Veterinary Microbiology 168 (2014) 50–59

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Pathogenicity and transmission of H5N1 avian influenza viruses in different birds Runyu Yuan a,b,c,d, Jin Cui a,b,c,d, Shuo Zhang a,b,c,d, Lan Cao a,b,c,d, Xiaoke Liu a,b,c,d, Yinfeng Kang a,b,c,d, Yafen Song a,b,c,d, Lang Gong a,b,c,d, Peirong Jiao a,b,c,d,**, Ming Liao a,b,c,d,* a

National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, China Key Laboratory of Animal Vaccine Development, Ministry of Agriculture, China Key Laboratory of Zoonosis Prevention and Control of Guangdong, China d College of Veterinary Medicine, South China Agricultural University, 483 Wushan Road, Guangzhou 510642, China b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 July 2013 Received in revised form 12 October 2013 Accepted 17 October 2013

In this study, we selected three H5N1 highly pathogenic avian influenza viruses (HPAIVs), A/Goose/Guangdong/1/1996 (clades 0), A/Duck/Guangdong/E35/2012 (clade 2.3.2.1) and A/Chicken/Henan/B30/2012 (clade 7.2) isolated from different birds in China, to investigate the pathogenicity and transmission of the viruses in terrestrial birds and waterfowl. To observe the replication and shedding of the H5N1 HPAIVs in birds, the chickens were inoculated intranasally with 106 EID50 of GSGD/1/96, 103 EID50 of DkE35 and CkB30, and the ducks and geese were inoculated intranasally with 106 EID50 of each virus. Meanwhile, the naive contact groups were set up to detect the transmission of the viruses in tested birds. Our results showed that DkE35 was highly pathogenic to chickens and geese, but not fatal to ducks. It could be detected from all the tested organs, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. GSGD/1/ 96 could infect chickens, ducks and geese, but only caused death in chickens. It could transmit to the chickens and ducks, but was not transmittable to geese. CkB30 was highly pathogenic to chickens, low pathogenic to ducks and not pathogenic to geese. It could be transmitted to the naive contact chickens, but not to the ducks or geese. Our findings suggested that H5N1 HPAIVs from different birds show different host ranges and tissue tropisms. Therefore, we should enhance serological and virological surveillance of H5N1 HPAIVs, and pay more attention to the pathogenic and antigenic evolution of these viruses. ß 2013 Elsevier B.V. All rights reserved.

Keywords: H5N1 Avian influenza virus Pathogenicity Transmission

1. Introduction Avian influenza viruses (AIVs) are RNA viruses belonging to the Orthomyxoviridae family and containing eight

* Corresponding author at: College of Veterinary Medicine, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou 510642, China. Tel.: +86 020 85280240; fax: +86 020 85285282. ** Corresponding author at: College of Veterinary Medicine, South China Agricultural University, 483 Wushan Road, Tianhe District, Guangzhou 510642, China. Tel.: +86 020 85283309; fax: +86 020 85280234. E-mail addresses: [email protected] (P. Jiao), [email protected] (M. Liao). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.10.013

gene segments of single-stranded, negative-sense RNA (Webster et al., 1992). Based on the antigenicity of hemagglutinin (HA) and neuraminidase (NA), avian influenza virus (AIV) can be divided into H1–H16 and N1–N9, respectively (Fouchier and Munster, 2009; Kaleta et al., 2005; Olsen et al., 2006; Webster et al., 1992). AIVs have different pathogenesis for the infections and distributions of lesions depending on the virus strain, especially as it relates to pathotypes. Low pathogenic avian influenza virus (LPAIV) can cause respiratory or gastrointestinal infection in birds. Highly pathogenic avian influenza virus (HPAIV) can cause multi-organ systemic infection in birds (Swayne and Halvorson, 2003).

R. Yuan et al. / Veterinary Microbiology 168 (2014) 50–59

H5N1 HPAIVs can be perpetuated in birds or mammals, such as aquatic birds, wild birds, domestic poultry, swine, tigers, leopards, cats, canines, mice, ferrets and humans. Aquatic birds are generally considered as the natural reservoir for AIVs, but some viruses are highly pathogenic when transmitted to domestic poultry. Moreover, aquatic birds can transmit H5N1 HPAIVs to domestic poultry or various mammalian species (Webster et al., 1992). In 1996, the H5N1 HPAIV (A/Goose/Guangdong/1/96) was isolated from sick geese in Guangdong, China (Chen et al., 1999). In 1997, the HA gene from GSGD/96-like H5N1 AIV was transmitted from birds to humans in Hong Kong, causing six deaths of 18 infected persons (Claas et al., 1998; Subbarao et al., 1998). Since the end of 2003, H5N1 viruses have begun to spread and have caused serious outbreaks in more than 60 countries including China, not only resulting in the destruction of hundreds of millions of poultry, but also causing serious infection and deaths of humans (WHO, 2013a). Moreover, the H5N1 influenza viruses have much antigenic variation, and some of them have the ability to infect mammals. As of August 2013, the cumulative number of confirmed human cases for avian influenza A (H5N1) reported to WHO are 637, including 378 deaths (WHO, 2013a). Therefore, H5N1 AIVs are zoonotic agents recognized as a continuing threat to both the poultry industry and human public health. According to antigenic characteristics, H5N1 viruses were divided into 10 clades (0–9) and several subclades (WHO/OIE/FAO H5N1 Evolution Working Group, 2008). Regular transmission of H5N1 HPAIVs between aquatic birds and domestic poultry has resulted in promoted genetic diversity of all clades known to circulate in domestic poultry in China (Duan et al., 2008; Vijaykrishna et al., 2008). Especially, viruses of clades 2.3.2, 2.3.4 and 7 predominantly cocirculated continuously in domestic birds and/or waterfowl in China since 2007 (Jiang et al., 2010; Li et al., 2010; Smith et al., 2009). Recently, several studies have suggested that clade 2.3.2 viruses were increasingly pathogenic to waterfowl (Sakoda et al., 2010). Meanwhile, H5N1 HPAIVs of clade 7 have been circulating in chickens in North China since 2005. In 2008, H5N1 HPAIV of clade 7.2 broke out in North China, such as Ningxia and Jiangsu, and caused a great deal of deaths in chickens. Interestingly, most H5N1 HPAIVs of clade 7 were isolated from chickens, which were high pathogenicity, but only a few viruses were isolated from waterfowl (WHO/ OIE, 2012). However, the movement and interaction of influenza virus between terrestrial birds and waterfowl have not been fully explored. To better understand the pathogenicity and transmission of the H5N1 HPAIVs in terrestrial birds and waterfowl, we selected three viruses isolated from the different birds. 2. Materials and methods 2.1. Viruses The two H5N1 HPAIVs A/Duck/Guangdong/E35/2012 (H5N1) (DkE35) and A/Chicken/Henan/B30/2012(H5N1) (CkB30) were isolated from cloacal swabs of apparently

