Neurological manifestations of acute SARS-CoV-2 infection in pediatric patients: a 3-year study (preprint)

Purpose This study analyzed the neurological manifestation profiles of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection across pandemic waves in pediatric patients.Methods This retrospective study analyzed the data of patients aged 0–18 years who were diagnosed with acute SARS-CoV-2 infection and admitted to a pediatric tertiary hospital between March 1, 2020, and February 28, 2023. The presence of neurological manifestations was established based on the symptoms noted in each patient chart. The relationships between neurological manifestations and pandemic waves or age groups were assessed using the chi-square test.Results This study included 1677 patients. Neurological manifestations were noted in 10% (n = 168) of patients with a 3.2 years median age (interquartile range: 1–11.92). Neurological manifestations were significantly associated with the pandemic waves (p = 0.006) and age groups (p < 0.001). Seizures were noted in 4.2% of cases and reached an increasing frequency over time (p = 0.001), but were not associated with age groups. Febrile seizures accounted for the majority of seizures. Headache was reported in 2.6% of cases and had similar frequencies across the pandemic waves and age groups. Muscular involvement was noted in 2% of cases and reached a decreasing frequency over time (p < 0.001) and showed different frequencies among the age groups.Conclusions Neurological manifestations of acute SARS-CoV-2 infection exhibit distinct patterns, depending on the pandemic wave and patient age group. The Wuhan and Omicron waves involved the nervous system more often than the other waves; however, this was probably through different mechanisms.

Virological characteristics of SARS-CoV-2 Omicron BA.5.2.48 (preprint)

With the prevalence of sequentially-emerged sublineages including BA.1, BA.2 and BA.5, SARS-CoV-2 Omicron infection has transformed into a regional epidemic disease. As a sublineage of BA.5, the BA.5.2.48 outbreak and evolved into multi-subvariants in China without clearly established virological characteristics, especially the pathogenicity. Though reduced airborne transmission and pathogenicity of former Omicron sublineages have been revealed in animal models, the virological characteristics of BA.5.2.48 was unidentified. Here, we evaluated the in vitro and in vivo virological characteristics of two isolates of the prevalent BA.5.2.48 subvariant, DY.2 and DY.1.1 (a subvariant of DY.1). DY.2 replicates more efficiently than DY.1.1 in HelahACE2+ cells and Calu-3 cells. The A570S mutation (of DY.1) in a normal BA.5 spike protein (DY.2) leads to a 20% improvement in the hACE2 binding affinity, which is slightly reduced by a further K147E mutation (of DY.1.1). Compared to the normal BA.5 spike, the double-mutated protein demonstrates efficient cleavage and reduced fusogenicity. BA.5.2.48 demonstrated enhanced airborne transmission capacity in hamsters than BA.2. The pathogenicity of BA.5.2.48 is greater than BA.2, as revealed in K18-hACE2 rodents. Under immune selection pressure, DY.1.1 shows stronger fitness than DY.2 in hamster turbinates. Thus the outbreaking prevalent BA.5.2.48 multisubvariants exhibites divergent virological features.

How do we respond to the next SARS CoV epidemic/pandemic? A bioinformatics approach with the promise of preventing or reducing the severity of future SARS CoV related pandemics (preprint)

In this work, we develop a Bayesian weighted scheme to generate evolutionary lineages of a particular viral protein sequence of interest and through a process of clustering and choosing representative lineages from the different clusters according to an evolutionary fitness objective function, we demonstrate it is possible to have anticipated the emergence of the SARS-CoV 2 (2019) strain from the SARS-CoV 1(2004) strain and having shown this retrospectively, we discuss the possibility of applying this approach along with continuous genomic surveillance of SARS-CoVs to prevent or reduce severity of future SARS-CoV related pandemics by being prepared with broad neutralization strategies for anticipated future lineages of SARS-CoVs identified through bioinformatics approaches such as that reported in this work.

