Molecular evolutionary characteristics of severe acute respiratory syndrome coronavirus 2 and the relatedness of epidemiological and socio-environmental factors.

After the first outbreak, SARS-CoV-2 infection continues to occur due to the emergence of new variants. There is limited information available on the comparative evaluation of evolutionary characteristics of SARS-CoV-2 among different countries over time, and its relatedness to epidemiological and socio-environmental factors within those countries. We assessed comparative Bayesian evolutionary characteristics for SARS-CoV-2 in eight countries from 2020 to 2022 using BEAST version 2.6.7. Additionally, the relatedness between virus evolution factors and both epidemiological and socio-environmental factors was analyzed using Pearson's correlation coefficient. The estimated substitution rates in the gene encoding S protein of SARS-CoV-2 exhibited a continuous increase from 2020 to 2022 and were divided into two distinct groups in 2022 (p value < 0.05). Effective population size (Ne) generally showed decreased patterns by time. Notably, the change rates of the substitution rates were negatively correlated with the cumulative vaccination rates in 2021. A strict and rapid vaccination policy in the United Arab Emirates dramatically reduced the evolution of the virus, compared to other countries. Also, the average yearly temperature in countries were negatively correlated with the substitution rates. The changes of six epitopes in SARS-CoV-2 were related to various socio-environmental factors. We figured out comparative virus evolutionary traits and the association of epidemiological and socio-environmental factors especially cumulative vaccination rates and average temperature.

Clostridium ventriculi in a cynomolgus monkey with acute gastric dilatation and rupture: A case report.

Acute gastric dilatation (AGD) is one of the most prevalent and life-threatening diseases in nonhuman primates worldwide. However, the etiology of this syndrome has not been determined. Recently, sudden death occurred in a 7-year-old female cynomolgus monkey with a history of fecal microbiota transplantation using diarrheic stools. The monkey had undergone surgery previously. On necropsy, gastric dilatation and rupture demonstrated a tetrad arrangement on histopathologic examination. On 16S rRNA sequencing, a high population of Clostridium ventriculi was identified in the duodenum adjacent to stomach but not in the colon. This paper is the first report of Clostridium ventriculi infection in a cynomolgus macaque with acute gastric dilatation and rupture.

Spatial transcriptome atlas reveals pulmonary microstructure-specific COVID-19 gene signatures in cynomolgus macaques.

Characterizing the host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the molecular level is necessary to understand viral pathogenesis and identify clinically relevant biomarkers. However, in humans, the pulmonary host response during disease onset remains poorly understood. Herein, we utilized a spatial transcriptome atlas to identify pulmonary microstructure-specific COVID-19 gene signatures during the acute phase of lung infection in cynomolgus macaques. The innate immune response to virus-induced cell death was primarily active in the alveolar regions involving activated macrophage infiltration. Inflamed vascular regions exhibited prominent upregulation of interferon and complement pathway genes that mediate antiviral activity and tissue damage response. Furthermore, known biomarker genes were significantly expressed in specific microstructures, and some of them were universally expressed across all microstructures. These findings underscore the importance of identifying key drivers of disease progression and clinically applicable biomarkers by focusing on pulmonary microstructures appearing during SARS-CoV-2 infection.

Comparative spatial transcriptomic profiling of severe acute respiratory syndrome coronavirus 2 Delta and Omicron variants infections in the lungs of cynomolgus macaques.

Recently emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants are generally less pathogenic than previous strains. However, elucidating the molecular basis for pulmonary immune response alterations is challenging owing to the virus's heterogeneous distribution within complex tissue structure. Here, we revealed the spatial transcriptomic profiles of pulmonary microstructures at the SARS-CoV-2 infection site in the nine cynomolgus macaques upon inoculation with the Delta and Omicron variants. Delta- and Omicron-infected lungs had upregulation of genes involved in inflammation, cytokine response, complement, cell damage, proliferation, and differentiation pathways. Depending on the tissue microstructures (alveoli, bronchioles, and blood vessels), there were differences in the types of significantly upregulated genes in each pathway. Notably, a limited number of genes involved in cytokine and cell damage response were differentially expressed between bronchioles of the Delta- and Omicron-infection groups. These results indicated that despite a significant antigenic shift in SARS-CoV-2, the host immune response mechanisms induced by the variants were relatively consistent, with limited transcriptional alterations observed only in large airways. This study may aid in understanding the pathogenesis of SARS-CoV-2 and developing a clinical strategy for addressing immune dysregulation by identifying potential transcriptional biomarkers within pulmonary microstructures during infection with emerging variants.

