Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerged H7N9 subtype, in vivo. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently, the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2 ؉/؉ wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with >1,000 50% lethal doses (LD 50 ) for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multibasic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerged H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage in vivo and, thus, that TMPRSS2 expression is essential for IAV replication in vivo. IMPORTANCEInfluenza A virus (IAV) is a leading pathogen that infects and kills many humans every year. We clarified that the infectivity and pathogenicity of IAVs, including a recently emerged H7N9 subtype, are determined primarily by a host protease, TMPRSS2. Our data showed that TMPRSS2 is the key host protease that activates IAVs in vivo through proteolytic cleavage of their HA proteins. Hence, TMPRSS2 is a good target for the development of anti-IAV drugs. Such drugs could also be effective for many other respiratory viruses, including the recently emerged Middle East respiratory syndrome (MERS) coronavirus, because they are also activated by TMPRSS2 in vitro. Consequently, the present paper could have a large impact on the battle against respiratory virus infections and contribute greatly to human health. Influenza A virus (IAV) is classified in the Orthomyxoviridae family and is a leading agent that affects and kills humans worldwide. IAV enters target cells via endocytosis, and virus-cell membrane fusion occurs at the late endosomes, thus releasing the viral genome to start virus replication. Membrane fusion is mediated by the hemagglutinin (HA) protein, which is synthesized as the inactive precursor HA 0 and cleaved by a host cell protease(s) to gain fusion activity. Proteolytic cleavage of HA 0 into the HA 1 and HA 2 subunits is essential for HA to express membrane fusion activity and, consequently, for IAV to acquire infectivity.The HA of low-pathogenic (LP) IAVs, for whic...

CD26/DPPIV is a multifunctional cell surface protein that is widely expressed in most cell types including T lymphocytes, on which it is a marker of activation. It is also present in serum and other body fluids in a truncated form (sCD26/DPPIV). It preferentially cleaves N-terminal dipeptides from polypeptides with proline or alanine in the penultimate position, and in doing so, regulates the activities of a number of cytokines and chemokines. Due in part to this ability to regulate the activity of biopeptides, it can act as a tumor suppressor or activator. It can associate with several proteins, among them fibroblast activating protein-alpha (FAP-alpha), plasminogen, adenosine deaminase (ADA), the tyrosine phosphatase CD45, and the chemokine receptor CXCR4. It can also bind to the extracellular matrix (ECM) and depending on the presence of other ligands, this process can either lead to increased or decreased invasive activity of the cells on which it is expressed. As a result of these characteristics, CD26/DPPIV plays an important role in tumor biology, and is useful as a marker for various cancers, with its levels either on the cell surface or in the serum being increased in some neoplasms and decreased in others. Our group has shown that CD26/DPPIV can be manipulated by such agents as CD26 cDNA-carrying plasmids, siRNA and monoclonal antibodies, resulting in both in vitro and in vivo inhibition of cell growth, enhanced sensitivity to selected chemotherapeutic agents, and enhanced survival of mouse xenograft models. These studies have demonstrated the utility of these tools as potential targeted therapies for specific cancers expressing CD26/DPPIV.

We have reported a novel bovine rotavirus, the AzuK-1 (G21P [29]) strain, isolated from an asymptomatic calf. We isolated another bovine rotavirus, the Dai-10 strain, bearing new G24P [33] genotypes, assigned by the Rotavirus Classification Working Group (RCWG), from an asymptomatic cow in Hyogo Prefecture, Japan in 2007. To gain an insight into the origins and evolution of these strains, we determined the complete ORF sequences of all 11 genes of the two strains. The NSP3 genes of both strains were confirmed to belong to a new NSP3 genotype, T9, by the RCWG. Genotype determination of AzuK-1 and Dai-10 strains revealed that eight gene segments of both strains possessed genotypes typically observed in bovine rotaviruses, with the exception of VP4, VP7 and NSP3 gene segments. Unexpectedly, phylogenetic analyses showed that VP6 and NSP2 gene segments of the AzuK-1 and Dai-10 strains were clustered with those of simian or canine/feline rotaviruses, rather than with those of bovine rotaviruses. These findings indicate the possibility that both strains originated by interspecies transmission and multiple reassortment events involving bovine, simian and canine/feline rotaviruses, resulting in the introduction of some genes into the genetic background of bovine rotaviruses. INTRODUCTIONGroup A rotaviruses are the major pathogens causing acute gastroenteritis in infants and a wide range of animals, including birds. Rotavirus-induced diarrhoea is a serious public health problem worldwide, responsible for more than 600 000 child deaths each year (Parashar et al., 2006). Likewise, in domestic animals, rotavirus-induced diarrhoea is a major problem causing significant economic losses (Dhama et al., 2009;Martella et al., 2010).Rotaviruses are members of the family Reoviridae. Rotaviruses possess a genome of 11 segments of dsRNA, which encode six viral structural proteins (VP1-VP4, VP6 and VP7) and six non-structural proteins (NSP1-NSP6). Because of the segmented nature of the genome, a reassortment event can occur in cells co-infected with two or more different strains (Estes & Kapikian, 2007;Palombo, 2002;Ramig, 1997). The rotavirus virion is a triple-layered icosahedral particle. The outer capsid is composed of VP7 and VP4. They elicit neutralizing antibodies independently. In a dual classification system, rotaviruses are classified into 24 G genotypes and 32 P genotypes based on the nucleotide sequences of VP7 and VP4 genes, respectively (Collins et al., 2010; Esona et al., 2010;Matthijnssens et al., 2006Matthijnssens et al., , 2008a Schumann et al., 2009; Solberg et al., 2009;Ursu et al., 2009). Recently, a new classification system has been established using nucleotide sequences of all of the 11 genomic RNA segments by the Rotavirus Classification Working Group (RCWG) (Matthijnssens et al., 2008b). In this system, the The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AB513836-AB513838 and AB573070-AB573086.Supplementary material is available with the online version of this paper. , 2...

Abstract. The expression of keratin 18 (K18) is restricted in humans primarily to a variety of single layered or simple epithelia. However, direct introduction of a cloned K18 gene into cultured, somatic cells by DNA transfection has been shown to result in the promiscuous expression of K18 even while the endogenous mouse form of K18 (Endo B) remains silent. To determine if the cloned K18 genomic DNA fragment contains sufficient information to be regulated appropriately when subjected to a normal developmental environment, and to determine if the cloned gene is expressed in diverse epithelia, the K18 gene, including 2.5 kb of 5' flanking sequence and 3.5 kb of 3' flanking sequence, has been introduced into the germ line of mice. Mice from all three resulting K18 transgenic lines express the gene in an appropriate tissue-specific pattern that includes hepatocytes, simple epithelia of the intestinal tract, ductal cells of several glands and epithelial cells of the thymus. No expression of K18 was found in muscle, heart, or in most of the brain even in mice carrying 18 copies of the K18 gene. In most tissues, the level of K18 RNA was directly proportional to copy number and was as efficiently expressed as the endogenous Endo B gene. The K18 protein was identified by both protein blotting methods and indirect immunofluorescence staining. No pathological consequences of overexpression of the K18 gene were observed. The cloned K18 gene appears to contain all cis-acting DNA sequences necessary for appropriate expression. In addition, diverse epithelial cell types are able to express this single human gene.