Abstract
Seventeen recombinant viruses were generated by a reverse genetic technique to elucidate the pathogenicity of highly pathogenic avian influenza viruses (HPAIVs) in chickens. The recombinant viruses generated possessed hemagglutinin (HA) and neuraminidase (NA) genes from an HPAIV. Other segments were combinations of the genes from an HPAIV and two low-pathogenic avian influenza viruses (LPAIVs) derived from chicken (LP) and wild bird (WB). Exchange of whole internal genes from an HPAIV with those of an LPAIV resulted in a significant extension of the survival time following intranasal infection of the chickens with the recombinants. Survival analysis demonstrated that the exchange of a gene segment affected survivability of the chickens with statistical significance. The analysis revealed three groups of recombinants with various gene constellations that depended upon the survivability of the infected chickens. Recombinants where the PA gene was exchanged from LP to WB in the LP gene background, LP (W/PA), did not kill any chickens. LP (W/PA) replicated less efficiently both in vitro and in vivo, suggesting that the intrinsic replication ability of LP (W/PA) affects pathogenicity; however, such a correlation was not seen for the other recombinants. Microarray analysis of the infected chicken lungs indicated that the expression of 7 genes, CD274, RNF19B, OASL, AC3HAV1, PLA2G6, GCH1, and USP18, correlated with the survivability of the chickens infected (P < 0.01). Further analysis of the functions of these genes in chickens would aid in the understanding of host gene responses following fatal infections by HPAIVs.
INTRODUCTION
The natural host of type A influenza virus is wild waterfowl. Most of these birds show no clinical sign when infected, and they serve as a reservoir of type A influenza viruses in nature. In 1996, a highly pathogenic avian influenza virus (HPAIV) of the H5N1 subtype was identified in geese in Guangdong Province, China, and the mortality rate of the infected geese was approximately 40% (50). In 1997, outbreaks of HPAI in chickens due to the H5N1 subtype were recognized in Hong Kong and were accompanied by human casualties due to the virus. HPAIVs of the H5N1 subtype isolated in Hong Kong were considered to be viruses reassorted with the Guangdong virus from geese in 1996 and other subtype influenza viruses (2). It was revealed that the hemagglutinin (HA) segment of A/Hong Kong/156/1997, a representative isolate of the Hong Kong human H5N1 virus, was derived from A/goose/Guangdong/1/1996 (H5N1), while the other segments were similar to the HA segment of the H9N2 subtype A/quail/Hong Kong/G1/1997 (HK97). In addition, those segments were suggested to have been derived from the A/teal/Hong Kong/W312/1997-like H6N1 virus. The mortality rates of poultry infected with the HK97 virus were much higher (70 to 100%) than those in the Guangdong outbreak (2), suggesting that reassortment of the internal genes contributed to the pathogenicity. The HK97 viruses are now recognized to be ancestors of the H5N1 subtype HPAIVs that have subsequently been circulating worldwide (16, 21). The virus has been evolving continuously, and on the basis of the nucleotide sequence identity, the HA gene of the H5N1 HPAIVs has been classified into 9 clades, with several subclades occurring in clade 2 (WHO [http://www.who.int/csr/disease/avian_influenza/guidelines/nomenclature/en/index.html]).
The molecular basis of the pathogenicity of HPAIV has not been fully elucidated. Multiple basic amino acids at the cleavage sites of HA proteins of the H5 and H7 subtypes are determinants of high mortality in chickens (23, 43, 48). They are responsible for the cleavability of the HA0 protein into the HA1 and HA2 subunits by a cellular proteinase(s) ubiquitously expressed in organs, allowing systematic infection in chickens (45). The PB2 protein present in mitochondria was recently found to increase the pathogenicity of HPAIV by inhibiting beta interferon (IFN-β) induction through interaction with mitochondrial antiviral signaling (MAVS) proteins (15). PB1-F2, transcribed from another translation frame of the PB1 gene, is also considered to take part in the expression of pathogenicity in hosts by inducing apoptosis of the infected cells through its C-terminal mitochondrion-targeted sequence (6, 14, 53). NS1 is also known to relate with the HPAIV pathogenicity by inhibiting antiviral reaction in hosts. NS1 inhibits type I interferon and the expression of the cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and innate and adaptive immunities (10, 38). Several animal experiments using reassortant viruses have determined viral factors for pathogenicity (24, 25, 32, 46, 54). Involvement of the PB2 segment in enhancing virulence in mice was demonstrated following infection of the recombinant virus containing the PB2 gene segment from the human influenza virus and others from the H5N1 subtype HPAIV without pathogenicity to the mice (29).
Microarray analysis has been used as a strategy to investigate the correlation between host gene response and pathogenicity by measuring mRNA expression levels of gene bulks in infected hosts. The microarray analysis indicated that of the innate immune response genes, the interferon response genes were strongly expressed in ferrets infected with the H5N1 HPAIV (4). A microarray analysis of chicken embryo fibroblasts infected with two H5N1 subtype HPAIVs of different characteristics revealed that expression of MEK2, PDCD10, Bcl-3, and IFN-α mRNAs was significantly different (42). Those studies demonstrated the usefulness of microarray analysis for identifying host genes involved in the manifestation of pathogenicity in infected hosts.
To investigate the influence of viral internal gene segments on pathogenicity in chickens, we generated 17 recombinant viruses possessing common surface antigens derived from an H5N1 subtype HPAIV having a combination of the internal gene segments from two low-pathogenic avian influenza viruses. These reassortant viruses showed mean survival times (MSTs), degrees of cyanosis, and even mortality rates different from those of the experimentally infected chickens, demonstrating the involvement of internal genes in the pathogenicity of HPAIV in chickens. We took advantage of the set of reassortant viruses with various pathogenicities to carry out a microarray analysis of the lungs obtained from the infected chickens. The purpose was to identify the group of host genes whose expression correlated with the survivability of the infected chickens. The pathogenicity of a viral infection is the sum of reactions by the host against infection and virulent viral factors. Thus, to explore a novel method to prevent HPAI in poultry, viral factors as well as the host gene response to pathogenic manifestations need to be specified.
MATERIALS AND METHODS
Viruses and cells.
