Background
Each of the known subtypes of the influenza A virus (H1 to H16 and N1 to N9) is circulating in water birds, especially in migratory ducks [
1]. A highly pathogenic avian influenza virus (HPAIV) is generated when a non-pathogenic virus brought in by migratory birds from nesting lakes in the north is transmitted to chickens via domestic ducks, geese, quails, turkeys, etc. and acquires pathogenicity for chickens with repeated multiple infections in the chicken population [
2‐
6]. The hemagglutinins (HAs) of HPAIVs differ from those of low pathogenic avian influenza viruses (LPAIVs) with a pair of di-basic amino acid residues at their cleavage site [
7]. This structure permits ubiquitous proteases such as furin and PC6, which recognize multiple basic amino acids, to cleave the HA, leading to systemic infection in chickens. By contrast, HAs of LPAIVs are cleaved only by trypsin-like proteases which are expressed in the cells lining the respiratory or intestinal tracts, so that the viruses cause only localized infections, resulting in mild or asymptomatic diseases. It is presently believed that the strains only with H5 or H7 HAs become HPAIVs during extensive infections in chicken populations [
8]. The reason why the subtypes of HPAIVs are restricted to H5 and H7 is not known although a model demonstrating that H5 HA is cleaved by furin through molecular docking analyses have been proposed [
9,
10].
H9N2 avian influenza virus strains have caused outbreaks in poultry, resulting in serious economic losses in Asia and the Middle East [
11‐
19]. The causal strains, however, are avirulent and none of them have multiple basic amino acid residues at the cleavage site of the HA [
12,
15]. No specific-pathogen-free chickens experimentally infected with H9N2 isolates from diseased chickens showed any clinical symptoms [
20]. Co-infection of H9N2 viruses with bacteria such as
Staphylococcus aureus and
Haemophilus paragallinarum or with attenuated coronavirus vaccine exacerbated the disease [
19,
21‐
23].
Since H9N2 viruses have been isolated not only from domestic birds but also from pigs and humans, the H9 virus has the potential to cause a next pandemic in humans [
17,
24‐
27]. It is important for controlling avian influenza and for preparing for pandemic influenza to assess whether the H9N2 virus aquires pathogenicity as H5 and H7 viruses. In the present study, we introduced a pair of di-basic amino acid residues into the cleavage site of the H9 and H5 HAs of non-pathogenic strains. These mutant H9 and H5 viruses were then serially passaged in the air sacs of chicks and their pathogenicity was assessed by inoculation to four-week-old chickens via intravenous and intranasal routes.
Discussion
Here, we demonstrated that the H9N2 influenza virus acquired intravenous pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the HA and serially passaged in chicks. Since rgY55sub-P10 (H9N2) killed 75% of chickens inoculated via intravenous route, the pathogenicity was comparable to that of HPAIVs (Table
1). On the other hand, chickens intranasally inoculated with rgY55sub-P10 (H9N2) did not show any clinical signs of disease (Table
4). These results are consistent with those of previous study showing that some H10 influenza viruses did not show intranasal pathogenicity for chicken while their intravenous pathogenicity index was over 1.2 and classified as HPAIV according to the definition by European Union [
29]. Amino acid changes during consecutive passages in the air sacs of chicks (Table
2) are considered to be responsible for the acquisition of intravenous pathogenicity, and their effects on the functions of viral proteins should be clarified further. Here we focused on two substitutions at positions 29 and 234 of the H9 HA molecule. It has been reported that residue 226, based on the H3 HA numbering (234 in the present study), relates to receptor specificity and cell tropism [
30]. Strain Y55 (H9N2) originally had a leucine at this position, and the change to glutamine after serial passages in the air sacs of chicks indicates that the passaged rgY55sub (H9N2) was further adapted to chicken. One of the asparagine-linked glycosylation sites on the HA of rgY55 (H9N2) lost a carbohydrate attachment with the substitution of Asn29His. The site locates sterically in the vicinity of the HA cleavage site, suggesting that the deletion of the carbohydrate chain affected the susceptibility of the HA to the host protease [
31]. This notion is also supported by the present finding that the rgY55sub viruses (H9N2) after six passages in the air sacs of chicks replicated in MDCK cells in the absence of trypsin (Table
1). Ohuchi et al. (1991) reported that the insertion of additional basic amino acids into the H3 HA cleavage site resulted in intracellular proteolytic cleavage. Other groups reported that H3 and H6 HAs tolerated amino acid mutations into their cleavage sites and the viruses with the mutated HAs replicated in MDCK and/or QT6 cells in the absence of trypsin [
32,
33]. The results in the present study are in agreement with these, namely, cleavage-based activation by an ubiquitous protease is not restricted to the H5 and H7 HAs.
rgVac1sub (H5N1) and rgVac1ins (H5N1) acquired marked intravenous and intranasal pathogenicity after a few passages in chicks (Table
1). It was reported that an avirulent H5 virus isolated from a swan became highly pathogenic in chickens after 24 consecutive passages in the air sacs, followed by five passages in the brains of chickens [
3]. The differences in time required for the viruses to become highly pathogenic between these studies depended on the amino acid motif at the HA cleavage site prior to passaging. rgVac1sub (H5N1) acquired an arginine at the HA cleavage site after only one passage in chickens (Figure
1B and Table
3), suggesting that an additional insertion of basic amino acid residues efficiently occurred in the serial basic amino acid residues at the cleavage site. One third of the chickens inoculated intranasally with rgVac1sub-P2 (H5N1) or rgVac1ins-P2 (H5N1) survived 14 days (Table
4). In addition, one of the birds was not susceptible to infection with rgVac1sub-P2 (H5N1), indicating that viral replication may depend on the presense of P3-like viruses in the inoculum.
