 0/2
Means � SD for the number of virus-positive mice that were sacrificed are shown.a Virus titers in the organs shown were determined.
Fig. 5. Histopathological observations of mouse lung infected with H5N5 WY11 virus. Small blood vessel passive congestion on day 2 post-inoculation (A);
small blood vessel passive congestion and fibrous pneumonia on day 4 post-inoculation (B); blood vessel passive congestion, fibrous pneumonia, and severe
interstitial pneumonia on day 6 post-inoculation (C); healthy lung of mice from the control group (D).
C.-G. Liu et al. / Veterinary Microbiology 167 (2013) 296–306 303
3.8
th(FruchchcovaThvam7.5Wththan4.2Than
4.
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1
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C.-G. Liu et al. / Veterinary Microbiology 167 (2013) 296–306304
. Cross-protection in chickens
The MHK513 vaccine provided only 75% protection toe immunized birds post-challenge with WY11 virusig. 6). Four chickens showed signs of depression andffled feathers on day 4 post-challenge; two of theseickens died on days 7 and 9, respectively, the other twoickens recovered on day 10 (Fig. 6). The chickens in PBSntrol group all died within 3 days, whereas none of theccinated birds died after challenge with MHK513 virus.e virus titers in the tracheal and cloacal swabs of theccinated chickens were listed in Table 5. The geometricean HI antibody titers of the chickens in group 1 were6 log 2 and 8.83 log 2 before and after challenge with
Y11, respectively, using the parental MHK513 virus ase detection antigen. When we used the WY11 virus ase detection antigen, the geometric means of the HItibody titers before and after challenge with WY11 were5 log 2 and 5.17 log 2, respectively (data not shown).ere were no signs of disease, detectable virus, or HItibodies in the chickens from group 4 (Fig. 6).
Discussion
Influenza A viruses are prone to reassort in naturalsts, resulting in the appearance of new subtypes of
influenza virus. Sometimes these reassortant viruses causeserious disease in hosts (Hatta et al., 2001). Here, wecharacterized three multiple reassortant H5N5 influenzaviruses isolated in Guangdong province of China in April2008. These viruses were the earliest H5N5 viruses everisolated in China, as documented by Gu et al. (2011).
The average percentage pairwise nucleotide distancesfor the HA genes of the three viruses and clade 2 viruseswere above the >1.5% divergence threshold. The isolatesshare a common node with clade 2.3.4 viruses and thebootstrap value of monophyletic grouping was 91(�60) atthe clade-defining node (1000 bootstrap replicates).Therefore, the three H5N5 isolates should be assigned tothe clade 2.3.4 according to specific clade definition criteriadeveloped by the WHO/OIE/FAO H5N1 Evolution WorkingGroup (WHO/OIE/FAO, 2008). Phylogenetic and nucleotidehomology analysis of the HA gene showed that theseviruses are clearly distinguishable from the HPAI virusespreviously isolated in China and are more related to A/duck/Hunan/211/2005 (H5N1).
Since 1997, H5N1 viruses from Hong Kong havereassorted with multiple other avian influenza virusesgiving rise to different genotypes (Hoffmann et al., 2000;Guan et al., 1999). Phylogenetic and sequence analysissuggest that the three H5N5 viruses are multiple-reassortant viruses among A/wild duck/Hunan/211/2005(H5N1), A/duck/Hunan/5613/2003 (H6N5), and possiblyother H5N1, H5N2, and H6N2 subtypes. However, it isworth mentioning that their M gene is closely related tothat of a human H5N1 strain (A/China/GD01/2006),suggesting that these H5N5 isolates might be reassortantsamong human and waterfowl influenza viruses. Thisfinding emphasizes the need for surveillance of influenzaviruses in aquatic birds.
Molecular analysis showed that the HA of the threeH5N5 viruses possess a series of basic amino acidinsertions at the cleavage site, PPREKRRKRGL (WY11),PLREKRRKRGL (WY19), and PLREKRRRKRGL(WY24), andno additional glycosylation site at amino acids 154–156 ofHA1, indicating that these three H5N5 viruses are HPAIviruses (Hatta et al., 2001). The 50% mortality rate amongthe challenged ducks and 25% mortality rate among the co-housed ducks suggest that the H5N5 viruses are highlypathogenic for ducks, a finding that is consistent with theresults of our sequence analysis of their HA gene.
The pathogenicity index value of 2.9 in chickensindicates that the WY11 virus is a highly pathogenic virus.The currently prevalent influenza virus MHK513 (clade
. 6. Percent survival of MHK513-vaccinated SPF chickens infected with
WY11 and MHK513 viruses. Chickens in group 1 and 3 were
ccinated with MHK513 (H5N1) vaccine. Chickens in group 2 and 4 were
ected with 0.3 mL of PBS. Two weeks post-vaccination, chicken in
ups 1 and 2 were challenged with the WY11 virus, and chickens in
up 3 and 4 were challenged with MHK513 virus.
ble 5
us titers of tracheal and cloacal swabs from vaccinated chickens post-infection with MHK and WY11 viruses.
