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During the coronavirus disease 2019 (COVID-19) pandemic, autopsies have provided valuable insights into the pathogenesis of COVID-19. The precise effect of this pandemic on autopsy procedures in Japan, especially in instances unrelated to COVID-19, has not yet been established. Therefore, we conducted a questionnaire survey from December 2020 to January 2021 regarding the status of pathological autopsy practices in Japan during the first year of the COVID-19 pandemic. The questionnaire was sent to 678 medical facilities with pathologists, of which 227 responded. In cases where a confirmed diagnosis of COVID-19 was not made at the time of autopsy, many facilities counted them as suspected COVID-19 cases if pneumonia was suspected clinically. At around half of the sites, autopsies were prohibited for suspected COVID-19 cases. In addition, the number of autopsies of non-COVID-19 cases during the pandemic period was also investigated, and a significant decrease was observed compared with the incidence in the pre-pandemic period. The COVID-19 pandemic has affected not only the autopsies of COVID-19 cases but also the entire practice of pathological autopsies. It is necessary to establish a system that supports the implementation of pathological autopsy practices during the pandemic of an emerging infectious disease.

To control the coronavirus disease 2019 (COVID-19) pandemic, there is a need to develop vaccines to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. One candidate is a nasal vaccine capable of inducing secretory IgA antibodies in the mucosa of the upper respiratory tract, the initial site of infection. However, regarding the development of COVID-19 vaccines, there is concern about the potential risk of inducing lung eosinophilic immunopathology as a vaccine-associated enhanced respiratory disease as a result of the T helper 2 (Th2)-dominant adaptive immune response. In this study, we investigated the protective effect against virus infection induced by intranasal vaccination of recombinant trimeric spike protein derived from SARS-CoV-2 adjuvanted with CpG oligonucleotides, ODN2006, in mouse model. The intranasal vaccine combined with ODN2006 successfully induced not only systemic spike-specific IgG antibodies, but also secretory IgA antibodies in the nasal mucosa. Secretory IgA antibodies showed high protective ability against SARS-CoV-2 variants (Alpha, Beta and Gamma variants) compared to IgG antibodies in the serum. The nasal vaccine of this formulation induced a high number of IFN-γ-secreting cells in the draining cervical lymph nodes and a lower spike-specific IgG1/IgG2a ratio compared to that of subcutaneous vaccination with alum as a typical Th2 adjuvant. These features are consistent with the induction of the Th1 adaptive immune response. In addition, mice intranasally vaccinated with ODN2006 showed less lung eosinophilic immunopathology after viral challenge than mice subcutaneously vaccinated with alum adjuvant. Our findings indicate that intranasal vaccine adjuvanted with ODN2006 could be a candidate that can prevent the infection of antigenically different variant viruses, reducing the risk of vaccine-associated enhanced respiratory disease.

Abstract COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro’s active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.

A high-flow nasal cannula (HFNC) therapy plays a significant role in providing respiratory support to critically ill patients with coronavirus disease 2019 (COVID-19); however, the dispersion of the virus owing to aerosol generation is a matter of concern. This study aimed to evaluate if HFNC disperses the virus into the air. Among patients with COVID-19 admitted to private rooms with controlled negative pressure, we enrolled those admitted within 10 days of onset and requiring oxygenation through a conventional nasal cannula or HFNC therapy. Of the 17 patients enrolled, we obtained 22 samples (11 in the conventional nasal cannula group and 11 in the HFNC group). Viral RNA was detected in 20 nasopharyngeal swabs, and viable viruses were isolated from three nasopharyngeal swabs. Neither viral RNA nor viable virus was detected in the air sample at 0.5 m regardless of the oxygen-supplementation device. We detected viral RNA in two samples in the conventional nasal cannula group but not in the HFNC therapy group in gelatin filters located 3 m from the patient and the surface of the ventilation. This study directly demonstrated that despite viral RNA detection in the nasopharynx, viruses may not be dispersed by HFNC therapy. This warrants further research to determine if similar results can be obtained under different conditions.