Viruses used in this study were the H5N1 subtype HPAIV (A/chicken/Yamaguchi/7/2004 [abbreviated HP]) (34), a low-pathogenicity avian influenza virus of the H5N3 subtype (A/whistling swan/Shimane/580/2002 [abbreviated Shimane]; kindly provided by T. Ito, Tottori University), and an H9N2 subtype virus (A/chicken/Yokohama/aq55/2001 [abbreviated Yokohama]) (33).
293T cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with l-glutamine (584 μg/ml), 10% fetal calf serum (FCS), 1% penicillin-streptomycin liquid (10,000 U/ml/10,000 μg/ml), and amphotericin B (Fungizone; 2.5 μg/ml). Madin-Darby canine kidney (MDCK) cells were cultured in minimum essential medium (MEM) supplemented as described above. Primary chicken kidney (CK) cells were cultured in DMEM supplemented with NaHCO3 (1.2 mg/ml), l-glutamine (292 μg/ml), 5% FCS, 1% penicillin-streptomycin liquid (10,000 U/ml/10,000 μg/ml), and amphotericin B (1 μg/ml). These cells were maintained at 37°C in 5% CO2.
Plasmid construction.
All eight segments of the viruses used in this study were amplified by PCR with KOD DNA polymerase (KOD plus, version 2; Toyobo) primers described by Hoffmann et al. (21a). The PCR products were cloned by a Zero Blunt TOPO PCR cloning kit (Invitrogen), followed by subcloning, after BsmBI or BsaI and phosphatase treatment, into a pHW2000 expression vector (kindly provided by E. Hoffmann, G. Neumann, Y. Kawaoka, G. Hobom, and R. G. Webster, St. Jude Children's Research Hospital, Memphis, TN). A DNA transfection system was used to generate the influenza A virus from eight plasmids (20). The identities of the inserted genes were confirmed against the original by nucleotide sequencing.
Production of recombinant viruses.
293T (4.0 × 105/well) cells and MDCK cells (1.0 × 105/well) were mixed and grown in DMEM in a six-well cell culture plate at 37°C under 5% CO2 1 day before transfection. Prior to the transfection, DMEM was replaced with Opti-MEM (Invitrogen). Eight plasmids (1 μg/μl each plasmid) were transfected into the mixed cells with TransIT-LT reagent (Invitrogen). Supernatant of the transfected cells was collected 48 h after transfection and transferred onto the MDCK cells with an infection medium (MEM supplemented with 0.4% bovine serum albumin, 1% penicillin-streptomycin liquid [10,000 U/ml/10,000 μg/ml], amphotericin B [2.5 μg/ml], and 3% MEM vitamin solution) for the replication of the recombinant viruses. At 48 to 72 h after the inoculation, the supernatant was collected and titrated to obtain the 50% egg infective dose (EID50) by the Reed and Muench method (40). Viruses with titers below 106 EID50s/100 μl were inoculated into eggs for propagation. HP and the recombinant viruses with the HA gene from HP were handled in the biosafety level 3 facilities at the National Institute of Animal Health in Japan.
Animal experiments.
Four-week-old specific-pathogen-free (SPF), white leghorn, L-M-6 strain chickens were obtained from Nisseiken Co., Ltd. Viruses at 106 EID50s/100 μl were inoculated intranasally. Six chickens were infected with HP and recombinant HP (rHP), and 15 chickens were infected with low-pathogenic avian influenza viruses derived from chicken (LP) and wild bird (WB). Clinical signs and survival rates were recorded for 7 days for chickens infected with HP, rHP, LP, and WB and for 10 days for chickens infected with the other recombinants. The survival rate and period of infection of the chickens were subjected to survival analysis, tracheal and cloacal swab specimens from the infected chickens were titrated, and the degree of cyanosis was recorded as described below. All animal experiments were carried out in the biosafety level 3 animal facilities at the National Institute of Animal Health in Japan. The experimental procedures and care of animals were approved by the Animal Experiment Committee of the National Institute of Animal Health.
Survival analysis.
Kaplan-Meier survival curves (27a) were plotted for the survival analysis using survival rate and period for each group of chickens. Differences in the Kaplan-Meier survival curves were analyzed by the log-rank test under the Bonferroni correction. In the survival analysis, survival curves for each group of chickens were compared against those obtained for chickens infected with HP, rHP, LP, and WB.
Titration of viruses in the respiratory and intestinal tracts.
Tracheal and cloacal swab specimens were collected from the infected chickens individually, and swabs were dipped into MEM containing 0.5% bovine serum albumin, 25 μg/ml of amphotericin B, 1,000 units/ml of penicillin, 1,000 μg/ml of streptomycin, 0.01 M HEPES, and 8.8 mg/ml of NaHCO3. Specimens were collected at 3, 5, 7, and 10 days postinfection (dpi) and at the death of the chickens. Swabs were removed from MEM and stored at −80°C until titration. The frozen samples were thawed and centrifuged at 3,000 rpm for 5 min at 4°C. The supernatant was used to calculate the viral values in the tracheal and cloacal swabs by EID50 determination.
Investigating intrinsic ability of viral replication in vitro.
Replication of representative viral recombinants from the different groups classified by the survival analysis was measured to investigate viral intrinsic ability. Primary chicken kidney cells were infected with the recombinants at 10−4 EID50/cell in the infection medium. Supernatants of the cells were collected preinfection and at 24 h postinfection (hpi), 48 hpi, 72 hpi, and 96 hpi and were frozen at −80°C until titration. Before titration, the frozen samples were thawed and centrifuged at 3,000 rpm for 5 min at 4°C. Tenfold serially diluted supernatant was inoculated into embryonated chicken eggs to calculate the EID50. Virus titers at each time point were analyzed statistically by analysis of variance (ANOVA) under the Bonferroni correction.
Scoring of cyanosis degree.
The degree of cyanosis at the comb, wattle, and legs was individually scored on a scale ranging from 0 to 3, as shown in the figure in the supplemental material. The highest score at each site during the observation period was averaged for each group, and the total score was the sum of the average at each site.
Microarray analysis.
