Arch Virol DOI 10.1007/s00705-014-2058-7
ORIGINAL ARTICLE
Experimental infection of dogs with H6N1 avian influenza A virus Kaihui Cheng • Zhijun Yu • Yuwei Gao • Xianzhu Xia • Hongbin He • Yuping Hua Hongliang Chai
•
Received: 28 November 2013 / Accepted: 15 March 2014 Ó Springer-Verlag Wien 2014
Abstract H6N1 avian influenza A viruses, which have spread across North America, Europe and Asia, have been shown to be infectious not only for birds but also for mammals. Because humans lack immunity to H6N1 avian influenza A viruses, the emergence of these viruses in humans would probably cause a pandemic. Replication of H6N1 avian influenza A viruses in dogs may facilitate their adaptation in humans because dogs are often in close contact with humans. However, the susceptibility of dogs to these viruses is unknown. To address this question, we infected beagles intranasally (i.n.) with an H6N1 avian influenza A virus that was isolated from a mallard. Inoculation of this virus into beagles resulted in the virus being
detectable in the lung and seroconversion with no clinical signs except for a fever at 1 day post-inoculation (dpi). In addition, the virus was transiently shed from the nose and in the feces of the infected beagles. Our results suggest that dogs can be subclinically infected with H6N1 avian influenza A viruses, which, like H7N9, have low pathogenicity in birds and may serve as an intermediate host to transfer this virus to humans. Certain actions may be taken to prevent the potential transmission of these viruses, including the development of H6N1 avian influenza vaccines for prevention.
Introduction K. Cheng and Z. Yu contributed equally to this work. K. Cheng H. He (&) Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, China e-mail: [email protected] K. Cheng Z. Yu Y. Gao (&) X. Xia Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute of Academy of Military Medical Sciences, Changchun 130122, China e-mail: [email protected] Z. Yu e-mail: [email protected] Z. Yu X. Xia (&) Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China e-mail: [email protected] Y. Hua H. Chai College of Wildlife Resources, Northeast Forestry University, Harbin 150040, China
H6N1 avian influenza A viruses have frequently been isolated from shorebirds and wild ducks in areas including North America [22] and Europe [11, 20] as well as in Asian areas including southeastern China [4], Taiwan [12], northeastern Japan [10] and Korea [5]. This virus subtype has been shown to be infectious not only for birds but also for mammals such as mice and ferrets [3, 13]. Several avian influenza A viruses, including H6N1, can replicate in the human upper respiratory tract and cause mild clinical symptoms in experimental infection [1]. In addition, a recent study showed significantly elevated antibody titers against H5, H6 and H7 avian influenza A viruses in United States veterinarians who had been exposed to birds, suggesting that human infections with H6 viruses can occur [15]. Moreover, an H6N1 influenza virus named A/teal/ Hong Kong/W312/97(H6N1), which was isolated from a green-winged teal during an H5N1 outbreak in Hong Kong in 1997 [9], possessed seven gene segments that were highly homologous to a human H5N1 virus named A/Hong
123
K. Cheng et al.
Kong/156/97(H5N1), suggesting that this virus might be the progenitor of the A/Hong Kong/156/97 (H5N1) virus [9], which killed humans and poultry in 1997. These facts raise concerns about whether H6N1 avian influenza A viruses have the potential to expand their host range into mammals, including humans. Because humans lack immunity to H6N1 avian influenza A viruses, the emergence of these viruses in humans would probably cause a pandemic. The often close contact between dogs and humans raises questions about the zoonotic potential and the role of dogs in transmission and adaptation of H6N1 avian influenza A viruses to mammals. Influenza virus infection of dogs was first reported in 2004 [13], and dogs experimentally inoculated with some avian influenza viruses such as H3N2 [17–19] and H5N1 [2, 7, 14] have been shown to be susceptible to infection and to shed virus, and this may contribute to the spread of these viruses and to pose a risk to humans. Therefore, dogs may have a role in adaptation of H6N1 avian influenza A viruses to mammals and humans. Several highly pathogenic avian influenza viruses (HPAIVs) and several low-pathogenic avian influenza viruses (LPAIVs) have been shown to be infectious not only for birds but also for humans. Dogs, especially pet dogs, are susceptible to these viruses and may contribute to the spread of these viruses and to pose a risk to humans. The susceptibility of dogs to HPAIVs such as H5N1 has been investigated, but their susceptibility to LPAIVs such as H6N1 is still unknown. Therefore, in this study, we have examined the susceptibility of beagles to a low-pathogenic H6N1 avian influenza A virus isolated from mallard.
Materials and methods Facility All animal studies were conducted in a biosafety level 2 ? laboratory approved by the Institute of Military Veterinary, Changchun, China. Virus An H6N1 avian influenza A virus named A/Mallard/SanJiang/275/2007(SJ/275) was isolated from an anal swab of a mallard in Sanjiang natural reserve of Heilongjiang province, China. The anal swab was vortexed with phosphate-buffered saline (PBS) and centrifuged for 10 min at 8,000 g. Virus from the supernatant was grown in the allantoic cavities of 10-day-old embryonated chicken eggs for 48 h at 37 °C, and allantoic fluids were tested for agglutinating activity using chicken erythrocytes as an indicator of virus replication in the eggs. Stock viruses were grown in the allantoic cavities of 10-day-old
123
embryonated chicken eggs for 48 h at 37 °C, and aliquots were stored at -70 °C until used. The titer of the SJ/275 stock virus was 8.25 log10 EID50/ml, and the 50 % mouse lethal dose (MLD50) was [6.5 log10 EID50. Genetic analysis The GenBank accession numbers of SJ/275 are KJ021045KJ021052. A sequence identity analysis for all eight segments of SJ/275 was conducted using nucleotide BLAST across two avian influenza virus sequence databases (GISAID, NCBI). To investigate molecular and epidemiological characteristics of SJ/275 and to determine the profile of genetic diversity, phylogenetic trees of eight genes were constructed using molecular evolutionary genetics analysis MegAlign version 5.0 [21] by the neighbor-joining (NJ) method to calculate distance. Bootstrap values were estimated for 1000 replicates. Animal studies and virus titrations We performed experimental viral infection in ordinary 16-week-old beagles that had been divided into a virusinoculated group (group I) and a non-virus-inoculated group (group NI). All of the dogs were healthy and showed no serological evidence of prior H6N1 avian influenza A virus infection. The group I beagles (n = 4), named D1, D2, D3 and D4, were inoculated i.n. with 2 ml of a virus preparation with a titer of 108 EID50/ml. The group NI beagles (n = 2), named D5 and D6, were inoculated i.n. with 2 ml of sterile PBS, and they were housed in a separate room and served as negative controls. Before they were inoculated, the dogs were sedated by intramuscular injection of 0.1 mg acepromazine malate per kg. Clinical signs of infection (sneezing, nasal discharge, and coughing) were monitored for 14 days. Rectal temperatures were monitored daily for 10 days, and excretion of virus was monitored daily from feces swabs, oropharynx swabs, and nasal lavage fluid for 7 days. At 3 dpi and 14 dpi, two beagles from group I (D1 and D4 at 3 dpi and D2 and D3 at 14 dpi) were humanely euthanized, and organs, including brain, spleen, kidney, liver, tonsil and different parts of their respiratory system were collected for virus titration in eggs. Briefly, embryonated eggs were inoculated with tenfold serial dilutions of each sample, and 72 hours after inoculation, the virus titer in the chicken egg allantoic cavity was tested for hemagglutinating activity using chicken erythrocytes as an indicator of virus replication in the eggs. Infectious virus titers were calculated from three replicates by the method of Reed and Muench [20]. Serum samples were collected at 14 dpi from D2, D3, D5 and D6.
