| Highly pathogenic avian influenza (HPAI) of the H5N1 subtype is considered endemic in China, Vietnam, Indonesia, India, Bangladesh and Egypt, which causes huge economic losses in poultry industry. Human infection is usually acquired by contact with H5N1 avian influenza virus (AIV) infected poultry. Although H5N1 AIV has not been capable of sustaining human-to-human transmission, the ability of the virus to undergo variation due to mutations and reassortment clearly poses its potential ability to undergo viral adaptation to the human species. Therefore, the H5N1 HPAI is a very important zoonosis. Hemagglutinin (HA) is one of the two main antigenic determinants on the viral surface and often undergoes N-linked glycosylation at several sites. A number of new clades and subclades of H5N1 AIVs have emerged in various regions with variable pattern of glycosylation sites in the HA protein. In this study, we used site-directed mutagenesis to modify potential glycosites on the HA of A/Mallard/Huadong/S/2005 (H5N1, Clade 2.3.4) and evaluated its effects on biological characteristics, pathogenicity and antigenicity of H5N1 AIVs.1. Generation and biological characteristics of H5N1 subtype mutant AIVs with different glycosylation patterns in the globular head of the HA protein.To understand the effect of glycosylation in the globular head of HA on the pathogenicity and antigenicity of H5N1 AIVs, we used site-directed mutagenesis to modify potential glycosites (144N,158N and 169N) on the HA of A/Mallard/Huadong/S/2005 and evaluated their biological characteristics. Results showed that loss of glycosylation 158N was the prerequisite for H5 AIV binding to the a2,6-linked receptor, and optimal binding preference with a2,6-linked receptor of H5 AIVs was obtained when HA harbored two glycosylation at residues 144N and 169N but lacked glycosylation 158N. Growth of viruses lacking glycosylation at either 158N or 169N was reduced in plaque size on MDCK cells, while additional glycosylation 144N of the HA protein could promote viral replication. The mutant viruses of first generation (P1) and fifth generation (P5) grew equally well in CEF cells, when compared to the wild-type virus. Thus, glycosylations in the global head of HA protein play an important role in receptor binding and replication of AIVs.2. Effect of glycosylation in the globular head of HA protein on the pathogenicity of H5N1 AIVs.To compare the virulence of mutant viruses and wild-type virus,6-week-old female BALB/c mice were inoculated intranasally with viruses at a dose of 10 EID50 or 106EID50. The viruses showed different virulence when the mice were infected with viruses at a dose of 104 EID50. The mutant viruses rS-144+/158-/169-and rS-144+/158-/169+, and wild-type virus showed a slight loss in body weight (-10%), while the other mutants showed a significant loss in body weight (>20%). Furthermore, all animals infected with the viruses rS-144-/158-/169+ or rS-144-/158+/169-were dead within 9 days. However, all viruses caused significant body weight loss and high mortality when the mice were infected with the viruses at a dose of 106 EID50. To test whether the mutant viruses possessed a different ability to spread systemically after respiratory tract infection, the mice were infected with 104 EID50 of each virus. All viruses were detectable in the lungs on days 3 and 6 p.i. Moreover, the mutant viruses rS-144-/158-/169+ and rS-144-/158+/169-were detectable with higher positive ratios and titers in the livers, spleens, kidneys and brains of the mice, exhibited an ability to spread systemically. In contrast, the wild-type virus was only detectable in the lungs of mice. Both mutant viruses lacking glycosylation at residue 158 or 169 induced significantly higher levels of IL-6, IL-8, CXCL10, MX-1, and IFN-β gene expression compared to wild-type virus. Thus, mutant viruses with loss of 158N or 169N glycosylation sites showed increased pathogenicity, systemic spread and pulmonary inflammation in mice.3. Effect of glycosylation in the globular head of HA protein on the antigenicity of H5N1 AIVs.To evaluate the effect of glycosylation on the antigenicity of the mutant viruses, chickens