Infectious virus shedding duration reflects secretory IgA antibody response latency after SARS-CoV-2 infection.

Infectious virus shedding from individuals infected with severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is used to estimate human-to-human transmission risk. Control of SARS-CoV-2 transmission requires identifying the immune correlates that protect infectious virus shedding. Mucosal immunity prevents infection by SARS-CoV-2, which replicates in the respiratory epithelium and spreads rapidly to other hosts. However, whether mucosal immunity prevents the shedding of the infectious virus in SARS-CoV-2-infected individuals is unknown. We examined the relationship between viral RNA shedding dynamics, duration of infectious virus shedding, and mucosal antibody responses during SARS-CoV-2 infection. Anti-spike secretory IgA antibodies (S-IgA) reduced viral RNA load and infectivity more than anti-spike IgG/IgA antibodies in infected nasopharyngeal samples. Compared with the IgG/IgA response, the anti-spike S-IgA post-infection responses affected the viral RNA shedding dynamics and predicted the duration of infectious virus shedding regardless of the immune history. These findings highlight the importance of anti-spike S-IgA responses in individuals infected with SARS-CoV-2 for preventing infectious virus shedding and SARS-CoV-2 transmission. Developing medical countermeasures to shorten S-IgA response time may help control human-to-human transmission of SARS-CoV-2 infection and prevent future respiratory virus pandemics.

Acute acalculous cholecystitis following extended administration of nirmatrelvir/ritonavir for persistent SARS-CoV-2 infection.

Immunocompromised patients with hematologic malignancies, particularly those treated with anti-CD20 antibodies such as rituximab and obinutuzumab, are known to be at risk of prolonged infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Prolonged administration or combination therapy with antiviral medications reportedly yields favorable outcomes in these patients. However, knowledge regarding the adverse events associated with such therapeutic approaches is limited. Herein, we report a case of acute acalculous cholecystitis (AAC) following extended administration of nirmatrelvir/ritonavir (NMV/r) in a 68-year-old Japanese man with persistent SARS-CoV-2 infection. The patient had received obinutuzumab and bendamustine for follicular lymphoma and was diagnosed with coronavirus disease 2019 (COVID-19) approximately one year after treatment initiation with these drugs. Subsequently, he was admitted to a different hospital, where he received antiviral drugs, monoclonal antibodies, and steroids. Despite these interventions, the patient relapsed and was subsequently transferred to our hospital due to persistent SARS-CoV-2 infection. Remdesivir administration was ineffective, leading to the initiation of extended NMV/r therapy. One week later, he exhibited elevated gamma-glutamyl transpeptidase (GGT) levels, and one month later, he developed AAC. Cholecystitis was successfully resolved via percutaneous transhepatic gallbladder drainage and administration of antibiotics. We speculate that extended NMV/r administration, in addition to COVID-19, may have contributed to the elevated GGT and AAC. During treatment of persistent SARS-CoV-2 infection with extended NMV/r therapy, patients should be carefully monitored for the appearance of findings suggestive of biliary stasis and the development of AAC.

Long-Term Renal Involvement in Association with Fontan Circulation.

Multiorgan dysfunction is a concern of Fontan patients. To clarify the pathophysiology of Fontan nephropathy, we characterize renal disease in the long-term observational study. Medical records of 128 consecutive Fontan patients [median age: 22 (range 15-37) years old] treated between 2009 and 2018 were reviewed to investigate the incidence of nephropathy and its association with other clinical variables. Thirty-seven patients (29%) showed proteinuria (n = 34) or < 90 mL/min/1.73 m2 of estimated glomerular filtration rate (eGFR) (n = 7), including 4 overlapping cases. Ninety-six patients (75%) had liver dysfunction (Forns index > 4.21). Patients with proteinuria received the Fontan procedure at an older age [78 (26-194) vs. 56 (8-292) months old, p = 0.02] and had a higher cardiac index [3.11 (1.49-6.35) vs. 2.71 (1.40-4.95) L/min/m2, p = 0.02], central venous pressure [12 (7-19) vs. 9 (5-19) mmHg, p < 0.001], and proportion with > 4.21 of Forns index (88% vs. 70%, p = 0.04) than those without proteinuria. The mean renal perfusion pressure was lower in patients with a reduced eGFR than those without it [55 (44-65) vs. 65 (45-102) mmHg, p = 0.03], but no other variables differed significantly. A multivariable analysis revealed that proteinuria was associated with an increased cardiac index (unit odds ratio 2.02, 95% confidence interval 1.12-3.65, p = 0.02). Seven patients with severe proteinuria had a lower oxygen saturation than those with no or mild proteinuria (p = 0.01, 0.03). Proteinuria or a decreased eGFR differentially occurred in approximately 30% of Fontan patients. Suboptimal Fontan circulation may contribute to the development of proteinuria and reduced eGFR.