Experimental infection of dogs
Serological analysis All of the serum samples were tested for antibodies against H6N1 avian influenza virus in hemagglutination-inhibition (HI) tests. HI tests were performed as described previously [23]. Briefly, the antisera were treated with trypsin, heat, and potassium metaperiodate to remove nonspecific inhibitors. Then, the required final dilution of the treated antiserum (1:10) was prepared and adsorbed with packed chicken erythrocytes. Twofold dilutions of the antiserum were prepared in PBS, and 25 ll of the diluted serum was used in each well of a microplate. Twenty-five microliters of virus containing 4 hemagglutination units was added to each well, and the microplate was incubated at room temperature for 30 min, after which 50 ll of 0.5 % chicken erythrocytes was added to each well. The results were read after incubation at room temperature for 60 min. HI antibody titers were expressed as the reciprocal of the highest serum dilution that completely inhibited hemagglutination.
Results Genetic analysis The PB2 gene of SJ/275 and the NP gene of SJ/275 showed that the highest nucleotide sequence similarity to A/duck/ Eastern China/1/2008(H6N1), at 98.5 % and 99.3 %, respectively. The PB1 gene of SJ/275 and the M gene of SJ/275 showed that the highest nucleotide sequence similarity to A/migratory duck/Hong Kong/MP2437/ 2003(H6N1), at 99.0 % and 99.4 %, respectively. The PA gene showed the highest nucleotide sequence similarity to A/chicken/Taiwan/165/99(H6N1), at 95.8 %. The HA gene showed the highest nucleotide sequence similarity to A/duck/Hokkaido/W159/2006(H6N1), at 98.6 %. The NA gene showed the highest nucleotide sequence similarity to A/greater white-fronted goose/California/44358-112/ 2007(H6N1), at 97.9 %. The NS gene showed the highest nucleotide sequence similarity to A/duck/Guizhou/1426/ 2006(H6N1), at 99.2 %. The results of phylogenetic analysis showed that the PB2, PB1, PA, NP, M, and NS genes of SJ/275 were clustered in the East Asia lineage (Fig. 1), whereas the HA and NA genes of SJ/275 were clustered in the North America lineage (Fig. 1). The results of phylogenetic analysis indicated that A/Mallard/SanJiang/275/ 2007 (H6N1) was a reassortant derived from A/duck/ Korea/DY97/2007(H4N3) (PB2 gene), A/duck/Hokkaido/ Vac-3/2007(H5N1) (PB1 gene), A/wild bird/Korea/YS109/ 2007(H4N6) (PA gene), A/northern pintail/Alaska/44203023/2006(H6N1) (HA gene), A/northern shoveler/Hong Kong/MPE2984/2008(H10N9) (NP gene), A/pintail/ Alberta/69/2005(H1N1) (NA gene), A/mandarin duck/
Korea/468/2011(H7N3) (M gene), and A/duck/Fujian/ 12035/2005(H6N6) (NS gene). The nucleotide sequences of the eight genes of SJ/275 were compared to those of recently reported H6 viruses (Tables 1 and 2). In addition, the world’s first case of human infection with H6N1 was reported recently in Taiwan, and the genetic differences between SJ/275 and that virus (A/Taiwan/2/2013(H6N1)) were also examined, revealing that nucleotide sequences of the eight segments of SJ/275 had only 82.8 % to 92.8 % identity to those of A/Taiwan/2/2013(H6N1) (Tables 1 and 2). Clinical findings Clinical signs, including sneezing, nasal discharge and coughing, were not observed in any of the infected beagles in this study within 14 dpi. Interestingly, at 1 dpi, all of the beagles in the infected group (group I) have a fever, with a rectal temperature of [39 °C (Fig. 2). However, at 3 dpi, the rectal temperatures had returned to normal \39 °C (Fig. 2). No clinical signs or fever were observed in the group-NI beagles. These results suggested a lack of clinical signs in the dogs inoculated with H6N1 avian influenza virus, although a transient rise in rectal temperature was observed in the early days after the inoculation. Virus replication Two beagles from group I (D1 and D4) were euthanized at 3 dpi, and their organs, including brain, spleen, kidney, liver, tonsil and different parts of their respiratory system, were collected to investigate virus replication in the beagles inoculated with H6N1 avian influenza virus. Virus was detected in the right upper lung and right middle lung of D1 (Fig. 3), and virus was detected in the left upper lung, left middle lung, and right middle lung of D4 (Fig. 3). However, no virus was detected in any of the other organs that were collected. The other two beagles from group I (D2 and D3) were euthanized at 14 dpi, but no viruses were detected from any of the organs collected except for the tonsil of D3 (viral titer, 100.75 EID50/ml). These results suggested that H6N1 avian influenza virus could replicate efficiently in dogs, but replication was limited to the lower respiratory tract. A previous study [17] indicated that large amounts of SAa2,3-gal are present on the surface of bronchial and bronchiolar epithelial cells of beagles and are rarely found on tracheal epithelial cells, whereas SAa2,6gal is not detected on tracheal, bronchial or bronchiolar epithelial cells. The H6N1 avian influenza A virus used in this study was found in our previous study to bind to both SAa2,3-gal and SAa2,6-gal (data not shown), and this property may thus enable the virus to replicate in the lower respiratory tract of beagles, as we observed in this study.
