Virus Genes DOI 10.1007/s11262-013-1014-z
Characterization of an avian influenza virus of subtype H4N6 isolated from ducks in the northern China Chuantian Xu • Mei Lu • Beixia Hu • Shaohua Yang • Qinghua Huang • Lin Zhang Xiumei Zhang
•
Received: 15 July 2013 / Accepted: 12 November 2013 Ó Springer Science+Business Media New York 2013
Abstract In 2010, an H4N6 avian influenza virus (AIV) was isolated and identified from healthy ducks in a waterfowl market in Shandong Province in the northern China. This virus was named A/duck/Shandong/1/2010 (H4N6) (DK/SD/1/2010 hereafter). The gene sequence of the virus was determined, and genetic and evolutionary analyses were conducted by combining related sequences in GenBank. Results indicated that seven genes of DK/SD/ 1/2010 (PB2, PB1, PA, HA, NP, M, and NS) originate from the Eurasian lineage. Another gene, the NA gene, originated from both the Eurasian and the North American lineages. The amino acid sequence near the cleavage site of DK/SD/1/2010 HA (PKKASR;GLF) corresponded to the characteristics of AIV of low pathogenicity. Animal inoculation tests showed that the virus cannot replicate in chickens and mice. Therefore, DK/SD/1/2010 may be a recombinant virus formed by influenza virus genes from different sources through complicated restructuring and evolution in ducks that is avirulent to chickens and mice. Keywords H4N6 avian influenza Evolutionary relationships Replicate Avirulent
C. Xu B. Hu S. Yang Q. Huang L. Zhang X. Zhang (&) Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences, Jinan 250100, People’s Republic of China e-mail: [email protected] M. Lu Weifang College of Education, Qingzhou 262500, People’s Republic of China
Introduction Avian influenza (AI), one of the most important epidemic diseases currently threatening aviculture in China and all over the world, poses significant implications for public health. Wild birds and waterfowl are believed to be the natural hosts and reservoirs of avian influenza viruses (AIVs). These birds usually remain healthy while carrying the viruses. Elucidating the mechanism governing the genetic evolution of AIV is a prerequisite for determining their hereditary variation mechanism and effectively preventing AI outbreaks. AIV can be divided into two types according to differences in pathogenicity: the first type is characterized by high pathogenicity, whereas the second is of low pathogenicity. H4N6, which belongs to the latter category, can be primarily isolated from waterfowl and wild birds in vivo. H4N6 can continuously exists undetected in ducks and geese, and infect chickens. Despite the low pathogenicity of H4N6 (primarily demonstrated in animals), this virus can result in declines in egg production, leading to significant economic losses to poultry farming. In 1999, Karasin et al. reported for the first time that H4N6 subtype AIV can be isolated from Canadian pigs with pneumonia. Similar results were also found in Russia and China [1–4], which indicates that virus subtypes can be transmitted among species. The H4N6 subtypes induce apoptosis in MDCK cells and inhibit the proliferation of such cells. Few domestic studies on influenza A virus subtypes H4N6 have been conducted. In the present work, sequence determination and analysis of the gene sequence of this virus subtype were conducted by monitoring and isolating an H4N6 virus strain from waterfowl. The pathogenicity of the virus was evaluated in specific pathogen-free (SPF) white leghorn chickens and 6-week-old female BABL/c
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Virus Genes
mice. Based on the source, characteristics, and evolutionary mechanism of the strain’s molecules, this study provides a theoretical foundation for further research on the outbreak of influenza subtype H4N6.
The chickens were then observed for 14 days for clinical signs of disease and death. Tracheal and cloacal swabs were collected on days 3, 5, and 7 postinoculation (p.i.) for virus titration in eggs. Sera were collected on day 21 p.i. for seroconversion confirmation.
Materials and methods
Mouse study
Virus isolation
Groups of eight 6-week-old female BALB/c mice (Beijing Experimental Animal Center, Beijing, China) were lightly anesthetized with CO2 and inoculated i.n. with 106 EID50 of virus in a volume of 50 ll. On day 3 p.i., three mice were euthanized and their lungs, spleens, and brains were collected under sterile conditions. The organs were ground and homogenized in 1 ml of cold PBS. Solid debris was pelleted by centrifugation at 4,0009g for 5 min, after which the homogenates were used to determine virus titration in 10-day-old embryonated chicken eggs. Virus titers were noted in units of log10 EID50 per 1 ml ± standard deviation. The five remaining mice were monitored daily for weight loss and mortality.
Genetic and phylogenetic analyses Viral RNA was extracted from allantoic fluid using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and reverse transcribed using a 12-nucleotide primer, 50 -AGCAAAAGCAGG-30 , with M-MLV reverse transcriptase (Life Technologies, Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C. PCR amplification was performed using fragmentspecific primers (primer sequences available upon request). The PCR products were purified with the QIA quick PCR Purification Kit (Qiagen) and sequenced using the CEQ DTCS Quick Start Kit on a CEQ 8000 DNA sequencer (Beckman Coulter, Fullerton, CA, USA). Sequence data were compiled with the SEQMAN program (DNASTAR, Madison, WI, USA). Phylogenetic analysis was carried out using the PHYLIP program of the CLASTALX software package (version 1.81) to implement a neighbor-joining algorithm. Chicken study Six-week-old SPF white leghorn chickens were either intranasally (i.n.) inoculated with 0.1 ml of 106 EID50 of virus or intravenously inoculated with 0.2 ml of a 1:10 dilution of bacteria-free allantoic fluid containing the virus.
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Results Virus identification An H4N6 AI virus (AIV), A/duck/Shandong/1/2010 (DK/ SD/1/2010) was isolated and identified from healthy ducks in a waterfowl market in Shandong Province in the northern China in 2010. DK/SD/1/2010 and reference virus CK/SD/SG1/2009 replicated in MDCK cells, and the mean titer reached the highest peak at 72 h.p.i. (Fig. 1). Genetic and evolutionary analysis Clone and sequence determination of the eight gene segments of DK/SD/1/2010 were performed. The sequences Viral titer (Log10PFU/ml)
Oropharyngeal and cloacal swabs of ducks were collected and kept in phosphate-buffered saline (PBS) containing 1,000 U/ml penicillin and 1,000 U/ml streptomycin. Solid debris was pelleted by centrifugation at 4,0009g for 5 min, and supernatants were inoculated into the allantoic cavities of 10-day-old embryonated chicken eggs. The eggs were incubated for 48–72 h at 37 °C. The isolated virus was identified by hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests with a panel of antisera provided by National Reference Laboratory for AI, Harbin Veterinary Research Institute, CAAS, China. The virus stock was grown in the allantoic cavities of 10-day-old embryonated SPF chicken eggs at 37 °C for 48 h. Afterward, the virus was aliquoted and stored at -70 °C for use in succeeding experiments. The kinetics of DK/SD/1/2010 and reference viruses’ replication were done in MDCK (Madin-Daby Canine Kidney) cells, and animal experiments were conducted in HEPA-filtered isolators.