51

healthy birds in live bird markets during 2012. A/Goose/ Guangdong/1/1996 (H5N1) (GsGD1/96) was conserved in our laboratory. All viruses were propagated in 10-day-old embryonated specific-pathogen-free (SPF) chicken eggs. Evaluation of 50% egg infective doses (EID50) was calculated by the Reed–Muench method (Thakur and Fezio, 1981). All experiments were carried out in animal biosafety level 3 (ABSL-3) facilities. 2.2. Sequence analysis The HA genes of the viruses used in this study were sequenced. Viral RNA was extracted from allantoic fluid by using Trizol LS Reagent (Life Technologies, Inc.) and transcribed into cDNA with SuperScript III reverse transcriptase (Invitrogen). PCR amplification was performed using fragment-specific primers that matched the conserved end sequence of the RNA fragments of the influenza virus. The PCR products were purified with the QIAquick PCR purification kit (QIAGEN) and sequenced using an automatic ABI Prism 3730 genetic analyzer (Applied Biosystems) according to the manufacturer’s instructions. DNA sequences were compiled and edited using Lasergene 7.1 (DNASTAR). A phylogenetic tree of the H5N1 influenza A viruses was generated by the distancebased neighbor-joining method using software MEGA 4 (Sinauer Associates, Inc., Sunderland, MA). The reliability of the tree was assessed by bootstrap analysis with 1000 replicates. Horizontal distances are proportional to genetic distance. The nucleotide sequences obtained in the present study are available from GenBank under the accession numbers (pending). 2.3. Animal experiments 2.3.1. Chicken study 6-week-old specific-pathogen-free White Leghorn chickens were housed in isolator cages and divided into 3 groups. Six chickens of the GsGD1/96 group were inoculated intranasally with 106 EID50 of GsGD1/96 viruses in a 0.2 ml volume. Six chickens of the DkE35 group were inoculated intranasally with 103 EID50 of DkE35 viruses in a 0.2 ml volume. Six chickens of the CkB30 group were inoculated intranasally with 103 EID50 of CkB30 viruses in a 0.2 ml volume. Three chickens were inoculated intranasally with 0.2 ml phosphate buffered saline (PBS) as a naive contact group housed with those inoculated with the GsGD1/96, DkE35 or CkB30 viruses, respectively. All chickens were observed for clinical symptoms for 14 days. On 3-day post-inoculated (DPI), three inoculated chickens in each group were euthanized to test the virus replication in organs, including lung, kidney, brain, heart, spleen and liver. Similar actions were performed on chickens that had died. Oropharyngeal and cloacal swabs were taken from chickens at 3, 5, 7, 9 and 11 DPI, and suspended in 1 ml PBS. All of the tissues and swabs were collected and titrated for virus infectivity in eggs. Seroconversion of the surviving birds on 14 DPI was confirmed by hemagglutinin inhibition (HI) test.

R. Yuan et al. / Veterinary Microbiology 168 (2014) 50–59

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2.3.2. Duck study Three groups of 3-week-old healthy ducks (Beijing ducks) purchased from a duck farm in Guangzhou were confirmed serologically negative for avian influenza by agar gel precipitation tests and HI assays. Six of nine ducks were inoculated intranasally with 106 EID50 of each virus in a 0.2 ml volume. The other three ducks were inoculated intranasally with 0.2 ml PBS as a naive contact group housed with those inoculated with the GsGD1/96, DkE35 or CkB30 viruses, respectively. Three inoculated ducks in each group were euthanized to test the virus replication in organs on 3 DPI, including lung, kidney, brain, heart, spleen, liver, colon, trachea and bursa of fabricius, and the six remaining birds were observed for 2 weeks. Swabs from the oropharynx and cloaca were collected for detection of viruses shedding at 3, 5, 7, 9 and 11 DPI. All of the tissues and swabs were collected and titrated for virus infectivity in eggs. Seroconversion of the surviving birds on 14 DPI was confirmed by HI test. 2.3.3. Goose study Three groups of 3-week-old healthy geese were purchased from a goose farm in Guangzhou and confirmed serologically negative for avian influenza by agar gel precipitation tests and HI assays. Six geese of each group were inoculated intranasally with 106 EID50 of each virus in a 0.2 ml volume. Three geese were inoculated with 0.2 ml PBS as a naive contact group housed with those inoculated with the GsGD1/96, DkE35 or CkB30 viruses, respectively. On 3 DPI, three inoculated geese in each group were euthanized to test the virus replication in organs, including lung, kidney, brain, heart, spleen, liver, colon, trachea and bursa of fabricius. Oropharyngeal and cloacal swab specimens were collected at 3, 5, 7, 9 and 11 DPI. All of the tissues and swabs were collected and titrated for virus infectivity in eggs. Seroconversion of the surviving birds on 14 DPI was confirmed by HI test. 3. Results 3.1. Phylogenetic analysis of the three H5N1 viruses To determine the molecular characteristic of the three viruses, the HA gene of each virus was sequenced. The

