TABLE 1.

Effect of HA glycosylation on agglutination of chicken erythrocytes and susceptibility to neuraminidase inhibitors in MDCK cells

HAaGlycan(s) at AsnbReciprocal HA titercNI assay, IC50 (nM)dMTT assay, EC50 (μM)eVirus yield reduction assayf
4°C37°CFoldOseltamivir carboxylateZanamivirFold0 μM0.01 μM1 μM100 μM
G012812810.8 ± 0.50.04 ± 0.010.05 ± 0.01>47.5 ± 0.216.0 ± 0.032.4 ± 0.122.0 ± 0.07
G194a12812811.0 ± 0.4<0.01<0.0117.3 ± 0.035.0 ± 0.064.2 ± 0.622.0 ± 0.50
G1,294a, 12964-12864-128≤22.0 ± 0.8<0.01<0.0118.3 ± 0.287.4 ± 0.126.3 ± 0.025.0 ± 0.03
G21291288-16≥81.3 ± 0.51.00 ± 0.020.80 ± 0.01>80g8.4 ± 0.126.7 ± 0.606.4 ± 0.125.8 ± 0.05
G316332-128<2>160.9 ± 0.6>100>100>10,0008.4 ± 0.078.2 ± 0.118.2 ± 0.347.9 ± 0.12
G1,394a, 1631282-4≥321.5 ± 0.5>100>100>10,0008.3 ± 0.238.1 ± 0.147.8 ± 0.497.8 ± 0.14
G2,3129, 163642-4≥161.0 ± 0.4>100>100>10,0008.0 ± 0.158.0 ± 0.177.5 ± 0.097.9 ± 0.45
G1,2,394a, 129, 1638-16<2>40.8 ± 0.3>100>100>10,0008.6 ± 0.347.8 ± 0.098.3 ± 0.047.9 ± 0.08
  • a Each genetically engineered reassortant virus contained the HA gene from A/Charlottesville/31/95 (H1N1) virus and remaining genes from the A/WSN/33 (H1N1) virus.

  • b Wild-type HA (G1,2,3) contained glycans attached at Asn 94a, 129, and 163; to abolish glycosylation motifs, single, double, or triple mutations were introduced into the HA gene (CH/95).

  • c The virus preparations were standardized for HA protein content by ELISA with the use of two anti-HA antibodies (a mouse MAb IVC102 and ferret polyclonal serum). The standardized virus preparations were then tested by a standard HA assay with guinea pig erythrocytes at 4°C. The viruses produced reciprocal HA titers in ranging from 128 to 256 HAU per 50 μl. The same virus preparations were then tested with chicken erythrocytes at 4°C and 37°C. A range of reciprocal HA titers determined in three experiments is shown.

  • d Viruses were tested by an NA activity inhibition assay against oseltamivir carboxylate. Average IC50 (inhibitory concentration 50%) values of three experiments are shown.

  • e Viruses were tested against either oseltamivir carboxylate or zanamivir by cell protection assay (MTT) at 48 h postinfection (MOI = 0.001). Average values of three experiments are shown. Fold change in average EC50s compared to that of the most drug susceptible reassortant virus (G1) is shown.

  • f MDCK cells were infected (MOI = 0.001) and overlaid with media containing zanamivir at indicated concentrations. At 48 h postinfection, the cell culture supernatants were harvested and subjected to the virus titration in MDCK cells. Results of three experiments are shown.

  • g A value of >100, against oseltamivir carboxylate was obtained.