Improvement of influenza A/Fujian/411/02 (H3N2) virus growth in embryonated chicken eggs by balancing the hemagglutinin and neuraminidase activities, using reverse genetics

Abstract

The H3N2 influenza A/Fujian/411/02-like virus strains that circulated during the 2003-2004 influenza season caused influenza epidemics. Most of the A/Fujian/411/02 virus lineages did not replicate well in embryonated chicken eggs and had to be isolated originally by cell culture. The molecular basis for the poor replication of A/Fujian/411/02 virus was examined in this study by the reverse genetics technology. Two antigenically related strains that replicated well in embryonated chicken eggs, A/Sendai-H/F4962/02 and A/Wyoming/03/03, were compared with the prototype A/Fujian/411/02 virus. A/Sendai differed from A/Fujian by three amino acids in the neuraminidase (NA), whereas A/Wyoming differed from A/Fujian by five amino acids in the hemagglutinin (HA). The HA and NA segments of these three viruses were reassorted with cold-adapted A/Ann Arbor/6/60, the master donor virus for the live attenuated type A influenza vaccines (FluMist). The HA and NA residues differed between these three H3N2 viruses evaluated for their impact on virus replication in MDCK cells and in embryonated chicken eggs. It was determined that replication of A/Fujian/411/02 in eggs could be improved by either changing minimum of two HA residues (G186V and V226I) to increase the HA receptor-binding ability or by changing a minimum of two NA residues (E119Q and Q136K) to lower the NA enzymatic activity. Alternatively, recombinant A/Fujian/411/02 virus could be adapted to grow in eggs by two amino acid substitutions in the HA molecule (H183L and V226A), which also resulted in the increased HA receptor-binding activity. Thus, the balance between the HA and NA activities is critical for influenza virus replication in a different host system. The HA or NA changes that increased A/Fujian/411/02 virus replication in embryonated chicken eggs were found to have no significant impact on antigenicity of these recombinant viruses. This study demonstrated that the reverse genetics technology could be used to improve the manufacture of the influenza vaccines.

FIG. 1.

HA receptor-binding affinity of recombinant viruses. The 6:2 A/Fujian, A/Sendai, A/Wyoming, and A/Fujian variants with V186 and I226 or L183 and A226 changes were adsorbed to MDCK cells at an MOI of 1.0 at 4°C or 33°C for 30 min, and the infected cells were washed three times (+) or left untreated (−). After 6 h of incubation at 33°C, the cells were processed for immunofluorescence staining. The percentage of infected cells (mean ± standard deviation) indicated in each image is an average for six images.

FIG. 2.

Growth kinetics of recombinant viruses in MDCK cells. MDCK cells were infected at an MOI of 1.0 at either 33°C or 4°C for 30 min and washed three times with PBS prior to the addition of the medium containing 1.0 μg/ml of trypsin. The infected cells were incubated at 33°C and, at the indicated time intervals, the culture supernatants were collected and the virus amount was determined by plaque assay on MDCK cells.

FIG. 3.

Receptor-binding sites in HA and NA of influenza H3N2 subtypes. The residues that were shown to increase the HA receptor-binding affinity and to decrease the NA enzymatic activity in relation to sialic acid (SIA)-binding sites are indicated. The HA monomer was modeled using 5HMG (49), and the NA monomer was modeled based on 2BAT (46) using WebLab ViewerLite 3.10 (Accelrys, San Diego, CA).