Specimens for microarray analysis were collected at 24 h postinfection since no significant differences in gene expression were seen at an earlier time in the preliminary study. Moreover, some of the chickens infected with recombinant viruses started to die after 24 h postinfection. For the microarray analysis, two chickens were infected with seven representative viruses. At 24 h postinfection, chickens infected with each virus were euthanized to collect lung tissue samples, which were individually homogenized with a Precellys 24 tissue homogenizer (Bertin). The lung homogenates were treated with TRIzol reagent (Invitrogen) to extract total RNA, which was then purified using a Qiagen RNeasy minikit (Qiagen). Total RNA was treated with DNase using an RNase-free DNase set (Qiagen). Total RNA from each lung tissue specimen was reverse transcribed to cDNA and then labeled with Cy3 and hybridized to a Nimblegen chicken genome array according to the manufacturer's protocol. A total of 38,681 chicken genes were loaded on the microarray chip. The hybridized gene chip was scanned, and the array data were collected. The chicken gene data were annotated with human orthologs using BLAST, based on the chicken reference sequence mRNAs of the National Center for Biotechnology Information (NCBI), assuming that chicken genes function similarly to the human orthologs. Survival analysis was performed on the array data to find genes whose expression correlates with the survival time that fits Cox's proportional hazards model. The analysis was performed using the survival analysis function of the BRB-Array Tools program (version 3.6.1), developed by Richard Simon and Amy Peng Lam. A parametric P value was calculated by the analysis, and the genes were extracted under P values of <0.05 and 0.01. The false discovery rate (FDR), which shows the possibility of pseudopositive results for probes, was not calculated because of the insufficient sample size in each group. Accumulation of genes correlated with the survival time. The categories of the biological process, based on the GeneOntology (GO) website (http://www.geneontology.org/), were analyzed by Fisher's exact test. Biological processes with a P value below 0.01 are listed in the Table S1 in the supplemental material. For the clustering of samples based on the expression of each probe extracted under a P value of <0.01 by survival analysis, the average of the expression values of two samples in each infected group was converted to a log2 ratio against the average of the control. Hierarchical clustering was performed by Pearson's correlation using MeV_4_6_1 software and the average linkage method as the linkage and distance matrix.
Real-time PCR for chicken USP18 and RNF19B.
cDNA was synthesized by reverse transcription (RT) from the total RNA used for the microarray analysis by a SuperScript III first-strand synthesis system (Invitrogen). RT-PCR was performed by the intercalation method using SYBR Premix Ex Taq (TaKaRa) and specific primers. Primer sequences for chicken USP18 and RNF19B were designed on the basis of the sequences with Gene ID accession numbers 418167 and 419669, respectively, which were available at NCBI. Those for chicken USP18 were 5′-CAACGTGGGAAGAGGAGAAA-3′ (forward) and 5′-ACTTCATGAGCGGAGAAGGA-3′ (reverse), and those for RNF19B were 5′-GGCTCCATCATCAGCTCCTA-3′ (forward) and 5′-ATTCTCTGCCATTTGGGTGT-3′ (reverse). The reaction was performed at 50°C for 2 min, at 95°C for 10 min, and with 40 cycles of 95°C for 15 s and 60°C for 60 s using an Applied Biosystems 7500 apparatus. The sample data from RT-PCR were calculated by the comparative threshold cycle (CT) method, using CT values of the beta-actin gene as the reference.
Microarray data accession number.
Raw microarray data were deposited in the Gene Expression Omnibus (GEO) of the NCBIm and the accession number is GSE32378.
RESULTS
Internal gene constellation of viruses with HA of a highly pathogenic form affects the survival time of infected chickens.
Reassortant viruses from three influenza viruses were generated by the reverse genetic technique to investigate the effects of the internal gene constellations on pathogenicity in chickens. Each internal genetic segment originated from Ck/Yamaguchi/7/2004 (H5N1; abbreviated HP), Ck/Yokohama/aq55/2001 (H9N2; Yokohama), and Whistling swan/Shimane/580/2002 (H5N3; Shimane). Chicken mortality following intranasal inoculation of the HP strain was 100%, with an MST of 2.0 days (see Table 2). It replicated well in the respiratory and intestinal organs (26, 37). The Yokohama and Shimane strains were low-pathogenic avian influenza viruses that did not kill chickens following intranasal infection. Yokohama replicates efficiently in the respiratory organs of chickens, while the Shimane strain does not replicate in chickens at all.
Table 2.
Survivability of each group of chickens infected with the reassortants
| Strain name | Mean survival time (days) | Survival rate (%) | Survival analysisa | Categorized groupb |
|---|---|---|---|---|
| HP | 2.00 | 0.00 | ***/**** | S |
| rHP | 2.00 | 0.00 | ***/**** | S |
| WB (L/PB1) | 2.00 | 0.00 | *** | S |
| LP (W/MNS) | 2.25 | 0.00 | *** | S |
| WB (L/PA) | 3.00 | 0.00 | ||
| LP (W/NS) | 3.00 | 0.00 | * | M |
| WB | 3.33 | 0.00 | */** | M |
| LP (W/PB2) | 3.33 | 0.00 | * | M |
| WB (L/M) | 3.75 | 0.00 | */** | M |
| LP | 3.87 | 6.67 | */** | M |
| LP (W/M) | 4.00 | 0.00 | * | M |
| LP (W/NP) | 4.33 | 0.00 | * | M |
| WB (L/NP) | 7.00 | 50.00 | */** | M |
| LP (W/PB1) | 7.50 | 50.00 | */** | M |
| WB (L/PB2) | 8.25 | 25.00 | */**/**** | L |
| WB (L/NS) | 9.25 | 75.00 | */**/**** | L |
| WB (L/MNS) | 9.25 | 50.00 | */**/**** | L |
| LP (W/PA) | 10.00 | 100.00 | */**** | L |
Statistical significance (P < 0.05) was observed against the result of HP (*), rHP (**), LP (***), or WB (****) by survival analysis.
Categorized group: M contained groups which differentiated from HP and rHP, while they did not differentiate from LP or WB. Virus groups differentiated from LB or WB and shorter MSTs were categorized into group S, while those with longer MSTs were categorized into group L.