In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622 (ensitrelvir), an inhibitor of SARS-CoV-2 main protease (M pro ; also known as 3C-like protease), decreases viral load and ameliorates disease severity in SARS-CoV-2–infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to submicromolar concentrations in cells. Oral administration of S-217622 demonstrated favorable pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern, including the highly pathogenic Delta variant and the recently emerged Omicron BA.5 and BA.2.75 variants. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase 3 clinical trial (clinical trial registration no. jRCT2031210350), has remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.

Plastic bronchitis (PB) is a rare and severe respiratory disease characterized by the formation of branching mucus casts, resulting in airway obstruction. PB can be divided into two types. Type 1 PB is primarily due to inflammatory casts caused by allergic diseases. In type 2 PB, mucinous casts are produced in association with congenital heart diseases. PB is also associated with viral respiratory infections, particularly influenza A (H1N1) pdm09 virus, which is the most common pathogen affecting pediatric patients. Herein, we report a severe case of PB type 1 caused by human bocavirus (HBoV) 1, affecting a child. Multiplex polymerase chain reaction (PCR) with a nasopharyngeal swab revealed the presence of respiratory syncytial virus and human parainfluenza virus 3.However, no viruses other than HBoV 1 were detected from mucus casts via real-time PCR. Consequently, we suggested that HBoV can cause PB in pediatric patients; direct and comprehensive PCR with bronchial casts may be useful for identifying etiologic agents.

BackgroundThe prolonged presence of infectious severe acute respiratory syndrome coronavirus (SARS-CoV-2) in deceased coronavirus disease 2019 (COVID-19) patients has been reported. However, infectious virus titers have not been determined. Such information is important for public health, death investigation, and handling corpses.AimThe aim of this study was to assess the level of SARS-CoV-2 infectivity in COVID-19 corpses.MethodsWe collected 11 nasopharyngeal swabs and 19 lung tissue specimens from 11 autopsy cases with COVID-19 in 2021. We then investigated the viral genomic copy number by real-time reverse transcription-polymerase chain reaction and infectious titers by cell culture and virus isolation.ResultsInfectious virus was present in 6 of 11 (55%) cases, 4 of 11 (36%) nasopharyngeal swabs, and 9 of 19 (47%) lung specimens. The virus titers ranged from 6.00E + 01 plaque-forming units (PFU)/mL to 2.09E + 06 PFU/g. In all cases in which an infectious virus was found, the time from death to discovery was within 1 day and the longest postmortem interval was 13 days.ConclusionCOVID-19 corpses may have high titers of infectious virus after a long postmortem interval (up to 13 days). Therefore, appropriate infection control measures must be taken when handling corpses.

The prevalence of the Omicron subvariant BA.2.75 is rapidly increasing in India and Nepal. In addition, BA.2.75 has been detected in at least 34 other countries and is spreading globally. However, the virological features of BA.2.75 are largely unknown. Here, we evaluated the replicative ability and pathogenicity of BA.2.75 clinical isolates in Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with BA.2, BA.5, or BA.2.75, the replicative ability of BA.2.75 in the lungs was higher than that of BA.2 and BA.5. Of note, BA.2.75 caused focal viral pneumonia in hamsters, characterized by patchy inflammation interspersed in alveolar regions, which was not observed in BA.5-infected hamsters. Moreover, in competition assays, BA.2.75 replicated better than BA.5 in the lungs of hamsters. These results suggest that BA.2.75 can cause more severe respiratory disease than BA.5 and BA.2 and should be closely monitored.

