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The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2.IMPORTANCEThe emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.

HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficientIfnar1−/−mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA.

Ebola virus (EBOV) VP30 regulates viral genome transcription and replication by switching its phosphorylation status. However, the importance of VP30 phosphorylation and dephosphorylation in other viral replication processes such as nucleocapsid and virion assembly is unclear. Interestingly, VP30 is predominantly dephosphorylated by cellular phosphatases in viral inclusions, while it is phosphorylated in the released virions. Thus, uncertainties regarding how VP30 phosphorylation in nucleocapsids is achieved and whether VP30 phosphorylation provides any advantages in later steps in viral replication have arisen. In the present study, to characterize the roles of VP30 phosphorylation in nucleocapsid formation, we used electron microscopic analyses and live cell imaging systems. We identified VP30 localized to the surface of protrusions surrounding nucleoprotein (NP)-forming helical structures in the nucleocapsid, suggesting the involvement in assembly and transport of nucleocapsids. Interestingly, VP30 phosphorylation facilitated its association with nucleocapsid-like structures (NCLSs). On the contrary, VP30 phosphorylation does not influence the transport characteristics and NCLS number leaving from and coming back into viral inclusions, indicating that the phosphorylation status of VP30 is not a prerequisite for NCLS departure. Moreover, the phosphorylation status of VP30 did not cause major differences in nucleocapsid transport in authentic EBOV-infected cells. In the following budding step, the association of VP30 and its phosphorylation status did not influence the budding efficiency of virus-like particles. Taken together, it is plausible that EBOV may utilize the phosphorylation of VP30 for its selective association with nucleocapsids, without affecting nucleocapsid transport and virion budding processes. IMPORTANCE Ebola virus (EBOV) causes severe fevers with unusually high case fatality rates. The nucleocapsid provides the template for viral genome transcription and replication. Thus, understanding the regulatory mechanism behind its formation is important for the development of novel therapeutic approaches. Previously, we established a live-cell imaging system based on the ectopic expression of viral fluorescent fusion proteins, allowing the visualization and characterization of intracytoplasmic transport of nucleocapsid-like structures. EBOV VP30 is an essential transcriptional factor for viral genome synthesis, and, although its role in viral genome transcription and replication is well understood, the functional importance of VP30 phosphorylation in assembly of nucleocapsids is still unclear. Our work determines the localization of VP30 at the surface of ruffled nucleocapsids, which differs from the localization of polymerase in EBOV-infected cells. This study sheds light on the novel role of VP30 phosphorylation in nucleocapsid assembly, which is an important prerequisite for virion formation.

A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS- and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10–17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure., Author summary SARS-CoV-2 infection presents a wide spectrum of clinical manifestations ranging from asymptomatic to fatal respiratory failure. The determinants for failure in viral control and/or fatal disease progression have not been elucidated fully. Both acquired immune effectors, antibodies and CD8+ T cells, are considered to contribute to viral control. However, it remains unknown whether a deficiency in either of these two arms is directly linked to failure in the control of SARS-CoV-2 replication. In the present study, to know the requirement of CD8+ T cells for viral control after the establishment of infection, we examined the effect of CD8+ cell depletion by monoclonal anti-CD8 antibody administration in the subacute phase on SARS-CoV-2 replication in cynomolgus macaques. Unexpectedly, our analysis revealed no significant impact of CD8+ cell depletion on viral replication, indicating that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but this study suggests that CD8+ T-cell dysfunction may not solely lead to viral control failure or fatal disease progression.

Significance This study directly demonstrates virulence-associated features of Helicobacter suis infection using isolates obtained from human stomachs. H. suis is the second-most-prevalent Helicobacter species in the human stomach, but diagnostic tests for Helicobacter pylori often fail to diagnose H. suis. H. suis does not have orthologs of either cytotoxin-associated gene A (CagA) or vacuolating cytotoxin A (VacA), two major virulence factors in H. pylori, suggesting that the molecular pathogenicity of H. suis and clinical manifestations caused by H. suis infection are different from those caused by H. pylori. This study paves the way for epidemiological research aimed at identifying the causal relationships between H. suis and gastric diseases and developing diagnostic methods., Helicobacter suis, a bacterial species naturally hosted by pigs, can colonize the human stomach in the context of gastric diseases such as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Because H. suis has been successfully isolated from pigs, but not from humans, evidence linking human H. suis infection to gastric diseases has remained incomplete. In this study, we successfully in vitro cultured H. suis directly from human stomachs. Unlike Helicobacter pylori, the viability of H. suis decreases significantly on neutral pH; therefore, we achieved this using a low-pH medium for transport of gastric biopsies. Ultimately, we isolated H. suis from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of H. suis yielded significant improvements in endoscopic and histopathological findings. Oral infection of mice with H. suis clinical isolates elicited gastric and systemic inflammatory responses; in addition, progression of gastric mucosal metaplasia was observed 4 mo postinfection. Because H. suis could be isolated from the stomachs of infected mice, our findings satisfied Koch’s postulates. Although further prospective clinical studies are needed, H. suis, like H. pylori, is likely a gastric pathogen in humans. Furthermore, comparative genomic analysis of H. suis using complete genomes of clinical isolates revealed that the genome of each H. suis isolate contained highly plastic genomic regions encoding putative strain-specific virulence factors, including type IV secretion system–associated genes, and that H. suis isolates from humans and pigs were genetically very similar, suggesting possible pig-to-human transmission.

Severe fever with thrombocytopenia syndrome (SFTS) caused by a species Dabie bandavirus (formerly SFTS virus [SFTSV]) is an emerging hemorrhagic infectious disease with a high case-fatality rate. One of the best strategies for preventing SFTS is to develop a vaccine, which is expected to induce both humoral and cellular immunity. We applied a highly attenuated but still immunogenic vaccinia virus strain LC16m8 (m8) as a recombinant vaccine for SFTS. Recombinant m8s expressing SFTSV nucleoprotein (m8-N), envelope glycoprotein precursor (m8-GPC), and both N and GPC (m8-N+GPC) in the infected cells were generated. Both m8-GPC- and m8-N+GPC-infected cells were confirmed to produce SFTSV-like-particles (VLP) in vitro, and the N was incorporated in the VLP produced by the infection of cells with m8-N+GPC. Specific antibodies to SFTSV were induced in mice inoculated with each of the recombinant m8s, and the mice were fully protected from lethal challenge with SFTSV at both 103 TCID50 and 105 TCID50. In mice that had been immunized with vaccinia virus strain Lister in advance of m8-based SFTSV vaccine inoculation, protective immunity against the SFTSV challenge was also conferred. The pathological analysis revealed that mice immunized with m8-GPC or m8-N+GPC did not show any histopathological changes without any viral antigen-positive cells, whereas the control mice showed focal necrosis with inflammatory infiltration with SFTSV antigen-positive cells in tissues after SFTSV challenge. The passive serum transfer experiments revealed that sera collected from mice inoculated with m8-GPC or m8-N+GPC but not with m8-N conferred protective immunity against lethal SFTSV challenge in naïve mice. On the other hand, the depletion of CD8-positive cells in vivo did not abrogate the protective immunity conferred by m8-based SFTSV vaccines. Based on these results, the recombinant m8-GPC and m8-N+GPC were considered promising vaccine candidates for SFTS., Author summary Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral hemorrhagic fever with a high case-fatality rate (approximately 5% to >40%). Indigenous SFTS has been reported in China, Japan, South Korea, and Vietnam. Thus, the development of an effective vaccine for SFTS is urgently needed. Vaccinia virus (VAC) was previously used as a vaccine for smallpox. Unfortunately, after these strains, the so-called second generation of VAC used during the eradication campaign was associated with severe adverse events, and the third generation of VAC strains such as LC16m8 (m8) and modified vaccinia Ankara (MVA) was established. m8 is confirmed to be highly attenuated while still maintaining immunogenicity. m8 is licensed for use in healthy people in Japan. At the present time, approximately 100,000 people have undergone vaccination with m8 without experiencing any severe postvaccine complications. At present, third-generation VAC strains are attractive for a recombinant vaccine vector, especially for viral hemorrhagic infectious diseases, such as Ebola virus disease, Lassa fever, Crimean-Congo hemorrhagic fever, and SFTS. We investigated the practicality of an m8-based recombinant vaccine for SFTS as well as other promising recombinant VAC-based vaccines for viral hemorrhagic infectious diseases.

