Arch Virol DOI 10.1007/s00705-014-2009-3

ORIGINAL ARTICLE

Differences in transmissibility and pathogenicity of reassortants between H9N2 and 2009 pandemic H1N1 influenza A viruses from humans and swine Liang He • Qiwen Wu • Kaijun Jiang • Zhiqiang Duan Jingjing Liu • Haixu Xu • Zhu Cui • Min Gu • Xiaoquan Wang • Xiaowen Liu • Xiufan Liu



Received: 14 October 2013 / Accepted: 26 January 2014 Ó Springer-Verlag Wien 2014

Abstract Both H9N2 subtype avian influenza and 2009 pandemic H1N1 viruses (pH1N1) can infect humans and pigs, which provides the opportunity for virus reassortment, leading to the genesis of new strains with potential pandemic risk. In this study, we generated six reassortant H9 viruses in the background of three pH1N1 strains from different hosts (A/California/04/2009 [CA04], A/Swine/ Jiangsu/48/2010 [JS48] and A/Swine/Jiangsu/285/2010 [JS285]) by replacing either the HA (H9N1-pH1N1) or both the HA and NA genes (H9N2-pH1N1) from an h9.4.2.5-lineage H9N2 subtype influenza virus, A/Swine/ Taizhou/5/08 (TZ5). The reassortant H9 viruses replicated to higher titers in vitro and in vivo and gained both efficient transmissibility in guinea pigs and increased pathogenicity in mice compared with the parental H9N2 virus. In addition, differences in transmissibility and pathogenicity were observed among these reassortant H9 viruses. The H9N2pH1N1viruses were transmitted more efficiently than the corresponding H9N1-pH1N1 viruses but showed significantly decreased pathogenicity. One of the reassortant H9 viruses that were generated, H9N-JS48, showed the highest virulence in mice and acquired respiratory droplet transmissibility between guinea pigs. These results indicate that coinfection of swine with H9N2 and pH1N1viruses may pose a threat for humans if reassortment occurs, emphasizing the importance of surveillance of these viruses in their natural hosts.

L. He  Q. Wu  K. Jiang  Z. Duan  J. Liu  H. Xu  Z. Cui  M. Gu  X. Wang  X. Liu  X. Liu (&) College of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou 225009, Jiangsu, People’s Republic of China e-mail: [email protected]

Introduction H9N2 subtype avian influenza viruses (AIVs) in poultry were first detected in turkey flocks in the United States in 1966 and have become prevalent in many countries and areas since the 1990s [1, 16]. After two decades of evolution, at least two lineages of H9N2 viruses have established themselves in poultry in Asia, the G1-like lineage (h9.4.1 lineage) represented by A/quail/Hong Kong/G1/ 1997 and the Y280-like lineage (h9.4.2 lineage) represented by A/duck/Hong Kong/Y280/1997 [11]. However, unlike other countries in Asia, the dominant lineage of H9 subtype AIVs in China in recent years has been the h9.4.2.5 lineage, which is distinct from the h9.4.1 lineage in its antigenic and genetic characteristics [20]. The endemicity of H9N2 influenza virus in poultry in China poses a threat to public health, as occasional human infections with this virus that are associated with flu-like disease have been reported from both Hong Kong and mainland China [3, 4, 26]. In recent years, most H9N2 isolates contain the amino acid mutation Q226L in the receptor-binding site of the HA protein, which facilitates their binding to the SAa2,6 receptor and their ability to replicate efficiently in mammalian hosts [36, 37]. However, there is no evidence suggesting that H9N2 influenza viruses are able to be transmitted from human to human. In 2009, a novel swine-origin H1N1 virus emerged and caused the first influenza pandemic of the 21st century [9, 33]. The 2009 pandemic H1N1 viruses (pH1N1) rapidly spread throughout the world with high transmissibility in humans and were able to infect a wide range of animal species, including pigs [27, 43]. Pigs are regarded as intermediate hosts or ‘‘mixing vessels’’ for reassortment of influenza A viruses, since they bear both SAa2,3 and SAa2,6 receptors for avian and human influenza viruses,

