Biological and genetic diversity of the GS/GD/1/96 and GS/GD/2/96 viruses.
GS/GD/1/96 and GS/GD/2/96 viruses were isolated from samples that were collected during the first H5N1 outbreak on a goose farm in Guangdong province. These two viruses exhibited different phenotypes with respect to causing lethality in chicken embryos. GS/GD/1/96 virus killed the inoculated embryos within 30 h, but GS/GD/2/96 did not lead to lethality up to 60 h after inoculation. We then tested their IVPI in SPF chickens as described in Materials and Methods. The IVPI for GS/GD/1/96 was 2.1, with 9 out of 10 chickens inoculated dying within the 10-day observation period (Table ). In contrast, the IVPI for GS/GD/2/96 was zero, as this virus did not cause any disease signs or deaths, though it was isolated from the cloacal swabs of 3 out of 10 chickens (Table ).
Disease and death caused by the H5N1 viruses in chickens after intravenous inoculationa
The GS/GD/1/96 virus replicated systemically when chickens were inoculated i.n., and virus could be detected on day 3 p.i. from any of the organs tested, including lung, bursa, kidney, brain, pancreas, and spleen (Table ). All of the birds died within the observation period. Virus was not detected from any organs or the swabs from the chickens that were inoculated with GS/GD/2/96 virus, however, though 3 of 10 chickens seroconverted (Table ). No disease signs or deaths were observed from any chicken infected by GS/GD/2/96 virus.
Since the viruses were isolated from geese, we also tested their replication and virulence in this avian species. As shown in Table , the GS/GD/1/96 virus could be detected in all of the tested organs of the geese killed on day 4 p.i. All eight of the geese showed disease signs, and one died during the 14-day observation period. All of the seven surviving geese seroconverted. In the GS/GD/2/96-inoculated geese, virus was detected from the swabs and the organs, including lung, bursa, kidney, pancreas, and spleen, but not from the brain. The titers were significantly lower than those of the GS/GD/1/96 virus-infected geese. No disease signs or deaths were observed in any geese during the observation period, and three out of eight geese seroconverted by day 14 p.i.
Replication and lethality of the H5N1 viruses in geese after i.n. inoculationa
Though the two viruses were isolated from the same goose flock, their virulence in chickens was quite different. To understand their genetic relationship, the open reading frames and the noncoding area of the RNA fragments (except the conserved end sequence) of the two viruses were sequenced. The sequencing results of our virus stocks indicated that these two viruses share identical PB2, HA, NA, M2, and NS2 genes at the nucleotide level, and both of the viruses have six basic amino acids in the connecting peptide of the HA gene. We detected a total of eight nucleotide differences between the genomes of the two viruses; two nucleotide differences in PB1 and one nucleotide in NP are silent mutations, while five of them coded for amino acid changes located in the PA, NP, M1, and NS1 genes (Table ). These data suggested that single or multiple-amino-acid combinations among these five differing amino acids in the PA, NP, M1, and NS1 genes contributed to the difference in virulence observed between the two viruses.
Amino acid differences between the GS/GD/1/96 and GS/GD/2/96 viruses
The rescued H5N1 viruses retained the biological properties of the wild-type viruses.
We inserted cDNAs of each full-length RNA segment of the GS/GD/1/96 and GS/GD/2/96 viruses into the vRNA-mRNA bidirectional expression plasmid pBD as described in Materials and Methods. Using these plasmids, we rescued the GS/GD/1/96 and GS/GD/2/96 viruses, designated R-GS/GD/1/96 and R-GS/GD/2/96, respectively. After confirmation by sequence analysis, we grew the virus stocks in 10-day-old, SPF embryonated chicken eggs and tested their replication and lethality in chickens.
The rescued R-GS/GD/1/96 virus, like its wild-type counterpart, was highly pathogenic for chickens, and the IVPI was 2.4 (Table ). The virus replicated systemically in chickens after i.n. inoculation and killed all of the chickens during the observation period (Table ). The rescued R-GS/GD/2/96 virus, like its wild-type counterpart, did not cause any illness or deaths in the chickens after i.v. inoculation (Table ) and did not replicate in any of the organs tested after i.n. inoculation (Table ).
In geese, R-GS/GD/1/96 replicated in the lung, bursa, kidney, and pancreas but not in the spleen and brain of the i.n.-inoculated animals (Table ). Virus shedding was also detected from both oropharyngeal and cloacal swabs (Table ). After i.n. inoculation in geese, the R-GS/GD/1/96 virus replicated systemically and caused illness in seven out of eight geese, and one animal died during the observation period. None of the geese infected by R-GS/GD/2/96 showed any disease signs, and they all stayed healthy during the observation period (Table ).
