TMPRSS2 Independency for Haemagglutinin Cleavage In Vivo Differentiates Influenza B Virus from Influenza A Virus

Influenza A and B viruses show clear differences in their host specificity and pandemic potential. Recent studies have revealed that the host protease TMPRSS2 plays an essential role for proteolytic activation of H1, H3, and H7 subtype strains of influenza A virus (IAV) in vivo. IAV possessing a monobasic cleavage site in the haemagglutinin (HA) protein replicates poorly in TMPRSS2 knockout mice owing to insufficient HA cleavage. In the present study, human isolates of influenza B virus (IBV) strains and a mouse-adapted IBV strain were analysed. The data showed that IBV successfully underwent HA cleavage in TMPRSS2 knockout mice, and that the mouse-adapted strain was fully pathogenic to these mice. The present data demonstrate a clear difference between IAV and IBV in their molecular mechanisms for spreading in vivo.


Results
Human isolate IBV strains are avirulent but spread similarly in wild-type (WT) and TMPRSS2 KO mice. Human TMPRSS2 (hTMPRSS2) has been shown to cleave IBV HA 11 . First, the cleavage activity of mouse TMPRSS2 (mTMPRSS2) for IBV HA was analysed and compared with that of hTMPRSS2. HeLa cells constitutively expressing mTMPRSS2 or hTMPRSS2 (HeLa/mTM2 and HeLa/hTM2, respectively) and the parental HeLa cells were infected with the B/Aichi/99[V] strain (Fig. 1A) or mouse-adapted (MA)-B/Ibaraki/85[V] strain ( Fig. 1B). At 12 and 24 hours post-infection (p.i.), polypeptides for IBV HA were detected by SDS-PAGE and immunoblotting. Cleaved forms of HA (HA 1 and HA 2 ) were clearly detected in HeLa/mTM2 and HeLa/hTM2 cells, but not in the parental HeLa cells, infected with either B/Aichi/99 [V] or MA-B/Ibaraki/85[V] (Fig. 1A,B). The cleavage efficiency was similar between HeLa/mTM2 and HeLa/hTM2 cells. When the culture media were supplemented with trypsin, cleaved forms of HA were detected in all three cell lines (Fig. 1A,B). These data indicated that mTMPRSS2 cleaves IBV HA as well as hTMPRSS2.
When challenged with B/Aichi/99[V] and B/Shiga/98 [Y], both WT and TMPRSS2 KO mice showed little, if any, body weight loss (data not shown). When challenged with B/Yamagata/88[Y] strain, both WT and TMPRSS2 KO mice showed very mild body weight loss ( Fig. 2A). Neither WT nor TMPRSS2 KO mice showed any clinical signs (data not shown). The viral titres in lung lavage fluids and lung homogenates of the mice were analysed. Since these IBV strains have not been adapted to grow in mice, the virus titres were generally low ( Fig. 2B,C). Nonetheless, the virus titres in TMPRSS2 KO mice were generally slightly lower than those in WT mice (Fig. 2B,C). However, most of the differences between WT and TMPRSS2 KO mice were not significant (P > 0.05), with only the data for the lung homogenates of B/Yamagata/88[Y] and B/Shiga/98[Y] at 2 days p.i. showing significant differences (P < 0.05) (Fig. 2B). Histopathological analyses demonstrated that the viruses spread moderately in the lungs of both WT and KO mice. Each strain caused slight bronchiolitis at 2 days p.i. and mild alveolitis at 6 days p.i. in both WT and KO mice (Fig. 3). The immunostaining patterns for IBV proteins were similar between WT and KO mice at 2 and 6 days p.i. (Fig. 3). Although these pathological conditions progressed to some extent, they remained mild (Fig. 3). Most importantly, the data revealed no significant differences in the histopathological changes and viral antigen patterns between WT and KO mice infected with IBV.
The mouse-adapted B/Ibaraki/2/85 strain is fully activated proteolytically in vivo and shows full pneumopathogenicity in TMPRSS2 KO mice. Next, the replication capacity and virulence of the MA-B/   Table 1. Seven amino acid changes were found in the HA protein. The residues were mapped on the IBV HA structure (PDB 2RFU). All of these residues were located in the globular head domain at positions far distant from the cleavage site ( Fig. 4). Both WT and KO mice showed severe body weight loss and required euthanasia, when challenged with MA-B/Ibaraki/85 [V]. Similar kinetics of body weight loss were observed for WT and KO mice (Fig. 5A). The survival rates were also similar between WT and TMPRSS2 KO mice (Fig. 5B). At 2 days p.i., the lungs of both WT and KO mice showed similar levels of bronchiolitis with slight alveolitis, and viral antigens were detected throughout the bronchial epithelia ( Fig. 5C). At 6 days p.i., moderate alveolitis had progressed in the lungs of both WT and KO mice with viral antigens spreading throughout the lungs (Fig. 5C). The virus titres in the lung lavage fluids and lung homogenates from both WT and KO mice were high during the observation period, although the titres in the lung lavage fluids in WT mice tended to be several-fold higher than those in KO mice (Fig. 6A). However, the differences were not significant (P < 0.05). The extent of activation in vivo of the progeny viruses was analysed. Even in KO mice, the infectivity titres were not restored significantly by in vitro trypsin treatment ( Table 2), indicating that the majority of MA-B/Ibaraki/85 [V] was produced in an almost fully-activated form in the lungs of both WT and KO mice. To obtain evidence of HA cleavage in vivo, lung lavage fluids collected from infected WT and KO mice (n = 3 for each) were analysed by SDS-PAGE and immunoblotting. Generally, the HA signal intensities were greater in WT mice than in KO mice (Fig. 6B), consistent with the greater amounts of progeny viruses in WT mice compared with KO mice (Fig. 6A). However, it was clearly shown that HA was already cleaved into subunits (HA 1 and HA 2 ) in both WT and KO mice (Fig. 6B). The NP signals were also greater in WT mice than in TMPRSS2 KO mice (Fig. 6B). The extents of cleavage estimated by the signal intensities of HA 0 and the cleaved HA subunits (HA 1 and HA 2 ) were similar in WT and TMPRSS2 KO mice (Fig. 6C). Taken together, in both WT and KO mice, MA-B/Ibaraki/85[V], whose HA possesses a monobasic cleavage site, was almost fully activated proteolytically and replicated in multiple steps in the lungs, resulting in pneumonia and exhibiting high pathogenicity.