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respectively [19]. Recently, reassortant viruses composed of pH1N1 and endemic swine influenza viruses have been isolated from pigs worldwide, and a reassortant of Eurasian avian-like and pH1N1 influenza virus isolates was demonstrated to be transmitted effectively from pig to pig and from pig to ferret [7, 8, 34, 45]. Using reverse genetics, recent studies also showed that there is high genetic compatibility between pH1N1 and other avian and human seasonal influenza viruses [25, 32, 35]. H9N2 viruses are also able to infect pigs, and we have reported the isolation of both H9N2 and pH1N1 viruses from pigs in the same area of eastern China [6, 40, 42]. Coinfection with H9N2 and pH1N1 viruses in pigs provides the opportunity for reassortment to occur, resulting in the generation of new strains with potential pandemic risk. Recent studies have demonstrated that G1-like-lineage H9 viruses with pH1N1 internal genes could acquire respiratory droplet transmissibility in ferrets with some adaptive amino acid changes in HA [21]. However, reassortment experiments using the currently predominant h9.4.2.5lineage H9N2 virus and pH1N1 influenza virus may be best for providing information about the natural reassortment situation in mainland China. So far, is not clear whether the surface genes from different lineages of the H9N2 subtype virus and the internal genes from pH1N1 are compatible. In this study, we generated six reassortant H9 viruses in the genetic background of three pH1N1 strains from different hosts by replacing either the HA (H9N1-pH1N1) or both the HA and NA genes (H9N2-pH1N1) from the h9.4.2.5lineage H9N2 virus A/Swine/Taizhou/5/08 and evaluated their replication, transmissibility and pathogenicity in vitro or in vivo.

passaged twice in specific-pathogen-free (SPF) chicken eggs and confirmed by sequencing. Phylogenetic analysis To assess the genetic relationships between TZ5 and other H9 influenza virus strains, reference gene sequences (HA and NA) were selected from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov), and a phylogenetic tree was constructed by the neighbour-joining method using MEGA 4.1 software. Bootstrap values were calculated based on 1000 replicates of the alignment. Receptor-binding analysis The receptor specificity of the parental H9N2 and pH1N1 viruses was analyzed by a solid-phase direct binding assay as described previously [2]. Briefly, synthetic sialylglycopolymers (GlycoTech), Neu5Aca2-3Galb1-4GlcNAcb (3’SLN)PAA-biotin and Neu5Aca2-3Galb1-4GlcNAcb (6’SLN)PAA-biotin, were serially diluted in phosphate-buffered saline (PBS) and added to the wells of 96-well streptavidincoated microtiter plates (Pierce). The plates were blocked with PBS containing 2 % skim milk powder, and 128 HA units of live virus was added per well. Mouse antisera against the virus were diluted in PBS and added to each well. Bound antibody was detected by sequential addition of HRP-conjugated rabbit anti-mouse IgG antibody and tetramethylbenzidine substrate solution. The reaction was stopped with 1 M H2SO4, and the absorbance at 450 nm was read. Each sample was determined in triplicate. Growth kinetics of reassortant viruses

Materials and methods Cells and viruses Human embryonic kidney cells (293T), MDCK cells and human alveolar epithelial cells (A549) were grown in DMEM (Invitrogen) containing 10 % FBS (Invitrogen). The human 2009 pandemic H1N1 (pH1N1) A/California/ 04/2009 (CA04) virus was kindly provided by Dr. Jinhua Liu of the China Agricultural University. The swine isolates of 2009 pandemic H1N1, A/Swine/Jiangsu/48/2010 (JS48) and A/Swine/Jiangsu/285/2010 (JS285), and the H9N2 virus, A/Swine/Taizhou/5/08 (TZ5), were isolated in Jiangsu Province of eastern China. Six reassortant viruses were generated by reverse genetics as reported previously [15]. The reassortant viruses were placed in the pH1N1 background by replacing the surface genes with those of the H9N2 virus TZ5 to produce H9N1-pH1N1 and H9N2pH1N1 (Fig. 3). All of the reassortant viruses were

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Monolayers of MDCK or A549 cells were cultured in sixwell plates and infected with parental and reassortant viruses at a multiplicity of infection (MOI) of 0.01. After inoculation at 37 °C for 1 h, cells were washed twice with PBS and incubated in minimum essential medium with 1 lg/ml tosyl-phenylalanyl-chloromethyl-ketone-treated trypsin (Sigma) at time zero. Aliquots of culture supernatants were collected at 12, 24, 48 and 72 h post-inoculation (h.p.i) and immediately stored at -80 °C for determination of virus titers. Viruses were titrated (log10TCID50) in MDCK cells by the method of Reed and Muench [29]. Animals Five-week-old Hartley strain female guinea pigs (300 to 350 g) and six-week-old female Balb/C mice were obtained from Vital River Laboratories (Beijing, China). Ketamine (20 mg/kg) and xylazine (1 mg/kg) were used to anesthetize animals by intramuscular injection. The animal