The NS1 gene, specifically amino acid 149, is crucial for the difference in virulence between GS/GD/1/96 and GS/GD/2/96 in chickens.
To identify the genes that contributed to the lethality of GS/GD/1/96 in chickens, we generated four single-gene recombinants, each containing one of the PA, NP, M, or NS genes derived from GS/GD/1/96; the remaining seven genes were derived from GS/GD/2/96. The pathogenicity of these recombinant viruses was tested in chickens. As shown in Table , after i.v. inoculation the recombinant viruses that contained the PA, NP, or M gene from GS/GD/1/96 virus in the GS/GD/2/96 background (GS/GD/2-1PA, GS/GD/2-1NP, and GS/GD/2-1PM, respectively) did not cause any disease signs and death in the chickens, though all of the chickens seroconverted by day 10 p.i. Only the recombinant virus that contained the NS1 gene of GS/GD/1/96 (GS/GD/2-1NS) induced disease signs in all of the inoculated chickens, and it killed three of them. The IVPI for GS/GD/2-1NS was 1.0 (Table ). When the GS/GD/2-1NS virus was inoculated into chickens i.n., it could be detected from lung, kidney, brain, and pancreas of the chickens killed on day 3 p.i. (Table ). Three chickens showed disease signs, and one of the animals died during the observation period. All of the chickens seroconverted by day 14 p.i.
We then tested the effect of individual genes derived from GS/GD/2/96 virus on the replication and virulence of GS/GD/1/96 virus. We again generated four single-gene recombinant viruses, each containing the PA, NP, M, or NS gene from GS/GD/2/96, and the remaining seven genes from GS/GD/1/96. The viruses that carried the PA, NP, or M gene of GS/GD/2/96 (GS/GD/1-2PA, GS/GD/1-2NP, and GS/GD/1-2M, respectively) were as virulent as the GS/GD/1/96 virus, with IVPI ranging from 1.9 to 2.4 (Table ). The recombinant virus bearing the NS1 gene from GS/GD/2/96, designated GS/GD/1-2NS, did not cause any disease signs and deaths in the inoculated chickens, and it yielded an IVPI of zero. All of the chickens were seroconverted by day 10 p.i. Virus was not detected from any tested organs when the chickens were i.n. inoculated with the recombinant virus GS/GD/1-2NS, and no disease signs or deaths were observed in any of the chickens. Two of 10 chickens seroconverted (Table ).
We also tested the replication and lethality of the eight rescued viruses in geese. Similar to the wild-type and rescued GS/GD/1/96 viruses, GS/GD/1-2PA and GS/GD/1-2M replicated in most of the tested organs, was shed through both oropharynx and cloacae (Table ), and caused illness in all of the inoculated geese; one or two animals died during the observation period. The recombinant virus GS/GD/1-2NP was detected from all of the tested organs, except the spleen, and virus was also shed through the cloacae (Table ). However, the titers of this virus in the lung were significantly lower than those from the wild-type and rescued GS/GD/1/96 virus-inoculated geese (Table ). Interestingly, the GS/GD/2-1NS virus did not exhibit the same virulence in geese as it did in chickens. It behaved more similarly to the less pathogenic GS/GD/2/96-based viruses.
The viruses GS/GD/2-1PA, GS/GD/2-1M, and GS/GD/2-1NS behaved in geese in a fashion similar to that of the wild-type and rescued GS/GD/2/96 viruses. These viruses could be detected from the undiluted samples of some of the organs tested, and all of the geese shed detectable viruses through both the oropharynx and cloaca. Seroconversion was observed in a portion of the geese inoculated with these viruses by day 14 p.i. (Table ). The recombinant virus GS/GD/2-1NP was detected from the diluted samples of several tested organs and the swabs, and the titers of this virus in the lung were significantly higher than those of the wild-type and rescued GS/GD/2/96 virus-inoculated geese. Seroconversion was observed in six out of eight geese inoculated with the GS/GD/2-1NP virus (Table ).
These results demonstrated that the NS1 gene plays an important role in determining the virulence of GS/GD/1/96 and GS/GD/2/96 in chickens, but it does not affect the replication and virulence of this pair of viruses in geese. There is one amino acid difference in the NS1 genes of GS/GD/1/96 and GS/GD/2/96 (Table ), which indicates that the amino acid alanine (Ala) at position 149 in the NS1 gene of GS/GD/1/96 is crucial for the ability of this virus to replicate in chickens. In geese, the NS1 gene does not seem to influence the replicative properties of these viruses, though our results suggest that sequences within the NP gene may influence replication levels.
The amino acid at position 149 of the NS1 gene affects the ability of GS/GD/1/96 and GS/GD//96 to antagonize IFN-α/β production in CEFs.