The recombinant virus designated rHP consists of genetic segments entirely from HP. The recombinants designated LP and WB possessed HA and NA segments from HP, while the rest of the internal gene segments were from Yokohama and Shimane (Table 1). The MST and survival rate of chickens infected intranasally with rHP were 2 dpi and 0%, respectively, which were similar to the values for HP. MSTs of LP and WB were 3.9 dpi and 3.3 dpi, respectively, and the respective survival rates of the infected chickens were 6.7% and 0% (Table 2). MSTs of the chickens infected with LP and WB were 1.3 to 1.9 days longer than those of the chickens infected with HP and rHP. It was clearly demonstrated that LP and WB were distinguishable from HP and rHP by the survival analysis. It thus appeared that differences in internal gene segments could affect the MST of chickens infected with viruses that have cleavable HA proteins. The survival analysis showed no statistically significant difference between the LP and WB infected chickens.
Table 1.
Constellations of genetic segments in each reassortant generated by reverse genetics
| Strain namea | Segmentb |
|||||||
|---|---|---|---|---|---|---|---|---|
| HA | NA | PB2 | PB1 | PA | NP | M | NS | |
| rHP | ||||||||
| LP | ||||||||
| WB | ||||||||
| LP (W/PB2) | ||||||||
| LP (W/PB1) | ||||||||
| LP (W/PA) | ||||||||
| LP (W/NP) | ||||||||
| LP (W/M) | ||||||||
| LP (W/NS) | ||||||||
| LP (W/MNS) | ||||||||
| WB (L/PB2) | ||||||||
| WB (L/PB1) | ||||||||
| WB (L/PA) | ||||||||
| WB (L/NP) | ||||||||
| WB (L/M) | ||||||||
| WB (L/NS) | ||||||||
| WB (L/MNS) | ||||||||
In the strain names, the parenthetical L and W represent LP and WB, respectively, and the exchanged gene segments are shown after the slash mark. LP or WB means that internal segments other than the parenthetical segment recombinant virus were used in the construction.
Dark gray shading, rHP segment derived from the corresponding gene from A/chicken/Yamaguchi/7/2004; no shading, LP segment derived from the corresponding gene from A/chicken/Yokohama/aq55/2001; light gray shading, WB segment derived from the corresponding gene from A/whistling swan/Shimane/580/2002.
To further investigate the effects of internal gene constellations on the MSTs and survival rates of chickens infected with recombinant viruses, one or two internal segments of LP and WB were cross exchanged to generate the 14 recombinant viruses shown in Table 1. The MSTs ranged from 2.0 to 10.0 days, with survival rates being from 0 to 100%. On the basis of the statistical differences obtained by the survival analysis, recombinant viruses were classified into three groups, designated groups S, M, and L (Table 2). Viruses that showed a statistically significant difference for LB or WB and that resulted in shorter MSTs were classified into group S, while those that resulted in longer MSTs were classified into group L. Nine recombinant viruses were classified into group M. Two viruses in group M showed relatively longer MSTs and higher survival rates than the rest of the recombinants in the group. The reason for the lack of statistical significance between the two viruses and the rest of group M in the survival analysis is possibly the effect of a censored variable on the 10-day observation period. Four viruses statistically distinguishable from LP or WB by the survival analysis were classified into group L.
Exchange of the PA gene in LP with that from WB, LP (W/PA), resulted in the extension of MST and increased the survival rate. Reversal exchange of WB to WB (L/PA) resulted in the shortening of MST, suggesting a negative effect of the PA gene of the Shimane strain on the pathogenicity in chickens. Exchange of the PB2 gene in WB with that from LP, WB (L/PB2), also resulted in the extension of MST and an increased survival rate. However, in the reversal exchange, LP (WB/PB2) had no effect on MST or survival rate. The PB1 gene derived from LP likely promotes pathogenicity. Reversal exchange of LP to LP (WB/PB1) tended to extend MST, although the survival analysis did not show any statistical significance between LP and LP (WB/PB1). The NS gene of the Yokohama strain appeared to negatively affect the pathogenicity of the WB recombinants.
Correlation between virus replication in vivo and in vitro and pathogenicity in infected chickens.
Virus titers in the tracheal and cloacal swabs were measured to examine whether virus replication in the respiratory and intestinal tracts correlate with MSTs (Table 3). Swab samples were taken from the trachea and cloaca of the live infected chickens on 3, 5, 7, and 10 dpi. In addition, the samples were taken from the chickens found dead at the time of the twice daily observations. The highest virus titers in the tracheal and cloacal swab samples from each chicken were observed on 2 to 3 dpi for group S, 2 to 6 dpi for group M, and 5 to 7 dpi for group L, indicating that the delay in viral replication in vivo likely correlates with the extension in the survival period. The highest virus titers in the tracheal or cloacal swab specimens collected from each infected chicken were averaged within the same group. Swab titers of rHP were statistically significantly higher in both the trachea and cloaca than those of LP (W/MNS) and WB (L/PB1), although they were all classified in group S, suggesting that virus replication in vivo alone does not determine the survival period. The highest titers of WB (L/PB1) in group S and LP (W/PA) in group L were similar; however, the kinetics for virus replication in vivo and the survival rates appeared to be different. The highest titer obtained for WB (L/PB1) was at 2 dpi, while that for LP (W/PA) was at 5 dpi, and all the chickens infected with LP (W/PA) survived during the observation time. The highest titer of WB (L/NP) was statistically significantly different from that of LP; although both belonged to group M, the survival period of WB (L/NP) was longer than that of LP (7 dpi and 3.87 dpi, respectively), and the survival rate of WB (L/NP) was higher than that of LP (50% and 6.67%, respectively). The tracheal swab specimen titer of WB was also statistically different from that of LP. The variance in titer of each of LP and WB likely caused the statistically significant difference between LP and WB; however, the highest titers did not result in much of a difference.
Table 3.
