Influenza H1N1 A/Solomon Island/3/06 virus receptor binding specificity correlates with virus pathogenicity, antigenicity, and immunogenicity in ferrets

Abstract

Influenza viruses attach to cells via a sialic acid moiety (sialic acid receptor) that is alpha2-3 linked or alpha2-6 linked to galactose (alpha2-3SAL or alpha2-6SAL); sialic acid acts as a receptor for the virus. Using lectin staining, we demonstrated that the alpha2-6SAL configuration is predominant in the respiratory tract of ferrets, including trachea, bronchus, and lung alveolus tissues. Recombinant wild-type (rWT) influenza A/Solomon Island/3/06 (SI06) (H1N1) viruses were constructed to assess the impact of the hemagglutinin (HA) variations (amino acids 190 or 226) identified in natural variants on virus replication in the upper and lower respiratory tract of ferrets, as well as virus antigenicity and immunogenicity. A single amino acid change at residue 226 (from Gln to Arg) in the HA of SI06 resulted in the complete loss of binding to alpha2-6SAL and a concomitant loss of the virus's ability to replicate in the lower respiratory tract of ferrets. In contrast, the virus with Gln226 in the HA protein has a receptor binding preference for alpha2-6SAL and replicates efficiently in the lungs. There was a good correlation between viral replication in the lungs of ferrets and disease symptoms. In addition, we also showed that the 190 and 226 residues affected viral antigenicity and immunogenicity. Our data emphasize the necessity of thoroughly assessing wild-type influenza viruses for their suitability as reference strains and for carefully selecting the HA antigen for vaccine production during annual influenza vaccine evaluation processes.

FIG. 1.

Hemagglutinin sequence alignment to compare the HA sequences of the H1N1 viruses that circulated from 1995 to 2007. Cold-adapted (ca) vaccine strains of A/New Caledonia/20/99 and A/Solomon Island/3/06 are also included. Residues 190 and 226 are highlighted in green or yellow, and three A/HK/2652/06-specific residues are highlighted in gray.

FIG. 2.

Detection of virus replication in the respiratory tract of ferrets. Ferrets were infected with rWT SI06-AQ or rWT SI06-DR intranasally, and three days later, the tissues were processed for immunohistochemistry staining using goat anti-influenza A virus polyclonal antibody followed by incubation with horseradish peroxidase-conjugated rabbit anti-goat antibody. Viral antigens in the infected cells are indicated in red.

FIG. 3.

Infected ferrets were monitored for changes in body weight and temperature. Ferrets in groups of three were inoculated with 7.0 log₁₀ PFU virus intranasally and monitored for their body weight daily (upper graph) and rectal temperature twice daily (lower graph) at a 6-h interval during the day. Each data value is the mean value for the three ferrets at that time point. The dotted line in the temperature graph represents the fever temperature.

FIG. 4.

Receptor distribution in the respiratory tract of ferrets. MAA1, MAA2, or SNA lectin was used to stain the tissues in the respiratory tract of ferrets to detect α2-3SAL (MAA1 and MAA2) or α2-6SAL (SNA). Madin-Darby canine kidney cells served as binding controls. Lectin-stained tissues are shown in red.

FIG. 5.

Virus binding to the respiratory tract tissues. FITC-labeled rWT SI06-AQ or rWT SI06-DR virus was incubated with ferret bronchus or lung tissues, followed by incubation with rabbit anti-FITC antibody and development with AEC substrate. The virus antigens bound to the tissues are shown in red. MDCK cells were used as binding controls.