Two fitness inference schemes compared using allele frequencies from 1,068,391 sequences sampled in the UK during the COVID-19 pandemic (preprint)

Throughout the course of the SARS-CoV-2 pandemic, genetic variation has contributed to the spread and persistence of the virus. For example, various mutations have allowed SARS-CoV-2 to escape antibody neutralization or to bind more strongly to the receptors that it uses to enter human cells. Here, we compared two methods that estimate the fitness effects of viral mutations using the abundant sequence data gathered over the course of the pandemic. Both approaches are grounded in population genetics theory but with different assumptions. One approach, tQLE, features an epistatic fitness landscape and assumes that alleles are nearly in linkage equilibrium. Another approach, MPL, assumes a simple, additive fitness landscape, but allows for any level of correlation between alleles. We characterized differences in the distributions of fitness values inferred by each approach and in the ranks of fitness values that they assign to sequences across time. We find that in a large fraction of weeks the two methods are in good agreement as to their top-ranked sequences, i.e., as to which sequences observed that week are most fit. We also find that agreement between ranking of sequences varies with genetic unimodality in the population in a given week.

Evaluation of Febrile Seizure Risk Following Ancestral Monovalent COVID-19 mRNA Vaccination Among U.S. Children Aged 2-5 Years (preprint)

Importance The United States Food and Drug Administration noted a potential safety concern for seizure in children aged 2-5 years receiving the ancestral monovalent COVID-19 mRNA vaccines. Objective To evaluate febrile seizure risk following monovalent COVID-19 mRNA vaccination among children aged 2-5 years. Design, Setting, and Participants The primary analysis evaluated children who had a febrile seizure outcome in the 0-1 days following COVID-19 vaccination. A self-controlled case series analysis was performed in three commercial insurance databases to compare the risk of seizure in the risk interval (0-1 days) to a control interval (8-63 days). Exposure Receipt of dose 1 and/or dose 2 of monovalent COVID-19 mRNA vaccinations. Main Outcomes and Measures The primary outcome was febrile seizure (0-1 day risk interval). Analysis A conditional Poisson regression model was used to compare outcome rates in risk and control intervals and estimate incidence rate ratios (IRR) and 95% confidence intervals (CIs). Meta-analyses were used to pool results across databases. Results The primary meta-analysis found a statistically significant increased incidence of febrile seizure, in the 0-1 days following mRNA-1273 vaccination compared to the control interval (IRR: 2.52, 95% CI: 1.35 to 4.69, risk difference (RD)/100,000 doses = 3.22 (95%CI -0.31 to 6.75)). For the BNT162b2 vaccination, the IRR was elevated but not statistically significant (IRR: 1.41, 95%CI: 0.48 to 4.11, RD/100,000 doses = -0.25 (95%CI -2.75 to 2.24). Conclusions and Relevance Among children aged 2-5 years, the analysis showed a small elevated incidence rate ratio of febrile seizures in the 0-1 days following the mRNA-1273 vaccination. Based on the current body of scientific evidence, the safety profile of the monovalent mRNA vaccines remains favorable for use in young children.

Effects of wearing a KF94 face mask on performance, perceptual, and physiological responses during a resistance exercise (preprint)

Wearing a face mask in indoor public places including fitness centers is an effective strategy to prevent the airborne transmission of COVID-19. However, only a few studies have been performed on wearing a mask during resistance exercise (RE) which is primarily performed in indoor fitness centers. This study aimed to investigate the effects of wearing a KF94 mask on exercise volume, perceptual parameters, and physiological responses during RE. Twenty young men participated in this randomized crossover trial. Participants performed moderate-intensity (1RM 60%) RE sessions in two different conditions (KF94 mask vs. no mask). Cardiorespiratory parameters, exercise volume, rating of perceived exertion (RPE), and dyspnea were measured during RE. Blood lactate concentration, blood pressure, arterial stiffness, and perceptual parameters were measured at pre-exercise and post-exercise. Exercise volume, ventilation volume, and ventilation efficiency parameters were lower with the KF94 mask than without the mask. However, RPE and dyspnea were higher with the KF94 mask than without the mask. Central arterial stiffness at post-exercise was higher with the KF94 mask than without the mask. Therefore, wearing a KF94 mask during RE affects exercise volume, perceptual parameters, and physiological responses, suggesting coaches need to modify RE manipulation variables while wearing a KF94 mask.