Virus titers in trachea and cloacal swabs at scheduled sampling date and at the day the chicken died
| Tissue and category | Strain name | Avg EID50 for tissue/swab specimen each day postinfection (EID50/ml)a |
Avg maximum virus titer (ml)b | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | |||
| Trachea | |||||||||||
| S | rHP | 6.86 ± 1.04 | − | − | − | − | − | − | − | − | 6.86 ± 1.04 |
| WB (L/PB1) | 2.70 ± 1.46 | − | − | − | − | − | − | − | − | 2.70 ± 1.46** | |
| LP (W/MNS) | 4.20 ± 0.88 | 1.32 | − | − | − | − | − | − | − | 3.48 ± 1.61** | |
| M | LP (W/NS) | + | 2.64 ± 0.39 | − | − | − | − | − | − | − | 2.64 ± 0.39 |
| WB | 2.95 ± 0.61 | 2.72 ± 1.27 | 2.42 | 3.69 ± 0.93 | 3.02 | − | − | − | − | 3.11 ± 1*** | |
| LP (W/PB2) | + | 1.20 ± 1.45 | 5.53 | − | − | − | − | − | − | 2.09 ± 1.45 | |
| WB (L/M) | + | 2.23 ± 0.78 | 3.76 | 5.32 | − | − | − | − | − | 3.51 ± 1.77 | |
| LP | + | 4.52 ± 1.13 | 5.44 ± 0.45 | 3.88 ± 1.73 | 3.70 | 0.32 | − | − | − | 4.88 ± 1.00 | |
| LP (W/M) | + | 0.31 ± 0.19 | 5.02 | 4.20 | − | − | − | − | − | 3.14 ± 2.58 | |
| LP (W/NP) | + | 2.09 ± 0.90 | 4.47 | 2.87 | − | − | − | − | − | 3.94 ± 1.68 | |
| WB (L/NP) | + | 1.54 ± 0.34 | + | 1.31 ± 0.97 | + | 0.20 | + | + | 0.20 | 1.62 ± 0.40*** | |
| LP (W/PB1) | + | 0.22 ± 0.05 | + | 3.52 ± 0.75 | + | 0.26 | + | + | 0.20 | 3.52 ± 0.75 | |
| L | WB (L/PB2) | + | 1.03 ± 0.58 | + | 2.43 ± 2.12 | + | 2.72 ± 1.95 | + | 1.02 | 0.20 | 3.34 ± 2.26 |
| WB (L/NS) | + | 0.51 ± 0.36 | + | 1.02 ± 0.62 | + | 0.20 ± 0 | + | + | 0.20 ± 0 | 1.08 ± 0.57 | |
| WB (L/MNS) | + | 0.20 ± 0 | + | 1.34 ± 1.16 | + | 0.56 ± 0.56 | + | + | 0.20 ± 0 | 1.34 ± 1.16 | |
| LP (W/PA) | + | 0.20 ± 0 | + | 3.11 ± 0.73 | + | 0.20 ± 0 | + | + | 0.20 ± 0 | 3.11 ± 0.73 | |
| Cloaca | |||||||||||
| S | rHP | 7.12 ± 0.44 | − | − | − | − | − | − | − | − | 7.12 ± 0.44 |
| WB (L/PB1) | 2.05 ± 1.02 | − | − | − | − | − | − | − | − | 2.05 ± 1.02** | |
| LP (W/MNS) | 2.33 ± 1.42 | 0.20 | − | − | − | − | − | − | − | 1.80 ± 1.57** | |
| M | LP (W/NS) | + | 0.76 ± 0.49 | − | − | − | − | − | − | − | 0.76 ± 0.49 |
| WB | 2.60 ± 0.82 | 0.83 ± 1.44 | 2.25 | 1.92 ± 0.68 | 0.53 | − | − | − | − | 1.91 ± 1.59 | |
| LP (W/PB2) | + | 0.67 ± 0.31 | 3.53 | − | − | − | − | − | − | 1.67 ± 0.31 | |
| WB (L/M) | + | 1.77 ± 1.53 | 1.28 | 1.87 | − | − | − | − | − | 2.19 ± 1.14 | |
| LP | + | 2.11 ± 1.24 | 5.78 ± 0.95 | 2.61 ± 1.02 | 0.20 | 1.70 | − | − | − | 3.04 ± 2.09 | |
| LP (W/M) | + | 1.56 ± 1.57 | 3.07 | 0.32 | − | − | − | − | − | 1.56 ± 1.57 | |
| LP (W/NP) | + | 2.31 ± 1.26 | 2.32 | 4.32 | − | − | − | − | − | 2.99 ± 1.53 | |
| WB (L/NP) | + | 1.77 ± 0.68 | + | 2.71 ± 1.31 | + | 1.26 | + | + | 0.37 | 2.36 ± 1.27 | |
| LP (W/PB1) | + | 0.37 ± 0.33 | + | 0.78 ± 0.50 | + | 0.20 | + | + | 0.20 | 0.87 ± 0.47 | |
| L | WB (L/PB2) | + | 1.11 ± 0.91 | + | 0.32 ± 0.18 | + | 0.22 ± 0.05 | + | 0.20 | 0.20 | 1.11 ± 0.91 |
| WB (L/NS) | + | 0.54 ± 0.52 | + | 2.02 ± 1.24 | + | 0.78 ± 0.69 | + | + | 0.87 ± 1.15 | 2.02 ± 1.24 | |
| WB (L/MNS) | + | 0.31 ± 0.16 | + | 2.77 ± 0.41 | + | 1.11 ± 1.52 | + | + | 0.20 ± 0 | 2.81 ± 0.48 | |
| LP (W/PA) | + | 0.25 ± 0.21 | + | 2.00 ± 0.54 | + | 0.49 ± 0.41 | + | + | 0.23 ± 0.05 | 2.00 ± 0.54 | |
−, all of chickens had already died; +, specimens were not collected since no chicken died on that day.
**, average maximum titer was significantly lower than that of rHP (P < 0.05); ***, average maximum titer was significantly lower than that of LP (P < 0.05).
Virus replication in vivo could be a reflection of the intrinsic ability of a virus to replicate; therefore, the levels of replication of the representative recombinants, rHP and LP (W/MNS) in group S, WB, LP, and LP (W/PB1) in group M, and WB (L/MNS) and LP (W/PA) in group L, were measured in vitro in primary chicken kidney (CK) cells (Fig. 1). For all the viruses, the peak virus titer was seen at 48 hpi and decreased gradually after 48 hpi. As expected, the titers of WB (L/MNS) and LP (W/PA) at 24 hpi were the lowest among the titers of the viruses examined. Also, the titers of rHP were the highest among the virus titers at all the examination times. The intrinsic ability of rHP, WB (L/MNS), and LP (W/PA) recombinant viruses to replicate may influence their survivability, which was linked to the pathogenicity in vivo. It is worth noting that LP (W/MNS) and LP replicated almost similarly in vitro, even though they were in different groups. These observations indicated that the replication ability of the HPAIVs could not solely determine their pathogenicity.
