Tmprss2 knock-out mice are resistant to H10 influenza A virus pathogenesis.

The surface protein haemagglutinin (HA) of influenza A viruses (IAV) needs to be cleaved by a host protease to become functional. Here, we investigated if IAV of the H10 subtype also requires TMPRSS2 for replication and pathogenesis in mice. We first showed in cell culture that TMPRSS2 is able to cleave H10-HA. When Tmprss2-/- deficient mice were infected with a re-assorted virus H10-HA, they did not lose body weight and no viral replication was observed in contrast to wild-type mice. Histopathological analysis showed that inflammatory lesions in the lung of Tmprss2-/- mice were reduced compared to wild-type mice. In addition, no viral antigen was detected in the lungs of Tmprss2-/- mice and no evidence for HA cleavage was observed. We conclude from these studies that TMPRSS2 activity is also essential for in vivo replication and pathogenesis of H10 IAV.

Exchange of amino acids in the H1-haemagglutinin to H3 residues is required for efficient influenza A virus replication and pathology in Tmprss2 knock-out mice.

The haemagglutinin (HA) of H1N1 and H3N2 influenza A virus (IAV) subtypes has to be activated by host proteases. Previous studies showed that H1N1 virus cannot replicate efficiently in Tmprss2-/- knock-out mice whereas H3N2 viruses are able to replicate to the same levels in Tmprss2-/- as in wild type (WT) mice. Here, we investigated the sequence requirements for the HA molecule that allow IAV to replicate efficiently in the absence of TMPRSS2. We showed that replacement of the H3 for the H1-loop sequence (amino acids 320 to 329, at the C-terminus of HA1) was not sufficient for equal levels of virus replication or severe pathology in Tmprss2-/- knock-out mice compared to WT mice. However, exchange of a distant amino acid from H1 to H3 sequence (E31D) in addition to the HA-loop substitution resulted in virus replication in Tmprss2-/- knock-out mice that was comparable to WT mice. The higher virus replication and lung damage was associated with increased epithelial damage and higher mortality. Our results provide further evidence and insights into host proteases as a promising target for therapeutic intervention of IAV infections.

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

UNLABELLED: Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is necessary for viral activation and infectivity. In humans and mice, members of the type II transmembrane protease family (TTSP), e.g., TMPRSS2, TMPRSS4, and TMPRSS11d (HAT), have been shown to cleave influenza virus HA for viral activation and infectivity in vitro Recently, we reported that inactivation of a single HA-activating protease gene,Tmprss2, in knockout mice inhibits the spread of H1N1 influenza viruses. However, after infection of Tmprss2 knockout mice with an H3N2 influenza virus, only a slight increase in survival was observed, and mice still lost body weight. In this study, we investigated an additional trypsin-like protease, TMPRSS4. Both TMPRSS2 and TMPRSS4 are expressed in the same cell types of the mouse lung. Deletion of Tmprss4 alone in knockout mice does not protect them from body weight loss and death upon infection with H3N2 influenza virus. In contrast,Tmprss2(-/-)Tmprss4(-/-)double-knockout mice showed a remarkably reduced virus spread and lung pathology, in addition to reduced body weight loss and mortality. 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 IMPORTANCE: Influenza epidemics and recurring pandemics are responsible for significant global morbidity and mortality. Due to high variability of the virus genome, resistance to available antiviral drugs is frequently observed, and new targets for treatment of influenza are needed. Host cell factors essential for processing of the virus hemagglutinin represent very suitable drug targets because the virus is dependent on these host factors for replication. We reported previously that Tmprss2-deficient mice are protected against H1N1 virus infections, but only marginal protection against H3N2 virus infections was observed. 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. Thus, TMPRSS4 represents another host cell factor that is involved in cleavage activation of H3N2 influenza viruses in vivo.

Lst1 deficiency has a minor impact on course and outcome of the host response to influenza A H1N1 infections in mice.

BACKGROUND: Previously, we performed a quantitative trait locus (QTL) mapping study in BXD recombinant inbred mice to identify host genetic factors that confer resistance to influenza A virus infection. We found Lst1 (leukocyte specific transcript 1) as one of the most promising candidate genes in the Qivr17-2 locus because it is non-functional in DBA/2 J mice. Several studies have proposed that LST1 plays a role in the immune response to inflammatory diseases in humans and has additional immune-regulatory functions. Here, we evaluated the relevance of LST1 for the host response to influenza A infection in B6-Lst1 (-/-) mutant mice. FINDINGS: To investigate the role of LST1, we infected B6-Lst1 (-/-) mutant and C57BL/6 N wild-type mice with a low-virulent influenza A virus (PR8M; H1N1). Lst1 deficient mice exhibited significantly increased body weight loss at days 5 and 6 after infection and slightly increased lethality compared to infected wild-type mice. Determination of viral loads, histopathological examination and analysis of immune cell composition in bronchoalveolar lavage of infected lungs did not reveal any obvious differences between KO and wild-type mice. CONCLUSIONS: The absence of Lst1 leads to a slightly more susceptible phenotype. However, deletion of Lst1 in DBA/2 J mice alone does not explain the high susceptibility of this strain to PR8M influenza infections.

Increased virulence of a PB2/HA mutant of an avian H9N2 influenza strain after three passages in porcine differentiated airway epithelial cells.

We analyzed the adaptation of influenza viruses to growth in differentiated airway epithelial cells of a new host by passaging an avian H9N2 virus three times in porcine precision-cut lung slices (PCLS). Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins, and two in the HA protein. In this study, we characterized the PB2 mutation G685R by generating recombinant H9N2 viruses containing the PB2 single mutation alone or in combination with one of the HA mutations (A190V or T212I). When analyzed in porcine cells - a tracheal cell line (NPTr) or PCLS - the PB2-685 mutant did not provide a growth advantage and had no effect on the ciliary activity which is a virulence marker of swine influenza viruses. Pathogenicity for mice was also not increased by the single PB2 mutation. However, both double mutants (HA-190+PB2-685 and HA-212+PB2-685) showed significantly increased virulence in mice. Therefore, the mutations in the HA and PB2 proteins may confer early adaptation of an avian H9N2 virus to a mammalian host. In conclusion, we expect that a broader ensemble of mutations will be required to render an H9N2 virus virulent for pigs.