9 8 7 6 5 4 3 2 1 0
DK/SD/1/2010 CK/SD/SG1/2009
24
36
48
60
72
84
Hours post infection
Fig. 1 MDCK cells in six-well plates were infected in duplicate with 100 PFU of DK/SD/1/2010 and CK/SD/SG1/2009 viruses, respectively. The viral yields (log10 PFU/ml) were measured at 24, 36, 48, 60, 72, and 84 h.p.i. Data are expressed as mean ± SD of viral titers (log10 PFU/mg) of each group of cells from two separate experiments
Virus Genes Table 1 The virus strains with the most homology with DK/ SD/l/2010
Gene
Nucleotide sequence compared
Virus with most closely related gene of interest
Homology (%)
GenBank Accession Number
PB2 PB1
28–2,259
A/wild bird/Korea/A323/2009 H10N1
98
JN817613
36–2,286
A/wild duck/Korea/CSM4-28/2010 H4N6
98.3
JX454696
PA
82–2,156
A/Mallard/Jiangxi/7376/2003 (H6N2)
98.2
HM145324
HA
213–1,708
A/avian/Japan/8kI0185/2008 (H4N6)
98.1
CY079219
NP
46–1,536
A/Spot-billedduck/Korean/546/2008(H6N1)
98.5
GQ414896
NA
28–1,332
A/avian/Japan/8kI0185/2008 (H4N6)
99.1
CY079221
M NS
33–969 16–861
A/pintail/Aomori/1130/2008 (H1N3) A/duck/Guangxi/912/2008 (H4N2)
98.1 98.1
AB546183 CY076896
were deposited in GenBank with accession numbers JN083378 to JN083385. Afterward, BLAST analysis of the eight gene segments was carried out. Table 1 shows the virus strains with the highest nucleotide homology, as well as their homologies. Nucleotide-sequence comparisons revealed that the PB1, HA, NA, NP, PA, PB2, M, and NS genes of this virus are highly homologous with those of the A/wild duck/Korea/CSM4-28/2010 (93.8 % homology), A/avian/Japan/8kI0185/2008 (98.1 % homology), A/avian/ Japan/8kI0185/2008 (99.1 % homology), A/Spot-billedduck/Korean/546/2008 (H6N1) (98.2 % homology), A/Mallard/Jiangxi/7376/2003 (H6N2) (98.5 % homology), A/wild bird/Korea/A323/2009 (H10N1) (98 % homology), A/avian/Japan/8kI0185/2008 (H4N6) (98.1 % homology), and A/duck/Guangxi/912/2008 (H4N2) (98.1 % homology), respectively, at the nucleotide level (Table 1). Based on the data in Table 2, we conclude that the eight gene segments of DK/SD/1/2010 share high homology with virus strains of diverse types, which indicates that the virus strain may be a reassortant of different subtypes of influenza viruses. Analysis of the amino acid sequence of the HA genes shows that PKKASR;GLF, the amino acid sequence near the cleavage site of the DK/SD/1/2010 HA genes, clearly differs from RERRRKKR;GLF, the amino acid sequence near the cleavage site of high-pathogenicity AIV HA. This result demonstrates that DK/SD/1/2010 displays the features of low-pathogenicity AIV. Nucleotide homologies of DK/SD/1/2010 and other domestic strains of H4 virus subtypes are shown in Table 2. DK/SD/1/2010 shares relatively low nucleotide homology with the six strains of H4 virus subtypes reported in domestic studies. In particular, the homology of the HA nucleotide, which is even lower, is between 86.1 and 87.2 % (Table 2). Compared with six domestic strains of H4 influenza subtypes, almost no variation is observed in the features of the amino acids near the binding site of the DK/SD/1/2010 HA gene receptor. Only the 226th and 228th amino acids of the HA genes for swine/Ontario/
Table 2 Comparison between DK/SD/l/2010 and H4 subtype AIV (%) H4 subtype AIV
PB2
PB1
PA
HA
NP
NA
M
NS
A/duck/ Nanchang/4165/2000 (H4N6)
96.1
93.5
91.5
87.2
94
/
97.4
96
A/duck/ PoyangLake/ FB13/2007 (H4N6)
/
/
/
86
/
/
/
/
A/mallard/ Poyang Lake/ 15/2007 (H4N6)
/
/
/
87
/
/
/
/
A/mallard/ Poyang Lake/ P17/2007 (H4N6)
/
/
/
86.8
/
/
/
/
A/mallard/Yan chen/2005 (H4N6)
91.7
93.3
96.5
86.3
94
/
97.5
97.3
A/mallard/ ZhaLong/88/ 2004 (H4N6)
91.3
93.5
90.8
86.8
93.8
/
97.9
97.6
A/duck/ Guangxi/912/ 2008 (H4N2)
94.9
96.2
96.5
86.1
92.7
44
97.3
98.1
/ No related sequence in GenBank
01911-1/99 changed from Q to L, and G to S, respectively (Table 3), which indicates that no significant variation occurs in the amino acids near the receptor binding site of isolated strains of the H4N6 AI subtype except swine/ Ontario/01911-1/99. In terms of evolutionary relationships, seven genes (PB2, PB1, PA, HA, NP, M, and NS) of DK/SD/1/2010 appear to originate from the Eurasian lineage; the NA gene originates from both the Eurasian and the North American
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Virus Genes Table 3 Amino acid in the characteristic sites of the HA gene of DK/SD/l/2010 Virus
Possible receptor-binding sites
H3 numbering system
98
134–137
153
155
183
186
190
194
195
226
228
H4 position
110
146–149
165
167
196
199
203
207
208
239
241
DK/SD/ly/2010 DK/GX/912/2008
Y Y
GKSGA GKSGA
W W
V V
H H
E E
E E
L L
Y Y
Q Q
G G
Mallard/poyang lake/15/2007
Y
GKSGA
W
V
H
E
E
L
Y
Q
G
Mallard/poyang lake/17/2007
Y
GKSGA
W
V
H
E
E
L
Y
Q
G
DK/Nanchang/4-165/2000
Y
GKSGA
W
V
H
E
E
L
Y
Q
G
Mallard/Yan chen/2005
Y
GKSGA
W
V
H
E
E
L
Y
Q
G
Mallard/ZhaLong/88/2004
Y
GKSGA
W
V
H
E
E
L
Y
Q
G
Swine/Ontario/01911-1/99
Y
GKSGA
W
V
H
E
E
L
Y
L
S
lineages (Fig. 2). However, seven genes (PB2, PB1, PA, HA, NP, M, and NS) of swine/Ontario/01911-1/99 probably come from the North American lineages, only NA gene originates from both the Eurasian and the North American lineages (Fig. 2). It is clear that DK/SD/1/2010 and swine/ Ontario/01911-1/99 viruses are different in terms of evolutionary relationships. Chicken study Results showed that the DK/SD/1/2010 virus is not lethal to chickens (Table 4). Oropharyngeal and cloacal swabs showed no viral shedding, although all of the birds that received DK/SD/1/2010 both intravenously and i.n. developed detectable antibodies. By contrast, viral shedding was detected in Oropharyngeal and cloacal swabs of chickens inoculated with reference strain A/Chicken/ Shandong/SG1/2009 (H9N2), and the mean titer reached 5.4 LgEID50 on day 3 p.i (Table 4). Mouse study We evaluated the potential of DK/SD/1/2010 with low pathogenicity to infect a mammalian host, as described previously [5]. After i.n. administration of 106 EID50 of the virus, DK/SD/1/2010-inoculated mice maintained a weight increase until about 14 d p.i., however, A/Chicken/Shandong/SG1/2009-inoculated mice exhibited caused a slight loss in body weight within 6 d after inoculation, and mice weights returned to normal 7 d after inoculation (data not provided) (Fig. 3). No virus was detected in the lungs, spleens, or brains of DK/SD/1/2010-inoculated mice, by contrast, A/Chicken/Shandong/SG1/2009 virus replicated in the lungs of the mice without adaptation, and the mean titer reached 4.1 LgEID50 on day 3 p.i. (Table 5), which indicates that DK/SD/1/2010 cannot infect mice.