Fig 1.
Kinetics of virus multiplication in chicken kidney (CK) cells. Viral replication of the representative recombinants from each category classified on the basis of survival analysis was observed to investigate the viral intrinsic ability in primary CK cells. After infection of 10−4 EID50/cell virus, samples were collected before infection and at 24 hpi, 48 hpi, 72 hpi, and 96 hpi to calculate the EID50. Significant differences were seen by the analysis for titers of WB (L/MNS) and LP (W/PA) against the titer of rHP at 24, 48, and 72 hpi.
Relation between virus replication, survival time, and cyanosis.
Reassortment of the internal genes was shown to affect the survival time of the infected chickens; thus, we decided to investigate whether or not survival time correlates with the expression of clinical signs. Chickens infected with each recombinant virus showed multiple clinical signs: depression, cyanosis of the exposed skin, and edema of the face and/or head. Among the several clinical signs, cyanosis, which was a visible clinical sign, was focused upon to investigate the correlation with the internal gene constellation and survivability of the chickens infected. The degree of cyanosis at the comb, wattle, and legs was scored from 0 to 3 on the basis of the severity (see the figure in the supplemental material). The average score for each of the three parts was recorded for each group, and the total score, which contained the average score of the three parts, was compared among all the infected groups (Table 4).
Table 4.
Clinical score of each group of chickens infected with the reassortants
| Categorized group | Strain name | Clinical score |
|||
|---|---|---|---|---|---|
| Comb | Wattle | Legs | Total | ||
| S | HP | 0.00 | 0.00 | 0.00 | 0.00 |
| S | rHP | 0.00 | 0.00 | 0.00 | 0.00 |
| S | WB (L/PB1) | 1.50 | 2.00 | 0.50 | 4.00 |
| S | LP (W/MNS) | 1.50 | 2.25 | 0.50 | 4.25 |
| WB (L/PA) | 1.75 | 1.75 | 1.50 | 5.00 | |
| M | LP (W/NS) | 1.00 | 1.67 | 1.00 | 3.67 |
| M | WB | 2.29 | 2.00 | 1.43 | 5.71 |
| M | LP (W/PB2) | 2.67 | 2.33 | 1.33 | 6.33 |
| M | WB (L/M) | 2.50 | 1.50 | 2.00 | 6.00 |
| M | LP | 1.86 | 0.71 | 2.00 | 4.14 |
| M | LP (W/M) | 2.33 | 2.00 | 1.67 | 6.00 |
| M | LP (W/NP) | 2.00 | 1.00 | 1.33 | 4.33 |
| M | WB (L/NP) | 2.00 | 1.50 | 1.75 | 5.25 |
| M | LP (W/PB1) | 2.50 | 1.50 | 2.75 | 6.75 |
| L | WB (L/PB2) | 1.50 | 0.75 | 2.00 | 4.25 |
| L | WB (L/NS) | 0.25 | 0.00 | 0.25 | 0.50 |
| L | WB (L/MNS) | 1.00 | 0.25 | 3.00 | 4.25 |
| L | LP (W/PA) | 0.67 | 0.00 | 2.00 | 2.67 |
These comparisons revealed that the expression of cyanosis was not directly correlated with survivability or the highest virus titer during the observation period. Of the viruses in group S, chickens infected with WB (L/PB1) and LP (W/MNS) expressed cyanosis at the comb, wattle, and legs (clinical scores, 1.5, 2 or 2.25, and 0.5, respectively), while HP and rHP did not induce cyanosis at all.
Next, the averages of the maximum virus titers and scores for the clinical signs were compared in order to examine whether viral replication and the expression of clinical signs are correlated. Although the averages of the highest titers of rHP in the tracheal and cloacal swabs were higher than those of the other viruses, no cyanosis was observed. In contrast, LP (W/PB2) and WB (L/NP) had relatively lower titers but high scores for clinical signs. The average of the highest titers was almost similar in the chickens infected with LP (W/MNS), WB, LP, and LP (W/PB1); however, the total clinical scores varied, ranging from 2.67 to 6.0. These results indicated that virus replication in vivo did not correlate with the degree of cyanosis. Clinical scores also did not appear to be proportional to the survival period. For the viruses in group L, the total score ranged from 0.5 to 4.25. WB (L/PB2) and WB (L/MNS) in the same group L had total clinical scores similar to those for WB (L/PB1) and LP (W/MNS) in group S.
Examination of the gene constellations and clinical scores indicated that the PB2, PB1, M, and NS segments might be involved in the expression of cyanosis. Exchange of the PB2 or PB1 gene in LP resulted in increased clinical scores by 2.19 and 2.61 for LP (W/PB2) and LP (W/PB1), respectively, compared to the values obtained for LP. In the reassortant obtained by reversal exchange, WB (L/PB2) and WB (L/PB1) decreased the score by 1.5 and 1.71, respectively, compared to that of WB. Comparison of the clinical scores of WB (L/NS), WB (L/MNS), and WB (L/M) against that of WB revealed that the L/NS segment suppressed cyanosis, while the L/M segment did the opposite. For the degree of cyanosis of the chickens infected with WB (L/NS), the clinical score was 0.5, and for WB (L/MNS), the score was 4.25; these scores were weaker than the score for WB (clinical score, 5.71). On the other hand, there was a slight increase in the clinical score from 5.71 to 6.00 when the chickens were infected with WB (L/M) compared to that when the chickens were infected with WB.
Correlation between gene expression and survival period of the infected chickens by microarray analysis.