The Impact of Inactivity During the COVID-19 Pandemic on the Physical Performance of High School Athletes (preprint)

Background/Objective This study was conducted during the 2019-2020 academic year to evaluate the impact of participation in school sports on students' Body Mass Index (BMI) and Assessing Levels of Physical Activity (ALPHA) test scores. Interrupted by the COVID-19 pandemic, which led to a suspension of in-person education, the study resumed in September 2021, refocusing on the effects of pandemic-induced inactivity on the physical fitness levels of the same cohort. Methods The study included twenty-nine male high school students, divided into thirteen athletes (participating in sports such as football, basketball, and track) and sixteen non-athletic counterparts. They underwent reassessment using the ALPHA test battery, evaluating cardiorespiratory, musculoskeletal, and motor skills fitness. Data were analyzed using independent and paired samples t-tests and a two-way repeated measures ANOVA to assess changes over time and between groups. Discriminant function analysis evaluated the ALPHA test's ability to classify students based on their athletic status pre- and post-pandemic. Results Initially, athlete students exhibited significantly better BMI, 20 m shuttle run, and 4 × 10 m speed run scores compared to their non-athlete peers. After the pandemic, only the 20 m shuttle run scores remained significantly higher for athletes, with diminished distinctions in other fitness areas. The classification accuracy of the ALPHA test battery decreased from 86.2% to 75.9% post-pandemic. Conclusion The enforced sedentary lifestyle due to the COVID-19 pandemic adversely affected all students, particularly diminishing health-related fitness parameters such as body composition, cardiorespiratory and musculoskeletal strength, and motor skills. Students previously engaged in regular physical activity, notably school athletes, experienced significant fitness declines. This highlights the urgent need for targeted interventions to encourage active lifestyles among youth in the post-pandemic phase, aiming to avert long-term adverse health outcomes.

Cross-neutralizing antibody against emerging Omicron subvariants of SARS-CoV-2 in infection-naïve individuals with homologous BNT162b2 or BNT162b2(WT+ BA.4/5) bivalent booster vaccination (preprint)

Since the emergence of SARS-CoV-2, different variants and subvariants successively emerged to dominate global virus circulation as a result of immune evasion, replication fitness or both. COVID-19 vaccines continue to be updated in response to the emergence of antigenically divergent viruses, the first being the bivalent RNA vaccines that encodes for both the Wuhan-like and Omicron BA.5 subvariant spike proteins. Repeated infections and vaccine breakthrough infections have led to complex immune landscapes in populations making it increasingly difficult to assess the intrinsic neutralizing antibody responses elicited by the vaccines. Hong Kong’s intensive COVID-19 containment policy through 2020–2021 permitted us to identify sera from a small number of infection naïve individuals who received 3 doses RNA vaccine BNT162b2 of vaccines encoding the Wuhan-like spike who were boosted with a fourth dose monovalent Wuhan-like (WT) vaccine or the bivalent Wuhan-like and BA.4/5 spike (WT + BA.4/5) expressing vaccine. While neutralizing antibody to wild-type virus was comparable in both vaccine groups, BNT162b2 bivalent vaccine elicited significantly higher plaque neutralizing antibodies to Omicron subvariants BA.5, XBB.1.5, XBB.1.16, XBB.1.9.1, XBB.2.3.2, EG.5.1, HK.3, BA.2.86 and JN.1, compared to BNT162b2 monovalent vaccine. The single amino acid substitution that differentiates the spike of JN.1 from BA.2.86 resulted in a profound antigenic change.

Taiwan Chingguan Yihau (NRICM101) prevents kainic acid-induced seizures in rats by modulating neuroinflammation and the glutamatergic system (preprint)