The species Keterah orthonairovirus is a member of the genus Orthonairovirus. Few studies have focused on this species, and there remains no treatment for Issyk-Kul fever, an infectious disease caused by a Keterah orthonairovirus. This study was performed to characterize this species using two viruses, Issyk-Kul virus (ISKV) and Soft tick bunyavirus (STBV), in cell culture and type I interferon receptor knockout (IFNAR-/-) mice and to evaluate the efficacy of serum transfusion using a mouse model of ISKV infection. The two viruses replicated in many kinds of mammal- and tick-derived cell lines but showed few different characteristics in tropism and antigenicity against anti-viral sera in cell culture. Neither virus caused clinical signs in wild-type mice, but both caused lethal infection in IFNAR-/- mice. ISKV caused more acute death than STBV in IFNAR-/- mice. In both viral infections in IFNAR-/- mice, macroscopic abnormalities were prominent in the liver. Similar levels of viral genome between ISKV- and STBV-infected IFNAR-/- mice were observed in blood, liver, lymphoid tissues and adrenal gland at moribund stages. Hematologic abnormalities in IFNAR-/- mice infected with these viruses, including leukopenia and thrombocytopenia, and biochemical abnormalities indicating liver damage were prominent. In addition, blood levels of many kinds of cytokines and chemokines such as granulocyte colony-stimulating factor, interleukin-6, tumor necrosis factor-α, interferon gamma-induced protein 10 and monocyte chemoattractant protein-1 were elevated. ISKV-immunized serum transfusion after infection delayed the time to death of IFNAR-/- mice. Thus, the present study showed that the species Keterah orthonairovirus could proliferate in most mammal-derived cell lines and cause severe liver lesions and death in IFNAR-/- mice and that serum transfusion might be effective in treatment against Issyk-Kul fever.

Enterovirus 71 (EV71) is a causative agent of hand-foot-mouth disease, and it sometimes causes severe neurological disease. Development of effective vaccines and animal models to evaluate vaccine candidates are needed. However, the animal models currently used for vaccine efficacy testing, monkeys and neonatal mice, have economic, ethical, and practical drawbacks. In addition, EV71 strains prepared for lethal challenge often develop decreased virulence during propagation in cell culture. To overcome these problems, we used a mouse model expressing human scavenger receptor B2 (hSCARB2) that showed lifelong susceptibility to EV71. We selected virulent EV71 strains belonging to the subgenogroups B4, B5, C1, C2, and C4 and propagated them using a culture method for EV71 without an apparent reduction in virulence. Here, we describe a novel EV71 vaccine efficacy test based on these hSCARB2 transgenic (Tg) mice and these virulent viruses. Adult Tg mice were immunized subcutaneously with formalin-inactivated EV71. The vaccine elicited sufficient levels of neutralizing antibodies in the immunized mice. The mice were subjected to lethal challenge with virulent viruses via intravenous injection. Survival, clinical signs, and body weight changes were observed for 2 weeks. Most immunized mice survived without clinical signs or histopathological lesions. The viral replication in immunized mice was much lower than that in nonimmunized mice. Mice immunized with the EV71 vaccine were only partially protected against lethal challenge with coxsackievirus A16. These results indicate that this new model is useful for in vivo EV71 vaccine efficacy testing. IMPORTANCE The development of new vaccines for EV71 relies on the availability of small animal models suitable for in vivo efficacy testing. Monkeys and neonatal mice have been used, but the use of these animals has several drawbacks, including high costs, limited susceptibility, and poor experimental reproducibility. In addition, the related ethical issues are considerable. The new efficacy test based on hSCARB2 Tg mice and virulent EV71 strains propagated in genetically modified cell lines presented here can overcome these disadvantages and is expected to accelerate the development of new EV71 vaccines.

Background: Helicobacter suis, a bacterial species naturally hosted by pigs, can colonize the human stomach in the context of gastric diseases, such as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Because H. suis has been successfully isolated from pigs, but not from humans, evidence linking human H. suis infection to gastric diseases has remained incomplete. The aim of this study is to elucidate the pathogenicity of H. suis by genetic characterization and through animals infected with H. suis isolated from human stomachs. Methods: H. suis from human gastric biopsies was isolated as previously described in pigs, with modifications. Mice were orally infected with H. suis for 4 months, and then pathogenesis was analyzed. Whole-genome analysis of H. suis isolates was performed by short-read and long-read sequencing, and their genomes were subjected to comparative analysis. Findings: H. suis was successfully isolated from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of H. suis yielded significant improvements in endoscopic and histopathological findings in all patients. H. suis infection in mice using clinical isolates elicited gastric and systemic inflammatory responses, and H. suis could be isolated from the stomachs of infected mice. Therefore, according to Koch’s postulates, these findings demonstrate that H. suis is a human gastric pathogen. Genomic analysis revealed high similarity between isolates from humans and pigs, suggesting possible horizonal transmission of H. suis. Interpretation: This is the first study to directly demonstrate virulence-associated features of H. suis infection using isolates obtained from human stomachs. Since H. suis would have been missed by routine diagnostic tests for Helicobacter pylori, large-scale epidemiological research on H. suis is needed to identify causal relationships with gastric diseases and develop novel diagnostic methods. Funding: MEXT/JSPS KAKENHI Grant numbers JP19H03474 for (HM) and JP20K08365 for (KT), and AMED Grant Numbers JP20fk0108148 (ER) and JP20fk0108133 (MS). Conflict of Interest: ER has received research funding from GlaxoSmithKline, outside the submitted work. All other authors declare no competing interests. Ethical Approval: Ethical approvals were obtained from all participating hospitals and the National Institute of Infectious Diseases (NIID). Written informed consent was obtained from all participants.

The spike (S) protein of coronavirus, which binds to cellular receptors and mediates membrane fusion for cell entry, is a candidate vaccine target for blocking coronavirus infection. However, some animal studies have suggested that inadequate immunization against severe acute respiratory syndrome coronavirus (SARS‐CoV) induces a lung eosinophilic immunopathology upon infection. The present study evaluated two kinds of vaccine adjuvants for use with recombinant S protein: gold nanoparticles (AuNPs), which are expected to function as both an antigen carrier and an adjuvant in immunization; and Toll‐like receptor (TLR) agonists, which have previously been shown to be an effective adjuvant in an ultraviolet‐inactivated SARS‐CoV vaccine. All the mice immunized with more than 0.5 µg S protein without adjuvant escaped from SARS after infection with mouse‐adapted SARS‐CoV; however, eosinophilic infiltrations were observed in the lungs of almost all the immunized mice. The AuNP‐adjuvanted protein induced a strong IgG response but failed to improve vaccine efficacy or to reduce eosinophilic infiltration because of highly allergic inflammatory responses. Whereas similar virus titers were observed in the control animals and the animals immunized with S protein with or without AuNPs, Type 1 interferon and pro‐inflammatory responses were moderate in the mice treated with S protein with and without AuNPs. On the other hand, the TLR agonist‐adjuvanted vaccine induced highly protective antibodies without eosinophilic infiltrations, as well as Th1/17 cytokine responses. The findings of this study will support the development of vaccines against severe pneumonia‐associated coronaviruses.

Tularemia is a severe infectious zoonotic disease caused by Francisella tularensis. Although F. tularensis is considered to be a potential biological weapon due to its high infectivity and mortality rate, no vaccine has been currently licensed. Recently, we reported that F. tularensis SCHU P9 derived ΔpdpC strain lacking the pathogenicity determinant protein C gene conferred stable and good protection in a mouse lethal model. In this study, the protective effect of ΔpdpC was evaluated using a monkey lethal model. Two cynomolgus macaques (Macaca fascicularis) intratracheally challenged with the virulent strain SCHU P9 were euthanized on 7 and 11 days post-challenge after the development of severe clinical signs. The bacterial replication in alveolar macrophages and type II epithelial cells in the lungs would cause severe pneumonia accompanied by necrosis. Conversely, two animals subcutaneously immunized with ΔpdpC survived 3 weeks after SCHU P9 challenge. Though one of the two animals developed mild symptoms of tularemia, bacterial replication was limited in the respiratory organs, which may be due to a high level of humoral and cellular immune responses against F. tularensis. These results suggest that the ΔpdpC mutant would be a safe and promising candidate as a live attenuated tularemia vaccine.