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Discussion Unlike high-pathogenicity AIV, low-pathogenicity AIV does not result in large-scale poultry deaths. However, lowpathogenicity AIV can also bring about significant economic losses in poultry farming generally manifested as declines in egg production, respiratory symptoms, and reductions in immunity in poultry, all of which can lead to immune failure. Immune failure, in turn causes infection of various etiologies, finally expressed as mixed or multiple infections. Given the damage caused by the low pathogenicity of some AIV subtypes, enhancing the control of such subtypes is essential in preventing human infection and large-scale epidemic outbreaks. A thorough understanding of the features, sources, transmission, and variations in the mechanism of H4 AIV subtypes with low pathogenicity is of great significance to public health. H4 AIV subtypes have spread to many countries, and related reports have been noted in America, Canada, Korea, Russia, and China, among others [3, 4, 6–10]. BLAST analysis of the eight gene segments (Table 1) indicated that the eight gene segments of DK/SD/1/2010 share high homology with virus strains of diverse types. In terms of evolutionary relationships, seven genes (PB2, PB1, PA, HA, NP, M, and NS) of DK/SD/1/2010 appear to originate from the Eurasian lineage; the NA gene originates from both the Eurasian and the North American lineages (Fig. 2). Thus, DK/SD/1/2010 may be a new virus restructured by AIVs of diverse sources in ducks. Several H4 AIV subtypes from Mainland China are found in the GenBank database, and the primary virus strains are as follows: A/duck/Poyang Lake/FB13/2007 (H4N6), A/mallard/poyangLake/15/2007 (H4N6), A/mallard/poyangLake/p17/2007 (H4N6), A/duck/nanchang/4165/2000 (H4N6), A/mallard/Yanchen/2005 (H4N6), A/mallard/ZhaLong/88/2004 (H4N6), and A/duck/Guangxi/
Virus Genes
A
HA Tree 0.001 0.004
0.011 0.013
0.004
0.004
0.009
0.024 0.015
duck/Nanchang/4-165/2000(H4N6) 0.037 0.025
0.025
0.023 0.009 0.047
0.009
0.014 0.061
0.015 0.054 0.031
C
NA Tree 0.000 -0.001
0.005 0.005
0.005 0.053 0.004
0.208 0.124 0.630
chicken/India/WB-NI/101006/2009(H4N6) avian/Japan/8KI0/85/2008(H4N6) duck /SD/l/2010 H4N6(H4N6) wild duck/Korea/7-D19/2005(H4N6) American black duck/NB/2007(H4N6)
North America lineage
D
duck/SD/1/2010 H4N6
NP Tree
wild duck/Korea/SH5-26/2008 H4N6 wild duck/Korea/CSM4-28/2010 H4N6
0.000 0.019
duck/Hong Kong/365/1978 H4N6
0.019
0.004
0.011
0.006
0.022
0.333 0.467
0.038
duck/Guizhou/888/2006 H6N5
0.036 0.033
0.003
0.033
spot-billed duck/Korea/546/2008 H6N1 wild bird/Korea/A323/2009 H10N1
0.007 mallard/ZhaLong/88/2004 H4N6 0.007 duck/Guangxi/912/2008 H4N2 0.007 duck/SD/1/2010 H4N6 -0.003 0.007 duck/Jiangsu/4/2010 H3N6 0.004 0.006 spot-billed duck/Korea/546/2008 H4N6 0.005 0.006 wild duck/Korea/SH5-26/2008 H4N6 0.005 0.009 0.006 mallard/Yan chen/2005 H4N6 0.011 avian/Japan/8KI0184/2008 H4N6 0.012 duck/Guizhou/888/2006 H6N5 0.028 0.003 0.000 Chicken/Nanchang/3-0128/2000 H4N6 -0.001 0.000 Quail/Nanchang/4-034/2000 H4N6 0.012 0.256 0.020 duck/Hong Kong/365/1978 H4N6 0.021 American black duck/N B/02396/2007 H4N6 0.021 Swine/Ontario/01911-1/99 H4N6 0.027 0.008 migratory duck/Jiang Xi/6568/2004H4N6 0.008 wild duck/Korea/CSM4-28/2010 H3N6 0.296
0.027
0.027
F
0.004
0.002 0.004 0.004 Eurasian
0.011
0.002
spot-billed duck/Korea/546/2008 H6N1 duck/SD/1/2010 H4N6 duck/Hong Kong/365/1978 H4N6 wild duck/Korea/CSM4-28/2010 H4N6 American black duck/N B/02396/2007 H4N6 Swine/Ontario/01911-1/99 H4N6
North America lineage
mallard/Jiangxi/7376/2003 H6N2 mallard/Netherlands/1/1999 H4N6
0.011
lineage 0.012
0.004
0.016
0.017
spot-billed duck/Korea/546/2008 H6N1 0.006 migratory duck/Jiang Xi/6568/2004 H4N6 0.006 mallard/Yan chen/2005 H4N6 0.005
Eurasian
wild duck/Korea/SH5-26/2008 H4N6 0.009 wild duck/Korea/7-D19/2005 H4N6 0.009 wild duck/Korea/CSM4-28/2010 PA H4N6
0.001 0.020 0.045
lineage
Duck/Nanchang/4-165/2000 H4N6 0.043
North America lineage
Swine/Ontario/01911-1/99 H4N6 0.029 duck/Hong Kong/365/1978 H4N6 0.029 American black duck/N B/02396/2007 H4N6 0.014
0.003
H PB2 Tree
0.002 0.003
0.004 duck/SD/1/2010 H4N6 0.004 wild bird/Korea/A323/2009 H10N1 spot-billed duck/Korea/546/2008 H6N1 0.000 DK/Nanchang/4-165/2000 H4N6 0.000 chicken/Nanchang/2-0527/2000 H4N6
0.006 0.009
0.001 0.011
Eurasian
duck/Hong Kong/365/1978 H4N6
0.018
Eurasian
mallard/ZhaLong/88/2004 H4N6
0.019
0.014
North America lineage
0.006
0.009
lineage
lineage
migratory duck/Jiang Xi/6568/2004 H4N6
0.005 duck/SD/1/2010 H4N6 0.005 duck/Guizhou/888/2006 H6N5
0.007
0.000 0.005 wild duck/Korea/CSM4-28/2010 H4N6 0.002 0.009 0.007 duck/SD/1/2010 H4N6 0.006 0.008 wild duck/Korea/SH5-26/2008 H4N6 0.018 0.012 DK/GX/912/2008 H4N2 0.035 duck/Jiangsu/4/2010 H3N6 0.005 0.021 duck/Nanchang/4-165/2000 H4N6 0.005 0.022 mallard/ZhaLong/88/2004 H4N6 0.021 migratory duck/Jiang Xi/6568/2004 H4N6 0.019 0.004 0.020 spot-billed duck/Korea/546/2008 H6N1 0.009 0.015 mallard/Yan chen/2005 H4N6 0.003 0.031 duck/Hong Kong/365/1978 H4N6 0.034 American black duck/N B/02396/2007 H4N6 0.040 Swine/Ontario/01911-1/99 PB1 H4N6 0.031
Eurasian
mallard/Yan chen/2005 H4N6
PA Tree
0.001
G PB1 Tree
quail/Nanchang/4-034/2000 H4N6 duck/Nanchang/d106/1997 H4N6
0.009
0.006
Chicken/Nanchang/2-0527/2000 H4N6 duck/Nanchang/4-165/2000 H4N6
0.018
pintail/Aomori/1130/2008 H1N3 0.038
0.000
0.018
0.007
duck/Nanchang/4-165/2000 H4N6
0.000
0.000
0.002
0.039 mallard/Yan chen/2005 H4N6 0.014 0.025 Swine/Ontario/01911-1/99 H4N6 0.031 wild duck/Korea/7-D19/2005 H4N6 0.031 American black duck/N B/02397/2007 H4N6 -0.006 0.505
E NS Tree
lineage
duck/Hong Kong/365/1978(H4N6)
0.002 mallard/Yan chen/2005(H4N6) 0.003 0.002 -0.001 mallard/ZhaLong/88/2004(H4N6) 0.005 0.003 wild duck/Korea/SH5-26/2008(H4N6) 0.004 0.001 aquatic bird/Korea/w20/2005(H4N6) 0.007 0.002 spot-billed duck/Korea/546/2008(H6N1) 0.003 avian/Japan/8KI0185/2008(H4N6) 0.006 0.003 migratory duck/Jiang Xi/6568/2004(H4N6) Eurasian 0.005 0.009 0.011 Chicken/Nanchang/4-008/2000(H4N6) lineage 0.008 mallard/Korea/KNU YP09/2009(H1N1) 0.006 0.022 Pintail/Aomori/1130/2008(H1N3) 0.007 0.006 duck/SD/1/2010(H4N6) 0.002 0.025 duck/Hong Kong/11/1977(H4N6) 0.025 duck/Guizhou/888/2006(H6N5) 0.002 0.011 American black duck/NB 023/2007(H4N6) North 0.011 Swine/Ontario/ 01911-1/99 (H4N6) 0.038 America lineage
avian/Japan/8KI0185/2008 H4N6 0.039
0.480
Eurasian
budgerigar/Hokkaido/1(H4N6)
0.018 Swine/Ontario/01911-1/99(H4N6)
0.006
0.052 0.092
duck/Taiwan/wb/917/2006(H4N6)
mallard/Yan chen/2005(H4N6)
0.034
0.035
duck/GX/912/2008(H4N2)
mallard/ZhaLong/88/2004(H4N6)
0.015
0.018
B M Tree mallard/PoyangLake/P17/2007(H4N6)
lineage
mallard/Yan chen/2005 H4N6 0.013
wild duck/Korea/CSM4-28/2010 H4N6
0.006
0.016 0.007
North America lineage
0.023
wild duck/Korea/SH5-26/2008 PB2 H4N6 0.004 migratory duck/Jiang Xi/6568 H4N6 0.004 avian/Japan/8KI0184/2008 H4N6 0.002 0.021 Swine/Ontario/01911-1/99 H4N6 0.017 wild duck/Korea/7-D19/2005 H4N6 0.017 American black duck/N B/02396/2007 H4N6 0.004
North America lineage
Fig. 2 Phylogenetic trees for the HA(A), M(B), NA(C), NP(D), NS(E), PA(F), PB1(G), and PB2(H) genes of the H4N6 influenza A viruses were generated with the PHYLIP program of the CLASTALX software package (version 1.81) by using the neighbor-joining algorithm. a Nucleotides 213–1,708 (1,495 bp) of HA, b 33–969
(936 bp) of M, c 28–1,332 (1,304 bp) of NA, d 46–1,536 (1,490 bp) of NP. e 16–861 (845 bp) of NS. f 82–2,156 (2,074 bp) of PA, g 36–2,286 (2,250 bp) of PB1, h 28–2,259 (2,231 bp) of PB2. Horizontal distances are proportional to the minimum number of nucleotide differences required to join nodes
912/2008 (H4N2). The gene sequences of these strains, however, have not been completed. According to the comparison results for the HA nucleotide sequence of the H4 influenza virus subtype, DK/SD/1/2010 does not have a
close relationship with other domestic H4 virus subtypes, and their HA nucleotide homology ranges from 86.1 to 87.2 % (Table 2), which suggests that DK/SD/1/2010 was distinctly different from the other domestic H4 strains,
123
Virus Genes Table 4 Pathotyping and replication of the DK/SD/1/2010 virus in chickens Virus
No. of days p.i.
