Impact of Reinfection with SARS-CoV-2 Omicron Variants in Previously Infected Hamsters.

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. IMPORTANCE The 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.

High-throughput isolation of SARS-CoV-2 nucleocapsid antibodies for improved antigen detection.

SARS-CoV-2 nucleocapsid protein (NP) is the main target for COVID-19-diagnostic PCR and antigen rapid diagnostic tests (Ag-RDTs). Ag-RDTs are more convenient than PCR tests for point-of-care testing or self-testing to identify the SARS-CoV-2 antigen. The sensitivity and specificity of this method depends mainly on the affinity and specificity of NP-binding antibodies; therefore, antigen-antibody binding is key elements for the Ag-RDTs. Here, we applied the high-throughput antibody isolation platform that has been utilized to isolate therapeutic antibodies against rare epitopes. Two NP antibodies were identified to recognize non-overlapping epitopes with high affinity. One antibody specifically binds to SARS-CoV-2 NP, and the other rapidly and tightly binds to SARS-CoV-2 NP with cross-reactivity to SARS-CoV NP. Furthermore, these antibodies were compatible with a sandwich enzyme-linked immunosorbent assay that exhibited enhanced sensitivity for NP detection compared to the previously isolated NP antibodies. Thus, the NP antibody pair is applicable to more sensitive and specific Ag-RDTs, highlighting the utility of a high-throughput antibody isolation platform for diagnostics development.

Subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques.

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.

A highly attenuated vaccinia virus strain LC16m8-based vaccine for severe fever with thrombocytopenia syndrome.

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.

Effectiveness of personal protective equipment in preventing severe acute respiratory syndrome coronavirus 2 infection among healthcare workers.

INTRODUCTION: Information on the effectiveness of personal protective equipment (PPE) for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among healthcare workers (HCWs), especially among HCWs with frequent contact with patients with SARS-CoV-2, is limited. METHODS: We conducted a prospective cohort study on 49 HCWs who worked in close contact with patients with SARS-CoV-2 infection. HCWs had blood samples taken every 2 weeks to test for SARS-CoV-2 antibodies using two different types of assay. RESULTS: Forty-nine participants (31 nurses, 15 doctors, 3 other workers) were enrolled. In total, 112 blood samples are obtained from participants. The median work days in 2 weeks was 9 (interquartile range (IQR): 5-10) days. In a single work day, 30 of the 49 participants (61.5%) had contact with patients with suspected or conformed SARS-CoV-2 at least 8 times, and approximately 60% of participants had more than 10 min of contact with a single patient. The median self-reported compliance to PPE was 90% (IQR: 80-100%). Seven participants tested positive for SARS-CoV-2 antibody using enzyme-linked immunosorbent assay (ELISA); however, none were seropositive for SARS-CoV-2 neutralizing antibody, so the positive ELISA results were assumed to be false-positive. CONCLUSIONS: The study provides evidence that appropriate PPE is sufficient to prevent infection amongHCWs. It is necessary to establish a system that provides a stable supply of PPE for HCWs to perform their duties.

TMPRSS2 Contributes to Virus Spread and Immunopathology in the Airways of Murine Models after Coronavirus Infection.

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.

Acute Respiratory Infection in Human Dipeptidyl Peptidase 4-Transgenic Mice Infected with Middle East Respiratory Syndrome Coronavirus.

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.

Gold nanoparticle-adjuvanted S protein induces a strong antigen-specific IgG response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs.

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.

VP1 Amino Acid Residue 145 of Enterovirus 71 Is a Key Residue for Its Receptor Attachment and Resistance to Neutralizing Antibody during Cynomolgus Monkey Infection.

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.

Intracerebral Inoculation of Mouse-Passaged Saffold Virus Type 3 Affects Cerebellar Development in Neonatal Mice.

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. IMPORTANCE Saffold 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 an in 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.

The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model.

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.

Effects of Toll-like receptor stimulation on eosinophilic infiltration in lungs of BALB/c mice immunized with UV-inactivated severe acute respiratory syndrome-related coronavirus vaccine.

UNLABELLED: 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. IMPORTANCE: Inactivated 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.

Establishment of a panel of in-house polyclonal antibodies for the diagnosis of enterovirus infections.