Genomic analysis of SARS-CoV-2 in forensic autopsy cases of COVID-19.

Numerous genomic analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been conducted, highlighting its variations and lineage transitions. Despite the importance of forensic autopsy in investigating deaths due to coronavirus disease 2019 (COVID-19), including out-of-hospital deaths, viral genomic analysis has rarely been reported due in part to postmortem changes. In this study, various specimens were collected from 18 forensic autopsy cases with SARS-CoV-2 infection. Reverse-transcription quantitative polymerase chain reaction revealed the distribution of the virus in the body, primarily in the respiratory organs. Next-generation sequencing determined the complete genome sequences in 15 of the 18 cases, although some cases showed severe postmortem changes or degradation of tissue RNA. Intrahost genomic diversity of the virus was identified in one case of death due to COVID-19. The accumulation of single-nucleotide variations in the lung of the case suggested the intrahost evolution of SARS-CoV-2. Lung of the case showed diffuse alveolar damage histologically and positivity for SARS-CoV-2 by immunohistochemical analysis and in situ hybridization, indicating virus-associated pneumonia. This study provides insights into the feasibility of genomic analysis of SARS-CoV-2 in forensic autopsy cases and the potential for uncovering important information in COVID-19 deaths, including out-of-hospital deaths.

Impact of the COVID-19 pandemic on pathological autopsy practices in Japan.

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.

The earliest enzyme replacement for infantile-onset Pompe disease in Japan.

BACKGROUND: Infantile-onset Pompe disease (IOPD) is the most severe phenotype of a lysosomal storage disorder caused by acid alpha-glucosidase (GAA) deficiency. An enzymatic newborn screening (NBS) program started regionally in Japan in 2013 for early enzyme replacement therapy (ERT). We report the ERT responses of the first NBS-identified Japanese IOPD case and of another case diagnosed prior to NBS, to discuss the problems of promptly starting ERT in Japan. METHODS: Acid alpha-glucosidase activity was measured by fluorometric assay in both patients. The diagnosis of IOPD was confirmed by next-generation followed by Sanger-method sequencing (patient 1) or direct sequencing of polymerase chain reaction (PCR)-amplified products (patient 2) of the GAA gene. RESULTS: A female infant identified by NBS had a novel out-of-frame (p.F181Dfs*6) variant and a reported pathogenic (p.R600C) variant, along with two pseudodeficiency variants. Enzyme replacement therapy was started at age 58 days when the infant had increased serum levels of creatine kinase and slight myocardial hypertrophy. Clinical and biochemical markers improved promptly. She has been alive and well without delayed development at age 14 months. Patient 2, a Japanese male, received a diagnosis of IOPD at age 5 months before the NBS era. He had a homozygotic variant of GAA (p.R608X), later registered as a cross-reactive immunological material (CRIM)-negative genotype, and developed a high titer of anti-rhGAA antibodies. The patient has survived myocardial hypertrophy with continuous respiratory support for 12 years of ERT. CONCLUSIONS: Enzyme replacement therapy should not be delayed over the age of 2 months for reversible cardiac function, although CRIM-negative cases may hamper turnaround time reduction.

High Salt Intake Worsens Aortic Dissection in Mice: Involvement of IL (Interleukin)-17A-Dependent ECM (Extracellular Matrix) Metabolism.