PI No positive/no sampled log10(EID50/mL)
Tracheal swabsa Cloacal swabsa
Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4
3 5/8 (4.32) 2/2 (6.54) 8/8 1/1 (5.95) 5/8 (3.79) 2/2 (6.83) 8/8 1/1 (6.68)
5 4/8 (5.44) – 8/8 – 5/8 (4.92) – 8/8 –
8 4/7 (4.76) – 8/8 – 7/7 (5.37) – 8/8 –
4 0/6 – 8/8 – 0/6 – 8/8 –
I: days post-infection; –: not done because the birds died.
Virus titers post-challenge.
2wAvccces
rpahmsmHli
Hr2Ar2Mvnththfob
vhssepvaincvelimntibHes
2(srHce
C.-G. Liu et al. / Veterinary Microbiology 167 (2013) 296–306 305
.3.2) could only provide 75% protection against challengeith the WY11 virus, suggesting that WY11 has evolved.ntigenic changes at sites 140 and 141 between the threeiruses and MHK513 are consistent with the results of thehicken cross-protection experiment. The antigenichange at site 83 between WY11 and EC008/EC031 alsoonfirmed that the China H5N5 viruses are continuouslyvolving. Taken together, these data suggest that vaccineelection to protect against these viruses will be difficult.
The HA of the H5N5 viruses has Q226 and G228 in itseceptor binding site, indicating that these virusesreferentially bind to avian-like NeuAca2, 3-Gal receptorsnd have not adapted to mammalian hosts. However, theigh virus titers in the lung and weight loss among Balb/cice, together with the HI antibody post-challenge data,
uggest that the WY11 virus could efficiently replicate inice without prior adaptation, which further implies that5N5 viruses have the potential to infect other mamma-an hosts, including humans.
Previous studies have shown that the substitutions274Y in N1 and E119V and R292K in N2 promote
esistance to common neuraminidase inhibitors (Tisdale,000; Ward et al., 2005), and the substitutions L26F, V27A,30T/S, S31N, and G34E in M2 are responsible foresistance to amantadine and rimantadine (Deyde et al.,009; Tosh et al., 2011; Hay et al., 1986; Hill et al., 2009).olecular analysis of NA showed that the three H5N5
iruses do not possess the substitutions that confereuraminidase inhibitor drug resistance, which suggestsat the H5N5 viruses are sensitive to NA inhibitors, as aree clade 2.3.4 viruses. The S31N resistance mutation wasund in M2, suggesting that the three H5N5 viruses may
e resistant to amantadine.The NS1 protein is known to play key roles in the
irulence of several influenza virus subtypes in differentosts (Obenauer et al., 2006; Basler et al., 2001). Theubstitution at position E92 of the NS1 protein has beenhown to increase virulence in pigs (Hatta et al., 2001; Seot al., 2004). The NS1 proteins of all of the H5N5 virusesossess D at position 92, suggesting that the China H5N5iruses may be virulent. Large-scale genome sequencenalysis of AIVs showed that the PDZ-binding motif (PBM)
NS1 consists of four C-terminal residues with theharacteristic X-S/T-X-V motif, which may represent airulence determinant (Obenauer et al., 2006; Jacksont al., 2008). The China H5N5 isolates contain the avian-ke NS1 C-terminal PBM of EPEV, suggesting that EPEVight also be a virulence determinant. Further studies are
eeded to prove this hypothesis. The amino acid substitu-ons at positions K627 and 701N in the PB2 protein haveeen correlated to the increased virulence of H7N7 and5N1 viruses in humans and mice, respectively (Gabrielt al., 2005; Gao et al., 2009; Nguyen et al., 2009); theseubstitutions were not found in the China H5N5 viruses.
In summary, the novel HPAI H5N5 viruses (WY11/19/4 and EC008/EC031) isolated in different parts of Chinaouthern and eastern) at different times are closely
elated to the clade 2.3.4 viruses. The emergence of the5N5 viruses will be monitored as clade 2.2 viruses
ontinue to evolve. Once these viruses become well-
be difficult. Therefore, it is important to conduct surveil-lance of influenza viruses in aquatic birds to control thespread of new viruses.
Competing interests
The authors have declared that they have no competinginterests.
Acknowledgments
This work was supported by the Modern Agro-industryTechnology Research System (CARS-43-10) and theChinese Special Fund for Agro-scientific Research in thePublic Interest (201303046), and the National NaturalScience Foundation of China (31072132).
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