Background Although fungi are found relatively easily by microscopic examination of pathological samples, identification of fungal genus and species in pathological samples is not easy because the morphological features of fungi are similar among genera and species. Objectives A multiple real-time PCR was developed for identification of fungal genus/species, and morphological characterizations of fungi were analyzed in pathological samples. Patients/methods Seventy-five formalin-fixed, paraffin-embedded samples morphologically proven to contain any fungus were examined. A multiple real-time PCR system was developed to identify 25 fungal genus/species in pathological samples. Morphology of fungus in the specimens was re-reviewed retrospectively based on the results of real-time PCR. Results Real-time PCR identified fungal genus/species in 56 of 75 (74.6%) specimens with histologically proven fungal infection. In 53 specimens of filamentous fungi, Aspergillus spp. (22 specimens), Cladosporium (8), Scedosporium apiospermum (4), Malassezia sympodialis (1) and Candida albicans (1) were identified. Pseudohyphae of Candida were confused with filamentous fungus in a case. Morphological observation suggested differences in the presence of septated or non-septated hyphae, the filament size, and the branch angle among genus/species of filamentous fungi; however, genus/species was not able to be determined by their morphological features. In 22 specimens of yeasts, real-time PCR allowed for the identification of Candida albicans (12 specimens), Candida glabrata (2), Cladosporium (2), Scedosporium apiospermum (2), Pichia kudriavzevii (1), and Aspergillus sydowii (1). Conclusions These data suggest that it is difficult to identify fungal genus/species by morphological features alone. Real-time PCR is useful to identify fungal genus/species in pathological samples.

The BA.2 sublineage of the SARS-CoV-2 Omicron variant has become dominant in most countries around the world; however, the prevalence of BA.4 and BA.5 is increasing rapidly in several regions. BA.2 is less pathogenic in animal models than previously circulating variants of concern (VOC). Compared with BA.2, however, BA.4 and BA.5 possess additional substitutions in the spike protein, which play a key role in viral infectivity, raising concerns that the infectivity and pathogenicity of BA.4 and BA.5 are higher than those of BA.2. Here, we evaluated the replicative ability and pathogenicity of authentic BA.4 and BA.5 isolates in wild-type Syrian hamsters and human ACE2 (hACE2) transgenic hamsters. In contrast to recent data with a recombinant chimeric virus possessing the spike protein of BA.4/BA.5 in the background of a BA.2 strain, we observed no obvious differences among BA.2, BA.4, and BA.5 isolates in growth ability or pathogenicity in hamsters, and less pathogenicity compared to a previously circulating Delta (B.1.617.2 lineage) isolate. In addition, in vivo competition experiments revealed that BA.5 outcompeted BA.2 in hamsters, whereas BA.4 and BA.2 exhibited similar fitness. These findings suggest that BA.4 and BA.5 have similar pathogenicity to BA.2 in rodents and that BA.5 possesses viral fitness superior to that of BA.2. Our study highlights the importance of using authentic isolates when evaluating virological features.

The BA.2 sublineage of the SARS-CoV-2 Omicron variant has become dominant in most countries around the world; however, the prevalence of BA.4 and BA.5 is increasing rapidly in several regions. BA.2 is less pathogenic in animal models than previously circulating variants of concern

Immunity to SARS-CoV-2 in COVID-19 cases has diversified due to complex combinations of exposure to vaccination and infection. Elucidating the drivers for upgrading neutralizing activity to SARS-CoV-2 in COVID-19 cases with pre-existing immunity will aid in understanding immunity to SARS-CoV-2 and improving COVID-19 booster vaccines with enhanced cross-protection against antigenically distinct variants. This study revealed that the magnitude and breadth of neutralization responses to SARS-CoV-2 infection in breakthrough infections are determined by upper respiratory viral load and vaccination-infection time interval, but not by the lineage of infecting viruses. Notably, the time interval, but not the viral load, may play a critical role in expanding the breadth of neutralization to SARS-CoV-2. This illustrates the importance of dosing interval optimization in addition to antigen design in the development of variant-proof booster vaccines.One-Sentence SummaryViral load and infection timing define the magnitude and breadth of SARS-CoV-2 neutralization after breakthrough infection.

Since the first case of COVID-19 was reported in Wuhan, China, in December 2019, the SARS-CoV-2 epidemic has spread all over the world and has become a significant public health issue. The development of treatments for COVID-19 is currently in progress; however, their effects remain limited, and the development of more effective therapeutics is desired. Thus, sufficient understanding of the pathophysiology of COVID-19 is essential to develop effective therapeutics for this disease. Pathological analyses in particular play an important role to demonstrate the causal link between an infectious disease and the pathogen and elucidate the mechanism of pathogenesis. As per pathological analyses to date, respiratory organs are identified as the major affected organs in most COVID-19 cases; also, various lesions were noted in other organs. Further, there have been increasing reports that show that the immune responses of the host contribute to the deterioration of the pathological condition of COVID-19, and a novel concept of MIS-C/MIS-A is also being established. Thus, in this article, we have provided an overview of the pathology of COVID-19 from a histopathological and immunological perspective focusing on the mechanisms of COVID-19 pathogenesis.