123
K. Cheng et al. Fig. 1 Phylogenetic trees of SJ/ 275 generated by the neighborjoining method with nucleotide sequences of the the eight genes (PB2, PB1, PA, HA, NP, NA, M, and NS). Bootstrapping with 1000 replicates was performed to determine the reliability of each internal node. Horizontal branch lengths are proportional to genetic distances between strains. The scale bar indicates nucleotide substitutions per site. The black circle indicates SJ/ 275
Virus shedding Nasal lavage fluid, oropharynx swabs and fecal swabs from the beagles of group I were tested for virus shedding (Fig. 4). Virus was detected in the fecal swabs and nasal
123
lavage fluid (Fig. 4A and C). The highest virus titer in nasal lavage fluid was 1.75 log10 EID50/ml at 1 dpi, but it could only be detected for two days. In contrast, virus was detected in fecal swabs until 4 dpi. However, no virus was be detected in oropharynx swabs within 7 days after
Experimental infection of dogs Fig. 1 continued
infection (Fig. 4B). These results indicate that this virus can be shed from dogs in feces and from the nose, and that shedding continues longer in feces than from the nose. Because the titer in nasal lavage fluid was very low, we cannot exclude the possibility that the inoculated virus was deposited in the nasal passages. These findings suggest that H6N1 avian influenza virus can be shed from dogs through the nasal route and the fecal route.
Serology Four beagles seroconverted, as shown using an HI test (Table 3). Two beagles (D2 and D3) that were inoculated with avian influenza virus had an HI titer of 20 (Table 3). However, no HI activity was detected in the serum of beagles in group NI. These results demonstrated that the beagles were infected with H6N1 avian influenza virus.
123
K. Cheng et al. Table 1 Nucleotide sequence identities (%) of the PB2, PB1, PA and HA genes of SJ/275 and recently reported H6N1 viruses PB2
PB1
PA
HA
A/Taiwan/2/2013
91.7
87.8
91.6
88.6
A/chicken/Taiwan/A2837/2013
91.1
87.4
91.6
89.8
A/chicken/Taiwan/TC128/2010
91.9
87.7
91.6
90.1
A/mallard/Nova Scotia/01013/2010
84.8
96.9
89.7
96.1
A/mallard/Ohio/11OS2240/2011
85.0
97.3
89.9
96.2
A/duck/Guangxi/GXd-5/2010
93.6
88.0
92.7
86.9
A/turkey/France/10-040/2010 A/mallard/California/2444/2010
94.6 84.8
88.3 96.7
93.4 90.6
93.0 96.2
Lung(right lower)
Lung(left middle)
Lung(left lower)
Lung(right upper)
Turbinate bone
Lung(right middle)
Trachea
4
log10EID50 /ml
Virus
Lung(left upper)
3
2
1
0
Table 2 Nucleotide sequence identities (%) of the NP, NA, M and NS genes of SJ/275 and recently reported H6N1 viruses Virus
NP
NA
M
NS
A/Taiwan/2/2013
89.5
82.8
91.0
92.8
A/chicken/Taiwan/A2837/2013
89.5
81.6
91.4
93.8
A/chicken/Taiwan/TC128/2010
89.9
82.3
91.9
93.7 93.8
A/mallard/Nova Scotia/01013/2010
89.1
95.5
93.2
A/mallard/Ohio/11OS2240/2011
90.0
95.1
93.3
94.0
A/duck/Guangxi/GXd-5/2010
90.5
83.8
92.9
93.7
A/turkey/France/10-040/2010
91.7
86.0
96.0
96.4
A/mallard/California/2444/2010
89.9
95.5
93.2
71.0
Body Temperature (ºC)
40.5 40.0
Group I Group NI
39.5 39.0 38.5 38.0 37.5 0
2
4
6
8
10
Days Post Inoculation Fig. 2 Rectal temperature in beagles after intranasal inoculation with H6N1 avian influenza A virus. Two beagles from group I (D1 and D4) were monitored from 0 to 3 dpi, and the other two beagles from group I (D2 and D4) were monitored from 0 to 10 dpi. The rectal temperatures of the beagles in group NI were also monitored from 0 to 10 dpi
Discussion Influenza virus infection of dogs was first reported in 2004 [6]. Since then, it has been demonstrated that several avian influenza A viruses, such as H3N2 and H5N1, can infect dogs [2, 6, 7, 14, 17–19]. Transmission of avian influenza
123
D1
D4
Fig. 3 Virus replication in beagles after inoculation with H6N1 avian influenza A virus. Tissues of two beagles which named from group I (D1 and D4) were collected at 3 dpi and homogenized, and virus titers were measured in embryonated eggs by EID50. Results are expressed as log10 EID50/ml. The dashed black lines indicate the lower limit of detection (0.5 log10 EID50/ml)
A viruses to a new mammalian hosts is of great concern, because it potentially allows the virus to cross new species barriers and to acquire pandemic potential. In our study, serological and pathological analysis provided evidence for cross-species infection with H6N1 avian influenza A virus. Our results suggested that dogs may be susceptible to H6N1 avian influenza A virus infection and that they can shed virus from the nose and in feces without showing apparent clinical signs, except a transient rise in rectal temperature. Prior to our study, the pathogenicity of animal H6 viruses was evaluated in mammals. In these studies, both mice and ferrets were susceptible to several avian influenza H6 viruses without prior adaptation [8, 9, 16]. H6N1 and H6N5 influenza viruses killed mice after inoculation and caused weight loss in those that survived inoculation, and these viruses replicated to high titers in the lungs and nasal turbinates. Notably, clinical signs of illness, including ruffled fur and hunching, could be observed as early as 1 dpi in mice inoculated with H6N1 influenza virus [8, 9, 16]. H6N2 and H6N9 influenza viruses could also replicated in the lungs and nasal turbinates of mice after inoculation; however, significant weight loss and illness were not observed [8]. In addition, H6N1, H6N2, H6N5, and H6N9 influenza viruses replicated in the lungs and nasal turbinate of ferrets after inoculation and caused transient weight loss or transient elevated body temperatures; however, the ferrets were not killed by these viruses [8, 16]. These findings demonstrated that mammals can be productively infected with animal H6 viruses.
Experimental infection of dogs
log10EID50/ml
2.0
A
Table 3 Antibody titers in serum of beagles measured in the hemagluttination-inhibition (HI) assay at 14 dpi
1.5
1.0
0.5
0.0 1
2
3
4
5
6
7
Days Post Inoculation 2.0
B
Beagle no.
Seroconversiona
D1
NDb
D2
20
D3
20
D4
ND
D5
\10
D6
\10
a
HI titers are presented as the reciprocal of the highest serum dilution that completely inhibited agglutination of chicken erythrocytes by H6N1 avian influenza A virus
log10 EID50/ml
b
Not done
1.5
contact with infected dogs, possibly promoting virus transmission to humans and leading to a pandemic. Therefore, efforts should be made to prevent dogs from being infected with H6N1 avian influenza A viruses, such as developing a novel H6N1 vaccine for dogs.
1.0
0.5
0.0 1
2
3
4
5
6
7
Days Post Inoculation 2.0
C
Acknowledgments This work was supported by the National Key Technologies R&D Program (No. 2013BAD12B04) and the Special Fund for Agro-Scientific Research in the Public Interest (No. 201303042).
log10EID50/ml
Conflict of interest
The authors declare no conflict of interest.