sequences were compared with representative H5N1 sequences obtained from GenBank. According to antigenic characteristics by the WHO, the HA gene GsGD1/96 belonged to clade 0. The HA gene of DkE35 belonged to clade 2.3.2.1 and shared 97.1% nucleotide similarity to A/Duck/Lao/471/2010 virus (Fig. 1). The HA gene of CkB30 viruses belonged to clade 7.2 and shared 95.9% nucleotide similarity to CK/HB/A-8/ 2009 virus (Fig. 1). Comparing with GsGD1/96 virus nucleotide, DkE35 and CkB30 nucleotide similarity were 91.3% and 91.0%. The DkE35 and CkB30 virus had a series of basic amino acids at the cleavage site of the HA (RRRKR#G-). The GsGD1/96 virus had a series of basic amino acids at the cleavage site of the HA (-RRRKKR#G-). The cleavage site of these three viruses was characteristic of highly pathogenic avian influenza viruses (Belser et al., 2009; Nobusawa et al., 1991). The receptor-binding pocket of HA1 of all the three viruses retained the amino acid residues Q226 and G228 (H3 numbering), which preferentially bind to the avian influenza virus receptor (Ha et al., 2001). The amino acid sequences of the three viruses revealed five potential N-linked glycosylation sites in HA1 (26 or 27, 39, 181 and 302) and two in HA2 (499 and 558). In addition, the GsGD1/96 virus had an extra potential Nlinked glycosylation site in HA1 (209), the DkE35 virus had an extra potential N-linked glycosylation site in HA1 (156), and the CkB30 virus had five extra potential N-linked glycosylation sites in HA1 (88, 142, 155, 169 and 251). 3.2. Pathogenicity and transmission of H5N1 HPAI viruses in chickens In order to investigate the pathogenicity of these three viruses in chickens, we inoculated intranasally with 106 EID50 of GsGD1/96 viruses, 103 EID50 of DkE35 viruses, 103 EID50 of CkB30 viruses in a volume of 200 ml in SPF chickens, respectively (Table 1). Then all the chickens were observed for 14 days after infection. All the chickens of the DkE35 group died within 5 DPI. All the chickens inoculated with CkB30 died within 8 DPI. Mortality in the group of GsGD1/96 chickens was 66.7%, and the rest of the inoculated chickens seroconverted (Table 1). The DkE35, GsGD1/96 and CkB30 viruses replicated systemically in chickens, which could be detected from all the tested organs on 3 DPI, including

Table 1 Replication and lethality in chickens of the H5N1 viruses after inoculated intranasally.a Strains

DkE35 GD1/96 CkB30

Titer (log10EID50) 7.00 8.63 6.52

Virus replication on 3 DPI (log10EID50/0.1ml)b in

Manifestations of chickens No. D/S/total 3/3/3 2/3/3 3/3/3

c

No. S.C./total – 1/3 –

d

Heart

Liver

Spleen

Lung

Kidney

Brain

6.67  0.88 2.00  0.35 4.33  0.52

7.13  0.38 3.63  0.18 4.83  0.63

7.13  0.38 2.63  1.60 4.67  0.14

8.33  0.14 5.75 5.50

6.83  2.32 6.00  0.35 5.67  0.52

8.17  0.80 4.00  0.71 4.91  0.29

a Six-week-old SPF chickens were inoculated intranasally (i.n.) with 106 EID50 of GsGD1/96 virus, 103 EID50 of DkE35 virus, 103 EID50 of CkB30 virus in a volume of 200 ml, respectively; three chickens in each group were euthanized on 3 DPI, and virus titer was determined in samples of heart, liver, spleen, lung, kidney and brain in eggs. b For statistical analysis, a value of 1.5 was assigned if the virus was not detected from the undiluted sample in three embryonated hen eggs (Sun et al., 2011). Virus titers are expressed as means  standard deviation in log10EID50/0.1 ml of tissue. c No. D/S/total shows the number of dead (D) and sick (S) as well as the total number of chickens from the observation period. The birds that showed disease signs, such as depression and ruffled feathers, but recovered at the end of the observation were counted as sick animals. d No. S.C./total shows the number of chickens that seroconverted out of the total number of chickens at the end of the observation period. –, all of the chickens died at the end of the observation.