Transmembrane serine protease TMPRSS2 activates the spike protein of highly pathogenic human coronaviruses such as severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV). In vitro, activation induces virus-cell membrane fusion at the cell surface. However, the roles of TMPRSS2 during coronavirus infection in vivo are unclear. Here, we used animal models of SARS-CoV and MERS-CoV infection to investigate the role of TMPRSS2. Th1-prone C57BL/6 mice and TMPRSS2-knockout (KO) mice were used for SARS-CoV infection, and transgenic mice expressing the human MERS-CoV receptor DPP4 (hDPP4-Tg mice) and TMPRSS2-KO hDPP4-Tg mice were used for MERS-CoV infection. After experimental infection, TMPRSS2-deficient mouse strains showed reduced body weight loss and viral kinetics in the lungs. Lack of TMPRSS2 affected the primary sites of infection and virus spread within the airway, accompanied by less severe immunopathology. However, TMPRSS2-KO mice showed weakened inflammatory chemokine and/or cytokine responses to intranasal stimulation with poly(I·C), a Toll-like receptor 3 agonist. In conclusion, TMPRSS2 plays a crucial role in viral spread within the airway of murine models infected by SARS-CoV and MERS-CoV and in the resulting immunopathology. IMPORTANCE Broad-spectrum antiviral drugs against highly pathogenic coronaviruses and other emerging viruses are desirable to enable a rapid response to pandemic threats. Transmembrane protease serine type 2 (TMPRSS2), a protease belonging to the type II transmembrane serine protease family, cleaves the coronavirus spike protein, making it a potential therapeutic target for coronavirus infections. Here, we examined the role of TMPRSS2 using animal models of SARS-CoV and MERS-CoV infection. The results suggest that lack of TMPRSS2 in the airways reduces the severity of lung pathology after infection by SARS-CoV and MERS-CoV. Taken together, the results will facilitate development of novel targets for coronavirus therapy.

Saffold virus (SAFV), a human cardiovirus, is occasionally detected in infants with neurological disorders, including meningitis and cerebellitis. We recently reported that SAFV type 3 isolates infect cerebellar glial cells, but not large neurons, in mice. However, the impact of this infection remained unclear. Here, we determined the neuropathogenesis of SAFV type 3 in the cerebella of neonatal ddY mice by using SAFV passaged in the cerebella of neonatal BALB/c mice. The virus titer in the cerebellum increased following the inoculation of each of five passaged strains. The fifth passaged strain harbored amino acid substitutions in the VP2 (H160R and Q239R) and VP3 (K62M) capsid proteins. Molecular modeling of the capsid proteins suggested that the VP2-H160R and VP3-K62M mutations alter the structural dynamics of the receptor binding surface via the formation of a novel hydrophobic interaction between the VP2 puff B and VP3 knob regions. Compared with the original strain, the passaged strain showed altered growth characteristics in human-derived astroglial cell lines and greater replication in the brains of neonatal mice. In addition, the passaged strain was more neurovirulent than the original strain, while both strains infected astroglial and neural progenitor cells in the mouse brain. Intracerebral inoculation of either the original or the passaged strain affected brain Purkinje cell dendrites, and a high titer of the passaged strain induced cerebellar hypoplasia in neonatal mice. Thus, infection by mouse-passaged SAFV affected cerebellar development in neonatal mice. This animal model contributes to the understanding of the neuropathogenicity of SAFV infections in infants.IMPORTANCESaffold virus (SAFV) is a candidate neuropathogenic agent in infants and children, but the neuropathogenicity of the virus has not been fully elucidated. Recently, we evaluated the pathogenicity of two clinical SAFV isolates in mice. Similar to other neurotropic picornaviruses, these isolates showed mild infectivity of glial and neural progenitor cells, but not of large neurons, in the cerebellum. However, the outcome of this viral infection in the cerebellum has not been clarified. Here, we examined the tropism of SAFV in the cerebellum. We obtained anin vivo-passaged strain from the cerebella of neonatal mice and examined its genome and its neurovirulence in the neonatal mouse brain. The passaged virus showed high infectivity and neurovirulence in the brain, especially the cerebellum, and affected cerebellar development. This unique neonatal mouse model will be helpful for elucidating the neuropathogenesis of SAFV infections occurring early in life.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection can manifest as a mild illness, acute respiratory distress, organ failure, or death. Several animal models have been established to study disease pathogenesis and to develop vaccines and therapeutic agents. Here, we developed transgenic (Tg) mice on a C57BL/6 background; these mice expressed human CD26/dipeptidyl peptidase 4 (hDPP4), a functional receptor for MERS-CoV, under the control of an endogenous hDPP4 promoter. We then characterized this mouse model of MERS-CoV. The expression profile of hDPP4 in these mice was almost equivalent to that in human tissues, including kidney and lung; however, hDPP4 was overexpressed in murine CD3-positive cells within peripheral blood and lymphoid tissues. Intranasal inoculation of young and adult Tg mice with MERS-CoV led to infection of the lower respiratory tract and pathological evidence of acute multifocal interstitial pneumonia within 7 days, with only transient loss of body weight. However, the immunopathology in young and adult Tg mice was different. On day 5 or 7 postinoculation, lungs of adult Tg mice contained higher levels of proinflammatory cytokines and chemokines associated with migration of macrophages. These results suggest that the immunopathology of MERS-CoV infection in the Tg mouse is age dependent. The mouse model described here will increase our understanding of disease pathogenesis and host mediators that protect against MERS-CoV infection. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) infections are endemic in the Middle East and a threat to public health worldwide. Rodents are not susceptible to the virus because they do not express functional receptors; therefore, we generated a new animal model of MERS-CoV infection based on transgenic mice expressing human DPP4 (hDPP4). The pattern of hDPP4 expression in this model was similar to that in human tissues (except lymphoid tissue). In addition, MERS-CoV was limited to the respiratory tract. Here, we focused on host factors involved in immunopathology in MERS-CoV infection and clarified differences in antiviral immune responses between young and adult transgenic mice. This new small-animal model could contribute to more in-depth study of the pathology of MERS-CoV infection and aid development of suitable treatments.

Rhinoviruses (RVs) are classified into three species: RV-A, B, and C. Unlike RV-A and -B, RV-C cannot be propagated using standard cell culture systems. In order to isolate RV-Cs from clinical specimens and gain a better understanding of their biological properties and pathogenesis, we established air&ndash, liquid-interface (ALI) culture methods using HBEC3-KT and HSAEC1-KT immortalized human airway epithelial cells. HBEC3- and HSAEC1-ALI cultures morphologically resembled pseudostratified epithelia with cilia and goblet cells. Two fully sequenced clinical RV-C isolates, RV-C9 and -C53, were propagated in HBEC3-ALI cultures, and increases in viral RNA ranging from 1.71 log10 to 7.06 log10 copies were observed. However, this propagation did not occur in HSAEC1-ALI cultures. Using the HBEC3-ALI culture system, 11 clinical strains of RV-C were isolated from 23 clinical specimens, and of them, nine were passaged and re-propagated. The 11 clinical isolates were classified as RV-C2, -C6, -C9, -C12, -C18, -C23, -C40, and -C53 types according to their VP1 sequences. Our stable HBEC3-ALI culture system is the first cultivable cell model that supports the growth of multiple RV-C virus types from clinical specimens. Thus, the HBEC3-ALI culture system provides a cheap and easy-to-use alternative to existing cell models for isolating and investigating RV-Cs.