Mode of virus administration (n)a
Virus isolation from swabsb Oropharyngeal No. of birds shedding virus
DK/SD/1/2010
Intravenous (8)
Intranasal (8)
A/Chicken/ Shandong/SG1/ 2009 (H9N2)d
Intravenous (8)
Intranasal (8)
Cloaca1 Titer (log10 EID50/ml)
No. of birds shedding virus
No. of survivors
No. of seroconverted birdsc
8
8
8
8
8
8
8
8
Titer (log10 EID50/ml)
3
0
0.5
0
0.5
5
0
0.5
0
0.5
7
0
0.5
0
0.5
3
0
0.5
0
0.5
5
0
0.5
0
0.5
7
0
0.5
0
0.5
3
8
5.2 ± 1.2
8
5.4 ± 1.4
5
8
4.8 ± 1.3
8
4.9 ± 1.3
7
8
3.1 ± 1.5
8
3.3 ± 1.5
3
8
4.8 ± 1.1
8
5.1 ± 1.3
5
8
4.9 ± 1.7
8
5.0 ± 1.2
7
8
3.0 ± 1.4
8
3.1 ± 1.5
a
Six-week-old specific-pathogen-free white leghorn chickens were inoculated with 0.2 ml of 1:10 diluted stock virus (9.6 log10 EID50/ml) intravenously or 106 log10 EID50 of the virus in a 0.1 ml volume i.n., and chickens were observed for 2 weeks p.i.
b
Oropharyngeal and cloacal swabs were collected for virus titration on days 3, 5, and 7 p.i.. A numeric value of 0.5 was assigned if virus was not isolated from a swab
c
Chickens were killed and sera were harvested 2 weeks p.i.. Seroconversion was confirmed by agar gel precipitin and HI tests
d
A/Chicken/Shandong/SG1/2009 was a reference strain, which was pathogenic in mice
Percentage of baseline Weight
115 110 105 100 95
CK/SD/SG1/2009 DK/SD/l/2010
90
Contol
85 1
2
3
4
5
6
7
8
9 10 11 12 13 14
Days after inoculation
Fig. 3 Comparison of weight changes in mice infected with A/duck/ Shandong/1/2010 and reference strain A/Chicken/Shandong/SG1/ 2009 (H9N2). Mice (five mice/group) were i.n. infected with 106 EID50 of virus. A/Chicken/Shandong/SG1/2009 was a reference strain, and was pathogenic in mice
therefore, it maybe a recent introduction of an H4 from wild birds into the domestic circuit, but more H4 strains identified were required. Our animal experiment indicated that the DK/SD/l/2010 virus cannot replicate in chickens and mice, which suggests that the virus cannot adapt in chickens and mice (Tables 4, 5; Fig. 3).
123
Influenza currently remains widespread and cannot be controlled effectively, primarily because variations in its proteins easily occur, eventually leading to vaccine failure [11]. Two forms of antigen variations for the influenza virus have been observed: antigenic drift and antigenic shift [12]. H4 AIV subtypes may again infect H5, H9, or other AIV subtypes after infection. NA and interior genes may undergo complicated restructuring, thereby generating new types of restructured viruses. In our study, this phenomenon appears again, DK/SD/1/2010 share high homology with virus strains of diverse types, included H1N3, H4N2, H4N6, H6N1, H6N2, and H10N1(Table 1). However, NA gene of DK/SD/1/2010 originates from both the Eurasian and the North American lineages (Fig. 2). All those above maybe explain that DK/SD/1/2010 displays limited nucleotide homology with other H4 strains. Thus, more isolations of H4N6 influenza virus and determination of its subtypes are necessary. Consistent enhancement of the detection of H4N6 AIV subtypes in ducks and other hosts poses significant implications for public health. Acknowledgments This study was supported by the Modern Agroindustry Technology Research System (CARS-42) and Chinese Special Fund for Agro-scientific Research in the Public Interest (201003012, 201303033). No competing interests exist.
Virus Genes Table 5 Replication and pathogenicity of the DK/SD/1/2010 virus in mice Virus
Virus titers in organs on day 3 p.i. (log10 EID50/ml)a Lung
Heart
No. of seroconverted miceb
MLD50 (log10 EID50)c
Brain
DK/SD/1/2010 (H4N6)
–
–
–
5
[6.5
A/Chicken/Shandong/SG1/2009(H9N2)d
4.1 ± 1.2
–
–
5
6.1
a
6
Groups of five 6-week-old BALB/c mice were infected i.n. with 10 EID50 of each virus in a 50-ll volume. Mice were killed on day 3 p.i., and organs were collected and homogenized. Clarified homogenates were titrated for virus infectivity in eggs at initial dilutions of 1:10 (lung) or 1:2 (other tissues). Homogenates were titrated without prior dilution if negative results were found at the lowest dilution. Virus titers were not measured in undiluted samples; virus was simply detected (?) or not detected (-)
b
Sera were collected on day 14 p.i., and seroconversion was determined by agar gel precipitation and HI tests
c
The mouse lethal dose (MLD50) was determined as described previously [13]
d
A/Chicken/Shandong/SG1/2009 (H9N2) was a reference strain, which was pathogenic in mice
References 1. S.S. Iamnikova, G.V. Kurinov, N.F. Lomakina, V.V. Kurinov, I.T. Fediakina, A.D. Zaberezhnyi, D.K. L’Vov, Infection of pigs with influenza A/H4 and A/H5 viruses isolated from wild birds on the territory of Russia. Vopr. Virusol. 53, 30–34 (2008) 2. A.I. Karasin, S. Carman, I.H. Brown, C.W. Olsen, Isolation and characterization of H4N6 avian influenza viruses from pigs with pneumonia in Canada. J. Virol. 774, 9322–9327 (2000) 3. A. Ninomiya, A. Takada, K. Okazaki, K.F. Shortridge, H. Kida, Seroepidemiological evidence of avian H4, H5, and H9 influenza A virus transmission to pigs in south-eastern China. Vet. Microbiol. 88, 107–114 (2002) 4. D.A. Senne, Avian influenza in the Western Hemisphere including the Pacific Islands and Australia. Avian Dis. 47, 798–805 (2003) 5. H. Chen, G. Deng, Z. Li, G. Tian, Y. Li, P. Jiao, L. Zhang, Z. Liu, R.G. Webster, K. Yu, The evolution of H5N1 influenza viruses in ducks in southern China Proc. Natl. Acad. Sci. USA 101(28), 10452–10457 (2004) 6. M.C. Cheng, M.S. Lee, Y.H. Ho, W.L. Chyi, C.H. Wang, Avian influenza monitoring in migrating birds in Taiwan during 1998-2007. Avian Dis. 54, 109–114 (2010) 7. B.A. Hanson, D.E. Stallknecht, D.E. Swayne, L.A. Lewis, D.A. Senne, Avian influenza viruses in Minnesota ducks during 1998–2000. Avian Dis. 47, 867–871 (2003)
8. G. Kayali, E.J. Ortiz, M.L. Chorazy, G.C. Gray, Evidence of previous avian influenza infection among US turkey workers. Zoonoses Public Health 2010(57), 265–272 (2010). doi:10.1111/ j.1863-2378.2009.01231.x 9. H.J. Lee, J.S. Kwon, D.H. Lee, Y.N. Lee, H.N. Youn, Y.J. Lee, M.C. Kim, O.M. Jeong, H.M. Kang, J.H. Kwon, J.B. Lee, S.Y. Park, I.S. Choi, C.S. Song, Continuing evolution and interspecies transmission of influenza viruses in live bird markets in Korea. Avian Dis. 54, 738–748 (2010) 10. J. Pasick, Y. Berhane, H. Kehler, T. Hisanaga, K. Handel, J. Robinson, D. Ojkic, F. Kibenge, M. Fortin, R. King, A. Hamel, D. Spiro, J. Parmley, C. Soos, E. Jenkins, A. Breault, D. Caswell, C. Davies, J. Rodrigue, K. McAloney, Survey of influenza A viruses circulating in wild birds in Canada 2005–2007. Avian Dis. 54(1 Suppl), 440–445 (2010) 11. R.G. Webster, W.J. Bean, O.T. Gorman, T.M. Chambers, Y. Kawaoka, Evolution and ecology of influenza A viruses. Microbiol. Rev. 56, 152–179 (1992) 12. J.S. Robertson, Sequence analysis of the haemagglutinin of A/Taiwan/1/86, a new variant of human influenza A (H1N1) virus. J. Gen. Virol. 68(Pt 4), 1205–1208 (1987) 13. F.X. Bosch, W. Garten, H.D. Klenk, R. Rott, Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of avian influenza viruses. Virology 113(2), 725–735 (1981)
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Characterization of an avian influenza virus of subtype H4N6 isolated from ducks in the northern China Chuantian Xu • Mei Lu • Beixia Hu • Shaohua Yang • Qinghua Huang • Lin Zhang Xiumei Zhang