The recent emergence of SARS-CoV-2 Omicron (B.1.1.529 lineage) variants possessing numerous mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies and antiviral drugs for COVID-19 against these variants

In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622, a novel inhibitor of SARS-CoV-2 main protease (Mpro, also known as 3C-like protease), decreases viral load and ameliorates the disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to sub-micromolar concentrations in cells. Oral administration of S-217622 demonstrated eminent pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern (VOCs), including the highly pathogenic Delta variant and the recently emerged Omicron variant. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase II/III clinical trial, possesses remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.

Since the first case of COVID-19 was reported from Wuhan, China in December 2019, SARS-CoV-2 has been spreading globally and has become major public health concern. At present, development of specific treatment for COVID-19 is in progress and several countermeasures have been subjected to clinical trials. However, efficacy of these countermeasures is limited. For development of effective medicines or vaccines against infectious diseases, it is mandatory to elucidate its etiology and pathogenesis by means of pathological analysis. Pathological studies revealed that the COVID-19 mainly affects respiratory tracts although other organs are also involved. In addition, immunological studies demonstrated that host immune response may exacerbates COVID-19 through systemic inflammation. In this review, we would like to overview pathology and immunology of COVID-19.

Despite the development and deployment of antibody and vaccine countermeasures, rapidly-spreading SARS-CoV-2 variants with mutations at key antigenic sites in the spike protein jeopardize their efficacy. The recent emergence of B.1.1.529, the Omicron variant1,2, which has more than 30 mutations in the spike protein, has raised concerns for escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in pre-clinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) program of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of multiple B.1.1.529 Omicron isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2) expressing mice and hamsters. Despite modeling and binding data suggesting that B.1.1.529 spike can bind more avidly to murine ACE2, we observed attenuation of infection in 129, C57BL/6, and BALB/c mice as compared with previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. Although K18-hACE2 transgenic mice sustained infection in the lungs, these animals did not lose weight. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease, and pathology with B.1.1.529 also were milder compared to historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from multiple independent laboratories of the SAVE/NIAID network with several different B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

SUMMARYBackgroundThe immune profile against SARS-CoV-2 has dramatically diversified due to a complex combination of exposure to vaccines and infection by various lineages/variants, likely generating a heterogeneity in protective immunity in a given population. To further complicate this, the Omicron variant, with numerous spike mutations, has emerged. These circumstances have created the need to assess the potential of immune evasion by the Omicron in individuals with various immune histories.MethodsThe neutralization susceptibility of the variants including the Omicron and their ancestor was comparably assessed using a panel of plasma/serum derived from individuals with divergent immune histories. Blood samples were collected from either mRNA vaccinees or from those who suffered from breakthrough infections by the Alpha/Delta with multiple time intervals following vaccination.FindingsThe Omicron was highly resistant to neutralization in fully vaccinated individuals without a history of breakthrough infections. In contrast, robust cross-neutralization against the Omicron were induced in vaccinees that experienced breakthrough infections. The time interval between vaccination and infection, rather than the variant types of infection, was significantly correlated with the magnitude and potency of Omicron-neutralizing antibodies.ConclusionsImmune histories with breakthrough infections can overcome the resistance to infection by the Omicron, with the vaccination-infection interval being the key determinant of the magnitude and breadth of neutralization. The diverse exposure history in each individual warrants a tailored and cautious approach to understanding population immunity against the Omicron and future variants.FundingThis study was supported by grants from the Japan Agency for Medical Research and Development (AMED).