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and sometimes causes severe or fatal neurological complications. The amino acid at VP1-145 determines the virological characteristics of EV71. Viruses with glutamic acid (E) at VP1-145 (VP1-145E) are virulent in neonatal mice and transgenic mice expressing human scavenger receptor B2, whereas those with glutamine (Q) or glycine (G) are not. However, the contribution of this variation to pathogenesis in humans is not fully understood. We compared the virulence of VP1-145E and VP1-145G viruses of Isehara and C7/Osaka backgrounds in cynomolgus monkeys. VP1-145E, but not VP1-145G, viruses induced neurological symptoms. VP1-145E viruses were frequently detected in the tissues of infected monkeys. VP1-145G viruses were detected less frequently and disappeared quickly. Instead, mutants that had a G-to-E mutation at VP1-145 emerged, suggesting that VP1-145E viruses have a replication advantage in the monkeys. This is consistent with our hypothesis proposed in the accompanying paper (K. Kobayashi, Y. Sudaka, A. Takashino, A. Imura, K. Fujii, and S. Koike, J Virol 92:e00681-18, 2018, https://doi.org/10.1128/JVI.00681-18) that the VP1-145G virus is attenuated due to its adsorption by heparan sulfate. Monkeys infected with both viruses produced neutralizing antibodies before the onset of the disease. Interestingly, VP1-145E viruses were more resistant to neutralizing antibodies than VP1-145G viruses in vitro A small amount of neutralizing antibody raised in the early phase of infection may not be sufficient to block the dissemination of VP1-145E viruses. The different resistance of the VP1-145 variants to neutralizing antibodies may be one of the reasons for the difference in virulence.IMPORTANCE The contribution of VP1-145 variants in humans is not fully understood. In some studies, VP1-145G/Q viruses were isolated more frequently from severely affected patients than from mildly affected patients, suggesting that VP1-145G/Q viruses are more virulent. In the accompanying paper (K. Kobayashi, Y. Sudaka, A. Takashino, A. Imura, K. Fujii, and S. Koike, J Virol 92:e00681-18, 2018, https://doi.org/10.1128/JVI.00681-18), we showed that VP1-145E viruses are more virulent than VP1-145G viruses in human SCARB2 transgenic mice. Heparan sulfate acts as a decoy to specifically trap the VP1-145G viruses and leads to abortive infection. Here, we demonstrated that VP1-145G was attenuated in cynomolgus monkeys, suggesting that this hypothesis is also true in a nonhuman primate model. VP1-145E viruses, but not VP1-145G viruses, were highly resistant to neutralizing antibodies. We propose the difference in resistance against neutralizing antibodies as another mechanism of EV71 virulence. In summary, VP1-145 contributes to virulence determination by controlling attachment receptor usage and antibody sensitivity.

Background Cases of acute respiratory tract infection caused by Pteropine orthoreovirus (PRV) of the genus Orthoreovirus (family: Reoviridae) have been reported in Southeast Asia, where it was isolated from humans and bats. It is possible that PRV-associated respiratory infections might be prevalent in Southeast Asia. The clinical course of PRV is not fully elucidated. Methods The virulence, pathology, and pathogenesis of two PRV strains, a human-borne PRV strain (isolated from a patient, who returned to Japan from Bali, Indonesia in 2007) and a bat-borne PRV (isolated from a bat [Eonycteris spelaea] in the Philippines in 2013) were investigated in BALB/c mice using virological, pathological, and immunological study methods. Results The intranasal inoculation of BALB/c mice with human-borne PRV caused respiratory infection. In addition, all mice with immunity induced by pre-inoculation with a non-lethal dose of PRV were completely protected against lethal PRV infection. Mice treated with antiserum with neutralizing antibody activity after inoculation with a lethal dose of PRV showed a reduced fatality rate. In this mouse model, bat-borne PRV caused respiratory infection similar to human-borne PRV. PRV caused lethal respiratory disease in an animal model of PRV infection, in which BALB/c mice were used. Conclusions The BALB/c mouse model might help to accelerate research on the virulence of PRV and be useful for evaluating the efficacy of therapeutic agents and vaccines for the treatment and prevention of PRV infection. PRV was shown for the first time to be a causative virus of respiratory disease on the basis of Koch’s postulations by the additional demonstration that PRV caused respiratory disease in mice through their intranasal inoculation with PRV., Author summary It is assumed that Pteropine orthoreovirus (PRV) is a causative agent of acute respiratory tract infection (RTI) in humans. PRV was isolated from patients and fruit bats in Southeast Asia. Furthermore, the genome of PRV was detected in patients with respiratory symptoms, suggesting that PRV causes RTI in humans. There is a potential for PRV to cause RTIs in tropical or sub-tropical regions in Southeast Asia more widely than thought. The virulence, pathology, and pathogenesis of PRV in BALB/c mice were elucidated in detail. To develop specific countermeasures and prophylactics against PRV infection, an animal model of lethal PRV infection is needed, and we developed such a model using BALB/c mice. This model allows investigation of the pathogenicity of PRV and evaluation of the efficacy of drugs and vaccines and might help to accelerate research on the virology of PRV.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV) is an emerging pathogen that causes severe respiratory illness. Whole UV-inactivated SARS-CoV (UV-V), bearing multiple epitopes and proteins, is a candidate vaccine against this virus. However, whole inactivated SARS vaccine that includes nucleocapsid protein is reported to induce eosinophilic infiltration in mouse lungs after challenge with live SARS-CoV. In this study, an ability of Toll-like receptor (TLR) agonists to reduce the side effects of UV-V vaccination in a 6-month-old adult BALB/c mouse model was investigated, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. Immunization of adult mice with UV-V, with or without alum, resulted in partial protection from lethal doses of SARS-CoV challenge, but extensive eosinophil infiltration in the lungs was observed. In contrast, TLR agonists added to UV-V vaccine, including lipopolysaccharide, poly(U), and poly(I·C) (UV-V+TLR), strikingly reduced excess eosinophilic infiltration in the lungs and induced lower levels of interleukin-4 and -13 and eotaxin in the lungs than UV-V-immunization alone. Additionally, microarray analysis showed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-V-immunized but not in UV-V+TLR-immunized mice. In particular, CD11b+cells in the lungs of UV-V-immunized mice showed the upregulation of genes associated with the induction of eosinophils after challenge. These findings suggest that vaccine-induced eosinophil immunopathology in the lungs upon SARS-CoV infection could be avoided by the TLR agonist adjuvants.IMPORTANCEInactivated whole severe acute respiratory syndrome-related coronavirus (SARS-CoV) vaccines induce neutralizing antibodies in mouse models; however, they also cause increased eosinophilic immunopathology in the lungs upon SARS-CoV challenge. In this study, the ability of adjuvant Toll-like receptor (TLR) agonists to reduce the side effects of UV-inactivated SARS-CoV vaccination in a BALB/c mouse model was tested, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. We found that TLR stimulation reduced the high level of eosinophilic infiltration that occurred in the lungs of mice immunized with UV-inactivated SARS-CoV. Microarray analysis revealed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-inactivated SARS-CoV-immunized mice. This study may be helpful for elucidating the pathogenesis underlying eosinophilic infiltration resulting from immunization with inactivated vaccine.

Influenza A virus is the respiratory pathogen responsible for influenza. Infection by the 2009 pandemic influenza A (H1N1) virus caused severe lower airway inflammation and pneumonia. Asthma is a chronic inflammatory disorder of the airways that affects the entire brachial tree, and was one of the commonest underlying medical conditions among patients hospitalized with the 2009 pandemic influenza virus infection. Although respiratory virus infections are the major causes of asthma exacerbation, the mechanism by which influenza exacerbates asthma is poorly understood. Animal models of disease comorbidity are crucial to understanding host-pathogen interactions and elucidating complex pathologies. Existing murine models of influenza virus infection in asthmatics show that asthmatic mice are highly resistant to influenza virus infection, which contradicts clinical observations in humans. Here, we developed a murine model of influenza virus/asthma comorbidity using NC/Nga mice, which are highly sensitive to allergic reactions such as atopic dermatitis and allergic airway inflammation. This model was then used to examine the impact of allergic airway inflammation on lung pathology in the 2009 pandemic influenza virus infected mice. The results showed that induction of acute allergic airway inflammation in pre-existing influenza virus infection had additive effects on exacerbation of lung pathology, which mirrors findings in human epidemiological studies. In contrast, pre-existing allergic airway inflammation protected from subsequent influenza virus infection, which was compatible with those of previous murine models of influenza virus infection in asthmatic mice. These variable outcomes of this murine model indicate that the temporal relation between allergic airway inflammation and influenza virus infection might play a critical role in asthma and influenza comorbidity. Thus, this murine model will further our understanding of how influenza virus infection affects an asthmatic host and may aid the development of strategies to improve treatments and outcomes for asthmatics harboring influenza virus infection.