•
Received: 15 July 2013 / Accepted: 12 November 2013 Ó Springer Science+Business Media New York 2013
Abstract In 2010, an H4N6 avian influenza virus (AIV) was isolated and identified from healthy ducks in a waterfowl market in Shandong Province in the northern China. This virus was named A/duck/Shandong/1/2010 (H4N6) (DK/SD/1/2010 hereafter). The gene sequence of the virus was determined, and genetic and evolutionary analyses were conducted by combining related sequences in GenBank. Results indicated that seven genes of DK/SD/ 1/2010 (PB2, PB1, PA, HA, NP, M, and NS) originate from the Eurasian lineage. Another gene, the NA gene, originated from both the Eurasian and the North American lineages. The amino acid sequence near the cleavage site of DK/SD/1/2010 HA (PKKASR;GLF) corresponded to the characteristics of AIV of low pathogenicity. Animal inoculation tests showed that the virus cannot replicate in chickens and mice. Therefore, DK/SD/1/2010 may be a recombinant virus formed by influenza virus genes from different sources through complicated restructuring and evolution in ducks that is avirulent to chickens and mice. Keywords H4N6 avian influenza Evolutionary relationships Replicate Avirulent
C. Xu B. Hu S. Yang Q. Huang L. Zhang X. Zhang (&) Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences, Jinan 250100, People’s Republic of China e-mail: [email protected] M. Lu Weifang College of Education, Qingzhou 262500, People’s Republic of China
Introduction Avian influenza (AI), one of the most important epidemic diseases currently threatening aviculture in China and all over the world, poses significant implications for public health. Wild birds and waterfowl are believed to be the natural hosts and reservoirs of avian influenza viruses (AIVs). These birds usually remain healthy while carrying the viruses. Elucidating the mechanism governing the genetic evolution of AIV is a prerequisite for determining their hereditary variation mechanism and effectively preventing AI outbreaks. AIV can be divided into two types according to differences in pathogenicity: the first type is characterized by high pathogenicity, whereas the second is of low pathogenicity. H4N6, which belongs to the latter category, can be primarily isolated from waterfowl and wild birds in vivo. H4N6 can continuously exists undetected in ducks and geese, and infect chickens. Despite the low pathogenicity of H4N6 (primarily demonstrated in animals), this virus can result in declines in egg production, leading to significant economic losses to poultry farming. In 1999, Karasin et al. reported for the first time that H4N6 subtype AIV can be isolated from Canadian pigs with pneumonia. Similar results were also found in Russia and China [1–4], which indicates that virus subtypes can be transmitted among species. The H4N6 subtypes induce apoptosis in MDCK cells and inhibit the proliferation of such cells. Few domestic studies on influenza A virus subtypes H4N6 have been conducted. In the present work, sequence determination and analysis of the gene sequence of this virus subtype were conducted by monitoring and isolating an H4N6 virus strain from waterfowl. The pathogenicity of the virus was evaluated in specific pathogen-free (SPF) white leghorn chickens and 6-week-old female BABL/c
123
Virus Genes
mice. Based on the source, characteristics, and evolutionary mechanism of the strain’s molecules, this study provides a theoretical foundation for further research on the outbreak of influenza subtype H4N6.
The chickens were then observed for 14 days for clinical signs of disease and death. Tracheal and cloacal swabs were collected on days 3, 5, and 7 postinoculation (p.i.) for virus titration in eggs. Sera were collected on day 21 p.i. for seroconversion confirmation.
Materials and methods
Mouse study
Virus isolation
Groups of eight 6-week-old female BALB/c mice (Beijing Experimental Animal Center, Beijing, China) were lightly anesthetized with CO2 and inoculated i.n. with 106 EID50 of virus in a volume of 50 ll. On day 3 p.i., three mice were euthanized and their lungs, spleens, and brains were collected under sterile conditions. The organs were ground and homogenized in 1 ml of cold PBS. Solid debris was pelleted by centrifugation at 4,0009g for 5 min, after which the homogenates were used to determine virus titration in 10-day-old embryonated chicken eggs. Virus titers were noted in units of log10 EID50 per 1 ml ± standard deviation. The five remaining mice were monitored daily for weight loss and mortality.
Genetic and phylogenetic analyses Viral RNA was extracted from allantoic fluid using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and reverse transcribed using a 12-nucleotide primer, 50 -AGCAAAAGCAGG-30 , with M-MLV reverse transcriptase (Life Technologies, Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C. PCR amplification was performed using fragmentspecific primers (primer sequences available upon request). The PCR products were purified with the QIA quick PCR Purification Kit (Qiagen) and sequenced using the CEQ DTCS Quick Start Kit on a CEQ 8000 DNA sequencer (Beckman Coulter, Fullerton, CA, USA). Sequence data were compiled with the SEQMAN program (DNASTAR, Madison, WI, USA). Phylogenetic analysis was carried out using the PHYLIP program of the CLASTALX software package (version 1.81) to implement a neighbor-joining algorithm. Chicken study Six-week-old SPF white leghorn chickens were either intranasally (i.n.) inoculated with 0.1 ml of 106 EID50 of virus or intravenously inoculated with 0.2 ml of a 1:10 dilution of bacteria-free allantoic fluid containing the virus.
123
Results Virus identification An H4N6 AI virus (AIV), A/duck/Shandong/1/2010 (DK/ SD/1/2010) was isolated and identified from healthy ducks in a waterfowl market in Shandong Province in the northern China in 2010. DK/SD/1/2010 and reference virus CK/SD/SG1/2009 replicated in MDCK cells, and the mean titer reached the highest peak at 72 h.p.i. (Fig. 1). Genetic and evolutionary analysis Clone and sequence determination of the eight gene segments of DK/SD/1/2010 were performed. The sequences Viral titer (Log10PFU/ml)
Oropharyngeal and cloacal swabs of ducks were collected and kept in phosphate-buffered saline (PBS) containing 1,000 U/ml penicillin and 1,000 U/ml streptomycin. Solid debris was pelleted by centrifugation at 4,0009g for 5 min, and supernatants were inoculated into the allantoic cavities of 10-day-old embryonated chicken eggs. The eggs were incubated for 48–72 h at 37 °C. The isolated virus was identified by hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests with a panel of antisera provided by National Reference Laboratory for AI, Harbin Veterinary Research Institute, CAAS, China. The virus stock was grown in the allantoic cavities of 10-day-old embryonated SPF chicken eggs at 37 °C for 48 h. Afterward, the virus was aliquoted and stored at -70 °C for use in succeeding experiments. The kinetics of DK/SD/1/2010 and reference viruses’ replication were done in MDCK (Madin-Daby Canine Kidney) cells, and animal experiments were conducted in HEPA-filtered isolators.
