SARS-CoV-2 mutations acquired during serial passage in human cell lines are consistent with several of those found in recent natural SARS-CoV-2 variants.

Since the outbreak of coronavirus disease (COVID-19) in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into diverse variants. Here, an early isolate of SARS-CoV-2 was serially passaged in multiple cell lines of human origin in triplicate, and selected mutations were compared to those found in natural SARS-CoV-2 variants. In the spike protein, Q493R and Q498R substitutions from passaged viruses were consistent with those in the B.1.1.529 (Omicron) variant. Y144del and H655Y substitutions from passaged viruses were also reported in B.1.1.7 (Alpha), P.1 (Gamma), and B.1.1.529 (Omicron) variants. Several single nucleotide polymorphisms (SNPs) found in first-passaged viruses have also been identified as selected mutation sites in serially passaged viruses. Considering the consistent mutations found between serially passaged SARS-CoV-2 and natural variants, there may be host-specific selective mutation patterns of viral evolution in humans. Additional studies on the selective mutations in SARS-CoV-2 experiencing diverse host environments will help elucidate the direction of SARS-CoV-2 evolution. Importance: SARS-CoV-2 isolate (SARS-CoV-2/human/KOR/KCDC03-NCCP43326/2020) was serially passaged in A549, CaCO2, and HRT-18 cells in triplicate. After 12 times of serial passages in each cell lines, several consistent selected mutations were found on spike protein between the serially passaged SARS-CoV-2 in human cell lines and recent natural variants of SARS-CoV-2 like omicron. On the non-spike protein genes, selected mutations were more frequent in viruses passaged in Caco-2 and HRT-18 cells (Colon epithelial-like) than in those passaged in A549 cells (Lung epithelial-like). In addition, several SNPs identified after one round of passaging were consistently identified as the selected mutation sites in serially passaged viruses. Thus, mutation patterns of SARS-CoV-2 in certain host environments may provide researchers information to understand and predict future SARS-CoV-2 variants.

Osteogenic Circulating Endothelial Progenitor Cells are Associated with Vascular Aging of the Large Arteries in Rheumatoid Arthritis.

Background and Aim: Rheumatoid arthritis is associated with both abnormal bone metabolism and accelerated vascular aging but a mechanistic link was lacking. This study aims to investigate the role of osteocalcin (OCN)-expressing circulating endothelial progenitor cells (EPCs) in vascular aging, as determined by arterial calcifications in rheumatoid arthritis. Methods: We performed flow cytometry studies in 145 consecutive patients with rheumatoid arthritis to determine osteogenic circulating levels of OCN-positive (OCN+) CD34+KDR+ and OCN+CD34+ versus conventional early EPC CD34+CD133+KDR+. Total calcium load of the thoracic aorta (ascending plus descending) and the carotid arteries were assessed by non-contrast computed tomography (CT) and contrast CT angiography. Results: Osteogenic EPCs OCN+CD34+KDR+ (P = 0.002) and OCN+CD34+ (P = 0.001), together with clinical parameters of age, history of hypertension, systolic blood pressure, serum levels of triglycerides, HbA1c and creatinine, use of leflunomide and brachial-ankle pulse-wave velocity (all P < 0.05), were associated with the clustered presence of aortic and carotid calcification. Multivariable analyses revealed that circulating OCN+CD34+KDR+ (B = 14.4 [95% CI 4.0 to 24.8], P = 0.007) and OCN+CD34+ (B = 9.6 [95% CI 4.9 to 14.3], P < 0.001) remained independently associated with increased aortic calcium load. OCN+CD34+ EPC (B = 0.8 [95% CI 0.1 to 1.5], P = 0.023), but not OCN+CD34+KDR+ EPC (B = 1.2 [95% CI -0.2 to 2.6], P = 0.09), was further independently associated with carotid calcium load. In comparison, conventional early EPC CD34+CD133+KDR+ had no significant association with aortic or carotid calcium load (P = 0.46 and 0.88, respectively). Conclusion: Circulating level of osteogenic EPC is associated with increased vascular aging in terms of calcification of the large arteries in patients with rheumatoid arthritis. The findings may suggest a role of the bone-vascular axis underlying vascular aging in rheumatic diseases. Further research is needed to characterize the mechanistic links and basis of these observations.