1.5
References
1.0
0.5
0.0 1d
2d
3d
4d
5d
6d
7d
Days Post Inoculation
Fig. 4 A Detection of virus in nasal lavage fluid from beagles after inoculation with the H6N1 avian influenza virus. B. Detection of virus in oropharynx swabs from beagles after inoculation with H6N1 avian influenza virus. C. Detection of virus in fecal swabs from beagles after inoculation with H6N1 avian influenza virus. Virus titers were measured in embryonated eggs by EID50 assay, and the results are expressed as log10 EID50/ml. The dashed black lines indicate the lower limit of detection (0.5 log10 EID50/ml)
In our study, the replication of H6N1 avian influenza A virus was restricted to the lower respiratory tract of dogs, with transient shedding virus from the nose, suggesting that the transmission capacity of H6N1 avian influenza virus is weak at present. However, the observation that dogs can be subclinically infected with H6N1 avian influenza A virus raised the concern that dogs may be become a new source of transmission of this virus, especially where the virus is circulating or has been detected and people are in close
1. Beare AS, Webster RG (1991) Replication of avian influenza viruses in humans. Arch Virol 119:37–42 2. Chen Y, Zhong G, Wang G, Deng G, Li Y, Shi J, Zhang Z, Guan Y, Jiang Y, Bu Z, Kawaoka Y, Chen H (2010) Dogs are highly susceptible to H5N1 avian influenza virus. Virology 405:15–19 3. Chen Z, Santos C, Aspelund A, Gillim-Ross L, Jin H, Kemble G, Subbarao K (2009) Evaluation of live attenuated influenza a virus h6 vaccines in mice and ferrets. J Virol 83:65–72 4. Chin PS, Hoffmann E, Webby R, Webster RG, Guan Y, Peiris M, Shortridge KF (2002) Molecular evolution of H6 influenza viruses from poultry in Southeastern China: prevalence of H6N1 influenza viruses possessing seven A/Hong Kong/156/97 (H5N1)-like genes in poultry. J Virol 76:507–516 5. Choi YK, Seo SH, Kim JA, Webby RJ, Webster RG (2005) Avian influenza viruses in Korean live poultry markets and their pathogenic potential. Virology 332:529–537 6. Crawford PC, Dubovi EJ, Castleman WL, Stephenson I, Gibbs EP, Chen L, Smith C, Hill RC, Ferro P, Pompey J, Bright RA, Medina MJ, Johnson CM, Olsen CW, Cox NJ, Klimov AI, Katz JM, Donis RO (2005) Transmission of equine influenza virus to dogs. Science 310:482–485 7. Giese M, Harder TC, Teifke JP, Klopfleisch R, Breithaupt A, Mettenleiter TC, Vahlenkamp TW (2008) Experimental infection and natural contact exposure of dogs with avian influenza virus (H5N1). Emerg Infect Dis 14:308–310 8. Gillim-Ross L, Santos C, Chen Z, Aspelund A, Yang CF, Ye D, Jin H, Kemble G, Subbarao K (2008) Avian influenza h6 viruses productively infect and cause illness in mice and ferrets. Journal of virology 82:10854–10863
123
K. Cheng et al. 9. Hoffmann E, Stech J, Leneva I, Krauss S, Scholtissek C, Chin PS, Peiris M, Shortridge KF, Webster RG (2000) Characterization of the influenza A virus gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1? J Virol 74:6309–6315 10. Jahangir A, Watanabe Y, Chinen O, Yamazaki S, Sakai K, Okamura M, Nakamura M, Takehara K (2008) Surveillance of avian influenza viruses in Northern pintails (Anas acuta) in Tohoku District, Japan. Avian Dis 52:49–53 11. Jonassen CM, Handeland K (2007) Avian influenza virus screening in wild waterfowl in Norway, 2005. Avian Dis 51:425–428 12. Lee MS, Chang PC, Shien JH, Cheng MC, Chen CL, Shieh HK (2006) Genetic and pathogenic characterization of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005. Avian Dis 50:561–571 13. Leneva IA, Goloubeva O, Fenton RJ, Tisdale M, Webster RG (2001) Efficacy of zanamivir against avian influenza A viruses that possess genes encoding H5N1 internal proteins and are pathogenic in mammals. Antimicrob Agents Chemother 45:1216–1224 14. Maas R, Tacken M, Ruuls L, Koch G, van Rooij E, StockhofeZurwieden N (2007) Avian influenza (H5N1) susceptibility and receptors in dogs. Emerg Infect Dis 13:1219–1221 15. Myers KP, Setterquist SF, Capuano AW, Gray GC (2007) Infection due to 3 avian influenza subtypes in United States veterinarians. Clin Infect Dis 45:4–9 16. Nam JH, Kim EH, Song D, Choi YK, Kim JK, Poo H (2011) Emergence of mammalian species-infectious and -pathogenic avian influenza H6N5 virus with no evidence of adaptation. J Virol 85:13271–13277
123
17. Song D, Kang B, Lee C, Jung K, Ha G, Kang D, Park S, Park B, Oh J (2008) Transmission of avian influenza virus (H3N2) to dogs. Emerg Infect Dis 14:741–746 18. Song D, Lee C, Kang B, Jung K, Oh T, Kim H, Park B, Oh J (2009) Experimental infection of dogs with avian-origin canine influenza A virus (H3N2). Emerg Infect Dis 15:56–58 19. Song D, Moon H, Jung K, Yeom M, Kim H, Han S, An D, Oh J, Kim J, Park B, Kang B (2011) Association between nasal shedding and fever that influenza A (H3N2) induces in dogs. Virol J 8:1 20. Suss J, Schafer J, Sinnecker H, Webster RG (1994) Influenza virus subtypes in aquatic birds of eastern Germany. Arch Virol 135:101–114 21. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 22. Wahlgren J, Waldenstrom J, Sahlin S, Haemig PD, Fouchier RA, Osterhaus AD, Pinhassi J, Bonnedahl J, Pisareva M, Grudinin M, Kiselev O, Hernandez J, Falk KI, Lundkvist A, Olsen B (2008) Gene segment reassortment between American and Asian lineages of avian influenza virus from waterfowl in the Beringia area. Vector Borne Zoonotic Dis 8:783–790 23. Yamanaka T, Nemoto M, Bannai H, Tsujimura K, Kondo T, Matsumura T, Muranaka M, Ueno T, Kinoshita Y, Niwa H, Hidari KI, Suzuki T (2012) No evidence of horizontal infection in horses kept in close contact with dogs experimentally infected with canine influenza A virus (H3N8). Acta Vet Scand 54:25
ORIGINAL ARTICLE
Experimental infection of dogs with H6N1 avian influenza A virus Kaihui Cheng • Zhijun Yu • Yuwei Gao • Xianzhu Xia • Hongbin He • Yuping Hua Hongliang Chai
•
Received: 28 November 2013 / Accepted: 15 March 2014 Ó Springer-Verlag Wien 2014
Abstract H6N1 avian influenza A viruses, which have spread across North America, Europe and Asia, have been shown to be infectious not only for birds but also for mammals. Because humans lack immunity to H6N1 avian influenza A viruses, the emergence of these viruses in humans would probably cause a pandemic. Replication of H6N1 avian influenza A viruses in dogs may facilitate their adaptation in humans because dogs are often in close contact with humans. However, the susceptibility of dogs to these viruses is unknown. To address this question, we infected beagles intranasally (i.n.) with an H6N1 avian influenza A virus that was isolated from a mallard. Inoculation of this virus into beagles resulted in the virus being
detectable in the lung and seroconversion with no clinical signs except for a fever at 1 day post-inoculation (dpi). In addition, the virus was transiently shed from the nose and in the feces of the infected beagles. Our results suggest that dogs can be subclinically infected with H6N1 avian influenza A viruses, which, like H7N9, have low pathogenicity in birds and may serve as an intermediate host to transfer this virus to humans. Certain actions may be taken to prevent the potential transmission of these viruses, including the development of H6N1 avian influenza vaccines for prevention.