9 8 7 6 5 4 3 2 1 0
DK/SD/1/2010 CK/SD/SG1/2009
24
36
48
60
72
84
Hours post infection
Fig. 1 MDCK cells in six-well plates were infected in duplicate with 100 PFU of DK/SD/1/2010 and CK/SD/SG1/2009 viruses, respectively. The viral yields (log10 PFU/ml) were measured at 24, 36, 48, 60, 72, and 84 h.p.i. Data are expressed as mean ± SD of viral titers (log10 PFU/mg) of each group of cells from two separate experiments
Virus Genes Table 1 The virus strains with the most homology with DK/ SD/l/2010
Gene
Nucleotide sequence compared
Virus with most closely related gene of interest
Homology (%)
GenBank Accession Number
PB2 PB1
28–2,259
A/wild bird/Korea/A323/2009 H10N1
98
JN817613
36–2,286
A/wild duck/Korea/CSM4-28/2010 H4N6
98.3
JX454696
PA
82–2,156
A/Mallard/Jiangxi/7376/2003 (H6N2)
98.2
HM145324
HA
213–1,708
A/avian/Japan/8kI0185/2008 (H4N6)
98.1
CY079219
NP
46–1,536
A/Spot-billedduck/Korean/546/2008(H6N1)
98.5
GQ414896
NA
28–1,332
A/avian/Japan/8kI0185/2008 (H4N6)
99.1
CY079221
M NS
33–969 16–861
A/pintail/Aomori/1130/2008 (H1N3) A/duck/Guangxi/912/2008 (H4N2)
98.1 98.1
AB546183 CY076896
were deposited in GenBank with accession numbers JN083378 to JN083385. Afterward, BLAST analysis of the eight gene segments was carried out. Table 1 shows the virus strains with the highest nucleotide homology, as well as their homologies. Nucleotide-sequence comparisons revealed that the PB1, HA, NA, NP, PA, PB2, M, and NS genes of this virus are highly homologous with those of the A/wild duck/Korea/CSM4-28/2010 (93.8 % homology), A/avian/Japan/8kI0185/2008 (98.1 % homology), A/avian/ Japan/8kI0185/2008 (99.1 % homology), A/Spot-billedduck/Korean/546/2008 (H6N1) (98.2 % homology), A/Mallard/Jiangxi/7376/2003 (H6N2) (98.5 % homology), A/wild bird/Korea/A323/2009 (H10N1) (98 % homology), A/avian/Japan/8kI0185/2008 (H4N6) (98.1 % homology), and A/duck/Guangxi/912/2008 (H4N2) (98.1 % homology), respectively, at the nucleotide level (Table 1). Based on the data in Table 2, we conclude that the eight gene segments of DK/SD/1/2010 share high homology with virus strains of diverse types, which indicates that the virus strain may be a reassortant of different subtypes of influenza viruses. Analysis of the amino acid sequence of the HA genes shows that PKKASR;GLF, the amino acid sequence near the cleavage site of the DK/SD/1/2010 HA genes, clearly differs from RERRRKKR;GLF, the amino acid sequence near the cleavage site of high-pathogenicity AIV HA. This result demonstrates that DK/SD/1/2010 displays the features of low-pathogenicity AIV. Nucleotide homologies of DK/SD/1/2010 and other domestic strains of H4 virus subtypes are shown in Table 2. DK/SD/1/2010 shares relatively low nucleotide homology with the six strains of H4 virus subtypes reported in domestic studies. In particular, the homology of the HA nucleotide, which is even lower, is between 86.1 and 87.2 % (Table 2). Compared with six domestic strains of H4 influenza subtypes, almost no variation is observed in the features of the amino acids near the binding site of the DK/SD/1/2010 HA gene receptor. Only the 226th and 228th amino acids of the HA genes for swine/Ontario/
Table 2 Comparison between DK/SD/l/2010 and H4 subtype AIV (%) H4 subtype AIV
PB2
PB1
PA
HA
NP
NA
M
NS
A/duck/ Nanchang/4165/2000 (H4N6)
96.1
93.5
91.5
87.2
94
/
97.4
96
A/duck/ PoyangLake/ FB13/2007 (H4N6)
/
/
/
86
/
/
/
/
A/mallard/ Poyang Lake/ 15/2007 (H4N6)
/
/
/
87
/
/
/
/
A/mallard/ Poyang Lake/ P17/2007 (H4N6)
/
/
/
86.8
/
/
/
/
A/mallard/Yan chen/2005 (H4N6)
91.7
93.3
96.5
86.3
94
/
97.5
97.3
A/mallard/ ZhaLong/88/ 2004 (H4N6)
91.3
93.5
90.8
86.8
93.8
/
97.9
97.6
A/duck/ Guangxi/912/ 2008 (H4N2)
94.9
96.2
96.5
86.1
92.7
44
97.3
98.1
/ No related sequence in GenBank
01911-1/99 changed from Q to L, and G to S, respectively (Table 3), which indicates that no significant variation occurs in the amino acids near the receptor binding site of isolated strains of the H4N6 AI subtype except swine/ Ontario/01911-1/99. In terms of evolutionary relationships, seven genes (PB2, PB1, PA, HA, NP, M, and NS) of DK/SD/1/2010 appear to originate from the Eurasian lineage; the NA gene originates from both the Eurasian and the North American
123
Virus Genes Table 3 Amino acid in the characteristic sites of the HA gene of DK/SD/l/2010 Virus
Possible receptor-binding sites
H3 numbering system
98
134–137
153
155
183
186
190
194
195
226
228
H4 position
110
146–149
165
167
196
199
203
207
208
239
241
DK/SD/ly/2010 DK/GX/912/2008
Y Y
GKSGA GKSGA
W W
V V
H H
E E
E E
L L
Y Y
Q Q
G G
Mallard/poyang lake/15/2007
Y
GKSGA
W
V
H
E
E
L
Y
Q
G
Mallard/poyang lake/17/2007
Y
GKSGA
W
V
H
E
E
L
Y
Q
G
DK/Nanchang/4-165/2000
Y
GKSGA
W
V
H
E
E
L
Y
Q
G
Mallard/Yan chen/2005
Y
GKSGA
W
V
H
E
E
L
Y
Q
G
Mallard/ZhaLong/88/2004
Y
GKSGA
W
V
H
E
E
L
Y
Q
G
Swine/Ontario/01911-1/99
Y
GKSGA
W
V
H
E
E
L
Y
L
S
lineages (Fig. 2). However, seven genes (PB2, PB1, PA, HA, NP, M, and NS) of swine/Ontario/01911-1/99 probably come from the North American lineages, only NA gene originates from both the Eurasian and the North American lineages (Fig. 2). It is clear that DK/SD/1/2010 and swine/ Ontario/01911-1/99 viruses are different in terms of evolutionary relationships. Chicken study Results showed that the DK/SD/1/2010 virus is not lethal to chickens (Table 4). Oropharyngeal and cloacal swabs showed no viral shedding, although all of the birds that received DK/SD/1/2010 both intravenously and i.n. developed detectable antibodies. By contrast, viral shedding was detected in Oropharyngeal and cloacal swabs of chickens inoculated with reference strain A/Chicken/ Shandong/SG1/2009 (H9N2), and the mean titer reached 5.4 LgEID50 on day 3 p.i (Table 4). Mouse study We evaluated the potential of DK/SD/1/2010 with low pathogenicity to infect a mammalian host, as described previously [5]. After i.n. administration of 106 EID50 of the virus, DK/SD/1/2010-inoculated mice maintained a weight increase until about 14 d p.i., however, A/Chicken/Shandong/SG1/2009-inoculated mice exhibited caused a slight loss in body weight within 6 d after inoculation, and mice weights returned to normal 7 d after inoculation (data not provided) (Fig. 3). No virus was detected in the lungs, spleens, or brains of DK/SD/1/2010-inoculated mice, by contrast, A/Chicken/Shandong/SG1/2009 virus replicated in the lungs of the mice without adaptation, and the mean titer reached 4.1 LgEID50 on day 3 p.i. (Table 5), which indicates that DK/SD/1/2010 cannot infect mice.