OBJECTIVE: Aortic dissection (AD) is a fatal disease that occurs suddenly without preceding clinical signs or symptoms. Although high salt intake is a proposed risk factor for cardiovascular diseases, the relationship between AD and high salt intake has not been clarified. We examined the effect of high-salt challenge on a mouse AD model. Approach and Results: AD was induced in male mice by continuous infusion of ß-aminopropionitrile and Ang II (angiotensin II). High-salt challenge exacerbated aortic wall destruction in AD. Deletion of Il17a (IL-17KO [IL (interleukin)-17A knockout]) did not affect the AD phenotype at baseline, but it abolished the high salt-induced worsening of the aortic destruction. Unexpectedly, aortas of IL-17KO mice exhibited global changes in ECM (extracellular matrix)-related genes without alteration of proinflammatory genes, altered architecture of collagen fibers, and reduced stiffness before AD induction. The aortas of IL-17KO mice were less sensitive to AD-inducing stimuli, as shown by the induction of phenotypic modulation markers SMemb and vimentin, suggesting a reduced stress response. The aortas of IL-17KO mice had a higher population of smooth muscle cells with nuclear-localized phosphorylated Smad2, indicative of TGFß (transforming growth factor-beta) signal activation. Consistently, pretreatment of smooth muscle cells in culture with IL-17A blunted the activation of Smad2 by TGFß1. CONCLUSIONS: These findings indicate that high salt intake has a worsening effect on AD in the context of high aortic wall stiffness, which is under the control of IL-17A through ECM metabolism. Therefore, salt restriction may represent a low-cost and practical way to reduce AD risk.

Portosystemic venous shunt in the patients with Fontan circulation.

Portosystemic venous shunt (PSVS) is a vascular anomaly between the portal and systemic veins, resulting in several critical complications. Although PSVS is often associated with congenital heart diseases, the clinical association between Fontan circulation and PSVS has not been elucidated. This study aimed to investigate the clinical features of Fontan patients with PSVS. Two hundred thirteen patients who underwent Fontan procedure are being followed up at Adult Congenital Heart Disease clinic in Kyushu University Hospital. Among them, 139 adult patients underwent cardiac catheterization between January 1, 2011 and September 30, 2019. Medical records were reviewed to investigate the laboratory, echocardiography, and cardiac catheterization findings, as well as clinical manifestations and outcomes. Eleven Fontan patients received the diagnosis of PSVS. The median age at cardiac catheterization was 25 (range 18-45) years. Fontan operation was performed using extracardiac conduit or lateral tunnel 22 (16-35) years previously. Ten patients presented with chronic heart failure [New York Heart Association class 2 (n = 5) and 3 (n = 5)]. The median level of peripheral oxygen saturation was 87 (70-95)%. Cardiac catheterization showed increased cardiac index [5.3 (2.72-14.3) L/min/m2] with or without high central venous pressure [18 (9-25) mmHg]. Although the pulmonary vascular resistance was within the normal range, the systemic vascular resistance was decreased [7.08 (1.74-18.6) Wood units]. Fontan patients complicated with PSVS had increased cardiac output. The presence of PSVS in Fontan circulation might be associated with unfavorable long-term outcome.

Procalcitonin levels predicting the infliximab response of immunoglobulin resistant Kawasaki disease.

OBJECTIVE: To search the predictive factors of infliximab resistance in intravenous immunoglobulin (IVIG)-resistant Kawasaki disease (KD) patients. STUDY DESIGN: Twenty-seven patients with KD who received infliximab after 4-5 g/kg of IVIG therapy from 2013 to 2015 were consecutively recruited in this study. They were divided into two groups: patients who responded to infliximab (infliximab-responsive group, n = 15) and patients who required additional therapy for the disease control (infliximab-resistant group, n = 12). We analyzed the clinical and laboratory parameters just before the infliximab treatment including serum levels of procalcitonin and cytokines with respect to the infliximab response. RESULTS: Serum procalcitonin concentration (P = 0.017), neutrophils to lymphocytes ratio (P = 0.013), and % neutrophils (P = 0.004) were higher, and serum sodium concentration (P = 0.017) was lower in infliximab-resistant group than those of infliximab-responsive group, respectively. Multivariate logistic regression analyses indicated that higher procalcitonin concentration (odds ratio [OR] 1.48, 95% confidence interval [CI] 1.00-5.00, P = 0.046) and lower sodium levels (OR 0.64, 95% CI 0.32-1.00, P = 0.047), but not other variables, were associated with infliximab-resistance. Serum procalcitonin concentrations positively correlated with the serum levels of interleukin-6, soluble tumor necrosis factor receptor type 1 and type 2, respectively. Analyses of the receiver operating characteristic (ROC) curve showed that the cut-off value of procalcitonin 2.0 ng/ml had 58.3% of sensitivity and 93.3% of specificity. ROC analysis yielded an area under the curve (AUC) of 0.739 to predict infliximab-resistance. CONCLUSION: Serum procalcitonin might be an effective biomarker to predict infliximab resistance in severe KD patients who are refractory to IVIG treatment.