Nurse-Led Consultation for Patients With Rheumatoid Arthritis at Low Disease Activity: A Randomized Noninferiority Trial.

OBJECTIVE: To determine the effectiveness of nurse-led consultations in patients with stable rheumatoid arthritis (RA) in Hong Kong. METHODS: The present work was a single-center, randomized, open-label, noninferiority trial. Patients who had rheumatoid arthritis (RA) with low disease activity (LDA) were randomized at a 1:1 ratio to attend a nurse-led consultation or rheumatologist follow-up visit for 2 years. The primary end point was the proportion of patients whose RA remained at LDA. Secondary end points included the proportion of patients with RA in disease remission and the scores recorded on the Leeds Satisfaction Questionnaire at 2 years, changes from baseline on the Disease Activity Score in 28 joints using the C-reactive protein level (DAS28-CRP), modified Sharp/van der Heijde score (SHS), Health Assessment Questionnaire disability index (HAQ DI), Short Form 36 (SF-36) physical component score, and 19-item Compliance Questionnaire for Rheumatology (CQR-19) score. RESULTS: Among 280 patients who were randomized equally to either attend nurse-led consultations or rheumatologist follow-up visits, 267 patients completed the study. In the nurse-led consultation and rheumatologist follow-up groups, 92.1% and 91.4% patients, respectively, remained at LDA at 2 years. The 95% confidence intervals (95% CIs) of the adjusted treatment difference were within the predefined noninferiority margin in both the intention-to-treat analysis (95% CI 5.75, 7.15) and the per-protocol analysis (95% CI 1.67, 7.47). Although the changes in DAS28-CRP score over 2 years were significantly different between the 2 treatment groups (P < 0.001), there were no significant changes from baseline in SHS, HAQ DI, SF-36 physical component scores, and CQR-19 scores. At the end of the study, more patients expressed satisfaction with nurse-led consultations. CONCLUSION: Nurse-led consultations were not inferior to rheumatologist follow-up visits in patients with stable RA.

Host- and Species-Dependent Quasispecies Divergence of Severe Acute Respiratory Syndrome Coronavirus-2 in Non-human Primate Models.

Recently, newly emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been continuously reported worldwide. However, the precise evaluation of SARS-CoV-2 microevolution in host is very limited because the exact genetic information of infected virus could not be acquired in human researches. In this report, we performed deep sequencing for seed virus and SARS-CoV-2 isolated in eight cynomolgus and rhesus macaques at 3 days postinoculation and evaluated single-nucleotide polymorphisms (SNPs) in SARS-CoV-2 by variant analysis. A total of 69 single-nucleotide variants (SNVs) were present in the 5'-untranslated region (UTR), 3'-UTR, ORF1ab, S, ORF3a, ORF8, and N genes of the seed virus passaged in VERO cells. Between those present on the seed virus and those on each SARS-CoV-2 isolated from the lungs of the macaques, a total of 29 variants was identified in 4 coding proteins (ORF1ab, S, ORF3a, and N) and non-coding regions (5'- and 3'-UTR). Variant number was significantly different according to individuals and ranged from 2 to 11. Moreover, the average major frequency variation was identified in six sites between the cynomolgus monkeys and rhesus macaques. As with diverse SNPs in SARS-CoV-2, the values of viral titers in lungs were significantly different according to individuals and species. Our study first revealed that the genomes of SARS-CoV-2 differ according to individuals and species despite infection of the identical virus in non-human primates (NHPs). These results are important for the interpretation of longitudinal studies evaluating the evolution of the SARS-CoV-2 in human beings and development of new diagnostics, vaccine, and therapeutics targeting SARS-CoV-2.