Properties of influenza A virus reassortants

research was approved by the Jiangsu Administrative Committee for Laboratory Animals (Permit number: SYXKSU-2007-0005) and complied with the guidelines of Jiangsu Laboratory Animal Welfare and Ethical of Jiangsu Administrative Committee of Laboratory Animals. Infection and transmission in guinea pigs To evaluate the replication of the parental and reassortant viruses in vivo, groups of three guinea pigs were anesthetized and inoculated intranasally with 106 50 % egg infectious doses (EID50) of the indicated viruses in a 300-ll volume (150 ll per nostril). The animals were euthanized 3 days post-inoculation (d.p.i), and nasal washes and lungs were collected for virus titration in eggs. Each transmission experiment involved three guinea pigs: one infected, one direct contact, and one respiratory droplet contact. The data shown in Fig. 5 represent cumulative results from three independent experiments for each virus. The procedure was as follows. On day zero, one guinea pig was inoculated intranasally with 106 EID50 of the indicated virus and housed in a cage inside an isolator. After 24 h, two naive guinea pigs were introduced into the isolator. The direct contact was introduced into the same cage as the infected guinea pig, while the respiratory droplet contact was placed into an adjacent cage (4 cm away), separated by a wire mesh wall. The airflow in the isolator was horizontal, with a speed of 0.1 m/s from the infected animals to the exposed animals. Nasal washes were collected at 2-day intervals for up to 2 weeks and were stored at -80 °C until used for performing EID50 titrations in eggs. Sera were collected from infected guinea pigs at 14 d.p.i and contact guinea pigs at 21 d.p.i for hemagglutinin inhibition (HI) antibody detection. These studies were performed under ambient conditions of 20–22 °C and 20–40 % relative humidity. Virulence and replication in mice To determine the 50 % mouse lethal dose (MLD50), groups of five 6-week-old female BALB/c mice were anesthetized and intranasally inoculated with 50 ll of 10-fold serial dilutions of parental and reassortant viruses in PBS. The MLD50 values were calculated by the method of Reed and Muench after a 14-day observation period and expressed as EID50. To determine the morbidity and mortality, ten groups of ten 6-week-old female BALB/c mice were infected intranasally with 106 EID50 of the indicated viruses in 50-ll volumes. Body weight was recorded daily until 14 d.p.i. Infected mice showing more than 25 % body weight loss were humanely euthanized and recorded as dead. To evaluate the replication ability of the parental and reassortant viruses in vivo, ten groups of six mice were inoculated with 106 EID50 of the indicated viruses, and

three mice from each infected group were euthanized humanely at 3 d.p.i and 5 d.p.i. The lungs were collected and homogenized in PBS at a ratio of 1:1 (g/ml). After centrifugation for 30 min at 8,000 rpm, virus titers in lungs were determined by inoculating chicken eggs with serial dilutions of the suspensions. Titers were calculated by the method of Reed and Muench and expressed as mean log10 EID50/ml ± standard deviation (SD). Statistical analysis Virus yields in vitro, virus titers in the nasal wash and lung of guinea pigs, and the lung titers of mice were compared by an unpaired, two-tailed t-test. The MLD50 value, mean days to death, and the weight changes of mice were compared by analysis of variance (one-way ANOVA). The probability of survival of infected mice was estimated by the Kaplan–Meier method [10]. A value of p \ 0.05 was considered significant. Statistical analysis was performed using PAWS Statistics 18 (SPSS Inc., USA).

Results Phylogenetic analysis Phylogenetic analysis of the HA gene showed that isolate TZ5 belonged to the h9.4.2.5 lineage, which has been dominant in mainland China in recent years, and was most closely related to chicken isolate A/chicken/Jiangsu/G12/2010, with 98.3 % similarity in the nucleotide sequence (Fig. 1a). For the NA gene, TZ5 belonged to the N2.1 lineage and showed the highest homology to A/chicken/Shandong/KD/2009, with 99.4 % similarity in the nucleotide sequence (Fig. 1b). These results indicated that swine H9N2 isolate TZ5 originated from chicken viruses circulating in eastern China. Both the HA and NA genes of TZ5 were distinct from that of the G1-like lineage in the genetic characterization. Receptor-binding analysis To evaluate the receptor-binding specificity of the parental viruses, a solid-phase direct binding assay was conducted. TZ5 and CA04 showed an absolute preference for a2, 6-sialoglycan (6’SLN) binding. However, JS48 preferred to bind to a2, 3-sialoglycan (3’SLN), and JS285 showed a preference for both 3’SLN and 6’SLN (Fig. 2). Compared to CA04, there is a Q226R mutation in the receptor-binding domain of HA of JS48 and JS285 (Table 1), which facilitates the binding of pH1N1 virus to the SAa2,3 receptor [41]. JS48 also has an N159D amino acid change in the Sa of HA (Table 1), which would further increased the preference for the SAa2,3 receptor [30].