Taiwan Chingguan Yihau (NRICM101) is a Traditional Chinese medicine (TCM) formula used to treat coronavirus disease 2019; however, its impact on epilepsy has not been revealed. Therefore, the present study evaluated the anti-epileptogenic effect of orally administered NRICM101 on kainic acid (KA)-induced seizures in rats and investigated its possible mechanisms of action. Sprague‒Dawley rats were administered NRICM101 (300 mg/kg) by oral gavage for 7 consecutive days before receiving an intraperitoneal injection of KA (15 mg/kg). NRICM101 considerably reduced the seizure behavior and electroencephalographic seizures induced by KA in rats. NRICM101 also significantly decreased the neuronal loss and glutamate increase and increased GLAST, GLT-1, GAD67, GDH and GS levels in the cortex and hippocampus of KA-treated rats. In addition, NRICM101 significantly suppressed astrogliosis (as determined by decreased GFAP expression); neuroinflammatory signaling (as determined by reduced HMGB1, TLR-4, IL-1β, IL-1R, IL-6, p-JAK2, p-STAT3, TNF-α, TNFR1 and p-IκB levels, and increased cytosolic p65-NFκB levels); and necroptosis (as determined by decreased p-RIPK3 and p-MLKL levels) in the cortex and hippocampus of KA-treated rats. The effects of NRICM101 were similar to those of carbamazepine, a well-recognized antiseizure drug. Furthermore, no toxic effects of NRICM101 on the liver and kidney were observed in NRICM101-treated rats. The results indicate that NRICM101 has antiepileptogenic and neuroprotective effects through the suppression of the inflammatory cues (HMGB1/TLR4, Il-1β/IL-1R1, IL-6/p-JAK2/p-STAT3, and TNF-α/TNFR1/NF-κB) and necroptosis signaling pathways (TNF-α/TNFR1/RIP3/MLKL) associated with glutamate level regulation in the brain and is innocuous. Our findings highlight the promising role of NRICM101 in the management of epilepsy.

Comparing care pathways between COVID-19 pandemic waves using electronic health records: a process mining case study (preprint)

Purpose: The COVID-19 pandemic caused rapid shifts in the workflow of many health services, but evidence of how this affected multidisciplinary care settings is limited. We propose a Process Mining approach that utilises timestamped data from Electronic Health Records to compare care provider patterns across pandemic waves.  Methods: We collected routine events from Scottish hospital episodes in adults with COVID-19 status and linked health provider inputs to generate standardised treatment logs. Conformance checking metrics were used to select the optimal model (Inductive Miner infrequent [IMi]) for downstream analysis. Visual diagrams from the discovered Petri Nets indicated the interactions on pre-coded provider and activity-level subsets. We used cross-log conformance checking and graph similarity to measure distances between adverse and less adverse groups across pandemic waves.  Results: We included 1,153 patients with COVID-19 (302 [26%] in Wave 1 and 851 [74%] in Wave 2) with 55,212 relevant care provider events. At the conformance checking stage, the IMi model, achieved good log fitness (LF=0.95) and generalisation (G=0.89), but limited precision (PR=0.27) across all granularity levels. More structured care procedures in Wave 1 were present, compared to mixed multidisciplinary patterns in Wave 2. Care activities differed in patients with extended stay (GED=348, PR=0.231 vs GED=197, PR=0.429 in shorter stays). Patients in out-of-hours care and intensive therapy were linked with more standardised patterns.  Conclusion: Process Mining can be incorporated alongside clinical oversight to provide visual and quantitative comparisons of care interactions in COVID-19 episodes and enhance further research in complex cases.

Functional and Morphological Disorders of Taste and Olfaction in COVID-19-Patients (preprint)

Objectives: To test the prevalence and evolution of acute olfactory and gustatory functional impairment and of their morphologic correlates in COVID-19 patients who require hospitalization due to COVID-19-related respiratory conditions. Key-words: COVID-19, taste, olfaction, electrogustometry, contact endoscopy Design: Electrogustometric (EGM) - thresholds at the tongue area supplied by the chorda tympani, at the soft palate and at the vallate papillae area were recorded bilaterally. Olfaction was examined by Sniffin’ sticks. The patients’ nasal and oral mucosa (fungiform papillae, fpap) were examined by contact endoscopy. Setting: Tertiary referral medical centre. Patients: 53 consecutive hospitalized patients (23 males, 30 females, age 42,54 ± 10, 95 yrs) with RT-PCR-confirmed COVID-19 diagnosis were included. Patients have been examined twice: just after hospital discharge and 4-6 weeks later. Main outcome measures: EGM-thresholds and taste strips, Schniffin-Sticks, Contact-Endoscopyesults: EGM-thresholds in patients were significantly higher at both instances than those of healthy subjects. EGM-thresholds at the second measurement were significantly lower than those at the first measurement. Accordingly, patient-reported gustatory outcomes were improved at the second measurement. The same pattern has been found using Sniffin’ sticks. Significant alterations in form and vascularization of fPap have been detected in patients, especially at the first instance. Conclusions: COVID-19 affects both gustatory and olfactory functions. It also affects in parallel the structure and vascularization of both nasal and oral mucosa, although the nasal mucosa to a much less, non-significant, extent. Our findings suggest that COVID-19 may cause a mild to profound neuropathy of multiple cranial nerves.