Discussion
Our data show a clear difference in the in vivo protease specificity between IAV and IBV. Many studies have been performed to identify the proteases responsible for HA activation, and many candidate proteases have been reported 12 . Several TTSPs, such as TMPRSS2 13,14 , HAT 13 22 suggested that IBV HA has higher sensitivity to an endoprotease(s), currently unidentified, than IAV HA, or that specific MDCK cell lines express an endoprotease that only activates IBV HA. Further analyses using cell cultures demonstrated that TMPRSS2 activates IBV HA as well as IAV HA 11 . Therefore, IBV strains may utilize TMPRSS2 for HA cleavage in vivo, similar to IAV strains. Nevertheless, our present data clearly demonstrate that TMPRSS2 is dispensable for HA cleavage and pathogenicity of IBV in mice. However, the virus titres in TMPRSS2 KO mice were slightly lower than those in WT mice. Therefore, potential anti-IAV drugs targeting TMPRSS2 are less effective for IBV. Under certain experimental conditions, H3N2 also showed the ability to use a TMPRSS2-independent HA activation mechanism through loss of an oligosaccharide at the HA stalk region 24 . The loss of this oligosaccharide likely improved the accessibility of an alternative host protease to the loop containing the HA cleavage site of IAV. The HA protein of IBV also possesses oligosaccharides at the stalk region and near the cleavage site 25 , but our data in the present study show that they do not limit the activation protease to TMPRSS2. The stalk oligosaccharide of IBV HA attached in vivo may be incompatible with the HA cleavage by TMPRSS2. Because the protease specificity of viruses determines virus tropism and pathogenicity 26,27 , identification of the protease(s) responsible for IBV HA cleavage may reveal a molecular basis for the different biological features between IAV and IBV, and contribute to the development of anti-IBV drugs.

Materials and Methods
Ethics statement. All experiments with animals were performed in strict accordance with the Animal Experimentation Guidelines of the National Institute of Infectious Diseases, and the protocol was approved by the Institutional Animal Care and Use Committee of the institute.
Mice. TMPRSS2 KO mice were reported previously 10    . The viral antigens of IBV were detected using a mouse antiserum against IBV NP (Lot. N179). This antiserum was obtained from BALB/c mice immunized intraperitoneally with inactivated IBV virions (Victoria lineage) using the Sigma adjuvant system (Sigma-Aldrich). The inflammation levels in individual mice were scored as follows: 0, no apparent changes; 1, minimal changes or bronchiolitis; 2, bronchiolitis and/or slight alveolitis; 3, mild alveolitis with neutrophils, monocytes/macrophages, or lymphocytes; 4, moderate alveolitis. The cut-off in body weight loss for euthanasia was 25%. Lung lavage fluids and lung homogenates were collected at 2, 4, and 6 days p.i. and subjected to a standard plaque assay.  WT   viruses to enter the cells. Trypsin was omitted to avoid HA cleavage before virus entry. At 24 hours p.i., the cell monolayers were additionally overlaid with MEM/1% agarose supplemented with 4.0 μ g/mL of trypsin to allow plaque formation. To determine the infectious titres including viruses that had not been activated in vivo but possessed an infectious potential, virus samples were treated with 2.0 μ g/mL of trypsin, and subjected to a standard plaque assay.

Analysis of infectious virus titres activated in vivo.
Immunoblotting. WT