Monocytes as suitable carriers for dissemination of dengue viral infection.

Dengue viruses (DENVs) exploit monocytes and macrophages for tropism and replication, therefore, establishing a long-term reservoir. However, their roles in dengue pathogenesis remains unclear. Here, using the human monocytic cell line THP-1, human primary monocytes, and non-human primate models, we show that DENV-infected monocytes represent suitable carriers for circulatory viral dissemination. Monocyte-derived macrophages expressing M2 surface markers at the gene level efficiently replicated, while the productivity of monocyte replication was low. However, attachment of DENVs to the cellular surface of monocytes was similar to that of macrophages. Furthermore, after differentiation with type-2 cytokines, DENV-attached monocytes could replicate DENVs. Productive DENV infection was confirmed by intravenous injection of DENVs into nonhuman primate model, in which, DENV attachment to monocytes was positively correlated with viremia. These results provide insight into the role of circulating monocytes in DENV infection, suggesting that monocytes directly assist in DENV dissemination and replication during viremia and could be applied to design antiviral intervention.

The Chimeric Adenovirus (Ad5/35) Expressing Engineered Spike Protein Confers Immunity against SARS-CoV-2 in Mice and Non-Human Primates.

Several COVID-19 platforms have been licensed across the world thus far, but vaccine platform research that can lead to effective antigen delivery is still ongoing. Here, we constructed AdCLD-CoV19 that could modulate humoral immunity by harboring SARS-CoV-2 antigens onto a chimeric adenovirus 5/35 platform that was effective in cellular immunity. By replacing the S1/S2 furin cleavage sequence of the SARS-CoV-2 Spike (S) protein mounted on AdCLD-CoV19 with the linker sequence, high antigen expression was confirmed in various cell lines. The high levels of antigen expression contributed to antigen-specific antibody activity in mice and non-human primates (NHPs) with a single vaccination of AdCLD-CoV19. Furthermore, the adenovirus-induced Th1 immune response was specifically raised for the S protein, and these immune responses protected the NHP against live viruses. While AdCLD-CoV19 maintained neutralizing antibody activity against various SARS-CoV-2 variants, it was reduced to single vaccination for ß and ο variants, and the reduced neutralizing antibody activity was restored with booster shots. Hence, AdCLD-CoV19 can prevent SARS-CoV-2 with a single vaccination, and the new vaccine administration strategy that responds to various variants can maintain the efficacy of the vaccine.

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.

Cynomolgus Macaque Model for COVID-19 Delta Variant.

With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, which are randomly mutated, the dominant strains in regions are changing globally. The development of preclinical animal models is imperative to validate vaccines and therapeutics against SARS-CoV-2 variants. The objective of this study was to develop a non-human primate (NHP) model for SARS-CoV-2 Delta variant infection. Cynomolgus macaques infected with Delta variants showed infectious viruses and viral RNA in the upper (nasal and throat) and lower respiratory (lung) tracts during the acute phase of infection. After 3 days of infection, lesions consistent with diffuse alveolar damage were observed in the lungs. For cellular immune responses, all macaques displayed transient lymphopenia and neutrophilia in the early stages of infection. SARS-CoV-2 Delta variant spike protein-specific IgM, IgG, and IgA levels were significantly increased in the plasma of these animals 14 days after infection. This new NHP Delta variant infection model can be used for comparative analysis of the difference in severity between SARS-CoV-2 variants of concern and may be useful in the efficacy evaluation of vaccines and universal therapeutic drugs for mutations.

Effect of Novel Polymer-Free Nitrogen-Doped Titanium Dioxide Film-Coated Coronary Stent Loaded With Mycophenolic Acid.

