SARS-CoV-2 Variants by Whole-Genome Sequencing in a University Hospital in Bangkok: First to Third COVID-19 Waves.

BACKGROUND: Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants emerged globally during the recent coronavirus disease (COVID-19) pandemic. From April 2020 to April 2021, Thailand experienced three COVID-19 waves, and each wave was driven by different variants. Therefore, we aimed to analyze the genetic diversity of circulating SARS-CoV-2 using whole-genome sequencing analysis. METHODS: A total of 33 SARS-CoV-2 positive samples from three consecutive COVID-19 waves were collected and sequenced by whole-genome sequencing, of which, 8, 10, and 15 samples were derived from the first, second, and third waves, respectively. The genetic diversity of variants in each wave and the correlation between mutations and disease severity were explored. RESULTS: During the first wave, A.6, B, B.1, and B.1.375 were found to be predominant. The occurrence of mutations in these lineages was associated with low asymptomatic and mild symptoms, providing no transmission advantage and resulting in extinction after a few months of circulation. B.1.36.16, the predominant lineage of the second wave, caused more symptomatic COVID-19 cases and contained a small number of key mutations. This variant was replaced by the VOC alpha variant, which later became dominant in the third wave. We found that B.1.1.7 lineage-specific mutations were crucial for increasing transmissibility and infectivity, but not likely associated with disease severity. There were six additional mutations found only in severe COVID-19 patients, which might have altered the virus phenotype with an inclination toward more highly pathogenic SARS-CoV-2. CONCLUSION: The findings of this study highlighted the importance of whole-genome analysis in tracking newly emerging variants, exploring the genetic determinants essential for transmissibility, infectivity, and pathogenicity, and helping better understand the evolutionary process in the adaptation of viruses in humans.

The Effects of CoronaVac and ChAdOx1 nCoV-19 in Reducing Severe Illness in Thailand: A Retrospective Cohort Study.

Two primary vaccines for coronavirus disease 2019 (COVID-19) have been rolled out in the mass vaccination campaign that started simultaneously with the spread of the delta variant. To explore the vaccines' effect on reducing viral load and disease severity, we conducted a retrospective cohort study in Thai patients aged ≥18 years who were confirmed COVID-19 positive by RT-PCR. Compared to unvaccinated patients, Ct values and the number of severe cases among vaccine regimens were analyzed. Ct values of vaccinated patients were not significantly different from unvaccinated patients, despite an increase of Ct values in a booster dose. The adjusted odd ratio for prevention of delta-related severe diseases was 0.47, 95% CI: 0.30-0.76 and 0.06, 95% CI: 0.01-0.45 after receiving one dose and two doses, respectively. No severe illness was found in booster-vaccinated individuals. Focusing on the vaccine types, one dose of ChAdOx1 nCoV-19 gave significant protection, whereas one dose of CoronaVac did not (0.49, 95% CI: 0.30-0.79, p = 0.003 vs. 0.28, 95% CI: 0.04-2.16, p = 0.223). Two-dose vaccination showed robust protective effects in all subpopulations regardless of vaccine type. Vaccinations with two primary vaccines could not reduce viral load in patients with COVID-19, but could prevent severe illness.

Performance of colorimetric reverse transcription loop-mediated isothermal amplification as a diagnostic tool for SARS-CoV-2 infection during the fourth wave of COVID-19 in Thailand.

BACKGROUND: COVID-19, which is caused by SARS-CoV-2 and its variants, poses an ongoing global threat, particularly in low-immunization coverage regions. Thus, rapid, accurate, and easy-to-perform diagnostic methods are in urgent demand to halt the spread of the virus. OBJECTIVES: We aimed to validate the clinical performance of the FastProof 30 min-TTR SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP) method using leftover RNA samples extracted from 315 nasopharyngeal swabs. The sensitivity and specificity of RT-LAMP were determined in comparison with reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Of 315 nasopharyngeal swabs, viral RNA was detected in 154 samples (48.9%) by RT-PCR assay. Compared with RT-PCR, overall sensitivity and specificity of RT-LAMP were 81.82% (95% CI: 74.81-87.57) and 100% (95% CI: 97.73-100), respectively. A 100% positivity rate was achieved in samples with cycle threshold (Ct) <31 for RT-PCR targeting the ORF1ab gene. However, samples with Ct >31 accounted for false-negative results by RT-LAMP in 28 samples. CONCLUSIONS: RT-LAMP reliably detected viral RNA with high sensitivity and specificity and has potential application for mass screening of patients with acute COVID-19 infection when viral load is high.

Trogocytosis with monocytes associated with increased α2,3 sialic acid expression on B cells during H5N1 influenza virus infection.

The immunopathogenesis of H5N1 virus has been studied intensively since it caused cross-species infection and induced high mortality to human. We previously observed the interaction between monocytes and B cells, which increased the susceptibility of B cell to H5N1 virus infection after a co-culture. Levels of α2,3 sialic acid (avian flu receptor) were also significantly increased on B cell surface in this co-culture model with unclear explanation. In this study, we aimed to determine the possible mechanism that responded for this increase in α2,3 sialic acid on B cells. Acquisition of α2,3 SA by B cells via cell contact-dependent trogocytosis was proposed. Results showed that the lack of α2,3 SA was detected on B cell surface, and B cells acquired membrane-bound α2,3 SA molecules from monocytes in H5N1-infected co-cultures. Occurrence of membrane exchange mainly relied on H5N1 infection and cell-cell contact as opposed to a mock infection and transwell. The increase in α2,3 SA on B cell surface mediated by trogocytosis was associated with the enhanced susceptibility to H5N1 infection. These observations thus provide the evidence that H5N1 influenza virus may utilize trogocytosis to expand its cell tropism and spread to immune cells despite the lack of avian flu receptor.