High incidence of ductal closure or narrowing at birth in patients with right ventricular outflow tract obstruction with normal orientation of the ductus arteriosus.

BACKGROUND: Ductal patency is mandatory to manage patients with ductal-dependent pulmonary circulation. The aim of this study is to elucidate the morphological and haemodynamic features of ductus arteriosus with right ventricular outflow tract obstruction, and investigate the appropriate perinatal management.Patients and methodsPatients with prenatal diagnosis of right ventricular outflow tract obstruction at our institution between 2010 and 2015 were included in the study. Reverse orientation of the ductus arteriosus is defined as an inferior angle of 90°. We retrospectively reviewed the shape and flow pattern of ductus arteriosus and the clinical characteristics of the cases. RESULTS: A total of 39 patients were enrolled. The shape was divided into normal orientation (n=15) and reverse orientation (n=24) of the ductus arteriosus. There was no significant difference in the type of oxygen saturation at birth and age at shunt operation between both the groups. However, the median narrowest diameter of ductus arteriosus in the normal orientation group was significantly smaller than that in the reverse orientation group (2.0 [1.0-5.4] versus 3.0 [1.3-4.4] mm, p<0.05). In two patients of the normal orientation group, ductus arteriosus had closed at birth, and one of whom died because of severe cyanosis. CONCLUSIONS: Normal orientation pattern might have high incidence of an early narrowing or closure of ductus arteriosus at birth. The critical patients need careful evaluation by repeated foetal echocardiography and further maternal interventions.

Activation of the AKT Pathway in the Ascending Aorta With Bicuspid Aortic Valve.

BACKGROUND: Dilatation of the ascending aorta affects those patients with bicuspid aortic valve (BAV), even after valvular surgery, possibly due to tissue fragility. The goal of the study was the molecular characterization of aorta with BAV compared to that with normal tricuspid aortic valve (TAV). Methods and Results: The subjects were patients who underwent surgery for aortic valve stenosis in 2013 and 2014. Nine patients with BAV and 13 with TAV were examined. There was no difference in the clinical characteristics or grade of aortic valve stenosis, but the diameters of the ascending aorta were significantly higher in the BAV group. The ascending aortic specimens were subjected to transcriptome analyses, which revealed the changes in receptor tyrosine kinase (RTK) pathway-related genes between TAV and BAV samples. Immunohistochemical study revealed higher staining of phosphorylated AKT (pAKT) in the media of the ascending aorta in the BAV group, regardless of the size of ascending aorta, whereas total AKT did not show such a difference. Immunofluorescence staining revealed the AKT activation was mainly in the medial vascular smooth muscle cells. CONCLUSIONS: The results showed that the RTK-AKT pathway in the medial layer of the ascending aorta is activated in aortae with BAV. Activation of this pathway may be associated with fragility and dilatation of the ascending aorta with BAV.

High incidence of progressive stenosis in aberrant left subclavian artery with right aortic arch.