R. Yuan et al. / Veterinary Microbiology 168 (2014) 50–59 A/duck/Lao/471/2010

96 78

A/tundra swan/Mongolia/1T/2010

99

Clade 2.3.2.1

A/Duck/Guangdong/E35/2012

68

A/Hubei/1/2010 A/chicken/Primorje/1/2008

100

Clade 2.3.2

grey heron/Hong Kong/3088/2007 83

A/Guangxi/1/2009

96

magpie robin/Hong Kong/1097/2008

71

Clade 2.3

magpie robin/Hong Kong/1897/2008

84

59

53

A/Chicken/Shantou/810/2005 A/duck/Hunan/127/2005 90

Clade 2.3.1

A/duck/Hunan/139/2005

100

100 A/chicken/Guiyang/3055/2005

Clade 2.3.3

A/duck/Guiyang/3242/2005 A/Anhui/1/2005

61

A/chicken/Malaysia/5223/2007

100

100 A/Chicken/Yunnan/447/2005 80

Clade 2.3.4

Clade 2.4

A/Chicken/Yunnan/493/2005

100 A/Indonesia/CDC594/2006

A/Indonesia/CDC595/2006

69

A/Indonesia/5/2005 99

Clade 2.1

A/Indonesia/7/2005

100

A/chicken/Indonesia/7/2003 A/chicken/Indonesia/11/2003

81 94

57

A/chicken/Guangdong/174/2004

94

Clade 2.5

A/chicken/Korea/ES/2003

100 A/chicken/Egypt/088S-NLQP/2008 93

A/chicken/Egypt/9403NAMRU3-CLEVB214/2007 A/goose/Hungary/3413/2007

37 100

Clade 2.2

A/Nigeria/6e/2007 A/Iraq/1/2006

38

91

A/Turkey/15/2006

96

A/Cambodia/JP52a/2005

A/chicken/Anhui/T5/2006 90

Clade 9

A/chicken/Fujian/1042/2005

85

A/Ck/HK/61.9/2002 A/Ck/HK/YU22/2002

100

100 68

Clade 8

A/duck/Guangxi/668/2004

Clade 5

A/goose/Guangxi/914/2004 A/blackbird/Hunan/1/2004

69

A/chicken/Yichang lung/1/04

95

Clade 6

A/goose/Guangdong/1/1996

100

Clade 0

A/Hong Kong/156/1997 A/Chicken/Hong Kong/FY77/2001

57 58

A/Chicken/Hong Kong/YU562/2001

Clade 3

A/goose/Fujian/bb/2003

98

Clade 1

A/Cambodia/R0405050/2007

100

A/goose/Guiyang/337/2006

Clade 4

A/Beijing/01/2003 A/chicken/Hebei/326/2005 100

Ck/Shanxi/2/2006 100

Clade 7

CK/SX/10/2006

97

CK/JS/18/2008

88

A/chicken/Henan/B30/2012

100 65

Clade 7.2

CK/HB/A-8/2009

0.005

Fig. 1. Phylogenetic relationships of the open reading frames of HA gene of representative H5 influenza viruses. Viruses highlighted with the black triangles (~) were characterized in this study. The tree was constructed using the neighbor-joining method of MEGA 4.0, with 1000 bootstrap trials to assign confidence to the groupings.

– ND (0/3)

e

d

c

b

Infected Contact CkB30

a

Infected Contact GD1/96

For statistical purposes, a value of 1.5 was assigned if virus was not detected from the undiluted sample in three embryonated hen’s eggs (Sun et al., 2011). Chickens inoculated with virus. Naive contact chickens housed with those inoculated. Not detected. All of the chickens died at the end of the observation.

– ND (0/3) – ND (0/3) – ND (0/3) 1.63  0.18 (1/2) ND (0/3) 2.63  0.18 (2/2) ND (0/3) 1.88  0.53 (1/2) ND (0/3) 1.96  0.46 (5/6) ND (0/3) 2.92  1.11 (5/6) ND (0/3)

2.63  0.18 (2/2) ND (0/3)

ND (0/1) ND (0/2) ND (0/1) ND (0/2) ND (0/1) 2.63  1.59 (1/2) ND (0/1) 2.13  0.88 (1/2) ND (0/1) 3.50 (2/2) 2.25 (1/1) 2.13  0.88 (1/2) 2.25  1.09 (2/3) 3.83  0.58 (3/3) 1.95  0.62 (3/6) 1.83  0.58 (1/3) 2.08  0.79 (4/6) 1.83  0.58 (1/3)

2.33  0.72 (2/3) 3.33  1.66 (2/3)

– – – – – – – – – – Infectedb Contactc DkE35

–e 2.50 (1/1) ND (0/2) ND (0/3) NDd (0/2) 2.17  1.15 (1/3)

– ND (0/1)

Oropharyngeal swabs Oropharyngeal swabs Cloacal swabs Oropharyngeal swabs

Cloacal swabs

Oropharyngeal swabs

Cloacal swabs

Oropharyngeal swabs

Cloacal swabs

11 day 9 day 7 day 5 day 3 day

Days post-inoculation (log10EID50/0.1 ml)  SDa Strains

Table 2 Virus titers in cloacal and oropharyngeal swabs from SPF chickens.

– –

R. Yuan et al. / Veterinary Microbiology 168 (2014) 50–59

Cloacal swabs

54

the lung, kidney, brain, heart, spleen and liver (Table 1). The DkE35, GsGD1/96 or CkB30 viruses replicated highly in lung; the mean titers were 8.33 log10EID50, 5.75 log10EID50 and 5.50 log10EID50, respectively (Table 1). The three viruses could also replicate in brain; the mean titers were from 4.00 log10EID50 to 8.17 log10EID50 (Table 1). Overall, DkE35 virus titers of the tested organs were significantly higher than the other two viruses. In other words, the DkE35 virus replicated more highly in the chickens. DkE35, GsGD1/96 and CkB30 viruses shedding from the inoculated chickens were detected in oropharyngeal and cloacal swabs at 3, 5, 7, 9 and 11 DPI. The DkE35 virus could not be tested from both oropharyngeal and cloacal swabs of inoculated chickens until they died within 5 DPI (Table 2). The GsGD1/96 virus was shed from the oropharynx in inoculated chickens within 7 DPI, whose virus titers were from 2.08 log10EID50 to 2.33 log10EID50. It also could be shed from the cloaca within 5 DPI; the virus titers were from 1.95 log10EID50 to 2.25 log10EID50 (Table 2). CkB30 virus shedding could be tested from both oropharyngeal and cloacal swabs at 3, 5 and 7 DPI. The virus titers were from 2.63 log10EID50 to 2.92 log10EID50 in oropharyngeal swabs and 1.63 log10EID5 to 1.96 log10EID50 in cloacal swabs (Table 2). To understand the horizontal transmission of these three viruses, three SPF chickens were inoculated intranasally with 0.2 ml phosphate buffered saline (PBS) as a naive contact group, which was housed with those inoculated with the GsGD1/96, DkE35 or CkB30 viruses. During the observed time, all the chickens in the naive contact group, housed with inoculated DkE35 chickens, died within 6 DPI. The virus could only be detected from oropharyngeal swabs at 3 and 5 DPI. The mean titers were from 2.17 log10EID50 to 2.50 log10EID50 (Table 2). One of the three naive contact chickens housed with GsGD1/96 died on 6 DPI; the rest of the naive contact chickens seroconverted on 14 DPI (Table 2). The virus could be detected from oropharyngeal and cloacal swabs within 9 DPI. The virus titers were from 1.83 log10EID50 to 3.33 log10EID50 in oropharyngeal swabs and 1.83 log10EID5 to 3.83 log10EID50 in cloacal swabs (Table 2). The naive contact chickens housed with CkB30 were not dead in 14 DPI, but seroconverted. Meanwhile, no virus in the naive contact chickens of CkB30 could be detected from oropharyngeal and cloacal swabs during the observation time (Table 2). Our results indicated that DkE35, GsGD1/96 and CkB30 were highly pathogenic to chickens, and could transmit between chickens by naive contact. 3.3. Pathogenicity and transmission of H5N1 HPAI viruses in ducks To evaluate the pathogenicity of the three viruses in ducks, we inoculated intranasally with 106 EID50 of each virus in a volume of 200 ml in ducks, and then observed them for 14 days after infection. No ducks died during the observation period. All the ducks seroconverted in the DkE35 and GsGD1/96 groups, but not in the CkB30 group (Table 3). The DkE35 virus