Since little is known about viral and host characteristics of breakthrough infections after COVID-19 vaccination, a nationwide investigation of breakthrough cases was initiated in Japan. 130 cases (90%+ received mRNA vaccines) were reported with respiratory specimens in 117 cases and sera in 68 cases. A subset of cases shed infectious virus regardless of symptom presence or viral lineages. Viral lineages for breakthrough infections matched both temporally and spatially with the circulating lineages in Japan with no novel mutations in spike receptor binding domain that may have escaped from vaccine-induced immunity were found. Anti-spike/neutralizing antibodies of breakthrough infections in the acute phase owing to vaccine-induced immunity were significantly higher than those from unvaccinated convalescent individuals but were comparable to vaccinated uninfected individuals, and followed by boosting in the convalescent phase. Symptomatic cases had low anti-spike/neutralizing antibodies in the acute phase with robust boosting in the convalescent phase, suggesting the presence of serological correlate for symptom development in COVID-19 vaccine breakthrough infections.

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 SummarySARS-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.

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.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global pandemic that began in December 2019. Lymphopenia is a common feature in severe cases of COVID-19; however, the role of T cell responses during infection is unclear. Here, we inoculated six cynomolgus monkeys, divided into two groups according to the CD3+ T cell population in peripheral blood, with two clinical isolates of SARS-CoV-2: one of East Asian lineage and one of European lineage. After initial infection with the isolate of East Asian lineage, all three monkeys in the CD3+ low group showed clinical symptoms, including loss of appetite, lethargy, and transient severe anemia with/without short-term fever, within 14 days post-infection (p.i.). By contrast, all three monkeys in the CD3+ high group showed mild clinical symptoms such as mild fever and loss of appetite within 4 days p.i. and then recovered. After a second inoculation with the isolate of European lineage, three of four animals in both groups showed mild clinical symptoms but recovered quickly. Hematological, immunological, and serological tests suggested that the CD3+ high and low groups mounted different immune responses during the initial and second infection stages. In both groups, anti-viral and innate immune responses were activated during the early phase of infection and re-infection. However, in the CD3+ low group, inflammatory responses, such as increased production of monocytes and neutrophils, were stronger than those in the CD3+ high group, leading to more severe immunopathology and failure to eliminate the virus. Taken together, the data suggest that the peripheral T lymphocyte population is associated with pneumonia severity in cynomolgus monkeys experimentally infected with SARS-CoV-2.Author summarySARS-CoV-2 infection causes an illness with clinical manifestations that vary from asymptomatic or mild to severe; examples include severe pneumonia and acute respiratory distress syndrome. Lymphopenia, which is common in severe COVID-19 cases, is characterized by markedly reduced numbers of CD4+ T cells, CD8+ T cells, B cells, and natural killer cells. Here, we showed that cynomolgus monkeys selected according to the T cell populations in peripheral blood have different outcomes after experimental infection with SARS-CoV-2. These findings will increase our understanding of disease pathogenesis and may facilitate the development of animal models for vaccine evaluation.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposi’s sarcoma as well as primary effusion lymphoma (PEL), an aggressive B-cell neoplasm which mostly arises in immunocompromised individuals. At present, there is no specific treatment available for PEL and its prognosis is poor. Lytic replication of KSHV is also associated with a subset of multicentric Castleman diseases. In this study, we found that the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA) induced KSHV reactivation in PEL cells in a dose-dependent manner. Next-generation sequencing analysis showed that more than 40% of all transcripts expressed in SBHA-treated PEL cells originated from the KSHV genome compared with less than 1% in untreated cells. Chromatin immunoprecipitation assays demonstrated that SBHA induced histone acetylation targeting the promoter region of the KSHV replication and transcription activator gene. However, there was no significant change in methylation status of the promoter region of this gene. In addition to its effect of KSHV reactivation, this study revealed that SBHA induces apoptosis in PEL cells in a dose-dependent manner, inducing cleavage of caspases and expression of proapoptotic factors, including Bim and Bax. These findings suggest that SBHA reactivates KSHV from latency and induces apoptosis through the mitochondrial pathway in PEL cells. Therefore, SBHA can be considered a new tool for induction of KSHV reactivation, and could provide a novel therapeutic strategy against PEL.ImportanceKaposi’s sarcoma and primary effusion lymphoma cells are latently infected with Kaposi’s sarcoma-associated herpesvirus (KSHV), whereas KSHV replication is frequently observed in multicentric Castleman disease. Although KSHV replication can be induced by some chemical reagents (e.g. 12-O-tetradecanoylphorbol-13-acetate), the mechanism of KSHV replication is not fully understood. We found that the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA) induced KSHV reactivation with high efficiency, through histone acetylation in the promoter of the replication and transcription activator gene, compared with 12-O-tetradecanoylphorbol-13-acetate. SBHA also induced apoptosis through the mitochondrial pathway in KSHV-infected cells, with a lower EC50than measured for viral reactivation. SBHA could be used in a highly efficient replication system for KSHVin vitro,and as a tool to reveal the mechanism of replication and pathogenesis of KSHV. The ability of SBHA to induce apoptosis at lower levels than needed to stimulate KSHV reactivation, indicates its therapeutic potential.