RIG-I-like receptors and Toll-like receptors (TLRs) play important roles in the recognition of viral infections. However, how these molecules contribute to the defense against poliovirus (PV) infection remains unclear. We characterized the roles of these sensors in PV infection in transgenic mice expressing the PV receptor. We observed that alpha/beta interferon (IFN-α/β) production in response to PV infection occurred in an MDA5-dependent but RIG-I-independent manner in primary cultured kidney cells in vitro . These results suggest that, similar to the RNA of other picornaviruses, PV RNA is recognized by MDA5. However, serum IFN-α levels, the viral load in nonneural tissues, and mortality rates did not differ significantly between MDA5-deficient mice and wild-type mice. In contrast, we observed that serum IFN production was abrogated and that the viral load in nonneural tissues and mortality rates were both markedly higher in TIR domain-containing adaptor-inducing IFN-β (TRIF)-deficient and TLR3-deficient mice than in wild-type mice. The mortality rate of MyD88-deficient mice was slightly higher than that of wild-type mice. These results suggest that multiple pathways are involved in the antiviral response in mice and that the TLR3-TRIF-mediated signaling pathway plays an essential role in the antiviral response against PV infection.

Poliomyelitis is an acute disease of the central nervous system (CNS) caused by poliovirus (PV), a human enterovirus that belongs to the family Picornaviridae. In humans, an infection is initiated by oral ingestion of the virus, followed by multiplication in the alimentary mucosa (2, 38), from which the virus spreads through the bloodstream. Viremia is considered essential for leading to paralytic poliomyelitis in humans. By use of a PV-sensitive mouse model, previous studies (9, 44) demonstrated that after intravenous inoculation, circulating PV crosses the blood-brain barrier at a high rate, and a neural dissemination pathway from the skeletal muscle without injury is not the primary route by which the circulating virus disseminates to the CNS. Along with the blood-brain barrier pathway of dissemination, a neural pathway has been reported for humans (30), primates (11), and PV-sensitive transgenic mice (Tg) carrying the human PV receptor (hPVR/CD155) gene (31, 34); this pathway appears to be important in causing provocation poliomyelitis (9). It has been proved that Tg carrying the hPVR gene (hPVR-Tg) are susceptible to all three PV serotypes, 1, 2, and 3 (22, 35), although mice without the hPVR gene are generally not susceptible to PV. This observation indicates that hPVR is the most important determinant of the host range of PV. After inoculation with PV by the intracerebral, intraspinal, intravenous, or intramuscular route (10, 20-22, 33-35), hPVR-Tg develop a flaccid paralysis in their limbs, which is clinically similar to human poliomyelitis. However, in contrast to its behavior in humans, PV does not replicate in the alimentary tracts of hPVR-Tg after oral administration, even in animals expressing high levels of hPVR in the intestinal epithelial cells (45). This result suggests that the expression of hPVR in the intestine is not solely responsible for the infection. It is also known that nonhuman primates are highly susceptible to PV by all routes except the oral route, yet the degree of oral susceptibility depends on the species (12). Thus, although oral infection is the most important route in humans, no adequate animal model has been established so far. After an oral infection with PV, the virus must overcome at least three barriers before it can start to replicate efficiently in the first target cells in the small intestine: (i) the gastric acid solution, by which PV may be inactivated; (ii) inappropriate distribution of hPVR, by which PV may not be ushered to the correct target cells; and (iii) innate immunity, including interferon (IFN) signaling, by which the replication of PV may be hampered in the target cells (7). To know why orally administered PV hardly causes any paralysis in animals other than humans, we have to verify each step (see Fig. ​Fig.7).7). In this report, barrier ii is defined as cell susceptibility and barrier iii is defined as cell permissivity. FIG. 7. Presumed PV dissemination routes and characteristics. The presumed barriers to orally ingested PV are shown. First, the ingested virus enters the stomach and suffers a low-pH environment (the first barrier). Second, the virus has to find the appropriate ... To control poliomyelitis, attenuated PV strains of all three serotypes have been developed and used effectively as oral polio vaccines (37, 39). The attenuated Sabin strains can replicate well only in the alimentary tracts of humans without showing neuropathogenicity, enough to elicit neutralizing antibodies against PV after oral administration. Picornaviruses are sensitive to IFNs (3, 5, 24, 28, 46). IFNs play an essential role in the innate immune antiviral response. Recently, Ida-Hosonuma et al. (13) found that deletion of the IFN-α/β receptor (IFNAR) gene in hPVR-Tg (hPVR-Tg/IfnarKO) resulted in the disruption of IFN-α/β signaling (27), which is an important determinant of the tissue tropism and pathogenicity of PV. Similarly, it has been reported that IFN-α/β plays an important role in the pathogenicity and tissue tropism of some viruses, including coxsackievirus and Theiler's virus in the Picornaviridae (6, 8, 26, 36, 42). These results suggest that not only hPVR (cell susceptibility) but also IFN-α/β (cell permissivity) contributes to the pathogenicity and tissue tropism of PV. In this paper, we have clarified the instability of the virus in the gastric environment, where the low pH of the gastric contents inactivates PV. Furthermore, using hPVR-Tg with or without IFNAR expression, we have shown that IFN-α/β plays a key role in preventing PV from replicating in the intestines of mice.

Highly pathogenic avian influenza virus (H5N1) is an emerging pathogen with the potential to cause great harm to humans, and there is concern about the potential for a new influenza pandemic. This virus is resistant to the antiviral effects of interferons and tumor necrosis factor-α. However, the mechanism of interferon-independent protective innate immunity is not well understood. The prophylactic effects of chitin microparticles as a stimulator of innate mucosal immunity against a recently obtained strain of H5N1 influenza virus infection were examined in mice. Clinical parameters and the survival rate of mice treated by intranasal application of chitin microparticles were significantly improved compared to non-treated mice after a lethal influenza virus challenge. Flow cytometric analysis revealed that the number of natural killer cells that expressed tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and that had migrated into the cervical lymph node was markedly increased (26-fold) after intranasal treatment with chitin microparticles. In addition, the level of IL-6 and interferon-gamma-inducible protein-10 (IP-10) in the nasal mucosa after H5N1 influenza virus challenge was decreased by prophylactic treatment with chitin microparticles. These results suggest that prophylactic intranasal administration of chitin microparticles enhanced the local accumulation of natural killer cells and suppressed hyper-induction of cytokines, resulting in an innate immune response to prevent pathogenesis of H5N1 influenza virus. J. Med. Virol. 79: 811–819, 2007. © 2007 Wiley-Liss, Inc.

Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV), is a recently identified emerging viral infectious disease. Despite the medical importance of this disease, there are currently neither vaccines nor effective therapeutics for SFTS. T-705, which is a pyrazine derivative, has shown broad antiviral activity against various RNA viruses. The present study demonstrated, for the first time to our knowledge, the efficacy of T-705 in treating SFTSV infection in a mouse lethal model. T-705 showed a high efficacy in the treatment of SFTSV infection in the mouse model, even when treatments were initiated after onset of the disease., Severe fever with thrombocytopenia syndrome virus (SFTSV) is the causative agent of SFTS, an emerging hemorrhagic fever. This disease has a high case fatality rate and is endemic to China, South Korea, and Japan. Because there are currently no effective therapeutics for SFTS, potent and safe antivirals are needed for the treatment of SFTS. The inhibitory effect of T-705 (favipiravir) on the replication of SFTSV in Vero cells was evaluated. Mice lacking the type I interferon receptor (IFNAR−/−) were used as an in vivo lethal model for SFTSV infection. T-705, which has been licensed as an anti-influenza drug in Japan, inhibits SFTSV replication both in vitro and in vivo. T-705 inhibited replication of SFTSV in Vero cells by 5 log units, with a 50% inhibitory concentration (IC50) and IC90 of 6.0 µM and 22 µM, respectively. Intraperitoneal or oral administration of T-705 for 5 days to IFNAR−/− mice infected with lethal SFTSV significantly improved survival rates (100% survival) without causing body weight loss and reduced the viral load in the serum. Ribavirin also inhibited SFTSV replication. However, it was less effective than T-705 both in vitro and in vivo. A time-of-drug-addition study revealed that therapeutic T-705 treatment of SFTSV infection in IFNAR−/− mice was effective. These results suggest that T-705 is a promising candidate for the treatment of SFTS. IMPORTANCE Severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV), is a recently identified emerging viral infectious disease. Despite the medical importance of this disease, there are currently neither vaccines nor effective therapeutics for SFTS. T-705, which is a pyrazine derivative, has shown broad antiviral activity against various RNA viruses. The present study demonstrated, for the first time to our knowledge, the efficacy of T-705 in treating SFTSV infection in a mouse lethal model. T-705 showed a high efficacy in the treatment of SFTSV infection in the mouse model, even when treatments were initiated after onset of the disease.