123
Discussion Unlike high-pathogenicity AIV, low-pathogenicity AIV does not result in large-scale poultry deaths. However, lowpathogenicity AIV can also bring about significant economic losses in poultry farming generally manifested as declines in egg production, respiratory symptoms, and reductions in immunity in poultry, all of which can lead to immune failure. Immune failure, in turn causes infection of various etiologies, finally expressed as mixed or multiple infections. Given the damage caused by the low pathogenicity of some AIV subtypes, enhancing the control of such subtypes is essential in preventing human infection and large-scale epidemic outbreaks. A thorough understanding of the features, sources, transmission, and variations in the mechanism of H4 AIV subtypes with low pathogenicity is of great significance to public health. H4 AIV subtypes have spread to many countries, and related reports have been noted in America, Canada, Korea, Russia, and China, among others [3, 4, 6–10]. BLAST analysis of the eight gene segments (Table 1) indicated that the eight gene segments of DK/SD/1/2010 share high homology with virus strains of diverse types. In terms of evolutionary relationships, seven genes (PB2, PB1, PA, HA, NP, M, and NS) of DK/SD/1/2010 appear to originate from the Eurasian lineage; the NA gene originates from both the Eurasian and the North American lineages (Fig. 2). Thus, DK/SD/1/2010 may be a new virus restructured by AIVs of diverse sources in ducks. Several H4 AIV subtypes from Mainland China are found in the GenBank database, and the primary virus strains are as follows: A/duck/Poyang Lake/FB13/2007 (H4N6), A/mallard/poyangLake/15/2007 (H4N6), A/mallard/poyangLake/p17/2007 (H4N6), A/duck/nanchang/4165/2000 (H4N6), A/mallard/Yanchen/2005 (H4N6), A/mallard/ZhaLong/88/2004 (H4N6), and A/duck/Guangxi/
Virus Genes
A
HA Tree 0.001 0.004
0.011 0.013
0.004
0.004
0.009
0.024 0.015
duck/Nanchang/4-165/2000(H4N6) 0.037 0.025
0.025
0.023 0.009 0.047
0.009
0.014 0.061
0.015 0.054 0.031
C
NA Tree 0.000 -0.001
0.005 0.005
0.005 0.053 0.004
0.208 0.124 0.630
chicken/India/WB-NI/101006/2009(H4N6) avian/Japan/8KI0/85/2008(H4N6) duck /SD/l/2010 H4N6(H4N6) wild duck/Korea/7-D19/2005(H4N6) American black duck/NB/2007(H4N6)
North America lineage
D
duck/SD/1/2010 H4N6
NP Tree
wild duck/Korea/SH5-26/2008 H4N6 wild duck/Korea/CSM4-28/2010 H4N6
0.000 0.019
duck/Hong Kong/365/1978 H4N6
0.019
0.004
0.011
0.006
0.022
0.333 0.467
0.038
duck/Guizhou/888/2006 H6N5
0.036 0.033
0.003
0.033
spot-billed duck/Korea/546/2008 H6N1 wild bird/Korea/A323/2009 H10N1
0.007 mallard/ZhaLong/88/2004 H4N6 0.007 duck/Guangxi/912/2008 H4N2 0.007 duck/SD/1/2010 H4N6 -0.003 0.007 duck/Jiangsu/4/2010 H3N6 0.004 0.006 spot-billed duck/Korea/546/2008 H4N6 0.005 0.006 wild duck/Korea/SH5-26/2008 H4N6 0.005 0.009 0.006 mallard/Yan chen/2005 H4N6 0.011 avian/Japan/8KI0184/2008 H4N6 0.012 duck/Guizhou/888/2006 H6N5 0.028 0.003 0.000 Chicken/Nanchang/3-0128/2000 H4N6 -0.001 0.000 Quail/Nanchang/4-034/2000 H4N6 0.012 0.256 0.020 duck/Hong Kong/365/1978 H4N6 0.021 American black duck/N B/02396/2007 H4N6 0.021 Swine/Ontario/01911-1/99 H4N6 0.027 0.008 migratory duck/Jiang Xi/6568/2004H4N6 0.008 wild duck/Korea/CSM4-28/2010 H3N6 0.296
0.027
0.027
F
0.004
0.002 0.004 0.004 Eurasian
0.011
0.002
spot-billed duck/Korea/546/2008 H6N1 duck/SD/1/2010 H4N6 duck/Hong Kong/365/1978 H4N6 wild duck/Korea/CSM4-28/2010 H4N6 American black duck/N B/02396/2007 H4N6 Swine/Ontario/01911-1/99 H4N6
North America lineage
mallard/Jiangxi/7376/2003 H6N2 mallard/Netherlands/1/1999 H4N6
0.011
lineage 0.012
0.004
0.016
0.017
spot-billed duck/Korea/546/2008 H6N1 0.006 migratory duck/Jiang Xi/6568/2004 H4N6 0.006 mallard/Yan chen/2005 H4N6 0.005
Eurasian
wild duck/Korea/SH5-26/2008 H4N6 0.009 wild duck/Korea/7-D19/2005 H4N6 0.009 wild duck/Korea/CSM4-28/2010 PA H4N6
0.001 0.020 0.045
lineage
Duck/Nanchang/4-165/2000 H4N6 0.043
North America lineage
Swine/Ontario/01911-1/99 H4N6 0.029 duck/Hong Kong/365/1978 H4N6 0.029 American black duck/N B/02396/2007 H4N6 0.014
0.003
H PB2 Tree
0.002 0.003
0.004 duck/SD/1/2010 H4N6 0.004 wild bird/Korea/A323/2009 H10N1 spot-billed duck/Korea/546/2008 H6N1 0.000 DK/Nanchang/4-165/2000 H4N6 0.000 chicken/Nanchang/2-0527/2000 H4N6
0.006 0.009
0.001 0.011
Eurasian
duck/Hong Kong/365/1978 H4N6
0.018
Eurasian
mallard/ZhaLong/88/2004 H4N6
0.019
0.014
North America lineage
0.006
0.009
lineage
lineage
migratory duck/Jiang Xi/6568/2004 H4N6
0.005 duck/SD/1/2010 H4N6 0.005 duck/Guizhou/888/2006 H6N5
0.007
0.000 0.005 wild duck/Korea/CSM4-28/2010 H4N6 0.002 0.009 0.007 duck/SD/1/2010 H4N6 0.006 0.008 wild duck/Korea/SH5-26/2008 H4N6 0.018 0.012 DK/GX/912/2008 H4N2 0.035 duck/Jiangsu/4/2010 H3N6 0.005 0.021 duck/Nanchang/4-165/2000 H4N6 0.005 0.022 mallard/ZhaLong/88/2004 H4N6 0.021 migratory duck/Jiang Xi/6568/2004 H4N6 0.019 0.004 0.020 spot-billed duck/Korea/546/2008 H6N1 0.009 0.015 mallard/Yan chen/2005 H4N6 0.003 0.031 duck/Hong Kong/365/1978 H4N6 0.034 American black duck/N B/02396/2007 H4N6 0.040 Swine/Ontario/01911-1/99 PB1 H4N6 0.031
Eurasian
mallard/Yan chen/2005 H4N6
PA Tree
0.001
G PB1 Tree
quail/Nanchang/4-034/2000 H4N6 duck/Nanchang/d106/1997 H4N6
0.009
0.006
Chicken/Nanchang/2-0527/2000 H4N6 duck/Nanchang/4-165/2000 H4N6
0.018
pintail/Aomori/1130/2008 H1N3 0.038
0.000
0.018
0.007
duck/Nanchang/4-165/2000 H4N6
0.000
0.000
0.002
0.039 mallard/Yan chen/2005 H4N6 0.014 0.025 Swine/Ontario/01911-1/99 H4N6 0.031 wild duck/Korea/7-D19/2005 H4N6 0.031 American black duck/N B/02397/2007 H4N6 -0.006 0.505
E NS Tree
lineage
duck/Hong Kong/365/1978(H4N6)
0.002 mallard/Yan chen/2005(H4N6) 0.003 0.002 -0.001 mallard/ZhaLong/88/2004(H4N6) 0.005 0.003 wild duck/Korea/SH5-26/2008(H4N6) 0.004 0.001 aquatic bird/Korea/w20/2005(H4N6) 0.007 0.002 spot-billed duck/Korea/546/2008(H6N1) 0.003 avian/Japan/8KI0185/2008(H4N6) 0.006 0.003 migratory duck/Jiang Xi/6568/2004(H4N6) Eurasian 0.005 0.009 0.011 Chicken/Nanchang/4-008/2000(H4N6) lineage 0.008 mallard/Korea/KNU YP09/2009(H1N1) 0.006 0.022 Pintail/Aomori/1130/2008(H1N3) 0.007 0.006 duck/SD/1/2010(H4N6) 0.002 0.025 duck/Hong Kong/11/1977(H4N6) 0.025 duck/Guizhou/888/2006(H6N5) 0.002 0.011 American black duck/NB 023/2007(H4N6) North 0.011 Swine/Ontario/ 01911-1/99 (H4N6) 0.038 America lineage
avian/Japan/8KI0185/2008 H4N6 0.039
0.480
Eurasian
budgerigar/Hokkaido/1(H4N6)