Molecular evolution of dengue virus types 1 and 4 in Korean travelers.

Dengue virus (DV) is a mosquito-borne virus that is endemic to many tropical and subtropical areas. Recently, the annual incidence of DV infection has increased worldwide, including in Korea, due to global warming and increased global travel. We therefore sought to characterize the molecular and evolutionary features of DV-1 and DV-4 isolated from Korean overseas travelers. We used phylogenetic analysis based on the full coding region to classify isolates of DV-1 in Korea into genotype I (43251, KP406802), genotype IV (KP406803), and genotype V (KP406801). In addition, we found that strains of DV-4 belonged to genotype I (KP406806) and genotype II (43257). Evidence of positive selection in DV-1 strains was identified in the C, prM, NS2A, and NS5 proteins, whereas DV-4 showed positive selection only in the non-structural proteins NS2A, NS3, and NS5. The substitution rates per site per year were 5.58 × 10-4 and 6.72 × 10-4 for DV-1 and DV-4, respectively, and the time of the most recent common ancestor was determined using the Bayesian skyline coalescent method. In this study, the molecular, phylogenetic, and evolutionary characteristics of Korean DV-1 and DV-4 isolates were evaluated for the first time.

Cardiovascular events prediction by left ventricular longitudinal strain and serum high-sensitivity troponin I in patients with axial spondyloarthritis.

OBJECTIVE: Patients with axial spondyloarthritis (SpA) are subjected to elevated cardiovascular risks, but assessment of early myocardial damage and clinical risk stratification remained obscure. The aim of this study was to evaluate the prognostic value of speckle-tracking strain analysis and serum high-sensitivity troponin I (hsTnI) in patients with axial SpA. METHODS: Two-dimensional speckle-tracking echocardiography was performed to derive longitudinal strain (LS), circumferential strain (CS), and radial strain (RS). Serum hsTnI was measured by validated immunoassay (Architect i1000SR Abbott) as indicator of subclinical myocardial damage. RESULTS: The mean Bath Ankylosing Spondylitis Disease Activity Index and median modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) were 3.8 and 15.5, respectively. Over a median follow-up of 81 months, major adverse cardiovascular events (MACE) occurred in 13% of subjects (n = 116). Univariate Cox regression showed that age, disease duration, Bath Ankylosing Spondylitis Functional Index, modified Schober test, mSASSS, hsTnI, interventricular septal thickness, E/E', LS, RS, and carotid intima-media thickness were significant predictors of MACE (all P < 0.05). After adjustment for age, sex, and statistically significant disease-related parameters, only subclinically raised hsTnI and impaired LS remained independent predictors for MACE. Kaplan-Meier analysis showed that combined impaired LS ≥ - 17.5% and hsTnI ≥ 3.0 pg/ml significantly predicted MACE (log-rank test P < 0.01; sensitivity 50%; specificity 90%; positive predictive value 43%; negative predictive value 92%). CONCLUSIONS: Depressed LS indicating subclinical left ventricular systolic dysfunction and elevated serum hsTnI both independently predicted MACE among young patients with axial SpA. Combined analysis of speckle-tracking-derived strain analysis and serum hsTnI improves risk stratification in these patients. Key Points • Both depressed longitudinal strain (LS) and elevated serum high-sensitivity troponin I (hsTnI) are promising independent predictors for cardiovascular (CV) events in axial SpA. • Importantly, patients with LS ≥ - 17.5% and hsTnI ≥ 3.0 pg/ml had the highest risk of incident MACE. • Axial SpA patients with concomitant impaired LS and raised hsTnI are at a high risk of CV events.

Cumulative Rheumatic Inflammation Modulates the Bone-Vascular Axis and Risk of Coronary Calcification.