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, occasionally causes severe neurological symptoms. We identified P-selectin glycoprotein ligand-1 (PSGL-1) as an EV71 receptor and found that an amino acid residue 145 in the capsid protein VP1 (VP1-145) defined PSGL-1-binding (PB) and PSGL-1-nonbinding (non-PB) phenotypes of EV71. However, the role of PSGL-1-dependent EV71 replication in neuropathogenesis remains poorly understood. In this study, we investigated viral replication, genetic stability, and the pathogenicity of PB and non-PB strains of EV71 in a cynomolgus monkey model. Monkeys were intravenously inoculated with cDNA-derived PB and non-PB strains of EV71, EV71-02363-EG and EV71-02363-KE strains, respectively, with two amino acid differences at VP1-98 and VP1-145. Mild neurological symptoms, transient lymphocytopenia, and inflammatory cytokine responses, were found predominantly in the 02363-KE-inoculated monkeys. During the early stage of infection, viruses were frequently detected in clinical samples from 02363-KE-inoculated monkeys but rarely in samples from 02363-EG-inoculated monkeys. Histopathological analysis of central nervous system (CNS) tissues at 10 days postinfection revealed that 02363-KE induced neuropathogenesis more efficiently than that induced by 02363-EG. After inoculation with 02363-EG, almost all EV71 variants detected in clinical samples, CNS, and non-CNS tissues, possessed a G to E amino acid substitution at VP1-145, suggesting a strong in vivo selection of VP1-145E variants and CNS spread presumably in a PSGL-1-independent manner. EV71 variants with VP1-145G were identified only in peripheral blood mononuclear cells in two out of four 02363-EG-inoculated monkeys. Thus, VP1-145E variants are mainly responsible for the development of viremia and neuropathogenesis in a non-human primate model, further suggesting the in vivo involvement of amino acid polymorphism at VP1-145 in cell-specific viral replication, in vivo fitness, and pathogenesis in EV71-infected individuals., Author Summary Recently, large outbreaks of hand, foot, and mouth disease, including fatal neurological cases in young children primarily because of enterovirus 71 (EV71) have been reported, particularly in the Asia Pacific regions where the disease poses a serious threat to public health. Based on mutational and structural analyses of EV71, we identified amino acid residue 145 of the capsid protein VP1 (VP1-145) as a critical molecular determinant for the binding of EV71 to a specific cellular receptor, human P-selectin glycoprotein ligand-1 (PSGL-1). VP1-145 is highly variable among EV71 isolates and has been identified as a potential neurovirulence determinant in humans and experimental mouse models. To elucidate the in vivo involvement of PSGL-1-depentent replication and pathogenesis, we investigated viral replication, genetic stability, and the pathogenicity of the PSGL-1-binding (PB) and PSGL-1-nonbinding (non-PB) strains of EV71 in a cynomolgus monkey model. After the intravenous inoculation with the PB strain, viruses found to be highly mutated at VP1-145 with resultant VP1-145E variants (non-PB) inducing viremia and neuropathogenesis, presumably in a PSGL-1-independent manner. VP1-145G variants were identified only in peripheral blood mononuclear cells from two PB-inoculated monkeys. Our study provides new insights into the interplay between virus, receptors, and host in EV71-infected individuals.

Human norovirus (NoV) strains cause a considerable number of outbreaks of gastroenteritis worldwide. Based on their capsid gene (VP1) sequence, human NoV strains can be grouped into two genogroups (GI and GII) and at least 14 GI and 17 GII genotypes (GI/1–14 and GII/1–17). Human NoV strains cannot be propagated in cell-culture systems, but expression of recombinant VP1 in insect cells results in the formation of virus-like particles (VLPs). In order to understand NoV antigenic relationships better, cross-reactivity among 26 different NoV VLPs was analysed. Phylogenetic analyses grouped these NoV strains into six GI and 12 GII genotypes. An antibody ELISA using polyclonal antisera raised against these VLPs was used to determine cross-reactivity. Antisera reacted strongly with homologous VLPs; however, a number of novel cross-reactivities among different genotypes was observed. For example, GI/11 antiserum showed a broad-range cross-reactivity, detecting two GI and 10 GII genotypes. Likewise, GII/1, GII/10 and GII/12 antisera showed a broad-range cross-reactivity, detecting several other distinct GII genotypes. Alignment of VP1 amino acid sequences suggested that these broad-range cross-reactivities were due to conserved amino acid residues located within the shell and/or P1-1 domains. However, unusual cross-reactivities among different GII/3 antisera were found, with the results indicating that both conserved amino acid residues and VP1 secondary structures influence antigenicity.

Poliovirus selectively replicates in neurons in the spinal cord and brainstem, although poliovirus receptor (PVR) expression is observed in both the target and nontarget tissues in humans and transgenic mice expressing human PVR (PVR-transgenic mice). We assessed the role of alpha/beta interferon (IFN) in determining tissue tropism by comparing the pathogenesis of the virulent Mahoney strain in PVR-transgenic mice and PVR-transgenic mice deficient in the alpha/beta IFN receptor gene (PVR-transgenic/Ifnarknockout mice). PVR-transgenic/Ifnarknockout mice showed increased susceptibility to poliovirus. After intravenous inoculation, severe lesions positive for the poliovirus antigen were detected in the liver, spleen, and pancreas in addition to the central nervous system. These results suggest that the alpha/beta IFN system plays an important role in determining tissue tropism by protecting nontarget tissues that are potentially susceptible to infection. We subsequently examined the expression of IFN and IFN-stimulated genes (ISGs) in the PVR-transgenic mice. In the nontarget tissues, ISGs were expressed even in the noninfected state, and the expression level increased soon after poliovirus infection. On the contrary, in the target tissues, ISG expression was low in the noninfected state and sufficient response after poliovirus infection was not observed. The results suggest that the unequal IFN response is one of the important determinants for the differential susceptibility of tissues to poliovirus. We consider that poliovirus replication was observed in the nontarget tissues of PVR-transgenic/Ifnarknockout mice because the IFN response was null in all tissues.

Transgenic mice bearing the human poliovirus receptor (TgPVR) are less susceptible to oral inoculation, although they are susceptible to parenteral inoculation. We investigated the susceptibility of TgPVR 21 line [Arch. Virol. 130 (1994) 351] to poliovirus through various mucosal routes. Intranasal inoculation of a neurovirulent Mahoney strain (OM1) caused flaccid paralysis with viral replication in the central nervous system at a dose of 106 cell culture infectious dose (CCID50), in contrast, no paralysis following oral or intragastric inoculation of the same dose. Intranasal inoculation of a vaccine strain, Sabin 1, at 106 CCID50, resulted in no paralysis. Initial replication of poliovirus in the nasal cavity was confirmed by virus isolation and detection of negative-stranded replicative intermediates by RT-PCR and viral antigens using a high-sensitive immunohistochemistry and genome/transcripts by in situ hybridization. Poliovirus-specific IgG antibodies were elevated in the sera of surviving TgPVR21. This model can be used as a mucosal infection model and for differentiation of neurovirulent and attenuated poliovirus strains.

Helicobacter pylori infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis-inducing protein that functions as a leading factor in H. pylori-mediated apoptosis induction. We purified the protein from H. pylori by separating fractions that showed apoptosis-inducing activity. This protein induced apoptosis of AGS cells in a dose-dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N-terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited gamma-glutamyl transpeptidase activity, the inhibition of which by 6-diazo-5-oxo-l-norleucine resulted in a complete loss of apoptosis-inducing activity. To the best of our knowledge, the apoptosis-inducing function is a newly identified physiological role for bacterial gamma-glutamyl transpeptidase. The apoptosis-inducing activity of the isogenic mutant gamma-glutamyl transpeptidase-deficient strain was significantly lower compared with that of the parent strain, demonstrating that gamma-glutamyl transpeptidase plays a significant role in H. pylori-mediated apoptosis. Our findings provide new insights into H. pylori pathogenicity and reveal a novel aspect of the bacterial gamma-glutamyl transpeptidase function.