Introduction K. Cheng and Z. Yu contributed equally to this work. K. Cheng H. He (&) Dairy Cattle Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, China e-mail: [email protected] K. Cheng Z. Yu Y. Gao (&) X. Xia Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute of Academy of Military Medical Sciences, Changchun 130122, China e-mail: [email protected] Z. Yu e-mail: [email protected] Z. Yu X. Xia (&) Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China e-mail: [email protected] Y. Hua H. Chai College of Wildlife Resources, Northeast Forestry University, Harbin 150040, China
H6N1 avian influenza A viruses have frequently been isolated from shorebirds and wild ducks in areas including North America [22] and Europe [11, 20] as well as in Asian areas including southeastern China [4], Taiwan [12], northeastern Japan [10] and Korea [5]. This virus subtype has been shown to be infectious not only for birds but also for mammals such as mice and ferrets [3, 13]. Several avian influenza A viruses, including H6N1, can replicate in the human upper respiratory tract and cause mild clinical symptoms in experimental infection [1]. In addition, a recent study showed significantly elevated antibody titers against H5, H6 and H7 avian influenza A viruses in United States veterinarians who had been exposed to birds, suggesting that human infections with H6 viruses can occur [15]. Moreover, an H6N1 influenza virus named A/teal/ Hong Kong/W312/97(H6N1), which was isolated from a green-winged teal during an H5N1 outbreak in Hong Kong in 1997 [9], possessed seven gene segments that were highly homologous to a human H5N1 virus named A/Hong
123
K. Cheng et al.
Kong/156/97(H5N1), suggesting that this virus might be the progenitor of the A/Hong Kong/156/97 (H5N1) virus [9], which killed humans and poultry in 1997. These facts raise concerns about whether H6N1 avian influenza A viruses have the potential to expand their host range into mammals, including humans. Because humans lack immunity to H6N1 avian influenza A viruses, the emergence of these viruses in humans would probably cause a pandemic. The often close contact between dogs and humans raises questions about the zoonotic potential and the role of dogs in transmission and adaptation of H6N1 avian influenza A viruses to mammals. Influenza virus infection of dogs was first reported in 2004 [13], and dogs experimentally inoculated with some avian influenza viruses such as H3N2 [17–19] and H5N1 [2, 7, 14] have been shown to be susceptible to infection and to shed virus, and this may contribute to the spread of these viruses and to pose a risk to humans. Therefore, dogs may have a role in adaptation of H6N1 avian influenza A viruses to mammals and humans. Several highly pathogenic avian influenza viruses (HPAIVs) and several low-pathogenic avian influenza viruses (LPAIVs) have been shown to be infectious not only for birds but also for humans. Dogs, especially pet dogs, are susceptible to these viruses and may contribute to the spread of these viruses and to pose a risk to humans. The susceptibility of dogs to HPAIVs such as H5N1 has been investigated, but their susceptibility to LPAIVs such as H6N1 is still unknown. Therefore, in this study, we have examined the susceptibility of beagles to a low-pathogenic H6N1 avian influenza A virus isolated from mallard.
Materials and methods Facility All animal studies were conducted in a biosafety level 2 ? laboratory approved by the Institute of Military Veterinary, Changchun, China. Virus An H6N1 avian influenza A virus named A/Mallard/SanJiang/275/2007(SJ/275) was isolated from an anal swab of a mallard in Sanjiang natural reserve of Heilongjiang province, China. The anal swab was vortexed with phosphate-buffered saline (PBS) and centrifuged for 10 min at 8,000 g. Virus from the supernatant was grown in the allantoic cavities of 10-day-old embryonated chicken eggs for 48 h at 37 °C, and allantoic fluids were tested for agglutinating activity using chicken erythrocytes as an indicator of virus replication in the eggs. Stock viruses were grown in the allantoic cavities of 10-day-old
123
embryonated chicken eggs for 48 h at 37 °C, and aliquots were stored at -70 °C until used. The titer of the SJ/275 stock virus was 8.25 log10 EID50/ml, and the 50 % mouse lethal dose (MLD50) was [6.5 log10 EID50. Genetic analysis The GenBank accession numbers of SJ/275 are KJ021045KJ021052. A sequence identity analysis for all eight segments of SJ/275 was conducted using nucleotide BLAST across two avian influenza virus sequence databases (GISAID, NCBI). To investigate molecular and epidemiological characteristics of SJ/275 and to determine the profile of genetic diversity, phylogenetic trees of eight genes were constructed using molecular evolutionary genetics analysis MegAlign version 5.0 [21] by the neighbor-joining (NJ) method to calculate distance. Bootstrap values were estimated for 1000 replicates. Animal studies and virus titrations We performed experimental viral infection in ordinary 16-week-old beagles that had been divided into a virusinoculated group (group I) and a non-virus-inoculated group (group NI). All of the dogs were healthy and showed no serological evidence of prior H6N1 avian influenza A virus infection. The group I beagles (n = 4), named D1, D2, D3 and D4, were inoculated i.n. with 2 ml of a virus preparation with a titer of 108 EID50/ml. The group NI beagles (n = 2), named D5 and D6, were inoculated i.n. with 2 ml of sterile PBS, and they were housed in a separate room and served as negative controls. Before they were inoculated, the dogs were sedated by intramuscular injection of 0.1 mg acepromazine malate per kg. Clinical signs of infection (sneezing, nasal discharge, and coughing) were monitored for 14 days. Rectal temperatures were monitored daily for 10 days, and excretion of virus was monitored daily from feces swabs, oropharynx swabs, and nasal lavage fluid for 7 days. At 3 dpi and 14 dpi, two beagles from group I (D1 and D4 at 3 dpi and D2 and D3 at 14 dpi) were humanely euthanized, and organs, including brain, spleen, kidney, liver, tonsil and different parts of their respiratory system were collected for virus titration in eggs. Briefly, embryonated eggs were inoculated with tenfold serial dilutions of each sample, and 72 hours after inoculation, the virus titer in the chicken egg allantoic cavity was tested for hemagglutinating activity using chicken erythrocytes as an indicator of virus replication in the eggs. Infectious virus titers were calculated from three replicates by the method of Reed and Muench [20]. Serum samples were collected at 14 dpi from D2, D3, D5 and D6.
