Virus Research ELSEVIER Virus Research 44 (1996) 79-95 Mutations in the hemagglutinin and matrix genes of a virulent influenza virus variant, A/FM/1/47-MA, control different stages in pathogenesis Cecilia A. Smeenk ~, Kathryn E. Wright ~, Bruce F. Burns b,c, Ajay J. Thaker ~, Earl G. Brown ~'* ~'Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada, KIH 81145 bDepartment of Pathology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada, KIH 8M5 CDepartment of Laboratory Medicine, Ottawa Civic Hospital, Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada, KIH 8M5 Received 9 January 1996; revised 5 April 1996; accepted 8 April 1996 Abstract The mouse adapted strain of influenza A/FM/1/47 virus, FM-MA, has increased virulence due to mutations in HA, MI and at least one other, unmapped, genome segment. Genetic reassortants that differ due to the HA or M1 mutations were used to define the role of these mutations in pathogenesis. Pathological changes in lungs of infected mice were assessed by hematoxylin phloxine saffron (HPS) staining, and viral infection was measured by fluorescent antibody staining of thin sections and flow cytometry of lung parenchymal cells. HA played a role in bronchiolar pathology by increasing necrosis of bronchiolar epithelium, peribronchiolar lymphocytes, and airway obstruction. The HA mutation was shown to be responsible for a 0.2 unit decreased in the pH optimum of fusion and controlled resistance to c~ and fl inhibitors of hemagglutination. Both these changes in biology may confer a replicative advantage in bronchioles seen in the first day of infection. Thus the HA mutation may have conferred a survival advantage in the extracellular lung environment. The M1 mutation resulted in improved growth in the lung and cultured cells and was associated with increases in recruitment of macrophages, spread of infection into the alveoli of the lung and interstitial pneumonia. Sequence analysis indicated that the unmapped mutation in the control of FM-MA virulence is either the K 482--*R substitution in the PB2 protein or the D 538---,G substitution in the PB1 protein. One or other of these mutations results in a growth advantage in infected lung but not in cultured cells as well as a further increased recruitment and infection of macrophages in the lung. Infection with virulent strains of influenza that induced increases in macrophage recruitment caused hypothermia in the mouse. Keywords: Pathogenesis; Hemagglutinin; PB2; Matrix; Macrophage; Replication control * Corresponding author. Tel.: + 1 613 5625800, ext. 8310; fax: 613-562-5452; e-mail: brown@labsunl.med.uottawa.ca 0168-1702/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII SO168-1702(96)01329- 9