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Properties of influenza A virus reassortants b Fig. 1 Phylogenetic trees of the HA (a) and NA (b) genes of H9N2

influenza virus A/Swine/Taizhou/5/08 (TZ5). Trees were constructed with sequences from TZ5 and reference strains from GenBank based on the open reading frame sequences. The sequences of TZ5 in the present study are marked by black triangles. The trees were generated using the neighbour-joining method in MEGA 4.1, with 1,000 bootstrap trials performed to assign confidence to the grouping

Fig. 2 Solid-phase receptor binding assay of the parental H9N2 virus TZ5 (a), human pH1N1 CA04 (b), swine pH1N1 JS48 (c), and swine pH1N1 JS285 (d). Direct binding of viruses to sialylglycopolymers

Generation and characterization of reassortant viruses in vitro Six reassortant viruses were generated by reverse genetics using surface genes from TZ5 together with internal genes from three pH1N1 viruses (CA04, JS48 or JS285), as

containing either 3’SLN-PAA or 6’SLN-PAA was measured. The data shown are representative of three independent binding experiments

Table 1 Protein-encoding sequence alignment of the three 2009 pandemic A (H1N1) influenza viruses Strain

PB2 64

PA 385

588

707

35

NP 70

224

269

451

547

100

NS1 102

107

123

205

207

215

CA04

M

I

T

A

F

A

P

K

A

D

V

G

E

I

N

D

P

JS48

M

V

I

T

L

V

S

K

S

E

I

G

K

V

N

N

P

JS285

I

I

I

A

F

V

S

R

S

E

I

R

E

V

S

D

S

Strain

NS2

82

106

248

264

HA (H3 number)

NA

26

48

90

132

159

200

202

206

208

226

324

68

75

CA04

E

T

P

S

N

T

V

S

R

Q

I

N

A

S

V

N

V

JS48

E

T

S

P

D

A

A

T

K

R

V

D

V

P

I

D

I

JS285

G

A

S

P

N

A

V

T

R

R

V

N

A

S

I

D

V

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L. He et al. Fig. 3 Genotypes of the reassortant H9 viruses. The genes of TZ5 (H9N2) are indicated in red, and three closely related pH1N1 viruses, CA04, JS48 and JS285, are indicated in gray, blue, and green, respectively. The reassortant H9N1-pH1N1 viruses were generated by replacing only the HA gene of TZ5 in the background of pH1N1, whereas the reassortant H9N2-pH1N1 viruses were generated by replacing both the HA and NA genes of TZ5

shown in Fig. 3. Compared to CA04, about 20 amino acid changes were found in six genes of JS48 and JS285 (Table 1). All of the reassortant viruses grew to similar titers in MDCK and A549 cells and reached their peak titers at 48 h.p.i. However, all of the reassortant H9 viruses replicated to significantly higher titres than the parental H9N2 and pH1N1 viruses throughout the course of infection in both cell lines (Fig. 4). These results indicated that the reassortant H9 viruses had acquired enhanced replication capability in vitro. Enhanced replication and transmission ability of the reassortant H9 viruses in guinea pigs We first evaluated the replication of the parental and reassortant viruses in guinea pigs. Groups of three guinea pigs were inoculated with 106 EID50 of each virus, and nasal washes and lung tissue from the guinea pigs were collected from the euthanized animals at 3 d.p.i for viral titration. All of the viruses replicated equally well in the nasal passages of guinea pigs. However, the reassortant H9 viruses replicated to significantly higher titers than the parental H9N2 virus TZ5 in the lung of guinea pigs (Table 2). In the transmission study, although the parental H9N2 virus TZ5 preferred to bind to the SAa2,6 receptor, it still failed to be transmitted to guinea pigs via direct contact and respiratory droplets. Unlike CA04, the parental pH1N1 viruses JS48 and JS285 lacked respiratory droplet transmissibility due to the Q226R mutation in HA (Fig. 5a), as has been demonstrated previously [41]. The six reassortant H9 viruses replicated to similar titers as the