Recent molecular investigations of chordoma show common expression of various receptor tyrosine kinases and activation of downstream signaling pathways contributing to tumor growth and progression. The transcription factor brachyury (also known as T) is important in notochord differentiation, and germline duplication of the gene is often found in familial chordomas. Nuclear expression of brachyury is consistent in chordoma and in benign notochordal cell tumor. Based on the molecular evidence, targeting of several kinds of molecular agents has been attempted for the treatment of uncontrolled chordomas and achieved partial response or stable condition in many cases.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare variant of fibrosarcoma that commonly occurs in soft tissue. Since its original description in 1995, studies have revealed cytogenetic similarities with low-grade fibromyxoid sarcoma (LGFMS) and hybrid tumor composed of SEF and LGFMS. We report a case of SEF with EWSR1 gene rearrangement. A 62-year-old man had complained of a soft tissue mass in the left part of the buttock for 4 months. Imaging studies revealed a 6-cm mass in the left gluteus muscle, which was excised. Microscopically, the tumor was composed of short, spindle-shaped tumor cells with bland oval or polygonal nuclei arranged in cords and clusters with intervening sclerosing collagen matrix. Immunohistochemically, the tumor cells were positive for epithelial membrane antigen and mucin 4. Fluorescence in situ hybridization demonstrated split signals for the EWSR1 gene. These findings were consistent with SEF. Some SEFs have been reported to have shared features with LGFMS; those lesions are termed “hybrid tumors”; however, recent studies revealed that there is a pure type of SEF, which harbors EWSR1 gene rearrangements, distinct from the chromosomal abnormalities seen in LGFMS. The present case is a histologically pure SEF with EWSR1 gene translocation; its cytogenetic profile suggests that pure type SEF is a separate entity distinct from LGFMS and hybrid tumor.

Summary Background The coronavirus disease 2019 (COVID-19) continues to spread around the world. In addition to community-acquired infections, nosocomial infections are also a major social concern. The likelihood of environmental contamination and transmission of the virus based on disease severity is unknown. Methods We collected nasopharyngeal, environmental and air samples from patients with COVID-19 admitted to the National Centre for Global Health and Medicine between January 29th and February 29th, 2020. The patients were classified by severity of disease. The collected samples were tested using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Results SARS-CoV-2 was not detected in a subset of 11 air samples. Of the 141 environmental samples collected from three patient bays and two single rooms, four samples tested positive for SARS-CoV-2 by real-time RT-PCR. Detections were made on the surface of a stethoscope used in the care of a patient with severe disease, on the intubation tube of a patient classified as critical (and on ventilator management), and on the surface of a gown worn by the nurse providing care. Conclusions Regardless of the patients' disease severity, SARS-CoV-2 was detected on very few environmental surfaces. However, detection of SARS-CoV-2 on stethoscopes used in the care of multiple patients and on the surface of gowns worn by clinical staff indicates that medical devices may be linked to the spread of infection.

Recent molecular investigations of chordoma show common expression of various receptor tyrosine kinases and activation of downstream signaling pathways contributing to tumor growth and progression. The transcription factor brachyury (also known as T) is important in notochord differentiation, and germline duplication of the gene is often found in familial chordomas. Nuclear expression of brachyury is consistent in chordoma and in benign notochordal cell tumor. Based on the molecular evidence, targeting of several kinds of molecular agents has been attempted for the treatment of uncontrolled chordomas and achieved partial response or stable condition in many cases.