Table 3 Replication and lethality in ducks of the H5N1 viruses after inoculated intranasally.a Strains

No. D/S/total DkE35 GD1/96 CkB30

Virus replication on 3 DPI (log10EID50/0.1ml)b in:

Manifestations of ducks c

0/1/3 0/0/3 0/0/3

No. S.C./total

d

3/3 3/3 0/3

Heart

Liver

Spleen

Lung

Kidney

Brain

Trachea

Colon

Bursa of fabricius

4.38  0.18 NDe ND

4.13  0.53 1.83  0.58 ND

2.88  0.53 ND ND

5.50 2.67  1.13 1.92  0.72

5.38  0.18 2.08  1.01 1.58  0.14

3.75  1.41 ND ND

2.50  0.63 ND 1.58  0.14

4.88  0.53 1.58  0.14 ND

4.00  0.71 1.58  0.14 ND

Table 4 Virus titers in cloacal and oropharyngeal swabs from ducks. Days post-inoculation (log10EID50/0.1 ml)  SDa

Strains

3 day

5 day

7 day

9 day

11 day

Oropharyngeal swabs

Cloacal swabs

Oropharyngeal swabs

Cloacal swabs

Oropharyngeal swabs

Cloacal swabs

Oropharyngeal swabs

Cloacal swabs

Oropharyngeal swabs

Cloacal swabs

DkE35

Infectedb Contactc

1.88  0.80 (2/6) 3.08  1.15 (3/3)

NDd (0/6) ND (0/3)

2.33  0.72 (2/3) 3.42  0.76 (3/3)

ND (0/3) 1.58  0.14 (1/3)

1.67  0.14 (2/3) ND (0/3)

ND (0/3) 1.58  0.14 (1/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

GD1/96

Infected Contact

2.04  0.62 (3/6) 1.92  0.72 (1/3)

ND (0/6) 1.92  0.72 (1/3)

1.92  0.29 (3/3) 2.58  0.29 (3/3)

ND (0/3) ND (0/3)

1.92  0.72 (2/3) 1.75  0.43 (1/3)

1.58  0.14 (2/3) ND (0/3)

1.92  0.52 (2/3) ND (0/3)

1.60  0.14 (2/3) ND (0/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

CkB30

Infected Contact

ND (0/6) ND (0/3)

ND (0/6) ND (0/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

ND (0/3) ND (0/3)

a b c d

R. Yuan et al. / Veterinary Microbiology 168 (2014) 50–59

a Three-week-old ducks were inoculated intranasally (i.n.) with 106 EID50 of each virus in a 0.2 ml volume; three ducks in each group were euthanized on 3 DPI, and virus titer was determined in samples of heart, liver, spleen, lung, kidney, brain, trachea, colon and bursa of fabricius in eggs. b For statistical analysis, a value of 1.5 was assigned if the virus was not detected from the undiluted sample in three embryonated hen eggs (Sun et al., 2011). Virus titers are expressed as means  standard deviation in log10EID50/0.1 ml of tissue. c No. D/S/total shows the number of dead (D) and sick (S) as well as the total number of ducks from the observation period. The ducks that showed disease signs, such as depression and ruffled feathers, but recovered at the end of the observation were counted as sick animals. d No. S.C./total shows the number of ducks that seroconverted out of the total number of ducks at the end of the observation period. e Not detected.

For statistical purposes, a value of 1.5 was assigned if virus was not detected from the undiluted sample in three embryonated hen’s eggs (Sun et al., 2011). Ducks inoculated with virus. Naive contact ducks housed with those inoculated. Not detected.

55

2.50  1.73 ND ND 2/3 3/3 0/3

No. S.C./total No. D/S/total

1/1/3 0/0/3 0/0/3 DkE35 GD1/96 CkB30

a Three-week-old geese were inoculated intranasally (i.n.) with 106 EID50 of each virus in a 0.2 ml volume; on 3 DPI, three geese in each group were euthanized, and virus titer was determined in samples of heart, liver, spleen, lung, kidney, brain, trachea, colon and bursa of fabricius in eggs. b For statistical analysis, a value of 1.5 was assigned if the virus was not detected from the undiluted sample in three embryonated hen eggs (Sun et al., 2011). Virus titers are expressed as means  standard deviation in log10EID50/0.1 ml of tissue. c No. D/S/total shows the number of dead (D) and sick (S) as well as the total number of geese from the observation period. The geese that showed disease signs, such as depression and ruffled feathers, but recovered at the end of the observation were counted as sick animals. d No. S.C./total shows the number of geese that seroconverted out of the total number of geese at the end of the observation period. e Not detected.