Neuropathological assessment of the olfactory bulb and tract in individuals with COVID-19 (preprint)

The majority of patients with Parkinson disease (PD) experience a loss in their sense of smell and accumulate insoluble alpha-synuclein aggregates in their olfactory bulbs (OB). Subjects affected by a SARS-CoV-2-linked illness (COVID-19) frequently experience hyposmia. We previously hypothesized that alpha-synuclein and tau misprocessing could occur following host responses to microbial triggers. Using semiquantitative measurements of immunohistochemical signals, we examined OB and olfactory tract specimens collected serially at autopsies between 2020 and 2023. Deceased subjects comprised 50 adults, which included COVID19+ patients (n=22), individuals with Lewy body disease (e.g., PD and dementia with Lewy bodies (DLB; n=6)), Alzheimer disease (AD; n=3), other non-synucleinopathy-linked degenerative diseases (e.g., progressive supranuclear palsy (PSP; n=2) and multisystem atrophy (MSA; n=1)). Further, we included neurologically healthy controls (HCO; n=9) and those with an inflammation-rich brain disorder as neurological controls (NCO; n=7). When probing for inflammatory changes focusing on anterior olfactory nuclei (AON) using anti-CD68 immunostaining, scores were consistently elevated in NCO and AD cases. In contrast, inflammation on average was not significantly altered in COVID19+ patients relative to controls, although anti-CD68 reactivity in their OB and tracts declined with progression in age. Mild-to-moderate increases in phospho-alpha-Syn and phospho-tau signals were detected in the AON of tauopathy- and synucleinopathy-afflicted brains, respectively, consistent with mixed pathology, as described by others. Lastly, when both sides were available for comparison in our case series, we saw no asymmetry in the degree of pathology of the left versus right OB and tracts. We concluded from our autopsy series that after a fatal course of COVID-19, microscopic changes, when present, in the rostral, intracranial portion of the olfactory circuitry generally reflected neurodegenerative processes seen elsewhere in the brain. In general, inflammation correlated best with the degree of Alzheimer's-linked tauopathy and declined with progression of age in COVID19+ patients.

GotGlycans: Role of N343 Glycosylation on the SARS-CoV-2 S RBD Structure and Co-Receptor Binding Across Variants of Concern (preprint)

Glycosylation of the SARS-CoV-2 spike (S) protein represents a key target for viral evolution because it affects both viral evasion and fitness. Successful variations in the glycan shield are difficult to achieve though, as protein glycosylation is also critical to folding and to structural stability. Within this framework, the identification of glycosylation sites that are structurally dispensable can provide insight into the evolutionary mechanisms of the shield and inform immune surveillance. In this work we show through over 45 s of cumulative sampling from conventional and enhanced molecular dynamics (MD) simulations, how the structure of the immunodominant S receptor binding domain (RBD) is regulated by N-glycosylation at N343 and how the structural role of this glycan changes from WHu-1, alpha (B.1.1.7), and beta (B.1.351), to the delta (B.1.617.2) and omicron (BA.1 and BA.2.86) variants. More specifically, we find that the amphipathic nature of the N-glycan is instrumental to preserve the structural integrity of the RBD hydrophobic core and that loss of glycosylation at N343 triggers a specific and consistent conformational change. We show how this change allosterically regulates the conformation of the receptor binding motif (RBM) in the WHu-1, alpha and beta RBDs, but not in the delta and omicron variants, due to mutations that reinforce the RBD architecture. In support of these findings, we show that the binding of the RBD to monosialylated ganglioside co-receptors is highly dependent on N343 glycosylation in the WHu-1, but not in the delta RBD, and that affinity changes significantly across VoCs. Ultimately, the molecular and functional insight we provide in this work reinforces our understanding of the role of glycosylation in protein structure and function and it also allows us to identify the structural constraints within which the glycosylation site at N343 can become a hotspot for mutations in the SARS-CoV-2 S glycan shield.