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Fig. 4 Replication of parental and reassortant viruses in MDCK (a) and A549 (b) cells. Cells were infected at an MOI of 0.01. Virus titers were determined in MDCK cells at appropriate time points. Each data point on the curve indicates the mean ± standard deviation of three independent experiments. *p \ 0.05

parental pH1N1 viruses in the inoculated guinea pigs and were cleared in a similar time frame (Fig. 5b, yellow bars). However, in contrast to the parental H9N2 virus, all of the

Properties of influenza A virus reassortants Table 2 Virus replication and seroconversion in guinea pigs inoculated with or exposed to parental and reassortant influenza viruses Virus

Virus titer (mean log10 EID50 ± SD/ml)a

Seroconversion (positive/total)b

Nasal wash

Inoculated (HI titer)

Lung

Direct contact (HI titer)

Aerosol contact (HI titer)

TZ5

5.1 ± 0.3

1.8 ± 0.5

3/3 (160-320)

0/3 (\10)

0/3 (\10)

CA04

5.4 ± 0.4

3.6 ± 0.3

3/3 (320)

3/3 (160-320)

3/3 (80-320)

JS48

5.2 ± 0.3

3.4 ± 0.4

3/3 (80-160)

1/3 (80)

0/3 (\10)

JS285

5.5 ± 0.2

3.5 ± 0.2

3/3 (160-320)

3/3 (80-160)

0/3 (\10)

H9N1-CA04

5.3 ± 0.3

3.5 ± 0.3*

3/3 (160-320)

3/3 (160-320)

3/3 (40-160)

H9N2-CA04

5.6 ± 0.2

3.5 ± 0.2*

3/3 (160-640)

3/3 (80-160)

3/3 (80-320)

H9N1-JS48 H9N2-JS48

5.2 ± 0.5 5.4 ± 0.4

3.3 ± 0.2* 3.8 ± 0.7*

3/3 (160) 3/3 (160-320)

3/3 (160) 3/3 (80-160)

1/3 (160) 3/3 (80-160)

H9N1-JS285

5.2 ± 0.4

3.5 ± 0.3*

3/3 (320)

2/3 (80,160)

0/3 (\10)

H9N2-JS285

5.9 ± 0.6

3.4 ± 0.5*

3/3 (160-320)

3/3 (160)

1/3 (80)

* p \ 0.05 compared with the value for the parental H9N2 virus (TZ5) a

Groups of three guinea pigs were inoculated intranasally with 106 EID50 of each virus and euthanized at 3 d.p.i for virus titration

b

Sera were collected from the animals 3 weeks after virus inoculation or exposure

reassortant H9 viruses replicated to significantly higher peak titers at 2 d.p.i (p \ 0.05). Differences were observed in the exposure groups of guinea pigs. As shown in Fig. 5b (red bars), all six reassortant H9 viruses were able to be transmitted to direct-contact guinea pigs, but virus was only detected in two of the three direct contact guinea pigs in the H9N1-JS285 group. Additionally, H9N2-CA04 was transmitted with peak titers at 3 days post-exposure (d.p.e) while others showed peak titers at 5 d.p.e. In the respiratory droplet contact groups, differences were apparent in the efficiency of transmission. H9N1-CA04, H9N2-CA04 and H9N2-JS48 were transmitted with 100 % efficiency. However, one of the three guinea pigs in the H9N1-CA04 group shed virus later (7 d.p.e.) with a peak titer of 3.5 log10 EID50, which was[1,000fold lower than the peak titer in the other H9N1-CA04exposed animals. One of the three guinea pigs in the H9N2JS48 group also shed virus later (5 d.p.e.) but reached a slightly higher peak titer at 9 d.p.e. Nasal virus shedding was found in only one respiratory-droplet-contact guinea pig of the H9N1-JS48 and H9N2-JS285 groups; however, H9N1JS285 failed to be transmitted to guinea pigs via respiratory droplets. The results of antibody detection further demonstrated that seroconversion occurred only in guinea pigs that were shedding the virus (Table 2). Taken together, reassortant H9 viruses showed enhanced replication and transmissibility in guinea pigs compared with the parental H9N2 virus, and the H9N2-pH1N1viruses were transmitted more efficiently than the H9N1- pH1N1 viruses. Enhanced replication and pathogenicity of reassortant H9 viruses in mice To evaluate the virulence of parental and reassortant viruses, we used a BALB/c mouse model to determine the