A novel rhabdovirus was isolated from the serum of a healthy Japanese wild boar (Sus scrofa leucomystax) and identified using the rapid determination system for viral nucleic acid sequences (RDV), next-generation sequencing, and electron microscopy. The virus was tentatively named wild boar rhabdovirus 1 (WBRV1). Phylogenetic analysis of the entire genome sequence indicated that WBRV1 is closely related to Tupaia rhabdovirus (TRV), which was isolated from cultured cells of hepatocellular carcinoma tissue of tree shrew. TRV has not been assigned to any genus of Rhabdoviridae till date. Analysis of the L gene indicated that WBRV1 belongs to the genus Vesiculovirus. These observations suggest that both TRV and WBRV1 belong to a new genus of Rhabdoviridae. Next-generation genome sequencing of WBRV1 revealed 5 open reading frames of 1329, 765, 627, 1629, and 6336 bases in length. The WBRV1 gene sequences are similar to those of other rhabdoviruses. Epizootiological analysis of a population of wild boars in Wakayama prefecture in Japan indicated that 6.5% were positive for the WBRV1 gene and 52% were positive for WBRV1-neutralizing antibodies. Furthermore, such viral neutralizing antibodies were found in domestic pigs in another prefecture. WBRV1 was inoculated intranasally and intraperitoneally into SCID and BALB/c mice and viral RNA was detected in SCID mice, suggesting that WBRV1 can replicate in immunocompromised mice. These results indicate this novel virus is endemic in wild animals and livestock in Japan.

Background: We previously reported that the purified Shiga toxins (Stx) Stx1 and Stx2, when coupled with liposomes, induced substantial production of anti-Stx1 and anti-Stx2 IgG antibody, respectively, in mice. The levels of anti-Stx antibody in the sera of mice immune to Stx-liposome correlated well with the protection against subsequent challenge with Stx. Furthermore, mice immunized with a mixture of Stx1-liposome and Stx2-liposome were successfully protected against oral infection with cytotoxin-producing Escherichia coli O157:H7. Methods: In this study, the induction of protection against Stx2 by Stx2-liposomes was evaluated in monkeys. Results: Stx2-liposomes induced a substantial amount of anti-Stx2 IgG antibodies as well as Stx2 neutralizing antibodies in monkeys. Test monkeys were successfully protected against challenge with lethal doses of Stx2. Moreover, these monkeys showed no apparent symptoms, while nonimmunized control monkeys died within 4 days with hemorrhagic gastroenteritis and renal disorder. In addition, as shown by other cases involving antigen-liposome conjugates, Stx2-liposome did not induce anti-Stx2 IgE antibody production, though it stimulated the production of a substantial amount of anti-Stx2 IgG antibodies. Conclusion: These results suggest that Stx-liposome conjugates may serve as candidate vaccines to induce protection against death caused by cytotoxin-producing E. coli infection.

Hantavirus infections are characterized by vascular hyperpermeability and neutrophilia. However, the pathogenesis of this disease is poorly understood. Here, we demonstrate for the first time that pulmonary vascular permeability is increased by Hantaan virus infection and results in the development of pulmonary edema in C.B-17 severe combined immunodeficiency (SCID) mice lacking functional T cells and B cells. Increases in neutrophils in the lung and blood were observed when pulmonary edema began to be observed in the infected SCID mice. The occurrence of pulmonary edema was inhibited by neutrophil depletion. Moreover, the pulmonary vascular permeability was also significantly suppressed by neutrophil depletion in the infected mice. Taken together, the results suggest that neutrophils play an important role in pulmonary vascular hyperpermeability and the occurrence of pulmonary edema after hantavirus infection in SCID mice. IMPORTANCE Although hantavirus infections are characterized by the occurrence of pulmonary edema, the pathogenic mechanism remains largely unknown. In this study, we demonstrated for the first time in vivo that hantavirus infection increases pulmonary vascular permeability and results in the development of pulmonary edema in SCID mice. This novel mouse model for human hantavirus infection will be a valuable tool and will contribute to elucidation of the pathogenetic mechanisms. Although the involvement of neutrophils in the pathogenesis of hantavirus infection has largely been ignored, the results of this study using the mouse model suggest that neutrophils are involved in the vascular hyperpermeability and development of pulmonary edema in hantavirus infection. Further study of the mechanisms could lead to the development of specific treatment for hantavirus infection.

A Japanese man suffered from acute respiratory tract infection after returning to Japan from Bali, Indonesia in 2007. Miyazaki-Bali/2007, a strain of the species of Nelson Bay orthoreovirus, was isolated from the patient's throat swab using Vero cells, in which syncytium formation was observed. This is the sixth report describing a patient with respiratory tract infection caused by an orthoreovirus classified to the species of Nelson Bay orthoreovirus. Given the possibility that all of the patients were infected in Malaysia and Indonesia, prospective surveillance on orthoreovirus infections should be carried out in Southeast Asia. Furthermore, contact surveillance study suggests that the risk of human-to-human infection of the species of Nelson Bay orthoreovirus would seem to be low.

We have previously reported that purified Shiga–like toxins (SLT), SLT–I and SLT–II coupled with liposomes induced a substantial amount of anti–SLT–I and anti–SLT–II IgG antibody production, respectively, in mice. The levels of anti–SLT antibody in the sera of SLT–liposome–immune mice correlated well with the protection against subsequent challenge with SLT. In this study, mice were immunized intraperitoneally with the mixture of SLT–I–liposome and SLT–II–liposome and protection against oral infection with cytotoxin–producing Escherichia coli O157:H7 was evaluated. All of the mice that received immunization with the mixture of SLT–I–liposome and SLT–II–liposome were protected against subsequent intravenous challenge with 10 LD50 of either SLT–I or SLT–II. Eight weeks after primary immunization, mice were inoculated intragastrically with 109 CFU of E. coli O157:H7 strain 96–60. All SLT–liposome–immune mice tested survived without any apparent symptom while control mice died within 5 days. In addition, as shown by other antigen–liposome conjugates, SLT–liposome induced undetectable anti–SLT IgE antibody production while they induced substantial amounts of anti–SLT IgG antibodies. These results suggest that SLT–liposome conjugate may serve as a candidate vaccine that induces protection against cytotoxin–producing E. coli infection.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. A recent epizootiological survey indicated that endemic foci of TBEV have been maintained in the southern part of Hokkaido until recently. In this study, we sought to isolate TBEV from wild rodents in the area. One virus, designated Oshima 08-As, was isolated from an Apodemus speciosus captured in Hokuto in 2008. Oshima 08-As was classified as the Far Eastern subtype of TBEV and formed a cluster with the other strains isolated in Hokkaido from 1995 to 1996. Thirty-six nucleotide differences resulted in 12 amino acid changes between Oshima 08-As and Oshima 5-10 isolated in 1995. Oshima 08-As caused high mortality and morbidity in a mouse model compared with Oshima 5-10. Although similar transient viral multiplication in the spleen was observed in the mice infected with Oshima 08-As and Oshima 5-10, greater viral multiplication with an inflammatory response was noted in the brains of mice infected with Oshima 08-As than those infected with Oshima 5-10. These data indicate that a few naturally occurring mutations affect the pathogenicity of the Oshima strains endemic in the southern part of Hokkaido.

Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. In pigs, infection resulted in a predominantly non-lethal respiratory disease; however, infection in humans resulted in over 100 deaths. Nipah virus has continued to re-emerge in Bangladesh and India, and person-to-person transmission appeared in the outbreak. Although a number of NiV vaccine studies have been reported, there are currently no vaccines or treatments licensed for human use. In this study, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins (rMV-HL-G and rMV-Ed-G). Vaccinated hamsters were completely protected against NiV challenge, while the mortality of unvaccinated control hamsters was 90%. We trialed our vaccine in a non-human primate model, African green monkeys. Upon intraperitoneal infection with NiV, monkeys showed several clinical signs of disease including severe depression, reduced ability to move and decreased food ingestion and died at 7 days post infection (dpi). Intranasal and oral inoculation induced similar clinical illness in monkeys, evident around 9 dpi, and resulted in a moribund stage around 14 dpi. Two monkeys immunized subcutaneously with rMV-Ed-G showed no clinical illness prior to euthanasia after challenge with NiV. Viral RNA was not detected in any organ samples collected from vaccinated monkeys, and no pathological changes were found upon histopathological examination. From our findings, we propose that rMV-NiV-G is an appropriate NiV vaccine candidate for use in humans.