The intranasal pathogenicity of the mutants of H9N2 virus was different from those of H5N1 mutants while these viruses replicated in MDCK cells in the absence of trypsin and killed chickens when inoculated via intravenous route (Tables
1 and
4). The viruses were recovered from the brain and the blood of some chickens infected with rgVac1 mutants (H5N1), and morbidity was closely associated with viral titers in the brain (Table
5). No viruses were recovered from the brain and the blood of chickens infected with rgY55 mutants (H9N2), indicating the reason why rgY55sub-P10 (H9N2) did not show intranasal pathogenicity. All the viruses passaged in the air sacs of chicks killed chicken embryos by 48 hours post allantoic inoculation (data not shown). rgVac1sub-P3 (H5N1) and rgVac1ins-P3 (H5N1) were more pathogenic to chicken embryos than rgY55sub-P10 (H9N2); the allantoic fluids obtained from the embryonated eggs inoculated with the H5N1 viruses passaged in the air sacs were turbid. It was reported that infection of a highly pathogenic virus was strictly confined to endothelial cells in chicken embryos or chickens [
34,
35]. Therefore, it is suggested that the difference of endotheliotropism between the H9N2 and H5N1 viruses passaged in the air sacs affected their intranasal pathogenicity. rgY55sub-P10 (H9N2) was not recovered from the brain and the blood of chickens although it caused systemic infection (Table
5), indicating that high levels of viremia followed by replication in the vascular endothelial cells was prerequisite for the virus to cross the blood-brain barrier and consequently replicated in the brain. This hypothesis is supported by the result that rgY55sub-P10 (H9N2) showed intravenous pathogenicity in chickens; direct injection of the virus to the blood vessels readily caused viremia, leading to invasion of the virus to the brain. (Table
1).
H9N2 viruses which have the PARSKR or PARSRR motifs at their HA cleavage site have been isolated from turkeys, ostriches, and chickens in Israel and quails in China [
14] although PARSSR motif has been found in most H9N2 isolates, indicating that such substitutions with basic amino acid residues occur in nature. If serine at the c-terminus of the HA1 of the H9 virus was substituted with lysine, the amino acid motif would be consistent with that of rgY55sub (H9N2) which acquired intravenous pathogenicity on consecutive passages in the air sacs of chicks. LPAI caused by H9N2 strains in poultry is now causing serious economic losses [
11‐
19], and its eradication is still difficult because of its low pathogenicity, frequently causing inapparent infections. The present study demonstrated that H9N2 viruses circulating in chicken flocks can acquire intravenous pathogenicity. It is predicted that co-infections of rgY55sub-P10 (H9N2) with bacteria exacerbate not only intravenous pathogenicity but intranasal pathogenicity in chickens as shown in a previous study [
21]. Therefore, continuous monitoring in poultry is important to prevent the emergence of pathogenic H9 viruses.
Materials and methods
Viruses
A/chicken/Yokohama/aq-55/2001 (Y55) (H9N2) isolated from chicken meat imported from China upon quarantine was kindly provided by Dr. M. Eto, Animal Quarantine Service (Yokohama, Kanagawa, Japan) [
36]. A/duck/Hokkaido/Vac-1/2004 (Vac1) (H5N1) was generated by the standard genetic reassortment procedure from non-pathogenic viruses, A/duck/Mongolia/54/2001 (H5N2) and A/duck/Mongolia/47/2001 (H7N1) [
37‐
39]. Viruses were propagated in ten-day-old embryonated chicken eggs for 48 hours at 35°C.
The complete nucleotide sequences of Y55 (H9N2) and Vac1 (H5N1) have been registered in GenBank/EMBL/DDBJ (Accession numbers: AB256671-AB256678 [
36] and AB259709-AB259716 [
37], respectively).