Microarray analysis of lungs from the chickens at 24 hpi was carried out to identify host genes whose expression correlated with the survival period of the infected chickens. Samples for the analysis were collected from two chickens infected by viruses from each group: HP, rHP, and LP (W/MNS) in group S; WB, LP, and WB (L/PB1) in group M; and LP (W/PA) in group L. Four representatives, HP, rHP, LP, and WB, from the survival analysis were selected as references for the microarray analysis, and two viruses, LP (W/MNS) and LP (W/PA), were examined as representatives from groups S and L, respectively. In addition, WB (L/PB1) was examined because the survivability was relatively higher for that virus than for LP and WB in group M. Four hundred eighty-four probes, representing 336 chicken genes, out of 38,681 probes were found to correlate with the survivability of the infected chickens in a survival analysis that fitted with Cox's proportional hazards model (P < 0.05). Among them, 292 genes were annotated to human genes using BLAST for the subsequent pathway analysis. Overrepresentation of genes for some biological processes in GeneOntology (GO) with the identified genes was evident by Fisher's exact test (P < 0.01; see Table S1 in the supplemental material). Several of those biological processes, such as response to exogenous double-stranded RNA (dsRNA), response to virus, inflammatory response, innate immune response, immune response, and pathogen-associated molecular pattern dependent on induction by a symbiont of the host innate immunity, were acceptable for influenza virus infection (13).
Forty-nine probes, annotated to 33 human genes, showed a stricter significance level (P < 0.01) in Cox's proportional hazards model (Fig. 2). Four genes classified as response to virus or immune response in the GeneOntology biological process were retrieved from these 33 human genes. Of these genes, those for the CD274 molecule (CD274), 2′-5′-oligoadenylate synthetase-like (OASL), and ring finger protein 19B (RNF19B) were classified as immune response, and those for the zinc finger CCCH type and antiviral 1 (ZC3HAV1) were classified as response to virus. Furthermore, three genes, those for phospholipase A2, group VI (PLA2G6), GTP cyclohydrolase 1 (GCH1), and ubiquitin-specific peptidase 18 (USP18), were identified on the basis of a search with the keywords infection, interferon, response to viruses, and viral reproduction using software for pathway analysis, Pathway studio, version 7. Expression of these seven genes was induced upon infection, and their levels of expression were higher in the chickens with shorter survival times. It was intriguing to note that USP18 is considered to suppress the antiviral response.
Fig 2.
Gene expression signatures associated with the survival period. The heat map shows the 33 signature genes extracted by the survival analysis of the Cox's proportional hazards model (P < 0.01). *, genes related to viral infection or immune response in GeneOntology; **, genes extracted by the software for pathway analysis on the basis of keywords such as infection, interferon, response to viruses, and viral reproduction.
In order to validate the feasibility of the observations made by the microarray analysis, levels of expression of USP18 and RNF19B as representative genes extracted by the microarray analysis were examined by real-time PCR analysis. Results of the real-time PCR strongly correlated with the levels of gene expression revealed by the microarray analysis, and this ensured the feasibility of the observations. The correlation coefficients (P values) of the gene expression level by the microarray analysis and the comparative gene expression level of the beta-actin gene in real-time PCR were 0.91 (P = 0.000001121) and 0.83 (P = 0.0000677) for USP18 and RNF19B, respectively.
Exploring genes involved in cyanosis.
The levels of gene expression examined by microarray analysis were compared to identify those genes among rHP, LP (W/MNS), and LP (W/PB1) that are involved in cyanosis. Among the viruses belonging to group S, rHP did not cause cyanosis in chickens, while LP (W/MNS) and LP (W/PB1) induced it. Thus, the gene expression levels examined by the microarray analysis were compared to identify those genes correlated with the expression of cyanosis (Table 4).
The gene expression values of two viruses [LP (W/MNS) and LP (W/PB1)] were compared against the value of rHP to extract genes that were significantly suppressed or induced (times < 0.5 or 2 < times). Five hundred sixty-one probes were extracted from all 38,681 probes. After that, the genes with rHP values within the range of 0.5 < times < 2 against the value of the control were excluded from the 561 probes to extract only genes ordinarily expressed and not correlated with cyanosis. As a result, 114 probes that were assumed to be correlated with the expression of cyanosis were extracted. The 114 probes, representing 67 chicken genes, were annotated to 48 human genes by BLAST. As for Fisher's exact test (P < 0.05), it was shown that none of the extracted genes accumulated in any particular GeneOntology biological process (see Table S2 in the supplemental material). As a result, from the analysis we were not able to identify genes that were possibly correlated with cyanosis.
DISCUSSION
In this study, the survivability and degree of cyanosis in chickens infected with recombinant viruses varied, even though they had an identical highly pathogenic form of the HA protein. Survival analysis of chickens infected with the various recombinant viruses revealed that the survivability of the chickens decreased following substitution of the PB1 gene segment from LP in the WB internal genetic constellation, while the PB2 gene segment from LP in the WB internal genetic constellation increased the survivability significantly. All the chickens infected with the PA gene segment from WB in the LP internal genetic constellation survived during the observation period. Polymerase of the influenza virus is a complex of the PA, PB1, and PB2 gene products, and it catalyzes RNA replication and transcription activity in the nucleus of infected cells. PB1 (amino acids [aa] 1 to 15) interacts with PA (aa 257 to 716) and PB1 (aa 685 to 757a) interacts with PB2 (aa 1 to 35) to form complexes (3). Comparison of the deduced amino acid sequences between the PB1 gene segments of LP and WB revealed two substitutions, one in the PA binding domain at aa 14 and the other in the PB2 binding site at aa 739. Those substitutions in PB1 of LP were identical to those observed in the H5N1 subtype HPAIVs, including HP. The amino acid at aa 14 in PB1 formed hydrophobic contacts with the amino acid at aa 670 in PA (39). The amino acid at aa 670 in PA and that between LP and WB were identical; therefore, substitution at aa 14 in PB1 was thought to affect the hydrophobic bond between PA and PB1. Wunderlich et al. reported that substitution from alanine to valine at aa 14 of PB1 increased binding between PB1 and PA (49). As PB1 of LP possesses valine at aa 14 while that of WB is alanine, PB1 of LP could contribute to increasing the binding between PB1 and PA, thus contributing to the decreased survivability. The substitutions in the PB2 binding site of PB1 at aa 739 and the PB1 binding region of PA (PA numbering, aa 261, 262, 272, 322, 323, 354, 391, 400, 405, 409, 543, 554, 596, 607, 643, and 716) were not reported to be amino acids relating to pathogenicity. Moreover, the amino acids in the PB1 binding site of PB2 were identical between LP and WB.