Colon

2.83  1.46 ND ND 1.83  0.58 ND ND

Trachea Brain

2.92  1.51 1.92  0.72 ND 2.75  2.17 ND ND

Kidney Lung

4.42  1.61 1.92  0.72 ND 3.33  1.23 ND ND 1.83  0.38 1.92  0.72 ND

Spleen Liver Heart

2.33  1.23 NDe ND

Virus replication on 3 DPI (log10EID50/0.1ml)b in

d c

Manifestations of geese Strains

replicated systemically in ducks, which could be detected from all the tested organs on 3 DPI, including the lung, kidney, brain, heart, spleen, liver, colon, trachea and bursa of fabricius (Table 3). The DkE35 virus replicated more highly in the lungs (5.50 log10EID50). The mean virus titers in the kidney, colon, heart, liver, bursa of fabricius, brain, spleen and trachea were 5.38, 4.88, 4.38, 4.13, 4.00, 3.75, 2.88 and 2.50 log10EID50, respectively. The GsGD1/96 virus titers were significantly lower than those of DkE35 in ducks. The GsGD1/96 virus replicated in some tested organs, including lung, kidney, liver, colon, and bursa of fabricius, but not in the brain (Table 3). CkB30 in inoculated ducks could only be detected in lung, kidney and trachea. The mean virus titers were 1.92, 1.58 and 1.58 log10EID50, respectively (Table 3). Although the three viruses could replicate in the tested organs, the DkE35 virus titers were significantly higher than the other two viruses. DkE35, GsGD1/96 and CkB30 viruses shedding were detected in oropharyngeal and cloacal swabs of the inoculated ducks at 3, 5, 7, 9 and 11 DPI. The DkE35 virus could only be isolated from the oropharynx within 7 DPI. The mean virus titers were from 1.67 log10EID50 to 2.33 log10EID50 (Table 4). The GsGD1/96 virus could be detected in both oropharyngeal and cloacal swabs within 9 DPI (Table 4). The mean virus titers in the oropharyngeal and cloacal swabs were from 1.92 log10EID50 to 2.04 log10EID50 and 1.50 to 1.60 log10EID50, respectively. However, no CkB30 virus could be isolated from oropharyngeal and cloacal swabs of the ducks during the test time. To understand the horizontal transmission of these three viruses in ducks, three were inoculated intranasally with 0.2 ml phosphate buffered saline (PBS) as a naive contact group housed with those inoculated with the three viruses described previously. The naive contact ducks, housed with the ducks inoculated DkE35, GsGD1/96 or CkB30, experienced no deaths during the test time. DkE35 viruses shedding of the naive contact group could be detected in the oropharyngeal and the cloacal swabs. The virus titers were from 3.08 log10EID50 to 3.42 log10EID50 within 5 DPI in oropharyngeal swabs and 1.58 log10EID5 at 5 and 7 DPI in cloacal swabs, respectively (Table 4). In the naive contact group of GsGD1/96, the virus titers of the oropharyngeal swabs were from 1.75 log10EID50 to 2.58 log10EID50 within 7 DPI. The virus titers of cloacal swabs could only be detected on 3 DPI (1.92 log10EID50) (Table 4). No virus of the CkB30 naive contact group could be isolated from oropharyngeal and cloacal swabs during the test time. Meanwhile, no naive contact ducks seroconverted in this group. According to these, the shedding time of GsGD1/96 virus was longest among the three viruses. However, the virus titers of DkE35 were the highest among the three viruses. In summary, these results indicate that DkE35 and GsGD1/96 viruses could infect ducks and transmit between ducks by naive contact. However, the CkB30 virus could replicate in only a few organs, in which the virus titers were very low, and could not transmit to the ducks by naive contact.

Bursa of fabricius

R. Yuan et al. / Veterinary Microbiology 168 (2014) 50–59

Table 5 Replication and lethality in geese of the H5N1 viruses after inoculated intranasally.a

56

ND (0/3) ND (0/3) ND (0/3) ND (0/3)

d

c

b

a

For statistical purposes, a value of 1.5 was assigned if virus was not detected from the undiluted sample in three embryonated hen’s eggs (Sun et al., 2011). Geese inoculated with virus. Naive contact geese housed with those inoculated. Not detected.

ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/6) ND (0/3) Infected Contact CkB30

ND (0/6) ND (0/3)

ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) ND (0/3) 2.33  0.52 (3/3) ND (0/3) 1.67  0.41 (1/6) ND (0/3) Infected Contact GD1/96

1.88  0.52 (3/6) ND (0/3)

ND (0/2) ND (0/2) NDd (0/2) ND (0/2) 1.88  0.53 (2/2) ND (0/2) 1.75 (2/2) ND (0/2) 2.13  0.88 (2/2) ND (0/2) 2.33  0.52 (3/3) 3.00  1.30 (2/3) 2.42  1.59 (2/3) 2.50  1.73 (1/3) 1.58  0.13 (2/6) ND (0/3) 3.04  0.84 (6/6) 1.92  0.72 (1/3) Infectedb Contactc DkE35

Oropharyngeal swabs

11 day

Cloacal swabs Oropharyngeal swabs

9 day

Cloacal swabs Oropharyngeal swabs

7 day

Oropharyngeal swabs

Cloacal swabs 5 day

Oropharyngeal swabs

Cloacal swabs 3 day

Days post-inoculation (log10EID50/0.1 ml)  SDa Strains

Table 6 Virus titers in cloacal and oropharyngeal swabs from geese.

To determine the pathogenicity in geese, we inoculated intranasally geese with 106 EID50 of each virus in a volume of 200 ml, and then observed the geese for 2 weeks after infection. In the inoculated group, DkE35 caused one of the three inoculated geese to die on 6 DPI, but no geese from the GsGD1/96 and CkB30 groups died during the 14-day observation period. All the surviving geese seroconverted in the DkE35 and GsGD1/96 groups, but not in the CkB30 group (Table 5). The DkE35 virus caused systemic infection in geese, including the spleen, brain, colon, kidney, bursa of fabricius, heart, liver and trachea. The mean virus titer was higher in the lungs (4.42 log10EID50). The GsGD1/96 virus could only replicate in the lung, liver and brain of the tested organs. The mean titers were 1.92 log10EID50 (Table 5). The CkB30 virus was not detected in any tested organs in the geese. Meanwhile, no geese seroconverted. Therefore, DkE35 replicated more highly than the other two viruses in the organs. GsGD1/96 could also replicate in a few organs, but CkB30 could not replicate in the tested organs of geese. DkE35, GsGD1/96 and CkB30 viruses shedding were detected in oropharyngeal and cloacal swabs of inoculated geese at 3, 5, 7, 9 and 11 DPI. In the DkE35 group, the virus titers of the oropharyngeal swabs were from 1.88 log10EID50 to 3.04 log10EID50 within 9 DPI. The virus titers of cloacal swabs were from 1.58 log10EID50 to 2.33 log10EID50 within 7 DPI (Table 6). In the GsGD1/96 group, the virus titers of the oropharyngeal swabs were from 1.88 log10EID50 to 2.33 log10EID50 within 5 DPI. The virus titers of cloacal swabs could only be detected at 3 DPI (1.67 log10EID50) (Table 6). However, the CkB30 virus was not detected from any swabs from the geese in the inoculated group. Generally, the DkE35 virus had the longest shedding time and the highest titers among the three viruses. The GsGD1/96 virus shedding could only be detected at 3 and 5 DPI and the titers were low. CkB30 virus shedding could not be detected in the inoculated group. To study the horizontal transmission of these three viruses, three geese were inoculated intranasally with 0.2 ml phosphate buffered saline (PBS) as a naive contact group housed with those inoculated with the viruses mentioned above. The DkE35 virus caused one of the three naive contact geese to die, and could be isolated from oropharyngeal and cloacal swabs during the observed period. The virus titers of the oropharyngeal swabs were from 1.92 log10EID50 to 2.50 log10EID50 within 5 DPI. And the virus titers of cloacal swabs could only be detected 3.00 log10EID50 at 5 DPI (Table 6). However, no virus in the naive contact groups of GsGD1/96 or CkB30 could be isolated from oropharyngeal and cloacal swabs. Also, no naive contact geese of the two viruses seroconverted. Therefore, DkE35 virus shedding could be tested in naive contact geese, but GsGD1/96 and CkB30 viruses shedding could not be detected. In the geese study, the DkE35 virus was higher pathogenicity, and could transmit by naive contact. The GsGD1/96 virus could infect geese but not transmit

