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The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases.

Kühn N, Bergmann S, Kösterke N, Lambertz RL, Keppner A, van den Brand JM, Pöhlmann S, Weiß S, Hummler E, Hatesuer B, Schughart K - J. Virol. (2016)

Bottom Line: In this study, we investigated an additional trypsin-like protease, TMPRSS4.Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virus in vivo Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality.Here we show that deletion of two host protease genes,Tmprss2 and Tmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection Genetics, Helmholtz Centre for Infection Research, and University of Veterinary Medicine Hannover, Braunschweig, Germany.

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Tmprss2−/−Tmprss4−/− double-knockout mice show reduced body weight loss and mortality after infection with H3N2 influenza A virus. (A) Eight- to 11-week-old female mice were infected with 2 × 103 FFU of mouse-adapted H3N2 influenza virus by intranasal application, and body weight and survival were monitored until day 14 p.i. In addition to mice that were found dead, mice with a weight loss of >30% of the starting body weight were euthanized and recorded as dead. (B) Numbers of infectious particles in lung homogenates were determined. Individual values, means and SEM are presented. (C) Relative lung weights were determined by weighing freshly prepared lungs and determining the percent ratio of lung weight to body weight. Individual values, means, and SEM are shown. (D) Hematological parameters were measured with a VetScan HM5 system, and the kinetics of relative numbers of lymphocytes (Lym), monocytes (Mon), and granulocytes (Gr) were determined. Homozygous Tmprss2−/−Tmprss4−/− knockout mice lost significantly less body weight than wild-type mice (e.g., P < 0.0001 at day 2 and P < 0.0001 at day 4; Mann-Whitney U test). Tmprss2−/−Tmprss4−/− mice showed significantly reduced mortality compared to that of wild-type mice (P < 0.0001; log rank test) as well as single-knockout mice. Viral loads were significantly higher in infected wild-type mice than in infected homozygous mutant mice at days 2, 4, and 6 p.i. (P < 0.01; Mann-Whitney U test). Furthermore, lung weights were significantly higher in infected wild-type mice than in infected Tmprss2−/−Tmprss4−/− knockout mice (P < 0.01). In the hemograms, lymphocyte numbers decreased until day 2 p.i., and granulocytes increased in wild-type mice on day 2 p.i. and, to a lesser degree, in Tmprss2−/−Tmprss4−/− knockout mice.
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Figure 4: Tmprss2−/−Tmprss4−/− double-knockout mice show reduced body weight loss and mortality after infection with H3N2 influenza A virus. (A) Eight- to 11-week-old female mice were infected with 2 × 103 FFU of mouse-adapted H3N2 influenza virus by intranasal application, and body weight and survival were monitored until day 14 p.i. In addition to mice that were found dead, mice with a weight loss of >30% of the starting body weight were euthanized and recorded as dead. (B) Numbers of infectious particles in lung homogenates were determined. Individual values, means and SEM are presented. (C) Relative lung weights were determined by weighing freshly prepared lungs and determining the percent ratio of lung weight to body weight. Individual values, means, and SEM are shown. (D) Hematological parameters were measured with a VetScan HM5 system, and the kinetics of relative numbers of lymphocytes (Lym), monocytes (Mon), and granulocytes (Gr) were determined. Homozygous Tmprss2−/−Tmprss4−/− knockout mice lost significantly less body weight than wild-type mice (e.g., P < 0.0001 at day 2 and P < 0.0001 at day 4; Mann-Whitney U test). Tmprss2−/−Tmprss4−/− mice showed significantly reduced mortality compared to that of wild-type mice (P < 0.0001; log rank test) as well as single-knockout mice. Viral loads were significantly higher in infected wild-type mice than in infected homozygous mutant mice at days 2, 4, and 6 p.i. (P < 0.01; Mann-Whitney U test). Furthermore, lung weights were significantly higher in infected wild-type mice than in infected Tmprss2−/−Tmprss4−/− knockout mice (P < 0.01). In the hemograms, lymphocyte numbers decreased until day 2 p.i., and granulocytes increased in wild-type mice on day 2 p.i. and, to a lesser degree, in Tmprss2−/−Tmprss4−/− knockout mice.

Mentions: Both TMPRSS2 and TMPRSS4 are able to cleave H3 hemagglutinin in vitro and are expressed in influenza virus target cells. Therefore, we generated Tmprss2−/−Tmprss4−/− double-knockout mice and infected them with 2 × 103 FFU H3N2 virus. Indeed, infected double-mutant mice showed a delayed and significantly reduced loss of body weight compared to wild-type mice or Tmprss2−/− or Tmprss4−/− single-knockout mice (Fig. 4A). Furthermore, viral loads were significantly lower in double-knockout mice than in wild-type mice after infection with 2 × 103 FFU H3N2 virus at days 2 to 6 p.i. (Fig. 4B). In addition, the relative weight of wild-type lungs increased considerably more from days 2 to 6 postinfection than that of knockout lungs (Fig. 4C), indicating less infiltration of immune cells and accumulation of fluid. In agreement with these observations, the relative number of granulocytes in peripheral blood increased to higher levels in wild-type mice than in double-knockout mice after infection with H3N2 virus (Fig. 4D), which represents an indicator of severe influenza virus infection (28). However, both wild-type and knockout mice showed similar degrees of lymphopenia during the first 2 days p.i., followed by an increase of lymphocytes in both knockout and wild-type mice (Fig. 4D). Similar granulocytosis was observed in both strains on days 2 to 6 p.i.


The Proteolytic Activation of (H3N2) Influenza A Virus Hemagglutinin Is Facilitated by Different Type II Transmembrane Serine Proteases.