Right aortic arch with aberrant left subclavian artery (RAA/aLSCA) is a rare aortic arch anomaly. The clinical association of aLSCA stenosis with RAA/aLSCA has not yet been fully elucidated. The aim of this study was to investigate the diagnosis, incidence, management and outcome of aLSCA stenosis in infants with prenatally diagnosed RAA/aLSCA. Ten fetuses who were diagnosed as having RAA/aLSCA in Kyushu University Hospital between January 2011 and December 2014 were enrolled. The maternal and child medical records were reviewed to investigate sex, gestational age at the fetal diagnosis, gestational age and body weight at birth, the findings of computed tomography (CT), Doppler ultrasonography of the vertebral artery and angiography, and the complications and outcomes of aLSCA stenosis. In 8 of 10 patients, aLSCA stenosis was identified on the first CT examination after birth. No patients had dysphagia or respiratory distress. The stenosis spontaneously resolved in 3 patients. In 4 of the 5 remaining patients, aLSCA stenosis progressed, including one case in which complete occlusion occurred-the case was associated with retrograde flow from the left vertebral artery supplying the distal LSCA. Balloon angioplasty was successfully used to treat stenosis in two cases. The subclavian steal phenomenon and developmental problems were not observed in any patients. aLSCA stenosis was identified in 80% of patients with RAA/aLSCA after birth. The early detection and elective treatment of stenotic lesions may be required to prevent complete occlusion during the development of the cardiovascular and cerebrovascular systems.

Medical equipment deployment in pediatric emergency prehospital medical units in Japan.

The deployment status of pediatric emergency equipment in ambulances in Japan is unknown. To investigate the status of and issues associated with prehospital emergency medical care for pediatric patients, we conducted a descriptive epidemiological study. We carried out a Web-based survey of 767 fire defense headquarters in Japan, of which 671 responded (valid response rate, 88%). Most of the fire defense headquarters equipped all of their ambulances with oxygen masks (82%), bag-valve masks (for neonates, 83%; for children, 84%), straight laryngoscope blades (for neonates, 47%; for children 68%), blood pressure cuffs for children (91%), oximeter probes (78%), and stiff neck collars (91%); but despite the need for other equipment such as nasopharyngeal and oropharyngeal airways, and Magill forceps, they were insufficiently deployed. In Japan, prehospital emergency medical equipment deployment does not meet the needs of pediatric patients. Minimum equipment standards need to be established for pediatric prehospital care.

Early progression of atherosclerosis in children with chronic infantile neurological cutaneous and articular syndrome.

OBJECTIVE: Chronic inflammation plays a key role in the development of atherosclerosis. Early progression of atherosclerosis has been reported in patients with RA. Cryopyrin-associated periodic syndromes (CAPS) are autosomal dominant autoinflammatory disorders caused by heterozygous NLRP3 gene mutations. Chronic infantile neurological cutaneous and articular (CINCA) syndrome is the most severe form of CAPS and patients display early onset of rash, fever, uveitis and joint manifestations. However, there has been no previous report on atherosclerosis in patients with CAPS. The objective of this study is to assess the development of atherosclerosis in patients with CINCA syndrome. METHODS: Intima-media thickness (IMT) of the carotid arteries, stiffness parameter ß, ankle brachial index (ABI) and pressure wave velocity (PWV) were evaluated by ultrasonography in 3 patients with CINCA syndrome [mean age 9.0 years (S.D. 5.3)] and 19 age-matched healthy controls [9.3 years (S.D. 4.3)]. RESULTS: The levels of carotid IMT, stiffness parameter ß and PWV in CINCA syndrome patients were significantly higher than those in healthy controls [0.51 mm (S.D. 0.05) vs 0.44 (0.04), P = 0.0021; 6.1 (S.D. 1.7) vs 3.9 (1.0), P = 0.0018; 1203 cm/s (S.D. 328) vs 855 (114), P = 0.017, respectively]. CONCLUSION: Patients with CINCA syndrome showed signs of atherosclerosis from their early childhood. The results of this study emphasize the importance of chronic inflammation in the development of atherosclerosis. Further analysis on atherosclerosis in young patients with CINCA syndrome may provide more insights into the pathogenesis of cardiovascular disease.

Evaluation of echogenicity of the heart in Kawasaki disease.