MLD50. As shown in Table 3, all of the reassortant H9 viruses were significantly more virulent for mice than parental H9N2 virus TZ5, except H9N2-CA04. Furthermore, all of the H9N1- pH1N1viruses were significantly more virulent (C10 fold decrease in MLD50 values) for mice than their respective H9N2-pH1N1viruses. The H9N1-JS48 was the most virulent strain, with an MLD50 of 104.5 EID50, whereas the H9N2-CA04 was not lethal to mice at inoculation doses as high as 107.0 EID50 (Table 3). To investigate their morbidity and mortality, mice were inoculated intranasally with 106.0 EID50 of each virus. Approximately 30 % weight loss was observed in the JS48, H9N1-JS48 and H9N1-JS285 groups of mice, and all of the mice died within 9 days. JS285 and H9N1-CA04 caused around 25 % weight loss, with a 60 and 80 % mortality rate, respectively. In contrast, 20 % weight loss were observed in the H9N2-JS48 and H9N2-JS285 groups of mice, and the mortality rates were 60 and 40 %, respectively. However, no morbidity or mortality was observed in mice infected with TZ5, CA04 and H9N2-CA04 (Fig. 6). Further statistical analysis indicated that all of the reassortant H9 viruses differed significantly from the parental H9N2 virus TZ5 in terms of body weight loss (5 d.p.i) and time to death of mice. The same significant differences were also observed in the mice infected with H9N1-pH1N1 versus the corresponding H9N2-pH1N1viruses (Table 3). To determine if there was a difference in the ability of these viruses to replicate in vivo, we inoculated mice with 106 EID50 of each virus and analyzed virus titers in lungs at 3 d.p.i and 5 d.p.i. All of the reassortant H9 viruses replicated as efficiently in mouse lungs as the parental pH1N1viruses without prior host adaptation. However, we observed significantly higher titers in the lungs of mice infected with all of the reassortant H9 viruses when

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L. He et al. Fig. 5 Transmission of the parental viruses (a) and reassortant H9 viruses (b) in guinea pigs. Groups of three guinea pigs were inoculated intranasally with 106 EID50 of each indicated virus. After 24 h of inoculation, three naive guinea pigs were placed in the same cage as the direct-contact group, and the other three naive guinea pigs were placed in an adjacent cage as respiratory droplet contact group. Nasal washes were collected every 2 days from all animals, beginning at 2 d.p.i. for the detection of virus shedding. Each pattern bar represents the virus titer from an individual animal. The dashed horizontal line indicates the lower limit of detection

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Properties of influenza A virus reassortants Table 3 Pathogenicity of parental and reassortant influenza viruses in BALB/c micea Virus

MLD50 (mean log10 EID50 ± SD)

Weight change at 5 d.p.i (%, mean ± SD)