Kühn N, Bergmann S, Kösterke N, Lambertz RL, Keppner A, van den Brand JM, Pöhlmann S, Weiß S, Hummler E, Hatesuer B, Schughart K - J. Virol. (2016)

Tmprss2−/−Tmprss4−/− double-knockout mice show reduced body weight loss and mortality after infection with H3N2 influenza A virus. (A) Eight- to 11-week-old female mice were infected with 2 × 103 FFU of mouse-adapted H3N2 influenza virus by intranasal application, and body weight and survival were monitored until day 14 p.i. In addition to mice that were found dead, mice with a weight loss of >30% of the starting body weight were euthanized and recorded as dead. (B) Numbers of infectious particles in lung homogenates were determined. Individual values, means and SEM are presented. (C) Relative lung weights were determined by weighing freshly prepared lungs and determining the percent ratio of lung weight to body weight. Individual values, means, and SEM are shown. (D) Hematological parameters were measured with a VetScan HM5 system, and the kinetics of relative numbers of lymphocytes (Lym), monocytes (Mon), and granulocytes (Gr) were determined. Homozygous Tmprss2−/−Tmprss4−/− knockout mice lost significantly less body weight than wild-type mice (e.g., P < 0.0001 at day 2 and P < 0.0001 at day 4; Mann-Whitney U test). Tmprss2−/−Tmprss4−/− mice showed significantly reduced mortality compared to that of wild-type mice (P < 0.0001; log rank test) as well as single-knockout mice. Viral loads were significantly higher in infected wild-type mice than in infected homozygous mutant mice at days 2, 4, and 6 p.i. (P < 0.01; Mann-Whitney U test). Furthermore, lung weights were significantly higher in infected wild-type mice than in infected Tmprss2−/−Tmprss4−/− knockout mice (P < 0.01). In the hemograms, lymphocyte numbers decreased until day 2 p.i., and granulocytes increased in wild-type mice on day 2 p.i. and, to a lesser degree, in Tmprss2−/−Tmprss4−/− knockout mice.
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Figure 4: Tmprss2−/−Tmprss4−/− double-knockout mice show reduced body weight loss and mortality after infection with H3N2 influenza A virus. (A) Eight- to 11-week-old female mice were infected with 2 × 103 FFU of mouse-adapted H3N2 influenza virus by intranasal application, and body weight and survival were monitored until day 14 p.i. In addition to mice that were found dead, mice with a weight loss of >30% of the starting body weight were euthanized and recorded as dead. (B) Numbers of infectious particles in lung homogenates were determined. Individual values, means and SEM are presented. (C) Relative lung weights were determined by weighing freshly prepared lungs and determining the percent ratio of lung weight to body weight. Individual values, means, and SEM are shown. (D) Hematological parameters were measured with a VetScan HM5 system, and the kinetics of relative numbers of lymphocytes (Lym), monocytes (Mon), and granulocytes (Gr) were determined. Homozygous Tmprss2−/−Tmprss4−/− knockout mice lost significantly less body weight than wild-type mice (e.g., P < 0.0001 at day 2 and P < 0.0001 at day 4; Mann-Whitney U test). Tmprss2−/−Tmprss4−/− mice showed significantly reduced mortality compared to that of wild-type mice (P < 0.0001; log rank test) as well as single-knockout mice. Viral loads were significantly higher in infected wild-type mice than in infected homozygous mutant mice at days 2, 4, and 6 p.i. (P < 0.01; Mann-Whitney U test). Furthermore, lung weights were significantly higher in infected wild-type mice than in infected Tmprss2−/−Tmprss4−/− knockout mice (P < 0.01). In the hemograms, lymphocyte numbers decreased until day 2 p.i., and granulocytes increased in wild-type mice on day 2 p.i. and, to a lesser degree, in Tmprss2−/−Tmprss4−/− knockout mice.
Mentions: Both TMPRSS2 and TMPRSS4 are able to cleave H3 hemagglutinin in vitro and are expressed in influenza virus target cells. Therefore, we generated Tmprss2−/−Tmprss4−/− double-knockout mice and infected them with 2 × 103 FFU H3N2 virus. Indeed, infected double-mutant mice showed a delayed and significantly reduced loss of body weight compared to wild-type mice or Tmprss2−/− or Tmprss4−/− single-knockout mice (Fig. 4A). Furthermore, viral loads were significantly lower in double-knockout mice than in wild-type mice after infection with 2 × 103 FFU H3N2 virus at days 2 to 6 p.i. (Fig. 4B). In addition, the relative weight of wild-type lungs increased considerably more from days 2 to 6 postinfection than that of knockout lungs (Fig. 4C), indicating less infiltration of immune cells and accumulation of fluid. In agreement with these observations, the relative number of granulocytes in peripheral blood increased to higher levels in wild-type mice than in double-knockout mice after infection with H3N2 virus (Fig. 4D), which represents an indicator of severe influenza virus infection (28). However, both wild-type and knockout mice showed similar degrees of lymphopenia during the first 2 days p.i., followed by an increase of lymphocytes in both knockout and wild-type mice (Fig. 4D). Similar granulocytosis was observed in both strains on days 2 to 6 p.i.

Bottom Line: In this study, we investigated an additional trypsin-like protease, TMPRSS4.Thus, our results identified TMPRSS4 as a second host cell protease that, in addition to TMPRSS2, is able to activate the HA of H3N2 influenza virus in vivo Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality.Here we show that deletion of two host protease genes,Tmprss2 and Tmprss4, strongly reduced viral spread as well as lung pathology and resulted in increased survival after H3N2 virus infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection Genetics, Helmholtz Centre for Infection Research, and University of Veterinary Medicine Hannover, Braunschweig, Germany.

Show MeSH
Related in: MedlinePlus