Survival analysis also revealed that the NS gene segment of LP was correlated with increasing survivability, as the survivability of the chickens infected with WB (L/NS) and WB (L/MNS) increased comparatively. Two viral proteins, NS1 and NS2, are translated from the NS gene. Leucine at aa 103 and isoleucine at aa 106 of the NS1 amino acid sequence enhance the replication ability of viruses in vitro and in vivo and mortality in vivo (7). Their substitution expanded viral tropism in the lungs and increased the extent of infection. This was unlikely the case in our study, because of the fact that NS1 of LP appeared to contribute to increasing survivability, even though it contained those substitutions. As the survival period of chickens infected with LP (W/MNS) was shorter than those of chickens infected with LP or WB, while that of WB (L/MNS) was longer than those of chickens infected with LP or WB, there is the possibility that a combination of the M and NS gene products is involved in the period of survival of the infected host. NS2 is another product translated from the NS gene. A previous study showed that the binding of NS2 and M1 proteins at amino acids 81 to 100 of NS2 and aa 76 to 115 of M1 was essential for nuclear exportation of progeny viral ribonucleoproteins by NS2 (44). Compared with the amino acids at aa 76 to 115 of M1 and aa 81 to 100 of NS2, two and seven substitutions were found between the sequences of LP and WB, respectively. Further study is necessary to elucidate the contributions of the NS and M gene product(s) to the period of survival of the infected chickens.
Two hundred ninety-two genes extracted by survival analysis that fitted Cox's proportional hazards model (P < 0.05) accumulated into several GOs by Fisher's exact test (P < 0.01) (see Table S1 in the supplemental material). Among the genes involved in six of the GOs that could be correlated with infection by the influenza virus described in the Results, the gene for Toll-like receptor 3 (TLR3) appeared in all six GOs, and two other genes, those for Toll-like receptor 6 (TLR6) and myeloid differentiation factor 88 (MYD88), were found among three GOs, namely, inflammatory response, innate immune response, and immune response. TLR6 was also seen in the pathogen-associated molecular pattern-dependent induction by symbiont of host, and MYD88 was found in the response to virus. TLR3 was a receptor-recognizing dsRNA produced by influenza virus infection (18), and MYD88 was related to the induction of interferon-sensitive response element (ISRE) that was activated following identification of the pathogen by TLR3 (17). TLR6 formed a complex with TLR2 and MYD88, and the complex played the role of a regulator of cytokine, chemokine, and interferon response by infection of the modified vaccinia virus Ankara (MVA), an attenuated double-stranded DNA poxvirus (8). Although TLR6 was found among four out of six GOs, the correlation between TLR6 and influenza viral infection remains unknown.
Several host genes that correlated with the survivability of the infected host were identified by microarray analysis. USP18, which negatively regulates the innate immune response against viral infections, was shown to be involved in such host genes (27). Studies on USP18-deficient mice showed that increasing the immunoactivity of ISG15 made the mice resistant against viral and bacterial infections (28, 41, 51), yet the mechanism of USP18 induction in infected hosts is controversial. One possible mechanism is a negative-feedback reaction against overexpression of the ISG15 gene, accompanied by a protective response after viral infection. Another possibility is a direct effect of the virus in the host to decrease resistance against infection with that virus. Previous research reported that the NS1 gene of the type B influenza virus directly inhibited E1 activity in ISG15 conjugation, and the ISG15 system, which induces IFN-α/β for antiviral response, was also inhibited (52). USP18 inhibits a signaling pathway that induces regulation of the ISRE promoter, and the IFN response is downregulated by this process (1). Induction of USP18 is considered advantageous for viruses to survive in hosts. As a higher expression of the USP18 gene was seen in chickens with a shorter survival period, induction of the gene as a direct effect of infection with a highly pathogenic virus was considered possible. On the contrary, of the genes correlating with survivability, RNF19B was considered to interact with HbcH8, which plays a similar role as E2 in the ISG15 system and might act as E3 in the ISG15 system (5, 9, 12, 22). RNF19B would play a conflicting role against USP18 in the ISG15 system, as it increases the conjugates of the target proteins and ISG15. These results suggested an important role of the ISG15 ubiquitin-like system in the survivability of chickens infected with HPAIVs. ZC3HAV1 is synonymous with poly(ADP-ribose) polymerase (PARP)-13 (ZAP). Hayakawa et al. had reported that the shorter isoform of ZAP (ZAPS) was a regulator of RIG-I signaling that triggered the activation of IRF3 and NF-κB transcription during the innate antiviral immune response. A novel function of ZAPS was recently elucidated, and it is thought to play an important role against influenza viral infection (19). CD274, OASL, and GCH1 are genes that were actively induced by interferon (11, 31, 36, 47). Among them, OASL was induced by IFN following influenza virus infection (36). Although PLA2G6 has been reported to play an antiviral role against HIV and adenovirus, involvement of this gene in the anti-influenza virus response has not been demonstrated (30).
This study demonstrated that the internal genes of viruses with the HA protein of a highly pathogenic form affected survivability, the clinical signs of the infected chickens, and the replication ability of the viruses. Functional scrutiny of the substitutions observed between the gene segments of LP and WB revealed contributions to the substitutions of internal genes in various pathotypes of HPAIVs. Various reassortant viruses involved in pathogenicity were produced by the reverse genetics method, and host genes correlated with survivability of the infected host were extracted by combined analysis, survival analysis, and microarray analysis using reassortant viruses with various pathogenicities. The analysis highlighted the relationship between survivability of the infected host and levels of expression of genes related to RIG-I signaling and the ISG15 ubiquitin-like system, such as ZC3HAV1 and USP18, respectively. The strategy utilized in this study is useful to identify host genes related to the pathotypes of viruses.
Supplementary Material
ACKNOWLEDGMENTS
We thank Hiroyuki Horiuchi and Masumi Sato for giving insightful comments and suggestions.
This study was supported by a grant-in-aid for scientific research from the Zoonoses Control Project of the Ministry of Agriculture, Forestry and Fisheries of Japan.
Footnotes
Published ahead of print 21 December 2011
Supplemental material for this article may be found at http://jvi.asm.org/.
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