Fitness models provide accurate short-term forecasts of SARS-CoV-2 variant frequency (preprint)

Genomic surveillance of pathogen evolution is essential for public health response, treatment strategies, and vaccine development. In the context of SARS-COV-2, multiple models have been developed including Multinomial Logistic Regression (MLR) describing variant frequency growth as well as Fixed Growth Advantage (FGA), Growth Advantage Random Walk (GARW) and Piantham parameterizations describing variant Rt. These models provide estimates of variant fitness and can be used to forecast changes in variant frequency. We introduce a framework for evaluating real-time forecasts of variant frequencies, and apply this framework to the evolution of SARS-CoV-2 during 2022 in which multiple new viral variants emerged and rapidly spread through the population. We compare models across representative countries with different intensities of genomic surveillance. Retrospective assessment of model accuracy highlights that most models of variant frequency perform well and are able to produce reasonable forecasts. We find that the simple MLR model provides ~0.6% median absolute error and ~6% mean absolute error when forecasting 30 days out for countries with robust genomic surveillance. We investigate impacts of sequence quantity and quality across countries on forecast accuracy and conduct systematic downsampling to identify that 1000 sequences per week is fully sufficient for accurate short-term forecasts. We conclude that fitness models represent a useful prognostic tool for short-term evolutionary forecasting.

Safety of Monovalent BNT162b2 (Pfizer-BioNTech), mRNA-1273 (Moderna), and NVX-CoV2373 (Novavax) COVID-19 Vaccines in US Children Aged 6 months to 17 years (preprint)

Importance Active monitoring of health outcomes after COVID-19 vaccination provides early detection of rare outcomes that may not be identified in prelicensure trials. Objective To conduct near-real-time monitoring of health outcomes following COVID-19 vaccination in the United States (US) pediatric population aged 6 months to 17 years. Design We evaluated 21 pre-specified health outcomes; 15 were sequentially tested through near-real-time surveillance, and 6 were monitored descriptively within a cohort of vaccinated children. We tested for increased rate of each outcome following vaccination compared to a historical comparator cohort. Setting This population-based study was conducted under the US Food and Drug Administration public health surveillance mandate using three commercial claims databases. Participants Children aged 6 months to 17 years were included if they received a monovalent COVID-19 vaccine dose before early 2023 and had continuous enrollment in a medical health insurance plan from the start of an outcome-specific clean window to the COVID-19 vaccination dose. Exposure Exposure was defined as receipt of a monovalent BNT162b2, mRNA-1273, or NVX-CoV2373 COVID-19 vaccine dose. The primary analysis evaluated dose 1 and dose 2 combined, and secondary analyses evaluated each dose separately. Follow-up time was censored at death, disenrollment, end of risk window, end of study period, or a subsequent dose administration. Main Outcomes Twenty-one prespecified health outcomes. Results The study included 4,102,016 enrollees aged 6 months to17 years. Thirteen of 15 outcomes sequentially tested did not meet the threshold for a statistical signal. In the primary analysis, myocarditis or pericarditis signals were detected following BNT162b2 vaccine in children aged 12-17 years old and seizures/convulsions signals were detected following vaccination with BNT162b2 and mRNA-1273 in children aged 2-4/5 years. However, in a post-hoc sensitivity analysis, the seizures/convulsions signal was sensitive to background rates selection and was not observed when 2022 background rates were selected instead of 2020 rates. Conclusions and Relevance Of the two signaled outcomes, the myocarditis or pericarditis signals are consistent with previously published reports. The new signal detected for seizures/convulsions among younger children should be further investigated in a robust epidemiological study with better confounding adjustment.