Pathologic studies of the heart in patients with Kawasaki disease (KD) revealed vasculitis, valvulitis, myocarditis, and pericarditis. However, there have been no studies on the quantitative determination of multi-site echogenicity of the heart in KD patients. It is also undetermined whether the degree of echogenicity of each site of the heart in patients with KD might be related to the response to intravenous immunoglobulin (IVIG) treatment. In 81 KD patients and 30 control subjects, we prospectively analyzed echogenicity of the heart. Echogenicity was measured in four sites: coronary artery wall (CAW), mitral valve (MV), papillary muscle (PM), and ascending aortic wall (AAo wall) by the calibrated integrated backscatters (cIBs). The cIB values of all measurement sites at acute phase in KD patients were significantly higher than those in control subjects (KD patients vs control subjects; CAW, 19.8 ± 6.2 dB vs 14.5 ± 2.0 dB, p < 0.05; MV, 23.3 ± 5.3 dB vs 16.0 ± 3.3 dB, p < 0.05; PM, 22.4 ± 5.1 dB vs 12.7 ± 1.9 dB, p < 0.05; AAo wall, 25.3 ± 5.6 dB vs 18.3 ± 3.4 dB, p < 0.05). The cIB values of CAW at the acute phase in IVIG nonresponders were significantly higher than those in responders. Conclusion: Echogenicity of the heart in KD patients at the acute phase increased not only in the coronary artery wall but also in other parts of the heart. Echogenicity of CAW might be helpful in determining the unresponsiveness of IVIG treatment.

Combination therapy with oral antiviral and anti-inflammatory drugs improves the efficacy of delayed treatment in a COVID-19 hamster model.

BACKGROUND: Pulmonary infection with SARS-CoV-2 stimulates host immune responses and can also result in the progression of dysregulated and critical inflammation. Throughout the pandemic, the management and treatment of COVID-19 has been continuously updated with a range of antiviral drugs and immunomodulators. Monotherapy with oral antivirals has proven to be effective in the treatment of COVID-19. However, treatment should be initiated in the early stages of infection to ensure beneficial therapeutic outcomes, and there is still room for further consideration on therapeutic strategies using antivirals. METHODS: We studied the therapeutic effects of monotherapy with the oral antiviral ensitrelvir or the anti-inflammatory corticosteroid methylprednisolone and combination therapy with ensitrelvir and methylprednisolone in a delayed dosing model of hamsters infected with SARS-CoV-2. FINDINGS: Combination therapy with ensitrelvir and methylprednisolone improved respiratory conditions and reduced the development of pneumonia in hamsters even when the treatment was started after 2 days post-infection. The combination therapy led to a differential histological and transcriptomic pattern in comparison to either of the monotherapies, with reduced lung damage and down-regulation of expression of genes involved in the inflammatory response. Furthermore, we found that the combination treatment is effective in case of infection with either the highly pathogenic delta or circulating omicron variants. INTERPRETATION: Our results demonstrate the advantage of combination therapy with antiviral and corticosteroid drugs in COVID-19 treatment from the perspective of lung pathology and host inflammatory responses. FUNDING: Funding bodies are described in the Acknowledgments section.

Characterization of Omicron BA.4.6, XBB, and BQ.1.1 subvariants in hamsters.

During the Omicron wave, previous variants such as BA.2, BA.4, and BA.5 were replaced by newer variants with additional mutations in the spike protein. These variants, BA.4.6, BQ.1.1, and XBB, have spread in different countries with different degrees of success. Here, we evaluated the replicative ability and pathogenicity of BA.4.6, BQ1.1, and XBB clinical isolates in male Syrian hamsters. Although we found no substantial differences in weight change among hamsters infected with these Omicron subvariants, the replicative ability of BQ.1.1 and XBB in lung tissue was higher than that of BA.4.6 and BA.5. Of note, BQ.1.1 was lethal in both male and female transgenic human ACE2 hamsters. In competition assays, XBB replicated better than BQ.1.1 in the nasal turbinate tissues of female hamsters previously infected with Omicron BA.2. These results suggest that newer Omicron subvariants in the XBB family are still evolving and should be closely monitored.

Changes in SARS-CoV-2 viral load and titers over time in SARS-CoV-2-infected human corpses.