The aim of this study was to establish a reliable method of virus detection for the diagnosis of critical enterovirus infections such as acute infective encephalitis, encephalomyelitis and myocarditis. Because histopathological and immunohistochemical analyses of paraffin-embedded tissues play an important role in recognizing infectious agents in tissue samples, six in-house polyclonal antibodies raised against three representative enteroviruses using an indirect immunofluorescence assay and immunohistochemistry were examined. This panel of polyclonal antibodies recognized three serotypes of enterovirus. Two of the polyclonal antibodies were raised against denatured virus particles from enterovirus A71, one was raised against the recombinant VP1 protein of coxsackievirus B3, and the other for poliovirus type 1 were raised against denatured virus particles, the recombinant VP1 protein and peptide 2C. Western blot analysis revealed that each of these antibodies recognized the corresponding viral antigen and none cross-reacted with non-enteroviruses within the family Picornaviridae. However, all cross-reacted to some extent with the antigens derived from other serotypes of enterovirus. Indirect immunofluorescence assay and immunohistochemistry revealed that the virus capsid and non-structural proteins were localized in the cytoplasm of affected culture cells, and skeletal muscles and neurons in neonatal mice experimentally-infected with human enterovirus. The antibodies also recognized antigens derived from recent clinical isolates of enterovirus A71, coxsackievirus B3 and poliovirus. In addition, immunohistochemistry revealed that representative antibodies tested showed the same recognition pattern according to each serotype. Thus, the panel of in-house anti-enterovirus polyclonal antibodies described herein will be an important tool for the screening and pathological diagnosis for enterovirus infections, and may be useful for the classification of different enterovirus serotypes, including coxsackieviruses A and B, echoviruses, enterovirus A71 and poliovirus.

Lethal canine distemper virus outbreak in cynomolgus monkeys in Japan in 2008.

Canine distemper virus (CDV) has recently expanded its host range to nonhuman primates. A large CDV outbreak occurred in rhesus monkeys at a breeding farm in Guangxi Province, China, in 2006, followed by another outbreak in rhesus monkeys at an animal center in Beijing in 2008. In 2008 in Japan, a CDV outbreak also occurred in cynomolgus monkeys imported from China. 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.

The comparison of pathogenicity among SARS-CoV-2 variants in domestic cats.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected or isolated from domestic cats. It is unclear whether cats play an important role in the SARS-CoV-2 transmission cycle. In this study, we examined the susceptibility of cats to SARS-CoV-2, including wild type and variants, by animal experiments. Cats inoculated with wild type, gamma, and delta variants secreted a large amount of SARS-CoV-2 for 1 week after the inoculation from nasal, oropharyngeal, and rectal routes. Only 100 TCID50 of virus could infect cats and replicate well without severe clinical symptoms. In addition, one cat inoculated with wild type showed persistent virus secretion in feces for over 28 days post-inoculation (dpi). The titer of virus-neutralizing (VN) antibodies against SARS-CoV-2 increased from 11 dpi, reaching a peak at 14 dpi. However, the omicron variant could not replicate well in cat tissues and induced a lower titer of VN antibodies. It is concluded that cats were highly susceptible to SARS-CoV-2 infection, but not to the Omicron Variant, which caused the attenuated pathogenicity.

Mpox Neutralizing Antibody Response to LC16m8 Vaccine in Healthy Adults.

BACKGROUND: Vaccination against mpox (formerly known as monkeypox), an infectious disease caused by the monkeypox virus (MPXV), is needed to prevent outbreaks and consequent public health concerns. The LC16m8 vaccine, a dried cell-cultured proliferative live attenuated vaccinia virus­based vaccine, was approved in Japan against smallpox and mpox. However, its immunogenicity and efficacy against MPXV have not been fully assessed. We assessed the safety and immunogenicity of LC16m8 against MPXV in healthy adults. METHODS: We conducted a single-arm study that included 50 participants who were followed up for 168 days postvaccination. The primary end point was the neutralizing antibody seroconversion rate against MPXVs, including the Zr599 and Liberia strains, on day 28. The secondary end points included the vaccine "take" (major cutaneous reaction) rate, neutralizing titer kinetics against MPXV and vaccinia virus (LC16m8) strains, and safety outcomes. RESULTS: Seroconversion rates on day 28 were 72% (36 of 50), 70% (35 of 50), and 88% (44 of 50) against the Zr599 strain, the Liberia strain, and LC16m8, respectively. On day 168, seroconversion rates decreased to 30% (15 of 50) against the Zr599 and Liberia strains and to 76% (38 of 50) against LC16m8. The vaccine "take" (broad definition) rate on day 14 was 94% (46 of 49). Adverse events (AEs), including common solicited cutaneous reactions, occurred in 98% (45 of 48) of participants; grade 3 severity AEs occurred in 16% (8 of 50). No deaths, serious AEs, or mpox onset incidences were observed up to day 168. CONCLUSIONS: The LC16m8 vaccine generated neutralizing antibody responses against MPXV in healthy adults. No serious safety concerns occurred with LC16m8 use. (Funded by the Ministry of Health, Labour and Welfare of Japan; Japan Registry of Clinical Trials number, jRCTs031220171.)

Association of major histocompatibility complex class I haplotypes with disease progression after simian immunodeficiency virus challenge in burmese rhesus macaques.

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A(+) (n = 6), E(+) (n = 6), B(+) (n = 4), and J(+) (n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A(+) animals, including two controllers, showed slower disease progression, whereas J(+) animals exhibited rapid progression. E(+) and B(+) animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8(+) T-cell responses were efficiently induced in A(+) animals, while Nef-specific CD8(+) T-cell responses were in A(+), E(+), and B(+) animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4(+) T-cell decline, and SIV-specific CD4(+) T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.