were vaccinated with 10 EID50 of each virus, then neutralization titers and HI titers of sera were determined against H5 AIVs S (clade 2.3.4), DT(clade 7.2), WX (clade 2.3.2.1) and ZJ (clade 2.3.4.6), respectively. In the neutralization assay, mutant viruses rS-144-/158+/169-and rS-144+/158+/169+ induced comparable neutralization antibody against homologous virus SY strain compared to the wild-type virus, and rS-144-/158+/169- induced remarkable higher neutralization antibody against heterologous viruses compared to the wild-type virus, indicating that rS-144-/158+/169- induced an cross-reaction neutralization antibody. Next, four-week-old chickens were immunized with the inactivated mutant virus rS-144-/158+/169-and wild-type virus to evaluate protective efficacy. These vaccines also provided complete clinical protection against H5 AIVs of S, DT or ZJ strain challenge with 106 EID50 of each virus. None of the vaccinated chickens developed disease signs or died. However, they only provided 70% protection against AIV WX strain challenge. The SPF chickens immunized with rS-144-/158+/169-showed a reduced percentage of virus shedding both in the oropharyngeal and cloacal swabs compared to the chickens immunized with the wild-type virus, particularly for DT challenge groups on day 7 post-challenge. Therefore, the rS-144-/158+/169- induced a better cross neutralization antibody and inhibition viral shedding than the wild type virus.4. Effect of glycosylation in the globular head of HA protein on cytokine production of H5N1 AIVs.To compare the gene expression of IL-6, IL-8, IL-18, IL-1β, CCL2, CXCL10, IFN-β, IFN-γ and TNF-a in influenza virus (rS-WT, rS△158 and rS△169)-infected cells, A549 cells were chosen as an infection model in vitro. The mutant rS△158 replicated to a titer that was slightly higher than that of WT and mutant rS△169 infections throughout the 72-h growth period. The mRNA levels of IL-8, CXCL10 and IFN-β determined by qRT-PCR were significantly up-regulated by the mutant influenza virus when compared with the wild-type virus infected cells. Cytokines up-regulated from 6 h p.i. onwards included IL-8, CCL2, CXCL10 and IFN-(3 whereas TNF-a was only up-regulated at 12 h p.i. To understand the mechanism of proinflammatory cytokine induction in H5N1 AIV-infected A549 cells, we investigated the activation of molecules involved in signaling pathways. Levels of activated NF-κB were increased significantly in rS△158-infected cells at 6 h p.i. and persisted at 12 h. IFN-a but not IFN-y-induced STAT1 phosphorylation was inhibited in all three AIVs infected cells and no significant difference was observed between different groups. Thus, the up-regulated cytokines in rS△158-infected cells is at least in part due to the activation of NF-κB pathway.5. Generation and biological characteristics of H5N1 subtype mutant AIVs with different glycosylation patterns in the stem of the HA protein.To understand the effect of glycosylation in the stem domain of HA on the biological characteristics of H5N1 AIVs, we used site-directed mutagenesis to generate different patterns (11N,23N and 286N) of stem glycans on the HA of A/Mallard/Huadong/S/2005. The results indicated that these three N-glycans were dispensable for the generation of replication-competent influenza viruses. However,N-glycans at Asn11 and either Asn23 or Asn268 were removed, and the cleavability of HA was almost completely blocked; thus, the growth rates of the mutant viruses in MDCK and CEF were significantly decreased compared with that of the wild-type virus. Moreover, mutant viruses lacking these oligosaccharides, particularly the N-glycan at Asn11, demonstrated significantly decreased thermostability and pH stability compared with the wild-type virus. Interestingly, mutant viruses without N-glycans induced reduced neutralizing antibodies against the wild-type virus, and most of the mutant viruses were more sensitive to neutralizing antibodies than the wild-type virus. Taken together, these data indicate that the glycosylation site 11 in HA stem play a critical role on HA cleavage, replication, thermostability, pH stability, and antigenicity of H5N1 AIVs. |