High viral titers of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been detected in human corpses long after death. However, little is known about the kinetics of infectious SARS-CoV-2 in corpses. In this case series study, we investigated the postmortem kinetics of infectious SARS-CoV-2 in human corpses by collecting nasopharyngeal swab samples at multiple time points from six SARS-CoV-2-infected patients after their death. SARS-CoV-2 RNA was detected by quantitative reverse transcription-polymerase chain reaction from nasopharyngeal swab samples collected from all six deceased patients. A viral culture showed the presence of infectious virus in one deceased patient up to 12 days after death. Notably, this patient had a shorter time from symptom onset to death than the other patients, and autopsy samples showed pathological findings consistent with viral replication in the upper respiratory tract. Therefore, this patient died during the viral shedding phase, and the amount of infectious virus in the corpse did not decrease over time up to the date of autopsy (12 days after death). The findings of this study indicate that the persistence of SARS-CoV-2 in corpses can vary among individuals and may be associated with the stage of the disease at the time of death. These important results complement many previously reported findings on the infectivity of SARS-CoV-2 at postmortem.

Recombinant spike protein vaccines coupled with adjuvants that have different modes of action induce protective immunity against SARS-CoV-2.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a glycoprotein, expressed on the virion surface, that mediates infection of host cells by directly interacting with host receptors. As such, it is a reasonable target to neutralize the infectivity of the virus. Here we found that a recombinant S protein vaccine adjuvanted with Alhydrogel or the QS-21-like adjuvant Quil-A effectively induced anti-S receptor binding domain (RBD) serum IgG and neutralizing antibody titers in the Syrian hamster model, resulting in significantly low SARS-CoV-2 replication in respiratory organs and reduced body weight loss upon virus challenge. Severe lung inflammation upon virus challenge was also strongly suppressed by vaccination. We also found that the S protein vaccine adjuvanted with Alhydrogel, Quil-A, or an AS03-like adjuvant elicited significantly higher neutralizing antibody titers in mice than did unadjuvanted vaccine. Although the neutralizing antibody titers against the variant viruses B.1.351 and B.1.617.2 declined markedly in mice immunized with wild-type S protein, the binding antibody levels against the variant S proteins were equivalent to those against wild-type S. When splenocytes from the immunized mice were re-stimulated with the S protein in vitro, the induced Th1 or Th2 cytokine levels were not significantly different upon re-stimulation with wild-type S or variant S, suggesting that the T-cell responses against the variants were the same as those against the wild-type virus. Upon Omicron XBB-challenge in hamsters, wild-type S-vaccination with Alhydrogel or AS03 reduced lung virus titers on Day 3, and the Quil-A adjuvanted group showed less body weight loss, although serum neutralizing antibody titers against XBB were barely detected in vitro. Collectively, recombinant vaccines coupled with different adjuvants may be promising modalities to combat new variant viruses by inducing various arms of the immune response.

In vitro and in vivo characterization of SARS-CoV-2 resistance to ensitrelvir.

Ensitrelvir, an oral antiviral agent that targets a SARS-CoV-2 main protease (3CLpro or Nsp5), is clinically useful against SARS-CoV-2 including its omicron variants. Since most omicron subvariants have reduced sensitivity to most monoclonal antibody therapies, SARS-CoV-2 resistance to other antivirals including main protease inhibitors such as ensitrelvir is a major public health concern. Here, repeating passages of SARS-CoV-2 in the presence of ensitrelvir revealed that the M49L and E166A substitutions in Nsp5 are responsible for reduced sensitivity to ensitrelvir. Both substitutions reduced in vitro virus growth in the absence of ensitrelvir. The combination of the M49L and E166A substitutions allowed the virus to largely evade the suppressive effect of ensitrelvir in vitro. The virus possessing Nsp5-M49L showed similar pathogenicity to wild-type virus, whereas the virus possessing Nsp5-E166A or Nsp5-M49L/E166A slightly attenuated. Ensitrelvir treatment of hamsters infected with the virus possessing Nsp5-M49L/E166A was ineffective; however, nirmatrelvir or molnupiravir treatment was effective. Therefore, it is important to closely monitor the emergence of ensitrelvir-resistant SARS-CoV-2 variants to guide antiviral treatment selection.