0.018 Swine/Ontario/01911-1/99(H4N6)
0.006
0.052 0.092
duck/Taiwan/wb/917/2006(H4N6)
mallard/Yan chen/2005(H4N6)
0.034
0.035
duck/GX/912/2008(H4N2)
mallard/ZhaLong/88/2004(H4N6)
0.015
0.018
B M Tree mallard/PoyangLake/P17/2007(H4N6)
lineage
mallard/Yan chen/2005 H4N6 0.013
wild duck/Korea/CSM4-28/2010 H4N6
0.006
0.016 0.007
North America lineage
0.023
wild duck/Korea/SH5-26/2008 PB2 H4N6 0.004 migratory duck/Jiang Xi/6568 H4N6 0.004 avian/Japan/8KI0184/2008 H4N6 0.002 0.021 Swine/Ontario/01911-1/99 H4N6 0.017 wild duck/Korea/7-D19/2005 H4N6 0.017 American black duck/N B/02396/2007 H4N6 0.004
North America lineage
Fig. 2 Phylogenetic trees for the HA(A), M(B), NA(C), NP(D), NS(E), PA(F), PB1(G), and PB2(H) genes of the H4N6 influenza A viruses were generated with the PHYLIP program of the CLASTALX software package (version 1.81) by using the neighbor-joining algorithm. a Nucleotides 213–1,708 (1,495 bp) of HA, b 33–969
(936 bp) of M, c 28–1,332 (1,304 bp) of NA, d 46–1,536 (1,490 bp) of NP. e 16–861 (845 bp) of NS. f 82–2,156 (2,074 bp) of PA, g 36–2,286 (2,250 bp) of PB1, h 28–2,259 (2,231 bp) of PB2. Horizontal distances are proportional to the minimum number of nucleotide differences required to join nodes
912/2008 (H4N2). The gene sequences of these strains, however, have not been completed. According to the comparison results for the HA nucleotide sequence of the H4 influenza virus subtype, DK/SD/1/2010 does not have a
close relationship with other domestic H4 virus subtypes, and their HA nucleotide homology ranges from 86.1 to 87.2 % (Table 2), which suggests that DK/SD/1/2010 was distinctly different from the other domestic H4 strains,
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Virus Genes Table 4 Pathotyping and replication of the DK/SD/1/2010 virus in chickens Virus
No. of days p.i.
Mode of virus administration (n)a
Virus isolation from swabsb Oropharyngeal No. of birds shedding virus
DK/SD/1/2010
Intravenous (8)
Intranasal (8)
A/Chicken/ Shandong/SG1/ 2009 (H9N2)d
Intravenous (8)
Intranasal (8)
Cloaca1 Titer (log10 EID50/ml)
No. of birds shedding virus
No. of survivors
No. of seroconverted birdsc
8
8
8
8
8
8
8
8
Titer (log10 EID50/ml)
3
0
0.5
0
0.5
5
0
0.5
0
0.5
7
0
0.5
0
0.5
3
0
0.5
0
0.5
5
0
0.5
0
0.5
7
0
0.5
0
0.5
3
8
5.2 ± 1.2
8
5.4 ± 1.4
5
8
4.8 ± 1.3
8
4.9 ± 1.3
7
8
3.1 ± 1.5
8
3.3 ± 1.5
3
8
4.8 ± 1.1
8
5.1 ± 1.3
5
8
4.9 ± 1.7
8
5.0 ± 1.2
7
8
3.0 ± 1.4
8
3.1 ± 1.5
a
Six-week-old specific-pathogen-free white leghorn chickens were inoculated with 0.2 ml of 1:10 diluted stock virus (9.6 log10 EID50/ml) intravenously or 106 log10 EID50 of the virus in a 0.1 ml volume i.n., and chickens were observed for 2 weeks p.i.
b
Oropharyngeal and cloacal swabs were collected for virus titration on days 3, 5, and 7 p.i.. A numeric value of 0.5 was assigned if virus was not isolated from a swab
c
Chickens were killed and sera were harvested 2 weeks p.i.. Seroconversion was confirmed by agar gel precipitin and HI tests
d
A/Chicken/Shandong/SG1/2009 was a reference strain, which was pathogenic in mice
Percentage of baseline Weight
115 110 105 100 95
CK/SD/SG1/2009 DK/SD/l/2010
90
Contol
85 1
2
3
4
5
6
7
8
9 10 11 12 13 14
Days after inoculation
Fig. 3 Comparison of weight changes in mice infected with A/duck/ Shandong/1/2010 and reference strain A/Chicken/Shandong/SG1/ 2009 (H9N2). Mice (five mice/group) were i.n. infected with 106 EID50 of virus. A/Chicken/Shandong/SG1/2009 was a reference strain, and was pathogenic in mice
therefore, it maybe a recent introduction of an H4 from wild birds into the domestic circuit, but more H4 strains identified were required. Our animal experiment indicated that the DK/SD/l/2010 virus cannot replicate in chickens and mice, which suggests that the virus cannot adapt in chickens and mice (Tables 4, 5; Fig. 3).
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Influenza currently remains widespread and cannot be controlled effectively, primarily because variations in its proteins easily occur, eventually leading to vaccine failure [11]. Two forms of antigen variations for the influenza virus have been observed: antigenic drift and antigenic shift [12]. H4 AIV subtypes may again infect H5, H9, or other AIV subtypes after infection. NA and interior genes may undergo complicated restructuring, thereby generating new types of restructured viruses. In our study, this phenomenon appears again, DK/SD/1/2010 share high homology with virus strains of diverse types, included H1N3, H4N2, H4N6, H6N1, H6N2, and H10N1(Table 1). However, NA gene of DK/SD/1/2010 originates from both the Eurasian and the North American lineages (Fig. 2). All those above maybe explain that DK/SD/1/2010 displays limited nucleotide homology with other H4 strains. Thus, more isolations of H4N6 influenza virus and determination of its subtypes are necessary. Consistent enhancement of the detection of H4N6 AIV subtypes in ducks and other hosts poses significant implications for public health. Acknowledgments This study was supported by the Modern Agroindustry Technology Research System (CARS-42) and Chinese Special Fund for Agro-scientific Research in the Public Interest (201003012, 201303033). No competing interests exist.
Virus Genes Table 5 Replication and pathogenicity of the DK/SD/1/2010 virus in mice Virus
Virus titers in organs on day 3 p.i. (log10 EID50/ml)a Lung
Heart
No. of seroconverted miceb
MLD50 (log10 EID50)c
Brain
DK/SD/1/2010 (H4N6)
–
–
–
5
[6.5
A/Chicken/Shandong/SG1/2009(H9N2)d
4.1 ± 1.2
–
–
5
6.1
a
6
Groups of five 6-week-old BALB/c mice were infected i.n. with 10 EID50 of each virus in a 50-ll volume. Mice were killed on day 3 p.i., and organs were collected and homogenized. Clarified homogenates were titrated for virus infectivity in eggs at initial dilutions of 1:10 (lung) or 1:2 (other tissues). Homogenates were titrated without prior dilution if negative results were found at the lowest dilution. Virus titers were not measured in undiluted samples; virus was simply detected (?) or not detected (-)
b
Sera were collected on day 14 p.i., and seroconversion was determined by agar gel precipitation and HI tests
c
The mouse lethal dose (MLD50) was determined as described previously [13]
d
A/Chicken/Shandong/SG1/2009 (H9N2) was a reference strain, which was pathogenic in mice
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