Reverse genetics-generated elastase-dependent swine influenza viruses are attenuated in pigs Aleksandar Masic, Lorne A. Babiuk3 and Yan Zhou Correspondence Yan Zhou yan.zhou@usask.ca Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E3, Canada Received 7 July 2008 Accepted 28 September 2008 Influenza A virus causes significant morbidity in swine, resulting in a substantial economic burden. Swine influenza virus (SIV) infection also poses important human public health concerns. It has been shown that conversion of the haemagglutinin (HA) cleavage site from a trypsin-sensitive motif to an elastase-sensitive motif resulted in attenuated viruses in mouse models. However, application of this attenuation approach in a natural host has not been achieved yet. Here, we report that using reverse genetics, we generated two mutant SIVs derived from strain A/SW/SK/ 18789/02 (H1N1). Mutant A/SW/SK-R345V carries a mutation from arginine to valine at aa 345 of HA. Similarly, mutant A/SW/SK-R345A encodes alanine instead of arginine at aa 345 of HA. Our data showed that both mutants are solely dependent on neutrophil elastase cleavage in tissue culture. These tissue culture-grown mutant SIVs showed similar growth properties in terms of plaque size and growth kinetics to the wild-type virus. In addition, SIV mutants were able to maintain their genetic information after multiple passaging on MDCK cells. Furthermore, mutant SIVs were highly attenuated in pigs. Thus, these mutants may have the potential to serve as live attenuated vaccines. INTRODUCTION Swine influenza virus (SIV) is a member of the family Orthomyxoviridae, genus Influenza A (Lamb & Krug, 2001). SIV is the causative agent of swine influenza, a highly contagious, acute viral disease of swine; it induces an acute respiratory tract infection and lung lesions. After an incubation period of 24–72 h, the disease begins suddenly, often appearing in many animals in the herd at the same time. Infection is clinically characterized by a high fever, sneezing, rhinitis with nasal discharge, laboured abdominal breathing and bronchial rales at auscultation. In general, morbidity rates may approach 100 %, while mortality is usually less than 1 %. SIV infections can be associated with secondary bacterial/viral infections and reproductive dis- orders that can result in abortions. Along with porcine reproductive and respiratory syndrome virus, SIV con- tributes significantly to post-weaning respiratory disease, causing economic losses due to decreased body condition and an increase in the number of days needed to reach market weight. Currently, H1N1, H3N2 and H1N2 are the dominant subtypes that cause disease in the North American swine population (Olsen, 2002). SIV infection also poses very important human public health concerns because it naturally infects pigs and can be transmitted to humans (Wells et al., 1991). Since pigs are able to support replication of swine, human and avian influenza viruses, it is very likely that genetic reassortments between these viruses could create novel influenza subtypes. Recently, avian/swine virus reassortant H2N3 influenza A viruses were isolated from diseased swine in the USA. The H2N3 virus has undergone some adaptation to the mammalian host and is able to transmit among pigs and ferrets (Ma et al., 2007). Data from SIV surveillance studies and characterization of influenza virus isolates from pigs are critical for the understanding of long-term evolutionary and epidemiological patterns of human influenza and pandemics (Wells et al., 1991). The genome of influenza A viruses consists of eight segmented RNAs of negative polarity. The crucial step for infection by influenza A virus is initial virus binding to the cells followed by receptor-mediated endocytosis and fusion of the viral envelope to endosomal membranes (Cross et al., 2001; Skehel & Wiley, 2000). Influenza A virus entry into cells is mediated by the viral surface glycoprotein haemagglutinin (HA). HA has three major roles during virus replication: (i) HA binds to sialic acid receptors on the cell surface; (ii) it allows penetration of the virus into the cytoplasm by mediating fusion between the viral and the endosomal membranes; and (iii) it is the main viral antigen against which neutralizing antibodies are produced (Lamb & Krug, 2001). HA is synthesized as a precursor, HA0, that consists of HA1 and HA2 (Skehel & Wiley, 2000). In order to be infectious, HA0 must be cleaved by host proteases into HA1 and HA2. Therefore, this process 3Present address: University of Alberta, 3–7 University Hall, Edmonton, AB T6G 2J9, Canada. Journal of General Virology (2009), 90, 375–385 DOI 10.1099/vir.0.005447-0 005447 G 2009 SGM Printed in Great Britain 375