Influenza viruses circulate in birds and poultry, with water fowl constituting the natural reservoir, and coinfection of swine with different influenza viruses is believed to play an important role in the emergence of pandemic viruses 
. TMPRSS2, TMPRSS4 and HAT were shown to activate influenza virus in transfected cells 
, but only for TMPRSS2 further evidence for a potential contribution to viral spread in humans was reported 
. In order to assess the role of TMPRSS2 in the influenza virus zoonosis, we tested whether this protease derived from chicken and swine is able to cleave HA. In parallel, we examined if TMPRSS2 of mouse origin facilitates HA proteolysis, since mice are commonly used as a model system for influenza virus spread and pathogenesis. Finally, cleavage and activation of HA by human and mouse HAT and TMPRSS4 were also evaluated.
Western blot analysis of transfected cells revealed that TMPRSS2, TMPRSS4 and HAT of all animal species tested cleaved the HA precursor HA0 and produced HA1 cleavage fragments identical to those observed for the human enzymes (). The slightly faster migration of HA1 fragments generated by TMPRSS2 compared to the other proteases is due to differential HA glycosylation 
. Cleavage resulted in HA activation, since lentiviral vectors produced in the protease expressing cells were fully infectious in the absence of trypsin treatment (). In contrast, no HA cleavage and activation was observed in cells transfected with empty vector or cells expressing human TMPRSS3 (), which was previously demonstrated not to process HA 
. Similarly, swine, chicken and mouse TMPRSS2 cleaved SARS-S into multiple fragments, as previously documented for human TMPRSS2 
, although some variation in cleavage efficiency was noted (). In contrast, trypsin digestion produced the S1 subunit, as expected 
. Of note, SARS-S was cleaved by TMPRSS2 upon coexpression of both proteins (cis cleavage, ) and upon mixing of SARS-S expressing cells with protease expressing cells (trans cleavage, Fig. S1
), although some variability in cleavage efficiency was noted in the latter setting. In agreement with SARS-S trans cleavage, expression of TMPRSS2 in target cells (TMPRSS2 panels) endogenously expressing very low amounts of viral receptor, ACE2 
, allowed efficient SARS-S-driven cell-cell fusion and fusion efficiency was not increased by the addition of trypsin. In contrast, SARS-S-driven fusion with control transfected cells (pcDNA panel) was inefficient and fusion efficiency was rescued by trypsin treatment (). Finally, transfection of ACE2 plasmid into target cells (ACE2 panel) also boosted cell-cell fusion and fusion efficiency was only modestly increased by trypsin, in agreement with our previous finding that receptor and protease expression on target cells can both limit SARS-S-mediated cell-cell fusion 
. In sum, these results demonstrate that cleavage-activation of influenza HA and SARS-S is conserved between human, porcine, avian and murine TMPRSS2 as well as human and murine HAT. Our observations also suggest that TMPRSS2 can support influenza virus spread in species integral to the influenza zoonosis, and that mice are suitable models to study the role of TMPRSS2, TMPRSS4 and HAT in viral spread and pathogenesis.
Proteolytic activation of influenza virus hemagglutinin and SARS spike protein is conserved between TMPRSS2 of human, porcine, avian and murine origin.
Binding of human influenza viruses to 2,6-linked sialic acids present on proteins and lipids on the host cell surface is critical for infectious viral entry into host cells 
. We assessed whether TMPRSS2 and HAT are coexpressed with 2,6-linked sialic acid human tissues. Immunostaining demonstrated the presence of 2,6-linked sialic acids on the surface of almost all cell types (, , ), in keeping with previous results 
, with the notable exception of vascular smooth muscle cells (data not shown), suggesting that expression of proteases, such as TMPRSS2 and HAT, but not 2,6-linked sialic acid is likely to be a major determinant of viral tropism. TMPRSS2 was expressed by epithelial cells at all sites examined in the aerodigestive tracts, as well as by many endothelial cells and myocytes of blood vessels, leucocytes (including alveolar macrophages) and smooth muscle cells (, , , ), indicating that TMPRSS2 could activate influenza virus in most permissive epithelia. HAT showed a distribution similar to TMPRSS2 (, , , ), but immunostaining of pneumocytes (alveolar epithelial cells) was weaker, implying low levels of protease expression at this site (). Unlike TMPRSS2, which appeared to be expressed by the majority of type 2 pneumocytes, HAT was expressed by fewer than 50% type 2 pneumocytes, but was additionally seen to be expressed by occasional type 1 pneumocytes (). Type 2 pneumocytes are defined by their morphology rather than a particular immunophenotype, being plump rather than flattened epithelial cells 
. All sections were examined by an experienced consultant pathologist (ES) in order to identify the cell types that were immunopositive. The exact intensities of staining for TMPRSS2 and HAT of various epithelial types in the aerodigestive tracts are summarized in . While TMPRSS2 expression by bronchial and intestinal smooth muscle cells was noted, these cells appeared negative for HAT, although some vascular smooth muscle cells were found to be positive (). Interestingly, TMPRSS2 but not HAT was expressed by cardiac myocytes (), suggesting that influenza myocarditis might be promoted by TMPRSS2 but not HAT. Notwithstanding, our data demonstrate the potential importance of both proteases in influenza infection.
Pulmonary and respiratory sinus expression of SARS-CoV and influenza virus activating proteases and receptors.
Tonsil and buccal mucosal expression of SARS-CoV and influenza virus activating proteases and receptors.
Ileal & myocardial expression of SARS-CoV and influenza virus activating proteases and receptors.
Expression pattern of influenza virus and SARS-coronavirus activating proteases and receptors in human tissues.
TMPRSS2 on target cells activates SARS-S on adjacent cells for cell-cell fusion and activates virion-associated SARS-S for infectious host cell entry 
. A wide range of sites demonstrated coexpression of ACE2 and TMPRSS2, and could thus support SARS-CoV spread (). Specifically, in the lung type 2, but not type 1 pneumocytes express both molecules, as do alveolar macrophages and the epithelial cells of intrapulmonary bronchi (). In the upper respiratory tract, the epithelium of the bronchi, larynx, nasal mucosa and respiratory sinuses () expresses both molecules, while ACE2 expression is absent from the trachea, vocal folds and epiglottis, although a previous study by Ren et al demonstrated ACE2 expression on the surface epithelium and mucus gland epithelium of trachea, similar to our findings in the larynx and bronchus 
. This suggests that ACE2 expression may be variable but widespread in the upper airway. The epithelia of the tonsil () and buccal mucosa () express both TMPRSS2 and ACE2. Additionally, ACE2 expression by some interstitial macrophages/dendritic cells in intra-alveolar septa of the lung, adjacent to TMPRSS2-expressing type 2 pneumocytes. In the gastrointestinal tract, epithelial co-expression of TMPRSS2 and ACE2 was identified at all sites examined, namely the oesophagus, stomach, ileum and colon. The two molecules were also expressed in cardiac myocytes. Furthermore, variable expression of both molecules by endothelial cells and myoctes of blood vessels, leucocytes and smooth muscle cells was seen (, , , ). Taken together, these results suggest that TMPRSS2 could promote SARS-CoV spread in important target sites, the gastrointestinal and respiratory tracts ().