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...

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In that outbreak, 46 monkeys died from severe pneumonia during a quarantine period. A CDV strain (CYN07-dV) was isolated in Vero cells expressing dog signaling lymphocyte activation molecule (SLAM). Phylogenic analysis showed that CYN07-dV was closely related to the recent CDV outbreaks in China, suggesting continuing chains of CDV infection in monkeys. In vitro, CYN07-dV uses macaca SLAM and macaca nectin4 as receptors as efficiently as dog SLAM and dog nectin4, respectively. CYN07-dV showed high virulence in experimentally infected cynomolgus monkeys and excreted progeny viruses in oral fluid and feces. These data revealed that some of the CDV strains, like CYN07-dV, have the potential to cause acute systemic infection in monkeys.

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is sometimes associated with serious neurological disorders. In this study, an attempt was made to identify molecular determinants of EV71 attenuation of neurovirulence in a monkey infection model. An infectious cDNA clone of the virulent strain of EV71 prototype BrCr was constructed; temperature-sensitive (ts) mutations of an attenuated strain of EV71 or of poliovirus (PV) Sabin vaccine strains were then introduced into the infectious clone. In vitro and in vivo phenotypes of the parental and mutant viruses were analysed in cultured cells and in cynomolgus monkeys, respectively. Mutations in 3D polymerase (3D pol ) and in the 39 non-translated region (NTR), corresponding to ts determinants of Sabin 1, conferred distinct temperature sensitivity to EV71. An EV71 mutant [EV71(S1-39)] carrying mutations in the 59 NTR, 3D pol and in the 39 NTR showed attenuated neurovirulence, resulting in limited spread of virus in the central nervous system of monkeys. These results indicate that EV71 and PV1 share common genetic determinants of neurovirulence in monkeys, despite the distinct properties in their original pathogenesis. INTRODUCTIONEnterovirus 71 (EV71) belongs to the genus Enterovirus of the family Picornaviridae and possesses a single-stranded, positive-sense RNA genome of approximately 7500 nt in length (Brown & Pallansch, 1995;Schmidt et al., 1974). Genetically, EV71 is classified as a species A human enterovirus along with some coxsackie A (CA) viruses, such as CA10 and CA16 (Brown & Pallansch, 1995;Pulli et al., 1995). As well as CA10 and CA16, EV71 causes hand, foot and mouth disease (HFMD) and herpangina, which are common and self-limiting diseases that typically occur in children. However, EV71 infection sometimes causes severe neurological diseases, such as brainstem encephalitis and polio-like paralysis (Chumakov et al., 1979; Wang et al., 2003), mainly in infants and young children (McMinn, 2002). A number of fatal encephalitis cases were reported in large-scale HFMD outbreaks in Malaysia in 1997 (Abubakar et al., 1999;Shimizu et al., 1999) and in Taiwan in 1998 (Ho et al., 1999Lin et al., 2003;Lu et al., 2002;Wang et al., 2002). Furthermore, sporadic HFMD cases with severe neurological manifestations have been reported in the Western Pacific region, e.g. in Australia, Singapore, Hong Kong and Japan (Ahmad, 2000;Chan et al., 2000; Fujimoto et al., 2002; Herrero et al., 2003;Komatsu et al., 1999;Lum et al., 1998;McMinn et al., 1999McMinn et al., , 2001b. Numerous factors (e.g. virus genotypes or specific mutations, herd protective immunity, individual immunity or association with other infectious agents) could lead HFMD to become a more serious disease. From molecular epidemiological studies of EV71, McMinn et al. (2001a) suggested that an amino acid change at position 170 of VP1 (from Ala to Val) is involved in the virulence of EV71. Non-structural proteins of EV71 (2A and 3C proteinases) were responsib...

Among the enteroviruses, polioviruses and enterovirus 71 (EV71) are two major neurotropic viruses causing serious neurological manifestations. While polioviruses are being eradicated globally by vaccination, EV71 still has the potential to cause a large outbreak such as that in Taiwan in 1998, in which there were many fatalities. In this study, we determined the neurovirulence of EV71 by neuropathological analysis of cynomolgus monkeys after experimental infection with five EV71 strains, which were isolated from individual patients with fatal encephalitis; meningitis; and hand, foot, and mouth disease. After intraspinal inoculation, the monkeys developed neurological manifestations within 1-6 days post-inoculation, irrespective of the inoculated strains. These manifestations included not only pyramidal tract signs such as flaccid paralysis, but also extrapyramidal tract signs such as tremor and ataxia. Histological and viral examinations confirmed virus replication in the spinal cord, brainstem, cerebellar cortex, and dentate nuclei, and cerebrum. The strains isolated during the 1970s and 1990s showed no particular differences with respect to neurotropism. Thus, it is clear that EV71 has a wider neurotropism than that of polioviruses.