Cloacal swabs

3.4. Pathogenicity and transmission of H5N1 HPAI viruses in geese

57 ND (0/2) ND (0/2)

R. Yuan et al. / Veterinary Microbiology 168 (2014) 50–59

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R. Yuan et al. / Veterinary Microbiology 168 (2014) 50–59

between geese by naive contact. However, the CkB30 virus was not pathogenic in geese and could not transmit between geese by naive contact. 4. Discussion The surveys indicated clades 2.3.2, 2.3.4 and 7 of the viruses cocirculated in domestic poultry and waterfowl in China from 2007 to 2009 (Jiang et al., 2010; Li et al., 2010; Smith et al., 2009). Clade 2.3.2 was first isolated from a dead Chinese pond heron in Hong Kong in 2004, it is widely distributed in Asia, particularly in China, Hong Kong, Korea, Vietnam, Laos, Bangladesh, Nepal, Mongolia, and the Tyva Republic; it is also distributed in Eastern Europe, mainly in Romania and Bulgaria. So, viruses of clade 2.3.2 have spread geographically and evolved genetically (WHO/OIE/ FAO H5N1 Evolution Working Group, 2012; European Commission Directorate-General for Health and Consumers, 2010). New fourth order clade 2.3.2.1 viruses have been circulating widely in China since 2010 and may cause a new wave of cross-continental spreading from Asia to Europe (Jiang et al., 2010; Li et al., 2010; Smith et al., 2009). This clade is already widely distributed in Asia and is being perpetuated in many birds, including chickens, ducks, geese and wild birds (WHO, 2013b). Since H5N1 HPAIV of clade 7 was first isolated in 2003, it has been circulating widely in Northern China. A new subclade 7.2 virus has been circulating in China and Vietnam since 2008, and causing great damage to the poultry industry (WHO, 2011). In the present study, we chose three H5N1 HPAIVs of different clades isolated in different birds in China. One was reported previously A/Goose/Guangdong/1/1996 (clades 0) H5N1 HPAIV isolated from sick geese in Guangdong during 1996 (Chen et al., 1999), and two circulating H5N1 HPAIVs, A/Duck/Guangdong/E35/2012 (clade 2.3.2.1) and A/Chicken/Henan/B30/2012 (clade 7.2), isolated from cloacal swabs of apparently healthy poultry in live bird markets during 2012. Our aim was to study the pathogenicity and transmission of the three H5N1 HPAIVs in terrestrial birds and waterfowl, so that we could understand the pathogenic evolution of H5N1 HPAIV from 1996 to 2012. Waterfowl play an important role in AIV ecology, because they can be infected by many subtype AIVs, including H5N1 viruses, but generally they do not show any symptoms of disease when infected (Sturm-Ramirez et al., 2005). These viruses replicate in the columnar epithelial cells, forming crypts in the colon, and are excreted in feces. Low-pathogenic avian influenza viruses (LPAIVs) and non-pathogenic avian influenza viruses (NPAIVs) circulating in waterfowl transmit to terrestrial birds such as quail, chickens and turkeys through domestic water birds such as ducks and geese. By the transmission, LPAIVs or NPAIVs may become highly pathogenic to the terrestrial birds. Since 2003, H5N1 HPAIVs have caused disease and death in waterfowl in China (Chen et al., 2004; Sun et al., 2011). In our study, the three H5N1 HPAIVs exhibited different biological properties with respect to pathogenicity and transmission in chickens, ducks and geese. DkE35 isolated from ducks was highly pathogenic for chickens and geese, but low pathogenic for ducks.