Background Multiple myeloma shows various morphological abnormalities such as plasmablastic morphology, cytoplasmic granules, vacuolation, and nuclear atypia. Although plasablastic morphology has been revealed as a poor prognostic factor, little is known about clinical significance of other abnormalities. Therefore we investigated a correlation between morphological abnormalities and clinical outcome. Methods Patient Selection A total of 76 multiple myeloma patients who hospitalized in National Center for Global Health and Medicine (NCGM), Japan, between 2010 and 2015 were enrolled in this study. Morphological Evaluation Bone marrow smears were obtained from each patient before initiation of treatments. We evaluated Greipp's morphological classification and morphological abnormalities of myeloma cells including cytoplasmic granules, vacuolation, and nuclear atypia. We also evaluated morphological abnormalities of other hemocytes. All specimens were observed at 400x magnification with an optical microscope. Clinical Information Clinical information obtained from medical records of NCGM includes gender, age, overall survival, Durie-Salmon staging system (DS), international staging system (ISS), types of M protein and light chain. The results of G-banded chromosomal analysis and fluorescent in situ hybridization (FISH) analysis were also collected from medical records. Statistical Analysis We performed survival analysis by generalized Wilcoxon test with EZR (version 1.31). Results Clinical Data Of all the patients, 46 (60.5%) are male. Mean age of the patients is 63.5 years old. Morphological Abnormality of Myeloma Cells According to Greipp's morphological classification, we identified 13 cases as mature type (17.1%), 28 cases as intermediate type (36.8%), 31 cases as immature type (40.8%), 4 cases as plasmablastic type (5.3%). We found cytoplasmic granules in 20 cases (26.3%), vacuolation in 8 cases (10.5%), atypical myeloma cells in 54 cases (71.1%), Russell bodies in 5 cases (6.6%), and Dutcher bodies in 3 cases (3.9%). Morphological Abnormality of Other Hematocytes As for myeloid cells, we found Auer rods in 1 case (1.3%), pseudo-Pelger-Huet anomaly in 2 cases (2.6%), neutrophils with ring-shaped nuclei in 2 cases (2.6%), megakaryocytes with multiple separated nuculeus in 3 cases (3.9%). Statistical Analysis We revealed that overall survival of granular group is significantly longer than non-granular group (p=0.04, Fig.1). Median overall survivals are 66.2 months (granular group) vs 54.5 months (non-granular group). However, there are no significance of vacuolation and nuclear atypia on overall survival. Conclusions A variety of morphological abnormalities were identified in multiple myeloma. We found that presence of cytoplasmic granules in myeloma cells correlates with good prognosis. Discussion Previous reports have shown that cytoplasmic granules of myeloma cells contain immunoglobulin however pathophysiological roles of them remain unclear. Further studies with molecular and cellular analysis should be carried out to elucidate roles of cytoplasmic granules. Disclosures Hagiwara: Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Eighteen outpatients with chronic renal failure undergoing hemodialysis (HD) were studied. Immediately before and after HD, the left and right ventricular function measured by electrocardiogram gated radionuclide ventriculography (RNV). By HD, body weight changed 58.6 +/- 7.50 kg to 57.2 +/- 6.80 kg and BUN level changed 67.9 +/- 29.00 mg/dl to 37.1 +/- 18.96 mg/dl and creatinine level changed 11.3 +/- 3.90 mg/dl to 6.8 +/- 2.48 mg/dl. Before HD, cardiac output was 8.08 +/- 1.50 l/min and cardiac index was 5.00 +/- 0.87 l/(min m2). Left ventricular function improved (LVEF changed 60.4 +/- 6.85% to 64.2 +/- 8.7%, LVEF/LVET changed 0.237 +/- 0.048%/ms to 0.254 +/- 0.021%/ms) between before and after HD, but there was not significant difference. Right ventricular function improved (RVEF changed 41.2 +/- 8.00% to 50.0 +/- 11.96, RVEF/RVET changed 0.167 +/- 0.028%/ms to 0.209 +/- 0.059%/ms) between before and after HD, and there was significant difference (p less than 0.05).