Cells were fixed and immunostained using an antibody against NP and an FITC-conjugated secondary antibody. and two recent Asian isolates, A/quail/Shantou/782/00 (H9-782) and A/quail/Shantou/2061/00 (H9-2061), made up of mono-, di-, and tribasic HA cleavage sites, respectively. All H9N2 isolates were activated by human proteases TMPRSS2 (transmembrane protease, serine S1 member 2) and Piceatannol HAT (human airway trypsin-like protease). Interestingly, H9-782 and H9-2061 were also activated by matriptase, Piceatannol a protease widely expressed in most epithelia with high expression levels in the kidney. Nephrotropism of H9N2 viruses has been observed in chickens, and here we found that H9-782 and H9-2061 were proteolytically activated in canine kidney (MDCK-II) and chicken embryo kidney (CEK) cells, whereas H9-Wisc was not. Computer virus activation was inhibited by peptide-mimetic inhibitors of matriptase, strongly suggesting that matriptase is responsible for HA cleavage in these kidney cells. Our MYH11 data demonstrate that H9N2 viruses with R-S-S-R or R-S-R-R cleavage sites are activated by matriptase in addition to HAT and TMPRSS2 and, therefore, can be activated in a wide range of tissues what may impact virus spread, tissue tropism and pathogenicity. INTRODUCTION Human influenza A viruses cause acute respiratory illness that affects millions of people during seasonal outbreaks and occasional pandemics and are therefore of major public health concern. Avian influenza A viruses are responsible for recurrent outbreaks in chickens and turkeys that may be connected with high morbidity and mortality and lead to serious economic losses in the poultry industry. Influenza A viruses belong to the family of and contain a segmented single-stranded RNA genome of unfavorable polarity that codes for 11 to 13 proteins (1). Based on antigenic criteria of the two surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), influenza A viruses are divided into 17 HA (H1 to H17) and 10 NA (N1 to N10) subtypes (2). Most subtypes circulate in wild aquatic birds, their natural reservoir, and are occasionally transmitted to other species, including poultry, pigs, and humans. Avian influenza viruses (AIV) differ in their pathogenicity and are classified as either low- or high-pathogenicity Piceatannol avian influenza viruses (LPAIV or HPAIV, respectively). LPAIV replicate primarily in the intestinal and also in the respiratory tract of birds, cause moderate or asymptomatic infections, and spread via the fecal-oral route. In contrast, HPAIV cause systemic infections in poultry, with mortality rates up to 100%. All HPAIV belong to the subtypes H5 and H7, but not all H5 and H7 viruses are highly pathogenic (3, 4). Influenza computer virus replication is initiated by the major viral surface glycoprotein hemagglutinin (HA), which binds to sialic acid-containing receptors and mediates fusion of the viral envelope with the endosomal membrane in order to Piceatannol release the computer virus genome into the target cell. HA is usually synthesized as a precursor protein, HA0, and has to be cleaved at a distinct arginine-glycine peptide bond by a host cell protease into the subunits HA1 and HA2 to gain its fusion capacity. Cleavage of HA0 is usually a prerequisite for any conformational switch at low pH in the endosome that triggers membrane fusion and is, therefore, essential for viral infectivity and spread. Depending on the amino acid sequence at the cleavage site, HAs vary in their susceptibility to different host cell proteases. Most LPAIV and mammalian viruses, including seasonal and pandemic human viruses, contain a single arginine (R) or rarely a lysine (K) at the HA cleavage site and are cleaved by trypsin (5). Relevant trypsin-like proteases are present in a restricted quantity of tissues, such as the respiratory or intestinal tract. We recognized the type II transmembrane serine proteases (TTSPs) HAT (human airway trypsin-like protease) and TMPRSS2 (transmembrane protease, serine S1 member 2) as HA-activating enzymes in the human airway epithelium (6). More recently, the related protease TMPRSS4 was shown to cleave HA with a monobasic cleavage site too (7). In contrast, HPAIV of subtypes H5 and H7 possess a multibasic HA cleavage site of the consensus sequence R-X-R/K-R that is activated by ubiquitous proteases furin and proprotein convertase 5/6 (PC5/6), supporting systemic infections (4, 8, 9). Within the last few years, influenza viruses of the subtype H9N2 have attracted particular attention. H9N2 viruses were first isolated from turkeys in the United States in 1966.