Moreover, it could transmit to chickens, ducks and geese by naive contact. GSGD/1/96 isolated from geese could infect some chickens, ducks and geese, but only caused chickens to die. Additionally, it could transmit to chickens and ducks, but could not transmit to the naive contact geese. CkB30 isolated from chicken was highly pathogenic to the inoculated chickens, low pathogenic to the inoculated ducks and non pathogenic to the inoculated geese. It could transmit to the naive contact chickens, but not to the naive contact ducks and geese. So, our findings showed that the pathogenicity and transmission in birds of the three viruses varied. This suggests that H5N1 HPAIVs from waterfowl were highly pathogenic to terrestrial birds and waterfowl, and might transmit among them; H5N1 HPAIVs from chickens were highly pathogenic to chickens, and might be low or none pathogenic to waterfowl, which might not transmit to waterfowl. Therefore, H5N1 HPAIVs isolated from different birds show different host ranges and tissue tropisms. Additionally, when the chickens were inoculated intranasally with 103 EID50 of GsGD1/96 (clade 0) virus, they did not show any disease symptoms, did not shed virus and did not seroconvert (data not shown). However, when inoculated intranasally with 103 EID50 of the two new isolated viruses DkE35 and CkB30, the chickens could be fatal and shed the viruses. Therefore, at the low dose of the viruses, the H5N1 virus isolated early from waterfowl was low pathogenic to chicken, but the H5N1 virus isolated recently from waterfowl was highly pathogenic and replicated systemically in the inoculated chickens. Interesting, clade 7 could be isolated from both domestic poultry and waterfowl in an early stage. However, few viruses of clade 7 and 7.2 could be isolated from waterfowl since 2008 (WHO, 2013). It seems that the H5N1 HPAIVs of clade 7 and 7.2 have been adapting to chickens. Vaccination is a major strategy used to control H5N1 AIVs in China. H5 vaccines, especially H5 inactivated vaccines, have been widely using to prevent and control the H5N1 AIVs in chickens, ducks, and geese in China. Previous studies have indicated that H5 inactivated vaccine has been proven to be efficacious in chickens, ducks, and geese (Kim et al., 2008; Tian et al., 2005). Since 2006, an inactivated vaccine Re-4, containing HA and NA genes of the CK/SX/2/06 virus (clade 7) and six internal genes from the PR8 virus, has been developed and widely used in Northern China (Chen, 2009). The Re-4 vaccine has played an important role for the control of the clade 7 outbreaks in China, as well. However, the new subclade 7.2 virus caused outbreaks in Ningxia and Jiangsu in 2008 and exhibited antigenic drift from the clade 7 isolated previously. Since 2010, a new fourth order clade 2.3.2.1 virus has been circulating in domestic poultry and waterfowl in China. In late 2012, a new H5N1 vaccine strain Re-6, which belonged to clade 2.3.2.1, was licensed to use in domestic poultry and waterfowl. However, vaccination comparisons of chickens and waterfowl with the Re-6 vaccine showed that chickens have a much more robust antibody response to the vaccine than waterfowl. In addition, differences in immune response to AIV vaccinations have been reported between chickens and waterfowl in a previous study (Magor, 2011). Thus, the immunity

R. Yuan et al. / Veterinary Microbiology 168 (2014) 50–59

effects of Re-6 vaccinations in chickens are better than in waterfowl. Although the vaccine could efficiently prevent and control the outbreaks of H5N1 AIVs in China, it also would accelerate the antigenic variation and newly emerging clades of the viruses. Thus, the control of H5N1 AIVs by vaccination still faces new challenges in different avian species. In conclusion, our findings illustrated that the three H5N1 HPAI viruses isolated from different birds had varying levels of pathogenicity and transmission in chickens, ducks and geese. Therefore, we should enhance serological and virological surveillance of H5N1 viruses, and pay more attention to the pathogenic and antigenic evolution of these viruses. Acknowledgements This work was supported by grants from the Natural Science Foundation of Guangdong Province (No. 10251064201000004, No. 10151064201000021), Natural Science Foundation of China (No. 31172343), the Science and Technology Projects of Guangdong Province (No. 2012B020306003), the Key Project of Agricultural Ministry (nycytx-42-G3-03), and High-level Talents in University Project of Guangdong Province (2010). References Belser, J.A., Szretter, K.J., Katz, J.M., Tumpey, T.M., 2009. Use of animal models to understand the pandemic potential of highly pathogenic avian influenza viruses. Adv. Virus Res. 73, 55–97. Chen, H., Yu, K., Bu, Z., 1999. Molecular analysis of hemagglutinin gene of a goose origin highly pathogenic avian influenza virus. Chin. Agric. Sci. 32, 87–92. Chen, H., 2009. H5N1 avian influenza in China. Sci. China Series C: Life Sci. 52, 419–427. Chen, H., Deng, G., Li, Z., Tian, G., Li, Y., Jiao, P., Zhang, L., Liu, Z., Webster, R.G., Yu, K., 2004. The evolution of H5N1 influenza viruses in ducks in southern China. Proc. Natl. Acad. Sci. USA 101, 10452–10457. Claas, E.C., Osterhaus, A.D., van Beek, R., De Jong, J.C., Rimmelzwaan, G.F., Senne, D.A., Krauss, S., Shortridge, K.F., Webster, R.G., 1998. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472–477. Duan, L., Bahl, J., Smith, G.J., Wang, J., Vijaykrishna, D., Zhang, L.J., Zhang, J.X., Li, K.S., Fan, X.H., Cheung, C.L., Huang, K., Poon, L.L., Shortridge, K.F., Webster, R.G., Peiris, J.S., Chen, H., Guan, Y., 2008. The development and genetic diversity of H5N1 influenza virus in China, 1996– 2006. Virology 380, 243–254. European Commission Directorate-General for Health and Consumers, 2010. Animal Disease Notifi cation System. Annual Report 2010. (cited 5 January 2012). Fouchier, R.A., Munster, V.J., 2009. Epidemiology of low pathogenic avian influenza viruses in wild birds. Rev. Sci. Tech. 28, 49–58. Ha, Y., Stevens, D.J., Skehel, J.J., Wiley, D.C., 2001. X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proc. Natl. Acad. Sci. USA 98, 11181– 11186. Jiang, W.M., Liu, S., Chen, J., Hou, G.Y., Li, J.P., Cao, Y.F., Zhuang, Q.Y., Li, Y., Huang, B.X., Chen, J.M., 2010. Molecular epidemiological surveys of H5 subtype highly pathogenic avian influenza viruses in poultry in China during 2007–2009. J. Gen. Virol. 91, 2491–2496. Kaleta, E.F., Hergarten, G., Yilmaz, A., 2005. Avian influenza A viruses in birds—an ecological, ornithological and virological view. Dtsch. Tierarztl. Wochenschr. 112, 448–456. Kim, J.K., Seiler, P., Forrest, H.L., Khalenkov, A.M., Franks, J., Kumar, M., Karesh, W.B., Gilbert, M., Sodnomdarjaa, R., Douangngeun, B., Govorkova, E.A., Webster, R.G., 2008. Pathogenicity and vaccine efficacy of

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