A major difference between vaccine and wild-type strains of measles virus (MV) in vitro is the wider cell specificity of vaccine strains, resulting from the receptor usage of the hemagglutinin (H) protein. Wild-type H proteins recognize the signaling lymphocyte activation molecule (SLAM) (CD150), which is expressed on certain cells of the immune system, whereas vaccine H proteins recognize CD46, which is ubiquitously expressed on all nucleated human and monkey cells, in addition to SLAM. To examine the effect of the H protein on the tropism and attenuation of MV, we generated enhanced green fluorescent protein (EGFP)-expressing recombinant wild-type MV strains bearing the Edmonston vaccine H protein (MV-EdH) and compared them to EGFP-expressing wild-type MV strains. In vitro , MV-EdH replicated in SLAM + as well as CD46 + cells, including primary cell cultures from cynomolgus monkey tissues, whereas the wild-type MV replicated only in SLAM + cells. However, in macaques, both wild-type MV and MV-EdH strains infected lymphoid and respiratory organs, and widespread infection of MV-EdH was not observed. Flow cytometric analysis indicated that SLAM + lymphocyte cells were infected preferentially with both strains. Interestingly, EGFP expression of MV-EdH in tissues and lymphocytes was significantly weaker than that of the wild-type MV. Taken together, these results indicate that the CD46-binding activity of the vaccine H protein is important for determining the cell specificity of MV in vitro but not the tropism in vivo . They also suggest that the vaccine H protein attenuates MV growth in vivo .

Measles is a highly contagious disease that causes temporary and severe immunosuppression in patients. Signaling lymphocyte activation molecule (SLAM) expressed on cells of the immune system functions as a receptor for measles virus (MV). In addition to SLAM, vaccine strains of MV also use a ubiquitously expressed complement regulatory protein, CD46, as a receptor, whereas wild-type (wt) MV strains do not use this receptor. However, recent studies have indicated that SLAM is not the sole receptor for wt MV strains. These strains have an intrinsic ability to enter both immune and epithelial cells using distinct receptor binding sites in their hemagglutinin (H) protein. Recently, a clear answer was obtained through the identification of an epithelial MV receptor, nectin4, expressed at adherens junctions, thereby greatly improving our knowledge of MV receptors. It is now clear that MV specifically targets two cell types, immune cells and epithelial cells, using SLAM and nectin4, respectively. MV loses the ability to use either SLAM or nectin4 when it possesses specific mutations in the H protein. However, nectin4-blind MV still infects SLAM-positive immune cells efficiently (SLAM-tropic), and conversely, SLAM-blind MV infects nectin4-positive epithelial cells efficiently (nectin4-tropic). In this regard, MV is intrinsically dual-tropic to immune cells and epithelial cells. Although many aspects and molecular mechanisms underlying immunosuppressive effects and a highly contagious nature of MV still remain to be elucidated, analyses of physiological functions of these two receptors would provide deep insights into MV pathogenesis.

The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.

SARS‐CoV grows in a variety of tissues that express its receptor, although the mechanism for high replication in the lungs and severe respiratory illness is not well understood. We recently showed that elastase enhances SARS‐CoV infection in cultured cells, which suggests that SARS development may be due to elastase‐mediated, enhanced SARS‐CoV infection in the lungs. To explore this possibility, we examined whether co‐infection of mice with SARS‐CoV and Pp, a low‐pathogenic bacterium which elicits elastase production in the lungs, induces exacerbation of pneumonia. Mice co‐infected with SARS‐CoV and Pp developed severe respiratory disease with extensive weight loss, resulting in a 33∼90% mortality rate. Mice with exacerbated pneumonia showed enhanced virus infection in the lungs and histopathological lesions similar to those found in human SARS cases. Intranasal administration of LPS, another elastase inducer, showed an effect similar to that of Pp infection. Thus, this study shows that exacerbated pneumonia in mice results from co‐infection with SARS‐CoV and a respiratory bacterium that induces elastase production in the lungs, suggesting a possible role for elastase in the exacerbation of pneumonia.

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also sometimes associated with serious neurological disorders. In this study, we characterized the antigenicity and tissue specificity of an attenuated strain of EV71 [EV71(S1-3′)], which belongs to genotype A, in a monkey infection model. Three cynomolgus monkeys were inoculated with EV71(S1-3′), followed by lethal challenge with the parental virulent strain EV71(BrCr-TR) via an intravenous route on day 45 postinoculation of EV71(S1-3′). Monkeys inoculated with EV71(S1-3′) showed a mild neurological symptom (tremor) but survived lethal challenge by virulent EV71(BrCr-TR) without exacerbation of the symptom. The immunized monkey sera showed a broad spectrum of neutralizing activity against different genotypes of EV71, including genotypes A, B1, B4, C2, and C4. For the strains examined, the sera showed the highest neutralization activity against the homotype (genotype A) and the lowest neutralization activity against genotype C2. The order of decreasing neutralization activity of sera was as follows: A > B1 > C4 > B4 > C2. To examine the tissue specificity of EV71(S1-3′), two monkeys were intravenously inoculated with EV71(S1-3′), followed by examination of virus distribution in the central nervous system (CNS) and extraneural tissues. In the CNS, EV71(S1-3′) was isolated only from the spinal cord. These results indicate that EV71(S1-3′) acts as an effective antigen, although this attenuated strain was still neurotropic when inoculated via the intravenous route.

To understand the pathogenesis and develop an animal model of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), the Frankfurt 1 SARS-CoV isolate was passaged serially in young F344 rats. Young rats were susceptible to SARS-CoV but cleared the virus rapidly within 3 to 5 days of intranasal inoculation. After 10 serial passages, replication and virulence of SARS-CoV were increased in the respiratory tract of young rats without clinical signs. By contrast, adult rats infected with the passaged virus showed respiratory symptoms and severe pathological lesions in the lung. Levels of inflammatory cytokines in sera and lung tissues were significantly higher in adult F344 rats than in young rats. During in vivo passage of SARS-CoV, a single amino acid substitution was introduced within the binding domain of the viral spike protein recognizing angiotensin-converting enzyme 2 (ACE2), which is known as a SARS-CoV receptor. The rat-passaged virus more efficiently infected CHO cells expressing rat ACE2 than did the original isolate. These results strongly indicate that host and virus factors such as advanced age and virus adaptation are critical for the development of SARS in rats.

The potential threat of smallpox as a bioweapon has led to the production and stockpiling of smallpox vaccine in some countries. Human monkeypox, a rare but important viral zoonosis endemic to central and western Africa, has recently emerged in the United States. Thus, even though smallpox has been eradicated, a vaccinia virus vaccine that can induce protective immunity against smallpox and monkeypox is still invaluable. The ability of the highly attenuated vaccinia virus vaccine strain LC16m8, with a mutation in the important immunogenic membrane protein B5R, to induce protective immunity against monkeypox in nonhuman primates was evaluated in comparison with the parental Lister strain. Monkeys were immunized with LC16m8 or Lister and then infected intranasally or subcutaneously with monkeypox virus strain Liberia or Zr-599, respectively. Immunized monkeys showed no symptoms of monkeypox in the intranasal-inoculation model, while nonimmunized controls showed typical symptoms. In the subcutaneous-inoculation model, monkeys immunized with LC16m8 showed no symptoms of monkeypox except for a mild ulcer at the site of monkeypox virus inoculation, and those immunized with Lister showed no symptoms of monkeypox, while nonimmunized controls showed lethal and typical symptoms. These results indicate that LC16m8 prevents lethal monkeypox in monkeys, and they suggest that LC16m8 may induce protective immunity against smallpox.

To develop a new live attenuated mumps vaccine, a wild mumps Y7 strain isolated from a patient who developed mild parotitis was treated with nitrosoguanidine and ultraviolet, followed by selection of a temperature-sensitive clone. The selected clone, Y125, showed stable temperature-sensitivity in Vero cells. Intraspinal inoculation of marmosets with the Y125 produced only minimal histopathological changes, while intracerebral inoculation of neonatal rats revealed that the Y125 did not cause hydrocephalus. Both these effects of the Y125 were similar to those of the non-neurovirulent Jeryl Lynn strain. Furthermore, subcutaneous inoculation of the Y125 induced high levels of neutralizing antibodies in all Cercopithecus monkeys examined. Although the safety and immunogenicity should be confirmed in further field trials in humans, the present results indicate that the Y125 could be a promising vaccine candidate.