Experimental infection of dogs
Serological analysis All of the serum samples were tested for antibodies against H6N1 avian influenza virus in hemagglutination-inhibition (HI) tests. HI tests were performed as described previously [23]. Briefly, the antisera were treated with trypsin, heat, and potassium metaperiodate to remove nonspecific inhibitors. Then, the required final dilution of the treated antiserum (1:10) was prepared and adsorbed with packed chicken erythrocytes. Twofold dilutions of the antiserum were prepared in PBS, and 25 ll of the diluted serum was used in each well of a microplate. Twenty-five microliters of virus containing 4 hemagglutination units was added to each well, and the microplate was incubated at room temperature for 30 min, after which 50 ll of 0.5 % chicken erythrocytes was added to each well. The results were read after incubation at room temperature for 60 min. HI antibody titers were expressed as the reciprocal of the highest serum dilution that completely inhibited hemagglutination.
Results Genetic analysis The PB2 gene of SJ/275 and the NP gene of SJ/275 showed that the highest nucleotide sequence similarity to A/duck/ Eastern China/1/2008(H6N1), at 98.5 % and 99.3 %, respectively. The PB1 gene of SJ/275 and the M gene of SJ/275 showed that the highest nucleotide sequence similarity to A/migratory duck/Hong Kong/MP2437/ 2003(H6N1), at 99.0 % and 99.4 %, respectively. The PA gene showed the highest nucleotide sequence similarity to A/chicken/Taiwan/165/99(H6N1), at 95.8 %. The HA gene showed the highest nucleotide sequence similarity to A/duck/Hokkaido/W159/2006(H6N1), at 98.6 %. The NA gene showed the highest nucleotide sequence similarity to A/greater white-fronted goose/California/44358-112/ 2007(H6N1), at 97.9 %. The NS gene showed the highest nucleotide sequence similarity to A/duck/Guizhou/1426/ 2006(H6N1), at 99.2 %. The results of phylogenetic analysis showed that the PB2, PB1, PA, NP, M, and NS genes of SJ/275 were clustered in the East Asia lineage (Fig. 1), whereas the HA and NA genes of SJ/275 were clustered in the North America lineage (Fig. 1). The results of phylogenetic analysis indicated that A/Mallard/SanJiang/275/ 2007 (H6N1) was a reassortant derived from A/duck/ Korea/DY97/2007(H4N3) (PB2 gene), A/duck/Hokkaido/ Vac-3/2007(H5N1) (PB1 gene), A/wild bird/Korea/YS109/ 2007(H4N6) (PA gene), A/northern pintail/Alaska/44203023/2006(H6N1) (HA gene), A/northern shoveler/Hong Kong/MPE2984/2008(H10N9) (NP gene), A/pintail/ Alberta/69/2005(H1N1) (NA gene), A/mandarin duck/
Korea/468/2011(H7N3) (M gene), and A/duck/Fujian/ 12035/2005(H6N6) (NS gene). The nucleotide sequences of the eight genes of SJ/275 were compared to those of recently reported H6 viruses (Tables 1 and 2). In addition, the world’s first case of human infection with H6N1 was reported recently in Taiwan, and the genetic differences between SJ/275 and that virus (A/Taiwan/2/2013(H6N1)) were also examined, revealing that nucleotide sequences of the eight segments of SJ/275 had only 82.8 % to 92.8 % identity to those of A/Taiwan/2/2013(H6N1) (Tables 1 and 2). Clinical findings Clinical signs, including sneezing, nasal discharge and coughing, were not observed in any of the infected beagles in this study within 14 dpi. Interestingly, at 1 dpi, all of the beagles in the infected group (group I) have a fever, with a rectal temperature of [39 °C (Fig. 2). However, at 3 dpi, the rectal temperatures had returned to normal \39 °C (Fig. 2). No clinical signs or fever were observed in the group-NI beagles. These results suggested a lack of clinical signs in the dogs inoculated with H6N1 avian influenza virus, although a transient rise in rectal temperature was observed in the early days after the inoculation. Virus replication Two beagles from group I (D1 and D4) were euthanized at 3 dpi, and their organs, including brain, spleen, kidney, liver, tonsil and different parts of their respiratory system, were collected to investigate virus replication in the beagles inoculated with H6N1 avian influenza virus. Virus was detected in the right upper lung and right middle lung of D1 (Fig. 3), and virus was detected in the left upper lung, left middle lung, and right middle lung of D4 (Fig. 3). However, no virus was detected in any of the other organs that were collected. The other two beagles from group I (D2 and D3) were euthanized at 14 dpi, but no viruses were detected from any of the organs collected except for the tonsil of D3 (viral titer, 100.75 EID50/ml). These results suggested that H6N1 avian influenza virus could replicate efficiently in dogs, but replication was limited to the lower respiratory tract. A previous study [17] indicated that large amounts of SAa2,3-gal are present on the surface of bronchial and bronchiolar epithelial cells of beagles and are rarely found on tracheal epithelial cells, whereas SAa2,6gal is not detected on tracheal, bronchial or bronchiolar epithelial cells. The H6N1 avian influenza A virus used in this study was found in our previous study to bind to both SAa2,3-gal and SAa2,6-gal (data not shown), and this property may thus enable the virus to replicate in the lower respiratory tract of beagles, as we observed in this study.