The activation of B cells enhances DC-SIGN expression and promotes susceptibility of B cells to HPAI H5N1 infection.

The interplay between highly pathogenic avian influenza (HPAI) H5N1 virus and immune cells has been extensively studied for years, as host immune components are thought to play significant roles in promoting the systemic spread of the virus and responsible for cytokine storm. Previous studies suggested that the interaction of B cells and monocytes could promote HPAI H5N1 infection by enhancing avian influenza virus receptor expression. In this study, we further investigate the relationship between the HPAI H5N1 virus, activated B cells, and DC-SIGN expression. DC-SIGN has been described as an important factor for mediating various types of viral infection. Here, we first demonstrate that HPAI H5N1 infection could induce an activation of B cells, which was associated with DC-SIGN expression. Using CD40L and recombinant IL-4 for B cell stimulation, we determined that DC-SIGN expressed on activated B cells was able to enhance its susceptibility to HPAI H5N1 infection. Our findings uncover the interplay between this H5N1 virus and B cells and provide important information in understanding how the virus overcomes our immune system, contributing to its unusual immunopathogenesis.

Mesenchymal Stromal Cells and Viral Infection.

Mesenchymal Stromal Cells (MSCs) are a subset of nonhematopoietic adult stem cells, readily isolated from various tissues and easily culture-expanded ex vivo. Intensive studies of the immune modulation and tissue regeneration over the past few years have demonstrated the great potential of MSCs for the prevention and treatment of steroid-resistant acute graft-versus-host disease (GvHD), immune-related disorders, and viral diseases. In immunocompromised individuals, the immunomodulatory activities of MSCs have raised safety concerns regarding the greater risk of primary viral infection and viral reactivation, which is a major cause of mortality after allogeneic transplantation. Moreover, high susceptibilities of MSCs to viral infections in vitro could reflect the destructive outcomes that might impair the clinical efficacy of MSCs infusion. However, the interplay between MSCs and virus is like a double-edge sword, and it also provides beneficial effects such as allowing the proliferation and function of antiviral specific effector cells instead of suppressing them, serving as an ideal tool for study of viral pathogenesis, and protecting hosts against viral challenge by using the antimicrobial activity. Here, we therefore review favorable and unfavorable consequences of MSCs and virus interaction with the highlight of safety and efficacy for applying MSCs as cell therapy.

The presence of monocytes enhances the susceptibility of B cells to highly pathogenic avian influenza (HPAI) H5N1 virus possibly through the increased expression of α2,3 SA receptor.

The highly pathogenic avian influenza (HPAI) H5N1 virus causes severe systemic infection in avian and mammalian species, including humans by first targeting immune cells. This subsequently renders the innate and adaptive immune responses less active, thus allowing dissemination of the virus to systemic organs. To gain insight into the pathogenesis of H5N1, this study aims to determine the susceptibility of human PBMCs to the H5N1 virus and explore the factors which influence this susceptibility. We found that PBMCs were a target of H5N1 infection, and that monocytes and B cells were populations which were clearly the most susceptible. Analysis of PBMC subpopulations showed that isolated monocytes and monocytes residing in whole PBMCs had comparable percentages of infection (28.97 ± 5.54% vs 22.23 ± 5.14%). In contrast, isolated B cells were infected to a much lower degree than B cells residing in a mixture of whole PBMCs (0.88 ± 0.34% vs 34.87 ± 4.63%). Different susceptibility levels of B cells for these tested conditions spurred us to explore the B cell-H5N1 interaction mechanisms. Here, we first demonstrated that monocytes play a crucial role in the enhancement of B cell susceptibility to H5N1 infection. Although the actual mechanism by which this enhancement occurs remains in question, α2,3-linked sialic acid (SA), known for influenza virus receptors, could be a responsible factor for the greater susceptibility of B cells, as it was highly expressed on the surface of B cells upon H5N1 infection of B cell/monocyte co-cultures. Our findings reveal some of the factors involved with the permissiveness of human immune cells to H5N1 virus and provide a better understanding of the tropism of H5N1 in immune cells.

Tropism of avian influenza A (H5N1) virus to mesenchymal stem cells and CD34+ hematopoietic stem cells.

The presence of abnormal hematologic findings such as lymphopenia, thrombocytopenia, and pancytopenia were diagnosed in severe cases of avian influenza A H5N1. Whether direct viral dissemination to bone marrow (BM) cells causes this phenomenon remains elusive. We explore the susceptibility of the two stem cell types; hematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs) isolated from human BM cells or cord blood, to infection with avian H5N1 viruses. For the first time, we demonstrated that the H5N1 virus could productively infect and induce cell death in both human stem cell types. In contrast, these activities were not observed upon human influenza virus infection. We also determined whether infection affects the immunomodulatory function of MSCs. We noted a consequent dysregulation of MSC-mediated immune modulation as observed by high cytokine and chemokine production in H5N1 infected MSCs and monocytes cocultures. These findings provide a better understanding of H5N1 pathogenesis in terms of broad tissue tropism and systemic spread.