Background: Titanium is commonly used in blood-exposed medical devices because it has superior blood compatibility. Mycophenolic acid inhibits the proliferation of vascular smooth muscle cells. This study examined the effect of a non-polymer TiO2 thin film-coated stent with mycophenolic acid in a porcine coronary overstretch restenosis model. Methods: Thirty coronary arteries in 15 pigs were randomized into three groups in which the coronary arteries were treated with a TiO2 film-coated stent with mycophenolic acid (NTM, n = 10), everolimus-eluting stent with biodegradable polymer (EES, n = 10), or TiO2 film-coated stent (NT, n = 10). A histopathologic analysis was performed 28 days after the stenting. Results: There were no significant intergroup differences in injury score, internal elastic lamina area, or inflammation score. Percent area stenosis was significantly smaller in the NTM and EES groups than in the NT group (36.1 ± 13.63% vs. 31.6 ± 7.74% vs. 45.5 ± 18.96%, respectively, p = 0.0003). Fibrin score was greater in the EES group than in the NTM and NT groups [2.0 (range, 2.0-2.0) vs. 1.0 (range, 1.0-1.75) vs. 1.0 (range, 1.0-1.0), respectively, p < 0.0001]. The in-stent occlusion rate measured by micro-computed tomography demonstrated similar percent area stenosis rates on histology analysis (36.1 ± 15.10% in NTM vs. 31.6 ± 8.89% in EES vs. 45.5 ± 17.26% in NT, p < 0.05). Conclusion: The NTM more effectively reduced neointima proliferation than the NT. Moreover, the inhibitory effect of NTM on smooth muscle cell proliferation was not inferior to that of the polymer-based EES with lower fibrin deposition in this porcine coronary restenosis model.

Germinal Center-Induced Immunity Is Correlated With Protection Against SARS-CoV-2 Reinfection But Not Lung Damage.

Germinal centers (GCs) elicit protective humoral immunity through a combination of antibody-secreting cells and memory B cells, following pathogen invasion or vaccination. However, the possibility of a GC response inducing protective immunity against reinfection following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unknown. We found GC activity was consistent with seroconversion observed in recovered macaques and humans. Rechallenge with a different clade of virus resulted in significant reduction in replicating virus titers in respiratory tracts in macaques with high GC activity. However, diffuse alveolar damage and increased fibrotic tissue were observed in lungs of reinfected macaques. Our study highlights the importance of GCs developed during natural SARS-CoV-2 infection in managing viral loads in subsequent infections. However, their ability to alleviate lung damage remains to be determined. These results may improve understanding of SARS-CoV-2-induced immune responses, resulting in better coronavirus disease 2019 (COVID-19) diagnosis, treatment, and vaccine development.

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.

Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates.

Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), various vaccines are being developed, with most vaccine candidates focusing on the viral spike protein. Here, we developed a previously unknown subunit vaccine comprising the receptor binding domain (RBD) of the spike protein fused with the tetanus toxoid epitope P2 (RBD-P2) and tested its efficacy in rodents and nonhuman primates (NHPs). We also investigated whether the SARS-CoV-2 nucleocapsid protein (N) could increase vaccine efficacy. Immunization with N and RBD-P2 (RBDP2/N) + alum increased T cell responses in mice and neutralizing antibody levels in rats compared with those obtained using RBD-P2 + alum. Furthermore, in NHPs, RBD-P2/N + alum induced slightly faster SARS-CoV-2 clearance than that induced by RBD-P2 + alum, albeit without statistical significance. Our study supports further development of RBD-P2 as a vaccine candidate against SARS-CoV-2. Also, it provides insights regarding the use of N in protein-based vaccines against SARS-CoV-2.

Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates.

The unprecedented and rapid spread of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) has motivated the need for a rapidly producible and scalable vaccine. Here, we developed a synthetic soluble SARS-CoV-2 spike (S) DNA-based vaccine candidate, GX-19. In mice, immunization with GX-19 elicited not only S-specific systemic and pulmonary antibody responses but also Th1-biased T cell responses in a dose-dependent manner. GX-19-vaccinated nonhuman primates seroconverted rapidly and exhibited a detectable neutralizing antibody response as well as multifunctional CD4+ and CD8+ T cell responses. Notably, when the immunized nonhuman primates were challenged at 10 weeks after the last vaccination with GX-19, they had reduced viral loads in contrast to non-vaccinated primates as a control. These findings indicate that GX-19 vaccination provides a durable protective immune response and also support further development of GX-19 as a vaccine candidate for 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.

A therapeutic neutralizing antibody targeting receptor binding domain of SARS-CoV-2 spike protein.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19.

Establishment and Characterization of Immortalized Miniature Pig Pancreatic Cell Lines Expressing Oncogenic K-RasG12D.