Background Rheumatic diseases are related to both abnormal bone turnover and atherogenesis, but a mechanistic link was missing. Methods and Results We investigated the effect of cumulative rheumatic inflammation ( CRI ) on risk of coronary calcification in a retrospective cohort of 145 rheumatoid arthritis patients. A time-adjusted aggregate CRI score was derived by conglomerating all quarterly biomarker encounters of serum C-reactive protein over 60 months immediately preceding computed tomography coronary angiography. Flow cytometry was performed to measure the osteocalcin-positive ( OCN +) CD 34+ KDR + and OCN + CD 34+ circulating endothelial progenitor cells ( EPCs ). Conventional early circulating EPCs CD 34+ CD 133+ KDR + was determined. Coronary calcification was defined as any Agatston score >0. 50% of patients (n=72/145) had coronary calcification. CRI score was associated with presence of coronary calcification ( P=0.004) (multivariable-adjusted: highest versus lowest quartile: odds ratio=5.6 [95% CI 1.1-28.9], P=0.041). Receiver operating characteristics curve revealed divergent behavior of OCN -expressing circulating EPCs ( OCN + CD 34+ EPCs : area under the curve=0.60, P=0.034; OCN + CD 34+ KDR + EPCs : area under the curve=0.59, P=0.053, positive predictors) versus conventional early EPCs ( CD 34+ CD 133+ KDR +: area under the curve=0.60, P=0.034, negative predictor) for coronary calcification, which persisted after multivariable adjustments ( OCN + CD 34+ KDR + [>75th percentile]: odds ratio=7.2 [95% CI 1.8-27.9], P=0.005; OCN + CD 34+ EPCs [>75th percentile]: odds ratio=6.0 [95% CI 1.5-23.3], P=0.010; CD 34+ CD 133+ KDR + [>75th percentile: odds ratio=0.3 [95% CI 0.1-1.0], P=0.053). Intriguingly, the CRI score was associated with increased OCN + CD 34+ EPCs (highest versus lowest quartile: B=+25.6 [95% CI 0.8-50.5] [×103/mL peripheral blood], P=0.043), but reduced CD 34+ CD 133+ KDR + EPCs (highest versus lowest quartile: B=-16.2 [95% CI -31.5 to -0.9], P=0.038). Conclusions Preceding 60 months of CRI is associated with increased risk of coronary calcification and altered OCN expression in circulating EPCs .

Osteogenic circulating endothelial progenitor cells are linked to electrocardiographic conduction abnormalities in rheumatic patients.

BACKGROUND: Osteogenic circulating endothelial progenitor cells (EPC) play a pathogenic role in cardiovascular system degeneration through promulgating vasculature calcification, but its role in conduction disorders as part of the cardiovascular degenerative continuum remained unknown. AIM: To investigate the role of osteocalcin (OCN)-expressing circulating EPCs in cardiac conduction disorders in the unique clinical sample of rheumatoid arthritis (RA) susceptible to both abnormal bone metabolism and cardiac conduction disorders. METHODS: We performed flow cytometry studies in 134 consecutive asymptomatic patients with rheumatoid arthritis to derive osteogenic circulating OCN-positive (OCN+) CD34+KDR+ vs. CD34+CD133+KDR+ conventional EPC. Study endpoint was the prespecified combined endpoint of electrocardiographic conduction abnormalities. RESULTS: Total prevalence of cardiac conduction abnormality was 9% (n = 12). All patients except one had normal sinus rhythm. One patient had atrial fibrillation. No patient had advanced atrioventricular (AV) block. Prevalence of first-degree heart block (>200 ms), widened QRS duration (>120 ms) and right bundle branch block were 6.7%, 2.1%, and 2.2% respectively. Circulating osteogenic OCN+ CD34+ KDR+ EPCs were significantly higher among patients with cardiac conduction abnormalities (p = 0.039). Elevated OCN+ CD34+ KDR+ EPCs> 75th percentile was associated with higher prevalence of cardiac conduction abnormalities (58.3% vs. 20.02%, p = 0.003). Adjusted for potential confounders, elevated OCN+ CD34+ KDR+ EPCs> 75th percentile remained independently associated with increased risk of cardiac conduction abnormalities (OR = 4.4 [95%CI 1.2-16.4], p = 0.028). No significant relation was found between conventional EPCs CD34+CD133+KDR+ and conduction abnormalities (p = 0.36). CONCLUSIONS: Elevated osteogenic OCN+ CD34+ KDR+ EPCs are independently associated with the presence of electrocardiographic conduction abnormalities in patients with rheumatoid arthritis, unveiling a potential novel pathophysiological mechanism.