123
K. Cheng et al. Fig. 1 Phylogenetic trees of SJ/ 275 generated by the neighborjoining method with nucleotide sequences of the the eight genes (PB2, PB1, PA, HA, NP, NA, M, and NS). Bootstrapping with 1000 replicates was performed to determine the reliability of each internal node. Horizontal branch lengths are proportional to genetic distances between strains. The scale bar indicates nucleotide substitutions per site. The black circle indicates SJ/ 275
Virus shedding Nasal lavage fluid, oropharynx swabs and fecal swabs from the beagles of group I were tested for virus shedding (Fig. 4). Virus was detected in the fecal swabs and nasal
123
lavage fluid (Fig. 4A and C). The highest virus titer in nasal lavage fluid was 1.75 log10 EID50/ml at 1 dpi, but it could only be detected for two days. In contrast, virus was detected in fecal swabs until 4 dpi. However, no virus was be detected in oropharynx swabs within 7 days after
Experimental infection of dogs Fig. 1 continued
infection (Fig. 4B). These results indicate that this virus can be shed from dogs in feces and from the nose, and that shedding continues longer in feces than from the nose. Because the titer in nasal lavage fluid was very low, we cannot exclude the possibility that the inoculated virus was deposited in the nasal passages. These findings suggest that H6N1 avian influenza virus can be shed from dogs through the nasal route and the fecal route.
Serology Four beagles seroconverted, as shown using an HI test (Table 3). Two beagles (D2 and D3) that were inoculated with avian influenza virus had an HI titer of 20 (Table 3). However, no HI activity was detected in the serum of beagles in group NI. These results demonstrated that the beagles were infected with H6N1 avian influenza virus.
123
K. Cheng et al. Table 1 Nucleotide sequence identities (%) of the PB2, PB1, PA and HA genes of SJ/275 and recently reported H6N1 viruses PB2
PB1
PA
HA
A/Taiwan/2/2013
91.7
87.8
91.6
88.6
A/chicken/Taiwan/A2837/2013
91.1
87.4
91.6
89.8
A/chicken/Taiwan/TC128/2010
91.9
87.7
91.6
90.1
A/mallard/Nova Scotia/01013/2010
84.8
96.9
89.7
96.1
A/mallard/Ohio/11OS2240/2011
85.0
97.3
89.9
96.2
A/duck/Guangxi/GXd-5/2010
93.6
88.0
92.7
86.9
A/turkey/France/10-040/2010 A/mallard/California/2444/2010
94.6 84.8
88.3 96.7
93.4 90.6
93.0 96.2
Lung(right lower)
Lung(left middle)
Lung(left lower)
Lung(right upper)
Turbinate bone
Lung(right middle)
Trachea
4
log10EID50 /ml
Virus
Lung(left upper)
3
2
1
0
Table 2 Nucleotide sequence identities (%) of the NP, NA, M and NS genes of SJ/275 and recently reported H6N1 viruses Virus
NP
NA
M
NS
A/Taiwan/2/2013
89.5
82.8
91.0
92.8
A/chicken/Taiwan/A2837/2013
89.5
81.6
91.4
93.8
A/chicken/Taiwan/TC128/2010
89.9
82.3
91.9
93.7 93.8
A/mallard/Nova Scotia/01013/2010
89.1
95.5
93.2
A/mallard/Ohio/11OS2240/2011
90.0
95.1
93.3
94.0
A/duck/Guangxi/GXd-5/2010
90.5
83.8
92.9
93.7
A/turkey/France/10-040/2010
91.7
86.0
96.0
96.4
A/mallard/California/2444/2010
89.9
95.5
93.2
71.0
Body Temperature (ºC)
40.5 40.0
Group I Group NI
39.5 39.0 38.5 38.0 37.5 0
2
4
6
8
10
Days Post Inoculation Fig. 2 Rectal temperature in beagles after intranasal inoculation with H6N1 avian influenza A virus. Two beagles from group I (D1 and D4) were monitored from 0 to 3 dpi, and the other two beagles from group I (D2 and D4) were monitored from 0 to 10 dpi. The rectal temperatures of the beagles in group NI were also monitored from 0 to 10 dpi
Discussion Influenza virus infection of dogs was first reported in 2004 [6]. Since then, it has been demonstrated that several avian influenza A viruses, such as H3N2 and H5N1, can infect dogs [2, 6, 7, 14, 17–19]. Transmission of avian influenza
123
D1
D4
Fig. 3 Virus replication in beagles after inoculation with H6N1 avian influenza A virus. Tissues of two beagles which named from group I (D1 and D4) were collected at 3 dpi and homogenized, and virus titers were measured in embryonated eggs by EID50. Results are expressed as log10 EID50/ml. The dashed black lines indicate the lower limit of detection (0.5 log10 EID50/ml)
A viruses to a new mammalian hosts is of great concern, because it potentially allows the virus to cross new species barriers and to acquire pandemic potential. In our study, serological and pathological analysis provided evidence for cross-species infection with H6N1 avian influenza A virus. Our results suggested that dogs may be susceptible to H6N1 avian influenza A virus infection and that they can shed virus from the nose and in feces without showing apparent clinical signs, except a transient rise in rectal temperature. Prior to our study, the pathogenicity of animal H6 viruses was evaluated in mammals. In these studies, both mice and ferrets were susceptible to several avian influenza H6 viruses without prior adaptation [8, 9, 16]. H6N1 and H6N5 influenza viruses killed mice after inoculation and caused weight loss in those that survived inoculation, and these viruses replicated to high titers in the lungs and nasal turbinates. Notably, clinical signs of illness, including ruffled fur and hunching, could be observed as early as 1 dpi in mice inoculated with H6N1 influenza virus [8, 9, 16]. H6N2 and H6N9 influenza viruses could also replicated in the lungs and nasal turbinates of mice after inoculation; however, significant weight loss and illness were not observed [8]. In addition, H6N1, H6N2, H6N5, and H6N9 influenza viruses replicated in the lungs and nasal turbinate of ferrets after inoculation and caused transient weight loss or transient elevated body temperatures; however, the ferrets were not killed by these viruses [8, 16]. These findings demonstrated that mammals can be productively infected with animal H6 viruses.
Experimental infection of dogs
log10EID50/ml
2.0
A
Table 3 Antibody titers in serum of beagles measured in the hemagluttination-inhibition (HI) assay at 14 dpi
1.5
1.0
0.5
0.0 1
2
3
4
5
6
7
Days Post Inoculation 2.0
B
Beagle no.
Seroconversiona
D1
NDb
D2
20
D3
20
D4
ND
D5
\10
D6
\10
a
HI titers are presented as the reciprocal of the highest serum dilution that completely inhibited agglutination of chicken erythrocytes by H6N1 avian influenza A virus
log10 EID50/ml
b
Not done
1.5
contact with infected dogs, possibly promoting virus transmission to humans and leading to a pandemic. Therefore, efforts should be made to prevent dogs from being infected with H6N1 avian influenza A viruses, such as developing a novel H6N1 vaccine for dogs.
1.0
0.5
0.0 1
2
3
4
5
6
7
Days Post Inoculation 2.0
C
Acknowledgments This work was supported by the National Key Technologies R&D Program (No. 2013BAD12B04) and the Special Fund for Agro-Scientific Research in the Public Interest (No. 201303042).
log10EID50/ml
Conflict of interest
The authors declare no conflict of interest.