Reverse genetics
Viral RNA was extracted from the allantoic fluid of embryonated chicken eggs infected with the Y55 and Vac1 strains using a commercial kit (TRIzol LS Reagent, Sigma-Aldrich, St. Louis, MO, U.S.A) and reverse-transcribed with the Uni12 primer [
40] and M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, U.S.A). PCR-based amplification of the full genomes of the eight gene segments was performed with universal primer sets [
41]. The PCR products were cloned into the vector pCR2.1-TOPO (Invitrogen) or pGEM-T Easy Vector (Promega, Mannheim, Germany). After confirmatory sequencing, T-vector clones were digested with
BsmBI and inserted into the vector pHW2000 [
42]. MDCK cells and 293T cells were maintained in Minimum Essential Medium (MEM, Nissui Pharmaceutical, Tokyo, Japan) containing 10% calf serum and D-MEM (Invitrogen) containing 10% FBS, respectively. Before transfection, confluent 293T and MDCK cells in 75 cm
2 flasks were trypsinized, and 10% of each cell line was mixed in 12 ml of Opti-MEM I (Invitrogen); 2 ml of the suspension was seeded into each well of six-well tissue culture plates (Nunc Inc., Naperville, IL). The cocultured 293T and MDCK cells were used for the transfection. TransIT-293 (Panvera, Madison, WI) was used to transfect cells according to the manufacturer's directions. Briefly, two microliters of TransIT-293 per microgram of DNA was mixed, incubated at room temperature for 45 minutes, and added to the cells. The transfection mixture was replaced with Opti-MEM I after six hours of incubation at 37°C. Thirty hours later, Opti-MEM I containing one microgram per microliter of trypsin was added. At 48 to 72 hours post-transfection, the culture supernatant was collected and propagated in ten-day-old embryonated chicken eggs.
Site-directed-mutagenesis
To generate H9 and H5 mutant viruses with basic amino acid residue substitutions (sub) or insertions (ins) at the HA cleavage site, mutations were introduced into the HA genes of the Y55 and Vac1 strains using a QuikChange II site-directed mutagenesis kit (Stratagene, Heidelberg, Germany) according to the manufacturer's instructions. The mutant viruses, rgY55sub (H9N2), rgVac1sub (H5N1), and rgVac1ins (H5N1), were rescued by reverse genetics as described above, and the entire genomes of the eight gene segments were sequenced to confirm the existence of the introduced mutations and the absence of undesired mutations.
Plaque assay
Ten-fold dilutions of viruses were inoculated onto confluent monolayers of MDCK cells and incubated at 35°C for one hour. Unbound viruses were removed by washing the cells with MEM. Cells were then overlaid with MEM containing 0.7% Bacto-agar (Difco, Sparks, MD) in the presence or absence of trypsin (5 μg/ml). After 48 hours of incubation at 35°C, cells were stained with 0.005% neutral red.
Consecutive passage in the air sacs of chicks
The caudal thoracic air sacs of three 3-day-old chicks were inoculated with 200 μl of each of the mutant Y55 and Vac1 viruses. The chicks were sacrificed, and their lungs and brains were collected at three days post-inoculation. Serial passages in the air sacs of three to six 3-day-old chicks were performed with 200 μl of a pooled 10% tissue suspension of infected organs. Brain samples were used as the inoculum when both samples (lungs and brains) tested positive for the virus. Isolates were identified by their parental strain's name, mutation (substitution or insertion), and number of passages. For example, the designation rgY55sub-P10 (H9N2) indicates that the amino acids at the HA cleavage site of the Y55 virus were substituted with basic amino acids as shown in Figure
1A, then passaged ten times in the air sacs. Passaged viruses were propagated in the allantoic cavities of ten-day-old embryonated chicken eggs for 48 hours at 35°C. The allantoic fluid was harvested and stored at -80°C.
Experimental infection of chickens with mutant virus strains
Four-week-old Boris Brown chickens were used to test the pathogenicity of the passaged viruses. Eight chickens were intravenously inoculated with 200 μl of each virus (1:10 diluted allantoic fluid), and examined for clinical signs at intervals of 24 hours over a period of ten days. Similarly, three chickens were infected intranasally with 100 μl of allantoic fluid containing each virus at a EID
50 of 10
6.5 and observed for 14 days. Specific antibodies against homologous viruses after 14 days of infection were detected in serum using the hemagglutination inhibition (HI) test and/or enzyme-linked immunosorbent assay (ELISA) as described previously [
43]. To study viral replication, each virus was inoculated into three chickens at an EID
50 of 10
6.5. The birds were euthenized three days post-challenge, and tissue and blood were collected aseptically. To make a 10% suspension with MEM, the tissue samples were homogenized using a Muti-Beads Shocker (Yasui Kikai, Osaka, Japan). These suspensions were serially diluted ten-fold with PBS and inoculated into ten-day-old embryonated eggs and incubated at 35°C for 48 hours. Viral titers were calculated by the method of Reed and Muench [
44] and expressed as EID
50 per gram and milliliter of tissue and blood, respectively.
All experiments were carried out in self-contained isolator units (Tokiwa Kagaku, Tokyo, Japan) at a BSL3 biosafety facility at the Graduate School of Veterinary Medicine, Hokkaido University, Japan. The experiments were performed according to the guidelines of the institutional animal care and use committee of the Graduate School of Veterinary Medicine.
Acknowledgements
The present study was supported by the Programs of Founding Research Centers for Emerging and Re-Emerging Infectious Diseases from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and Solution-Oriented Research for Science and Technology (SORST) from the Japan Science and Technology Agency (JST).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HK is the leader of the study group. KS carried out the experiments and wrote the manuscript. SA helped in passaging study. KS, MO, YS, and HK designed the experiments and analyzed the data. All authors read and approved the final manuscript.