Ten-year progression of coronary artery, carotid artery, and aortic calcification in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with increased vascular calcification, although the rate of progress of calcification is uncertain. The aim of the study was to evaluate the progression of and the predictors for calcification in different vascular beds over 10 years. The 10-year actual coronary calcium score (CS) and 10-year predicted coronary CS, based on the pattern of the general population, were compared. Calcification of the coronary and carotid artery and the aorta was assessed by multi-detector computed tomography. Significant CS progression was determined by the difference between the square root of baseline and square root of follow-up calcium score (i.e., SQRT method). The 10-year predicted coronary CS was based on the mathematical formula derived by the Heinz Nixdorf Recall Study. A total of 49 patients (54 ± 11 years, 90% female) had a follow-up scan after 10.0 ± 0.2 years. The CS in all vascular beds was significantly increased; 55% of the patients had a significant progression of CS in the coronary, 29% in the carotid, and 80% in the aorta. Age and systolic blood pressure (SBP) were independently associated with calcification progression in all vascular beds. Importantly, the absolute increase in 10-year actual coronary CS was significantly higher than that predicted. In patients with RA, calcification in all vascular beds significantly increased over 10 years and was independently associated with age and SBP. Importantly, the absolute increase in 10-year actual coronary CS progression was significantly higher than that predicted.

Left ventricular myocardial dysfunction and premature atherosclerosis in patients with axial spondyloarthritis.

OBJECTIVE: To evaluate left ventricular (LV) function and carotid intima-media thickness (IMT) in patients with axial SpA in relationship to underlying disease severity. METHODS: We recruited 104 patients with axial SpA and 50 controls. Detailed transthoracic echocardiography was performed and analysed with two-dimensional speckle tracking strain analysis for systolic function and tissue Doppler-derived E/E' for diastolic function assessment. Carotid IMT was measured by ultrasonography to evaluate subclinical atherosclerosis. Radiological severity of patients with axial SpA was assessed by the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS). RESULTS: Despite a similar LV ejection fraction [62.7% (s.d. 3.9) vs 62.8% (s.d. 3.8), P = 0.83], patients with axial SpA had impaired LV myocardial longitudinal strain (LS), circumferential strain (CS) and radial strain (RS) compared with controls [-18.1% (s.d. 2.4) vs -20.1% (s.d. 2.5), -17.2% (s.d. 2.2) vs -20.3% (s.d. 2.9) and 37.1% (s.d. 8.6) vs 43.2% (s.d. 10.9), respectively; all P < 0.01]. In addition, patients with axial SpA had a greater E/E' [7.9 (s.d. 2.5) vs 7.0 (s.d. 1.7), P < 0.01] and carotid IMT [0.78 mm (s.d. 0.19) vs 0.69 mm (s.d. 0.10), P < 0.01] than controls. After adjusting for potential confounding factors, axial SpA remained independently associated with LV myocardial strains, E/E' and carotid IMT. Importantly, multivariate linear regression analysis demonstrated that mSASSS was independently associated with LV longitudinal strain, E/E' and carotid IMT. CONCLUSION: Our study demonstrated that patients with axial SpA had impaired LV systolic and diastolic function and increased carotid IMT. Importantly, mSASSS was independently associated with LV longitudinal strain, E/E' and carotid IMT after adjusting for confounding factors. Speckle tracking echocardiography may be a useful tool for early detection of impaired LV function in patients with SpA and carotid IMT examination can provide valuable assessment of subclinical atherosclerosis in patients with SpA.