1.5
References
1.0
0.5
0.0 1d
2d
3d
4d
5d
6d
7d
Days Post Inoculation
Fig. 4 A Detection of virus in nasal lavage fluid from beagles after inoculation with the H6N1 avian influenza virus. B. Detection of virus in oropharynx swabs from beagles after inoculation with H6N1 avian influenza virus. C. Detection of virus in fecal swabs from beagles after inoculation with H6N1 avian influenza virus. Virus titers were measured in embryonated eggs by EID50 assay, and the results are expressed as log10 EID50/ml. The dashed black lines indicate the lower limit of detection (0.5 log10 EID50/ml)
In our study, the replication of H6N1 avian influenza A virus was restricted to the lower respiratory tract of dogs, with transient shedding virus from the nose, suggesting that the transmission capacity of H6N1 avian influenza virus is weak at present. However, the observation that dogs can be subclinically infected with H6N1 avian influenza A virus raised the concern that dogs may be become a new source of transmission of this virus, especially where the virus is circulating or has been detected and people are in close
1. Beare AS, Webster RG (1991) Replication of avian influenza viruses in humans. Arch Virol 119:37–42 2. Chen Y, Zhong G, Wang G, Deng G, Li Y, Shi J, Zhang Z, Guan Y, Jiang Y, Bu Z, Kawaoka Y, Chen H (2010) Dogs are highly susceptible to H5N1 avian influenza virus. Virology 405:15–19 3. Chen Z, Santos C, Aspelund A, Gillim-Ross L, Jin H, Kemble G, Subbarao K (2009) Evaluation of live attenuated influenza a virus h6 vaccines in mice and ferrets. J Virol 83:65–72 4. Chin PS, Hoffmann E, Webby R, Webster RG, Guan Y, Peiris M, Shortridge KF (2002) Molecular evolution of H6 influenza viruses from poultry in Southeastern China: prevalence of H6N1 influenza viruses possessing seven A/Hong Kong/156/97 (H5N1)-like genes in poultry. J Virol 76:507–516 5. Choi YK, Seo SH, Kim JA, Webby RJ, Webster RG (2005) Avian influenza viruses in Korean live poultry markets and their pathogenic potential. Virology 332:529–537 6. Crawford PC, Dubovi EJ, Castleman WL, Stephenson I, Gibbs EP, Chen L, Smith C, Hill RC, Ferro P, Pompey J, Bright RA, Medina MJ, Johnson CM, Olsen CW, Cox NJ, Klimov AI, Katz JM, Donis RO (2005) Transmission of equine influenza virus to dogs. Science 310:482–485 7. Giese M, Harder TC, Teifke JP, Klopfleisch R, Breithaupt A, Mettenleiter TC, Vahlenkamp TW (2008) Experimental infection and natural contact exposure of dogs with avian influenza virus (H5N1). Emerg Infect Dis 14:308–310 8. Gillim-Ross L, Santos C, Chen Z, Aspelund A, Yang CF, Ye D, Jin H, Kemble G, Subbarao K (2008) Avian influenza h6 viruses productively infect and cause illness in mice and ferrets. Journal of virology 82:10854–10863
123
K. Cheng et al. 9. Hoffmann E, Stech J, Leneva I, Krauss S, Scholtissek C, Chin PS, Peiris M, Shortridge KF, Webster RG (2000) Characterization of the influenza A virus gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1? J Virol 74:6309–6315 10. Jahangir A, Watanabe Y, Chinen O, Yamazaki S, Sakai K, Okamura M, Nakamura M, Takehara K (2008) Surveillance of avian influenza viruses in Northern pintails (Anas acuta) in Tohoku District, Japan. Avian Dis 52:49–53 11. Jonassen CM, Handeland K (2007) Avian influenza virus screening in wild waterfowl in Norway, 2005. Avian Dis 51:425–428 12. Lee MS, Chang PC, Shien JH, Cheng MC, Chen CL, Shieh HK (2006) Genetic and pathogenic characterization of H6N1 avian influenza viruses isolated in Taiwan between 1972 and 2005. Avian Dis 50:561–571 13. Leneva IA, Goloubeva O, Fenton RJ, Tisdale M, Webster RG (2001) Efficacy of zanamivir against avian influenza A viruses that possess genes encoding H5N1 internal proteins and are pathogenic in mammals. Antimicrob Agents Chemother 45:1216–1224 14. Maas R, Tacken M, Ruuls L, Koch G, van Rooij E, StockhofeZurwieden N (2007) Avian influenza (H5N1) susceptibility and receptors in dogs. Emerg Infect Dis 13:1219–1221 15. Myers KP, Setterquist SF, Capuano AW, Gray GC (2007) Infection due to 3 avian influenza subtypes in United States veterinarians. Clin Infect Dis 45:4–9 16. Nam JH, Kim EH, Song D, Choi YK, Kim JK, Poo H (2011) Emergence of mammalian species-infectious and -pathogenic avian influenza H6N5 virus with no evidence of adaptation. J Virol 85:13271–13277
123
17. Song D, Kang B, Lee C, Jung K, Ha G, Kang D, Park S, Park B, Oh J (2008) Transmission of avian influenza virus (H3N2) to dogs. Emerg Infect Dis 14:741–746 18. Song D, Lee C, Kang B, Jung K, Oh T, Kim H, Park B, Oh J (2009) Experimental infection of dogs with avian-origin canine influenza A virus (H3N2). Emerg Infect Dis 15:56–58 19. Song D, Moon H, Jung K, Yeom M, Kim H, Han S, An D, Oh J, Kim J, Park B, Kang B (2011) Association between nasal shedding and fever that influenza A (H3N2) induces in dogs. Virol J 8:1 20. Suss J, Schafer J, Sinnecker H, Webster RG (1994) Influenza virus subtypes in aquatic birds of eastern Germany. Arch Virol 135:101–114 21. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 22. Wahlgren J, Waldenstrom J, Sahlin S, Haemig PD, Fouchier RA, Osterhaus AD, Pinhassi J, Bonnedahl J, Pisareva M, Grudinin M, Kiselev O, Hernandez J, Falk KI, Lundkvist A, Olsen B (2008) Gene segment reassortment between American and Asian lineages of avian influenza virus from waterfowl in the Beringia area. Vector Borne Zoonotic Dis 8:783–790 23. Yamanaka T, Nemoto M, Bannai H, Tsujimura K, Kondo T, Matsumura T, Muranaka M, Ueno T, Kinoshita Y, Niwa H, Hidari KI, Suzuki T (2012) No evidence of horizontal infection in horses kept in close contact with dogs experimentally infected with canine influenza A virus (H3N8). Acta Vet Scand 54:25
