RESISTANCE OF INFLUENZA A AND B VIRUSES TO 4- GUANIDINO-Neu5Ac2en. N. Y. Chirgadze¹, J. M. Colacino¹, K. A. Staschke¹, A. Baxter¹, G. Air², A. Bansal², E. Garman³, J. Tang¹, W. J. Hornback¹, J. E Munroe¹, W. G. Laver⁴, ¹Lilly Research Laboratories, Indianapolis, USA, ²University of Alabama, Birmingham, USA, ³University of Oxford, UK, ⁴Australian National University, Canberra, AU

The reassortant influenza viruses, A/NWS-G70c (N9 neuraminidase [NA]) and B/HK/8/73 (HG) (B/Lee/40 NA), were selected for resistance to 4-Guanidino-Neu5Ac2en (4-GuDANA). The NA of resistant viruses was >200-fold more resistant to 4-GuDANA than was the NA of parental viruses. Resistant A and B viruses displayed 5% and 0.5%, respectively, of the parental NA activity yet both were able to undergo multicycle replication in MDCK cells and grow to equal titer in embryonated eggs. The expression by these viruses of NA activity in MDCK cells over a 72 hour period was extremely low relative to that of parental viruses. Sequence analysis revealed a single mutation in the NA gene leading to the change of a conserved Glu 119 (N9 numbering) to Gly for both A and B viruses. Glu 119 lies in a pocket beneath the active site of the enzyme and has been shown to interact with the C-4 guanidinium moiety of 4-GuDANA. The NA from 4- GuDANA^r A/NWS-G70c has been crystallized. Although these crystals grew to only 0.2 mm in the largest dimension, data from low temperature (100K) X-ray diffraction experiments were collected with a merging R-value on intensities of 6.2% to 2.0 Å resolution. These data revealed the absence of the glutamate residue at amino acid position 119. We have been unable to obtain NA crystals of X-ray diffraction quality from the 4- GuDANA^r B virus. In addition to the above viruses, wild-type influenza B/Lee/40 was selected for resistance to 4-GuDANA and the identical nucleotide change leading to the Glu to Gly alteration in the NA was found. Attempts to select a reassortant N2 virus (A/NWS-Tokyo) for resistance to 4- GuDANA have so far been unsuccessful. In preliminary experiments, 4- GuDANA^r A/NWS-G70c was able to induce pyrexia in ferrets indicating that viruses with low-levels of an altered NA retain pathogenicity, at least in this model of infection.