Identification of amino acids in HA and PB2 critical for the transmission of H5N1 avian influenza viruses in a mammalian host

Abstract

Since 2003, H5N1 influenza viruses have caused over 400 known cases of human infection with a mortality rate greater than 60%. Most of these cases resulted from direct contact with virus-contaminated poultry or poultry products. Although only limited human-to-human transmission has been reported to date, it is feared that efficient human-to-human transmission of H5N1 viruses has the potential to cause a pandemic of disastrous proportions. The genetic basis for H5N1 viral transmission among humans is largely unknown. In this study, we used guinea pigs as a mammalian model to study the transmission of six different H5N1 avian influenza viruses. We found that two viruses, A/duck/Guangxi/35/2001 (DKGX/35) and A/bar-headed goose/Qinghai/3/2005(BHGQH/05), were transmitted from inoculated animals to naïve contact animals. Our mutagenesis analysis revealed that the amino acid asparagine (Asn) at position 701 in the PB2 protein was a prerequisite for DKGX/35 transmission in guinea pigs. In addition, an amino acid change in the hemagglutinin (HA) protein (Thr160Ala), resulting in the loss of glycosylation at 158-160, was responsible for HA binding to sialylated glycans and was critical for H5N1 virus transmission in guinea pigs. These amino acids changes in PB2 and HA could serve as important molecular markers for assessing the pandemic potential of H5N1 field isolates.

Figure 1. Transmisson of H5N1 avian influenza viruses in guinea pigs.

Groups of three guinea pigs were inoculated i.n. with 10⁶EID₅₀ of test virus and, 24 hours after the inoculation, three contact guinea pigs were placed in each cage. Nasal washes were collected every two days from all animals beginning 2 days p.i. for detection of virus shedding. (A) DKGX/22 virus; (B) DKGX/17 virus; (C) DKSH/13 virus; (D) DKGX/35 virus; (E) DKGD/22 virus; and (F) BHGQH/3 virus. Each color bar represents the virus titer from an individual animal. The dashed blue lines in these panels indicate the lower limit of detection.

Figure 2. Replication and transmission of DKGX/35 PB2 mutants.

(A) Groups of two guinea pigs were inoculated i.n. with 10⁶EID₅₀ of test virus and then euthanized on day 3 p.i. Organs were collected for virus titration in eggs. (B) Transmission of the 22/PB2-701N virus in guinea pigs. (C) Transmission of the 35/PB2-701D virus in guinea pigs. The dashed blue lines in these panels indicate the lower limit of detection.

Figure 3. Hemagglutination assays of H5N1 influenza viruses using cRBCs with different treatments.

The upper panel shows the hemagglutination by two HA units of each virus while the lower panel shows the HA titers of test viruses with 0.5% cRBCs treated as follows: cBRCs, untreated; Desial cRBCs, treated with VCNA; α-2,3 cRBC, VCNA treated and resialylated with α-2,3 glycans; α-2,6 cRBC,VCNA treated and resialylated with α-2,6 glycans.

Figure 4. Glycan binding specificity of H1N1 and H5N1 viruses.

(A) H1N1 human influenza BC/05 virus. (B) DKGX/35 virus. (C) 35/HA-226L/228S. (D) 35/HA-160T.

Figure 5. Replication and transmission of DKGX/35 HA mutants.

(A) Groups of two guinea pigs were inoculated i.n. with 10⁶EID₅₀ of test virus and euthanized on day 3 p.i. Organs were collected for virus titration in eggs. (B) Transmission of 35/HA-226L/228S in guinea pigs. (C) Transmission of 35/HA-160T in guinea pigs. The dashed blue lines in these panels indicate the lower limit of detection.

Figure 6. Western blot analyses of H5N1 avian influenza HA1 protein.

Lysates of H5N1 viruses treated with or without PNGase F were incubated with chicken anti-H5N1 antiserum. Binding was visualized with 3,30-diaminobenzidine after incubation with peroxidase-conjugated secondary antibodies. The locations of marker proteins are indicated on the left.

Figure 7. Receptor-binding preference and transmission of BHGQH/3 and its HA mutant.

Receptor-binding preference of r-BHGQH/3 (A) and BHGQH/3-160T (B) were performed by dose-dependent direct binding assay as described in the text. (C) and (D) Transmisson of H5N1 duck viruses in guinea pigs. (C) r-BHGQH/3-inoculated group. (D) BHGQH/3-160T-inoculated group. The dashed blue lines in these panels indicate the lower limit of detection.

Figure 8. Receptor distribution in the respiratory system of guinea pigs.

(A) Alveoli. Red staining indicates MAAII-binding α-2,3 glycan. (B) Tracheal mucosa of guinea pig. Red staining indicates the presence of α-2,3 glycan and green staining indicates the presence of α-2,6 glycan. (C) Nasal mucosa (respiratory region) of guinea pig. (D) Nasal mucosa (olfactory region) of guinea pig. Green staining indicates the presence of α-2,6 glycan.