Results from a preclinical study in rodents and a Phase 1/2, randomized, double-blind, placebo-controlled, parallel-group study of COVID-19 vaccine S-268019-a in Japanese adults.

BACKGROUND: In early 2020, developing vaccines was an urgent need for preventing COVID-19 from a contingency perspective. METHODS: S-268019-a is a recombinant protein-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a modified recombinant spike protein antigen adjuvanted with agatolimod sodium, a Toll-like receptor-9 agonist. In the preclinical phase, it was administered intramuscularly twice at a 2-week interval in 7-week-old mice. Immunogenicity was assessed, and the mice were challenged intranasally with mouse-adapted SARS-CoV-2 at 2 and 8 weeks, respectively, after the second immunization. After confirming the preclinical effect, a Phase 1/2, randomized, parallel-group clinical study was conducted in healthy adults (aged 20-64 years). All participants received 2 intramuscular injections at various combinations of the antigen and the adjuvant (S-910823/agatolimod sodium, in µg: 12.5/250, 25/250, 50/250, 25/500, 50/500, 100/500, 10/500, 100/100, 200/1000) or placebo (saline) in an equivalent volume at a 3-week interval and were followed up until Day 50 in this interim analysis. RESULTS: In the preclinical studies, S-268019-a was safe and elicited robust immunoglobulin G (IgG) and neutralizing antibody responses in mice. When challenged with SARS-CoV-2, all S-268019-a-treated mice survived and maintained weight until 10 days, whereas all placebo- or adjuvant-treated (without antigen) mice died within 6 days. In the Phase 1/2 trial, although S-268019-a was well tolerated in adult participants, was safe up to Day 50, and elicited robust anti-spike protein IgG antibodies, it did not elicit sufficient neutralizing antibody levels. CONCLUSIONS: The S-268019-a vaccine was not sufficiently immunogenic in Japanese adults despite robust immunogenicity and efficacy in mice. Our results exemplify the innate challenges in translating preclinical data in animals to clinical trials, and highlight the need for continued research to overcome such barriers. (jRCT2051200092).

Saturation time of exposure interval for cross-neutralization response to SARS-CoV-2: Implications for vaccine dose interval.

Evaluating the serum cross-neutralization responses after breakthrough infection with various SARS-CoV-2 variants provides valuable insight for developing variant-proof COVID-19 booster vaccines. However, fairly comparing the impact of breakthrough infections with distinct epidemic timing on cross-neutralization responses, influenced by the exposure interval between vaccination and infection, is challenging. To compare the impact of pre-Omicron to Omicron breakthrough infection, we estimated the effects on cross-neutralizing responses by the exposure interval using Bayesian hierarchical modeling. The saturation time required to generate saturated cross-neutralization responses differed by variant, with variants more antigenically distant from the ancestral strain requiring longer intervals of 2-4 months. The breadths of saturated cross-neutralization responses to Omicron lineages were comparable in pre-Omicron and Omicron breakthrough infections. Our results highlight the importance of vaccine dosage intervals of 4 months or longer, regardless of the antigenicity of the exposed antigen, to maximize the breadth of serum cross-neutralization covering SARS-CoV-2 Omicron lineages.

Lethal severe fever with thrombocytopenia syndrome virus infection causes systemic germinal centre failure and massive T cell apoptosis in cats.

Introduction: Severe fever with thrombocytopenia syndrome (SFTS) is a fatal viral disease characterized by high fever, thrombocytopenia, leukopenia, and multi-organ haemorrhage. Disruption of the humoral immune response and decreased lymphocyte numbers are thought to contribute to the disease severity. These findings have been obtained through the analysis of peripheral blood leukocytes in human patients, whereas analysis of lymph nodes has been limited. Thus, in this study, we characterized the germinal centre response and apoptosis in the lymph nodes of cats with fatal SFTS, because SFTS in cats well mimics the pathology of human SFTS. Methods: Lymph node tissue sections collected during necropsy from seven fatal SFTS patients and five non-SFTS cases were used for histopathological analysis. Additionally, lymph node tissue sections collected from cats with experimental infection of SFTS virus (SFTSV) were also analysed. Results: In the lymphoid follicles of cats with SFTS, a drastic decrease in Bcl6- and Ki67-positive germinal centre B cells was observed. Together, the number of T cells in the follicles was also decreased in SFTS cases. In the paracortex, a marked increase in cleaved-caspase3 positivity was observed in T cells. These changes were independent of the number of local SFTS virus-positive cell. Furthermore, the analysis of cats with experimental SFTSV infection revealed that the intrafollicular Bcl6- and CD3-positive cell numbers in cats with low anti-SFTSV antibody production were significantly lower than those in cats with high anti-SFTSV antibody production. Discussion: These results suggest that dysfunction of the humoral response in severe SFTS was caused by the loss of germinal centre formation and massive apoptosis of T cells in the lymph nodes due to systemically circulating viruses.