Transmissibility, infectivity, and immune resistance of the SARS-CoV-2 BA.2.86 variant (preprint)

In September 2023, the SARS-CoV-2 XBB descendants, such as XBB.1.5 and EG.5.1 (originally XBB.1.9.2.5.1), are predominantly circulating worldwide. Unexpectedly, however, a lineage distinct from XBB was identified and named BA.2.86 on August 14, 2023. Notably, BA.2.86 bears more than 30 mutations in the spike (S) protein when compared to XBB and the parental BA.2, and many of them are assumed to be associated with immune evasion. Although the number of reported cases is low (68 sequences have been reported as of 7 September 2023), BA.2.86 has been detected in several continents (Europe, North America and Africa), suggesting that this variant may be spreading silently worldwide. On 17 August 2023, the WHO designated BA.2.86 as a variant under monitoring. Here we show evidence suggesting that BA.2.86 potentially has greater fitness than current circulating XBB variants including EG.5.1. The pseudovirus assay showed that the infectivity of BA.2.86 was significantly lower than that of B.1.1 and EG.5.1, suggesting that the increased fitness of BA.2.86 is not due to the increased infectivity. We then performed a neutralization assay using XBB breakthrough infection sera to address whether BA.2.86 evades the antiviral effect of the humoral immunity induced XBB subvariants. The 50% neutralization titer of XBB BTI sera against BA.2.86 was significantly (1.4-fold) lower than those against EG.5.1. The sera obtained from individuals vaccinated with 3rd-dose monovalent, 4th-dose monovalent, 4th-dose BA.1 bivalent, and 4th-dose BA.5 bivalent mRNA vaccines exhibited very little or no antiviral effects against BA.2.86. Moreover, the three monoclonal antibodies (Bebtelovimab, Sotrovimab and Tixagevimab), which worked against the parental BA.2, did not exhibit antiviral effects against BA.2.86. These results suggest that BA.2.86 is one of the most highly immune evasive variants ever.

Subphenotypes of Self-Reported Symptoms and Outcomes in Long COVID: a prospective cohort study with latent class analysis. (preprint)

Objective: To characterize subphenotypes of self-reported symptoms and outcomes(SRSOs) in Post-acute sequelae of COVID-19(PASC). Design: Prospective, observational cohort study of PASC subjects. Setting: Academic tertiary center from five clinical referral sources. Participants: Adults with COVID-19 [≥] 20 days before enrollment and presence of any new self-reported symptoms following COVID-19. Exposures: We collected data on clinical variables and SRSOs via structured telephone interviews and performed standardized assessments with validated clinical numerical scales to capture psychological symptoms, neurocognitive functioning, and cardiopulmonary function. We collected saliva and stool samples for quantification of SARS-CoV-2 RNA via qPCR. Primary and Secondary outcomes of measure: Description of PASC SRSOs burden and duration, derivation of distinct PASC subphenotypes via latent class analysis (LCA), and relationship between viral load with SRSOs and PASC subphenotypes. Results: Baseline data for 214 individuals were analyzed. The study visit took place at a median of 197.5 days after COVID-19 diagnosis, and participants reported ever having a median of 9/16 symptoms (interquartile range 6-11) after acute COVID, with muscle-aches, dyspnea, and headache being the most common. Fatigue, cognitive impairment, and dyspnea were experienced for a longer time. Participants had a lower burden of active symptoms (median 3, interquartile range 1-6) than those ever experienced (p<0.001). Unsupervised LCA of symptoms revealed three clinically-active PASC subphenotypes: a high burden constitutional symptoms (21.9%) , a persistent loss/change of smell and taste (20.6%) , and a minimal residual symptoms subphenotype (57.5%). Subphenotype assignments were strongly associated with self-assessments of global health, recovery and PASC impact on employment (p<0.001). Viral persistence (5.6% saliva and 1% stool samples positive) did not explain SRSOs or subphenotypes. Conclusions: We identified distinct PASC subphenotypes and highlight that although most symptoms progressively resolve, specific PASC subpopulations are impacted by either high burden of constitutional symptoms or persistent olfactory/gustatory dysfunction, requiring prospective identification and targeted preventive or therapeutic interventions.