In recent decades, many studies on the treatment and prevention of pancreatic cancer have been conducted. However, pancreatic cancer remains incurable, with a high mortality rate. Although mouse models have been widely used for preclinical pancreatic cancer research, these models have many differences from humans. Therefore, large animals may be more useful for the investigation of pancreatic cancer. Pigs have recently emerged as a new model of pancreatic cancer due to their similarities to humans, but no pig pancreatic cancer cell lines have been established for use in drug screening or analysis of tumor biology. Here, we established and characterized an immortalized miniature pig pancreatic cell line derived from primary pancreatic cells and pancreatic cancer-like cells expressing K-rasG12D regulated by the human PTF1A promoter. Using this immortalized cell line, we analyzed the gene expression and phenotypes associated with cancer cell characteristics. Notably, we found that acinar-to-ductal transition was caused by K-rasG12D in the cell line constructed from acinar cells. This may constitute a good research model for the analysis of acinar-to-ductal metaplasia in human pancreatic cancer.

Assessment of Hand Motor Function in a Non-human Primate Model of Ischemic Stroke.

Ischemic stroke results from arterial occlusion and can cause irreversible brain injury. A non-human primate (NHP) model of ischemic stroke was previously developed to investigate its pathophysiology and for efficacy testing of therapeutic candidates; however, fine motor impairment remains to be well-characterized. We evaluated hand motor function in a cynomolgus monkey model of ischemic stroke. Endovascular transient middle cerebral artery occlusion (MCAO) with an angiographic microcatheter induced cerebral infarction. In vivo magnetic resonance imaging mapped and measured the ischemia-induced infarct lesion. In vivo diffusion tensor imaging (DTI) of the stroke lesion to assess the neuroplastic changes and fiber tractography demonstrated three-dimensional patterns in the corticospinal tract 12 weeks after MCAO. The hand dexterity task (HDT) was used to evaluate fine motor movement of upper extremity digits. The HDT was modified for a home cage-based training system, instead of conventional chair restraint training. The lesion was localized in the middle cerebral artery territory, including the sensorimotor cortex. Maximum infarct volume was exhibited over the first week after MCAO, which progressively inhibited ischemic core expansion, manifested by enhanced functional recovery of the affected hand over 12 weeks after MCAO. The total performance time decreased with increasing success rate for both hands on the HDT. Compensatory strategies and retrieval failure improved in the chronic phase after stroke. Our findings demonstrate the recovery of fine motor skill after stroke, and outline the behavioral characteristics and features of functional disorder of NHP stroke model, providing a basis for assessing hand motor function after stroke.

Transient Lymphopenia and Interstitial Pneumonia With Endotheliitis in SARS-CoV-2-Infected Macaques.

Using a reliable primate model is critical for developing therapeutic advances to treat humans infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we exposed macaques to high titers of SARS-CoV-2 via combined transmission routes. We observed acute interstitial pneumonia with endotheliitis in the lungs of all infected macaques. All macaques had a significant loss of total lymphocytes during infection, which were restored over time. These data show that SARS-CoV-2 causes a coronavirus disease 2019 (COVID-19)-like disease in macaques. This new model could investigate the interaction between SARS-CoV-2 and the immune system to test therapeutic strategies.

Prevalence and characterization of Clostridium perfringens isolated from feces of captive cynomolgus monkeys (Macaca fascicularis).

Clostridium perfringens is ubiquitous in the environment and the gastrointestinal tract of warm-blooded animals. While part of the gut microbiome, abnormal growth of C. perfringens causes histotoxic, neurologic, and enteric diseases in a variety of animal species, including humans, due to the production of toxins. There is extremely limited information on C. perfringens infection in non-human primates. Presently, 10 strains were successfully isolated from 126 monkeys and confirmed by molecular and biochemical analyses. All isolates were genotype A based on molecular analysis. Alpha toxin was identified in all isolates. Beta 2 toxin was detected in only three isolates. No other toxins, including enterotoxin, beta, iota, epsilon, and net B toxin, were identified in any isolate. All isolates were highly susceptible to ß-lactam antibiotics. Double hemolysis and lecithinase activity were commonly observed in all strains. Biofilm formation, which can increase antibiotic resistance, was identified in 90% of the isolates. The data are the first report the prevalence and characteristics of C. perfringens isolated from captive cynomolgus monkeys.