The NS1 protein of influenza virus has previously been shown to act as an IFN-α/β antagonist (10
). To explore if the difference of the replication and virulence of GS/GD/1/96 and GS/GD/2/96 viruses was directly correlated with the ability of these viruses to inhibit the IFN-α/β system, the production of IFN in cells infected with different viruses was investigated. VSV is very sensitive to type I IFN; when the host cells were pretreated with IFN-α/β, VSV growth was inhibited (5
). To assay IFN production, supernatants from influenza-infected cells were tested for their ability to inhibit GFP expression and viral replication of a recombinant VSV-GFP virus. Supernatants from the mock-infected CEFs and GS/GD/1/96-infected CEFs did not inhibit the GFP expression (Fig. ), and the VSV-GFP virus grew to the titers of 103.5
50% tissue culture infectious doses (TCID50
) and 106
at 12 and 24 h p.i., respectively (Fig. ). This indicated that GS/GD/1/96 infection did not lead to induced IFN production, or rather that IFN production was antagonized by this virus. Supernatants from GS/GD/2-1NS-infected CEFs partially inhibited VSV-GFP replication (Fig. ), and the virus grew to 101
at 12 and 24 h p.i., respectively. However, the supernatants from CEFs infected with R-GS/GD/2/96 and R-GS/GD/1-2NS completely prevented the replication of the VSV-GFP virus (Fig. ), indicating that the viruses bearing the GS/GD/2/96-like NS1 gene were not able to antagonize the production of IFN in these cells.
FIG.1. Induction of IFN-α/β in CEFs or GEFs infected with different influenza viruses. (A) CEFs or GEFs were infected with the tested influenza viruses, and the UV-treated supernatants were used to examine IFN-mediated inhibition of VSV-GFP replication (more ...)
The VSV-GFP viral system was also used to examine the ability of the influenza viruses to antagonize IFN production in GEF cells. The supernatants from GS/GD/1/96 and GS/GD/2-1NS virus-infected GEFs did not inhibit the growth of VSV-GFP (Fig. ). Again, this indicated that viruses bearing the GS/GD/1/96-like NS1 gene were able to antagonize IFN production in GEF cells. The growth of VSV-GFP in GEFs was inhibited by the GS/GD/2/96 and GS/GD/1/96-2NS virus-infected GEFs supernatant 12 h p.i., but the viruses grew to the titers of 102.5 TCID50 at 24 h p.i.(Fig. ). This indicated that unlike the scenario in CEFs, viruses with the GS/GD/2/96-like NS1 gene are also able to antagonize the production of IFN to a certain extent in GEFs.
To examine the effect of influenza virus infection on the synthesis of IFN-α/β mRNA, CEFs were infected with various influenza viruses, and the cells were harvested for RNA extraction 20 h after infection. The cDNAs of chicken IFN-α/β were amplified by using primer pairs specific to either the IFN-α or the IFN-β gene. As shown in Fig. , the cDNAs of both IFN-α and IFN-β genes were amplified from the R-GS/GD/2/96- and R-GS/GD/1-2NS-inoculated CEFs but not from the CEFs inoculated with the R-GS/GD/1/96 and R-GS/GD/2-1NS viruses. The effect of these viruses on IFN-α/β mRNA production is consistent with the effect of these viruses in the IFN-α/β bioassay.
In order to compare the levels of viral proteins in the different virus-infected cells, CEFs were infected with the rescued wild-type viruses and two NS reassortant viruses at an MOI of 2. Cells were lysed at 12 h p.i., and the cell extracts were analyzed by Western blotting (Fig. ). Western blot analysis revealed that the levels of the NS1 protein in the cells infected with virus containing the GS/GD/1/96 NS gene are considerably increased in comparison to the NS1 protein levels seen in cells infected with the virus containing the GS/GD/2/96 NS gene (Fig. ). However, no significant difference in the levels of other viral proteins was detected among the different virus-infected cells.
FIG. 2. Levels of virus proteins determined by Western blotting. Lysates of mock-infected cells or cells infected with R-GS/GD/1/96, R-GS/GD/2-1NS, R-GS/GD/2/96, or R-GS/GD/1-2NS were incubated with mouse anti-truncated NS1 antiserum (I) or chicken antiserum (more ...)
These results indicated that viruses bearing the NS1 gene from GS/GD/1/96 have a greater ability to antagonize the IFN-α/β response in CEFs than viruses bearing the NS1 gene from GS/GD/2/96. Since the NS1 genes of these two viruses differ only at the amino acid at position 149, it would indicate that the Ala149 residue present in the NS1 gene of GS/GD/1/96 is important for this virus to be able to antagonize the host IFN-α/β response and to replicate with lethality in chickens.