Competitive fitness and homologous recombination of SARS-CoV-2 variants of concern (preprint)

SARS-CoV-2 variants continue to emerge and cocirculate in humans and wild animals. The factors driving the emergence and replacement of novel variants and recombinants remain incompletely understood. Herein, we comprehensively characterized the competitive fitness of SARS-CoV-2 wild type (WT) and three variants of concern (VOCs), Alpha, Beta and Delta, by coinfection and serial passaging assays in different susceptible cells. Deep sequencing analyses revealed cell-specific competitive fitness: the Beta variant showed enhanced replication fitness during serial passage in Caco-2 cells, whereas the WT and Alpha variant showed elevated fitness in Vero E6 cells. Interestingly, a high level of neutralizing antibody sped up competition and completely reshaped the fitness advantages of different variants. More importantly, single clone purification identified a significant proportion of homologous recombinants that emerged during the passage history, and immune pressure reduced the frequency of recombination. Interestingly, a recombination hot region located between nucleotide sites 22995 and 28866 of the viral genomes could be identified in most of the detected recombinants. Our study not only profiled the variable competitive fitness of SARS-CoV-2 under different conditions, but also provided direct experimental evidence of homologous recombination between SARS-CoV-2 viruses, as well as a model for investigating SARS-CoV-2 recombination.

Phylogeny and evolution of the SARS-CoV-2 spike gene from December 2022 to February 2023 (preprint)

Background: By the end of 2022, new variants of SARS-CoV-2, such as BQ.1.1.10, BA.4.6.3, XBB, and CH.1.1, emerged with higher fitness than BA.5. Methods: The file (spikeprot0304), which contains spike protein sequences, isolates collected before March, 4, 2023, was downloaded from Global Initiative on Sharing All Influenza Data (GISAID). A total of 188 different spike protein sequences were chosen, of which their isolates were collected from December 2022 to February 2023. These sequences did not contain undetermined amino acid X, and each spike protein sequence had at least 100 identical isolate sequences in GISAID. Phylogenetic trees were reconstructed using IQ-TREE and MrBayes softwares. A median-join network was reconstructed using PopART software. Selection analyses were conducted using site model of PAML software. Results: The phylogenetic tree of the spike DNA sequences revealed that the majority of variants belonged to three major lineages: BA.2 (BA.1.1.529.2), BA.5 (BA.1.1.529.5), and XBB. The median network showed that these lineages had at least six major diversifying centers. The spike DNA sequences of these diversifying centers had the representative accession IDs (EPI_ISL_) of 16040256 (BN.1.2), 15970311 (BA.5), 16028739 (BA.5.11), 16028774 (BQ.1), 16027638 (BQ.1.1.23), and 16044705 (XBB.1.5). Selection analyses revealed 26 amino-acid sites under positive selection. These sites included L5, V83, W152, G181, N185, V213, H245, Y248, D253, S255, S256, G257, R346, R408, K444, V445, G446, N450, L452, N460, F486, Q613, Q675, T883, P1162, and V1264. Conclusion: The spike proteins of SARS-CoV-2 from December 2022 to February 2023 were characterized by a swarm of variants that were evolved from three major lineages: BA.2 (BA.1.1.529.2), BA.5 (BA.1.1.529.5), and XBB. These lineages had at least six diversifying centers. Selection analysis identified 26 amino acid sites were under positive selection. Continued surveillance and research are necessary to monitor the evolution and potential impact of these variants on public health.

Epidemiology of Covid-19 (preprint)

We provide a summary of various epidemiological parameters related to COVID-19 such as incubation period, serial interval and other parameters. Understanding these parameters is important for developing prevention strategies. SARS-CoV-2 can be transmitted by droplets and close contact, but there is evidence of airborne transmission. Aerosol-generating procedures have been identified as one of the specific risk factors for healthcare workers. Super-spreading events refer to situations where a small number of individuals cause the majority of infections. The basic reproductive number (R0) and the spread parameter (k) are used to characterise the transmissibility of the disease. Estimated values for R0 range from 2 to 3 and the estimated value for k is 0.1.The duration of infectiousness depends on viral load and shedding. Viral load varies according to factors such as clinical spectrum, type of variant and vaccination status. The relationship between viral load and infectivity is not fully understood.With regard to the frequency of symptoms and signs of COVID-19, fever, cough, fatigue and dyspnoea are common. The prevalence of olfactory and gustatory dysfunction (OGD) varies between studies and countries. Age and comorbidities are factors associated with olfactory dysfunction.Estimates of the proportion of asymptomatic patients range from 6% to 96%. Asymptomatic transmission is considered likely and is important for control measures.We reviewed the quantitative semiology of COVID-19 is reported on sensitivity, specificity and likelihood ratios of signs.Finally, we also review risk factors for COVID-19 (including health care workers), co-infections, and epidemiology of variants..