Day of death (mean ± SD)c

Virus titer (mean log10 EID50 ± SD/g) 3 d.p.i

5 d.p.i

TZ5

[6.5

?4.8 ± 2.0

[14

3.8 ± 0.4

2.5 ± 0.4

CA04

[6.5

-11.4 ± 2.1

[14

5.8 ± 0.4

4.9 ± 0.2

JS48

4.4 ± 0.3

-24.4 ± 3.8

5.6 ± 0.4

6.4 ± 0.3

5.6 ± 0.2 5.5 ± 0.4

JS285

6.2 ± 0.2

-25.9 ± 1.8

8.4 ± 1.5

6.4 ± 0.5

H9N1-CA04

5.5 ± 0.1*4°b

-14.6 ± 7.7*°

8.3 ± 1.2*4°

6.2 ± 0.4*

5.7 ± 0.4*

H9N2-CA04

[6.5°

[14°

5.5 ± 0.7*

5.0 ± 0.7*

H9N1-JS48 H9N2-JS48

4.5 ± 0.2*° 5.8 ± 0.2*4°

-24.2 ± 4.3*° -13.5 ± 4.4*4°

4.5 ± 0.3*D° 9.6 ± 1.2*4°

6.8 ± 0.5* 6.1 ± 0.5*

5.8 ± 0.5* 5.3 ± 0.2*

H9N1-JS285

5.1 ± 0.1*4°

-25.6 ± 3.1*°

4.2 ± 0.3*4°

6.8 ± 0.3*

5.5 ± 0.2*

11.1 ± 1.2*°

6.1 ± 0.5*

5.0 ± 0.6*

H9N2-JS285

6.2 ± 0.3°

-3.3 ± 4.4*4°

4

-14.5 ± 5.6* °

a

Groups of five BALB/c mice were inoculated intranasally with 10-fold serial dilutions of each virus for MLD50 detection. Groups of 16 BALB/c mice were inoculated intranasally with 106 EID50 of each virus; Ten mice from each group were used to determine average body weight changes and survival rates, and the other six mice were euthanized at 3 d.p.i and 5 d.p.i for virus titration * p \ 0.05 compared with the value for the parental H9N2 virus (TZ5). D p \ 0.05 compared with the value for the corresponding parental pH1N1 virus. ° p \ 0.05 compared with the value for the corresponding reassortant virus

b,

c

The mean day of death of mice was determined by the Kaplan–Meier method

reassortant H9 viruses showed enhanced replication and pathogenicity in mice compared with the parental H9N2 virus, and the H9N1-pH1N1viruses were more virulent than the H9N2-pH1N1 viruses. Discussion

Fig. 6 Average body weight changes (a) and survival rates (b) of BALB/c mice infected with the parental and reassortant viruses. Sixweek-old female BALB/c mice (n = 10/group) were inoculated intranasally with 50 ll containing 106 EID50 of the indicated virus, and each group was monitored daily for 14 days. The results are expressed as the mean ± SD

compared with parental H9N2 virus TZ5. H9N1pH1N1viruses replicated to a slightly higher titer than their correpsonding H9N2-pH1N1viruses, but this difference was not significant (Table 3). These results indicate that

Although H9N2 subtype influenza A virus is considered to be one of the candidate pandemic strains and occasional human infections have been reported, still little is known about its transmissibility in mammals. The fact that both H9N2 and pH1N1 viruses are able to infect pigs provides an opportunity for the reassortment and genesis of new strains with pandemic potential risk. The triple reassortant internal gene constellation of pH1N1 is considered to be stable and able to reassort with other influenza viruses [22]. Most of the reassortant pH1N1 viruses isolated in nature provide evidence that their internal gene constellation fits with the surface genes from other endemic swine influenza viruses [8, 13, 17, 24, 34, 45]. Previous studies also showed that the internal genes of pH1N1 provide a transmission advantage for the reassortant viruses, regardless of the surface genes [18, 21, 28, 45]. In the present study, we generated six reassortant viruses composed of the surface genes of an h9.4.2.5-lineage H9N2 and the internal genes of pH1N1 strains from both humans and swine to determine their compatibility in terms of replication ability, transmissibility and pathogenicity. Our in vitro study showed that all of the reassortant H9 viruses replicated to a higher titer than both the parental

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H9N2 and the corresponding pH1N1 viruses in MDCK and A549 cells at each time point, indicating that H9N2 AIV is able to gain the ability to replicate better in mammalian cells through reassortment. However, all of the H9N1pH1N1 and H9N2-pH1N1 viruses grew equally well, indicating that the mutations in the internal genes (especially polymerase genes) of swine pH1N1 isolates and different NA origin did not affect the replication of the reassortant viruses. In contrast, previous studies indicated that the reassortant viruses of G1-like lineage H9N1pH1N1 grew to significantly higher titres in vitro than the corresponding H9N2-pH1N1virus [21, 28], suggesting that the compatibility between pH1N1 and H9N2 viruses belonging to different HA lineages may be different. We also tested the ability of these reassortant H9 viruses to replicate in guinea pigs and mice. We found that the titers of the reassortant viruses in vivo were similar to those of the parental pH1N1 viruses but still higher than that of the parental H9N2 virus. These results further demonstrate that H9N2 AIV is able to adapt to mammals through reassortment with the pH1N1 virus. However, different transmission efficiencies and virulence were observed among these reassortant viruses, indicating that growth kinetics is not always consistent with transmissibility and pathogenicity. In the transmission study, we did not find that the parental H9N2 virus TZ5 could be transmitted between guinea pigs although it preferred to bind to the SAa2,6 receptor. In contrast, although the parental pH1N1 viruses JS48 and JS285 exhibited an increased preference for the SAa2,3 receptor when compared with CA04, they still could be transmitted to guinea pigs via direct contact. These results indicate that both preference for the SAa2,6 receptor and the gene constellation are important for the influenza virus to acquire transmissibility in mammals. All of the reassortant H9 viruses were transmitted efficiently to guinea pigs via direct contact, except for the H9N1-JS285 direct contact group, with 67 % efficiency and low peak titers. In the respiratory droplet contact groups, however, the H9N2-pH1N1viruses were transmitted more efficiently than the H9N1-pH1N1 viruses. These viruses differ only in the origin of their NA segment. A previous study indicated that the balance between HA and NA is critical for efficient respiratory-droplet transmission of influenza A viruses [39], suggesting that H9N2-pH1N1viruses with surface genes of shared origin are fitter than H9N1-pH1N1 viruses with surface genes of mixed origin. Recently, Kimble et al. demonstrated that two mutations (T189A and G192R) in the G1-like lineage H9 were needed for the reassortant H9N1-pH1N1 virus to acquire respiratory droplet transmissibility [21]. However, these two amino acid changes were absent in the HA of TZ5 belonging to h9.2.4.5. Interestingly, both H9N1-CA04 and H9N1-JS48 in this study gained respiratory transmissibility without adaptive

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amino acid substitutions in HA, indicating that the HA of h9.2.4.5 is fitter than the G1-like lineage H9 for reassortment with pH1N1, and that the transmission and compatibility between H9 and N1 are dependent on the linage of H9 to some extent. Compared to H9N1-CA04, decreased respiratory droplet transmission was detected in the H9N1JS48 group, though H9N2-JS48 was transmitted as efficiently as H9N2-CA04. The difference in the N1 proteins of CA04 and JS48 was only six mutations (Table 1), indicating that these small changes result in decreased fitness of this H9 and N1 gene combination for respiratory droplet transmission. In contrast, H9N2-JS285 was transmitted to only one out of three guinea pigs in the respiratory droplet contact group, suggesting that small changes in the internal genes also affect the efficiency of respiratory droplet transmission. The reassortant H9N1-JS285 virus with a less-fit H9 gene from H9N2 and an N1 gene from pH1N1 failed to be transmitted between guinea pigs via respiratory droplets. Pathologically, the reassortant H9 viruses also showed increased virulence in mice compared with the parental H9N2 virus, and the H9N1-pH1N1virus was more virulent than the corresponding H9N2-pH1N1 virus, which is consistent with the results of Sun et al. [35]. This finding shows that there is no necessary connection between transmissibility and pathogenicity. Despite their high genetic similarity, many pH1N1 strains vary considerably in their virulence in animal models [23, 31, 38]. Three parental pH1N1 viruses in this study also showed diverse pathogenicity in mice. Furthermore, a previous study demonstrated that introduction of known molecular markers determining pathogenicity in mammalian models, such as PB2 E627 K/ D701 N/E667G and PB1-F2, does not change the virulence of pH1N1 [12, 14, 44]. These findings indicate that there may be some unrecognized molecular mechanisms for pH1N1 to acquire pathogenicity in mammals. In the present study, differences in pathogenicity were also detected in separate H9N1-pH1N1 and H9N2-pH1N1groups, indicating that the internal gene constellation of pH1N1 virus does contribute to the virulence of the reassortant viruses and that the molecular markers of pH1N1 also fit with the surface genes of influenza viruses of other subtypes. Among these reassortant viruses, H9N1-JS48 deserves particular attention, as it was the most pathogenic one in mice and acquired respiratory droplet transmissibility between guinea pigs. Genetic reassortment and mutation are two major molecular mechanisms for influenza virus to evolve and adapt [5]. However, it is unknown if the internal genes of H9N2 contributed to the transmissibility or if other swine influenza viruses would participate in the reassortment. As the real situation in nature is much more complicated, further adaptive reassortment and mutation would be needed for the H9N2 subtype influenza virus to

Properties of influenza A virus reassortants

cross species barriers to adapt to swine and humans. Our results highlight the potential pandemic risk of H9N2 and pH1N1 virus coinfection and reassortment in pigs, emphasizing the importance of surveillance of these viruses in their natural hosts. Acknowledgments This study was supported by the Major State Basic Research Development Program (973 Program) (No. 2011CB505003), the National Natural Science Foundation of China (31101827), and the Research and Innovation Project of College Students in Jiangsu Province (No. CXZZ12_0914). We are also grateful to Jinhua Liu for kindly providing the 2009 pandemic H1N1 virus A/California/04/2009.

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Differences in transmissibility and pathogenicity of reassortants between H9N2 and 2009 pandemic H1N1 influenza A viruses from humans and swine.

Both H9N2 subtype avian influenza and 2009 pandemic H1N1 viruses (pH1N1) can infect humans and pigs, which provides the opportunity for virus reassort...
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