Spike S2 Subunit: Possible Target for Detecting Novel SARS-CoV-2 Variants with Multiple Mutations.

Novel SARS-CoV-2 variants have multiple mutations that may impact molecular diagnostics. The markedly conserved S2 subunit may be utilized to detect new variants. A comparison of 694 specimens (2019-2022) in Thailand using a commercial RT-PCR kit and the kit in combination with S2 primers and a probe was performed. Delayed amplification in ORF1ab was detected in one BA.4 omicron, whereas no amplification problem was encountered in the S2 target. There were no statistically significant differences in mean Ct value between the target genes (E, N, ORF1ab, and S2) and no significant differences in mean Ct value between the reagents. Furthermore, 230,821 nucleotide sequences submitted by 20 representative counties in each region (Jan-Oct 2022) have been checked for mutations in S2 primers and probe using PrimerChecker; there is a very low chance of encountering performance problems. The S2 primers and probe are still bound to the top five currently circulating variants in all countries and Thailand without mismatch recognition (Jun-Nov 2023). This study shows the possible benefits of detecting S2 in combination with simultaneously detecting three genes in a kit without affecting the Ct value of each target. The S2 subunit may be a promising target for the detection of SARS-CoV-2 variants with multiple mutations.

IP-10 and complement activation as friend or foe in COVID-19.

INTRODUCTION: The Innate immune system senses danger signals of COVID-19 infection and produce an orchestration of cellular, complement and cytokines cascades. These led to the approach using immunosuppressive agents. It is intriguing whether certain biomarkers can aid the proper administration of such drugs. METHODS: Plasma specimens of 58 COVID-19 patients with differing severity, from very mild illness (group A), mild (group B), moderate (group C), and severe/critical illness (group D) were assayed for cyto-chemokines and terminal complement complex (SC5b-9) during the course of diseases. None received anti-IL-6 therapy, there was no mortality in this cohort. RESULTS: IP-10 and RANTES levels were dominant cytokines. IP-10 levels increased significantly in all groups when compared between pre-nadir and nadir phases (group A, p =0.428; group B =0.034; group C =0.159; group D <0.001) and in groups B and D when compared between nadir and recovery phases (p <0.001). RANTES levels were elevated in all groups across all phases with no significant differences. SC5b-9 levels increased significantly as compared to healthy controls [pre-nadir- group A versus healthy, p =0.122; group B-D versus healthy, p =0.021); nadir-group A versus healthy, p =0.003; group B-D versus healthy, p <0.001; recovery phase (p <0.001)] but not between groups A and B-D at pre-nadir (p=0.606). CONCLUSION: The absence of significant pro-inflammatory responses and early elevation of IP-10 levels and complement activation may be favorable and necessary for viral elimination in COVID-19 patients. Expression of distinct cyto-chemokines during each clinical phase may be useful for guiding proper therapeutic interventions on alleviating thrombo-inflammation responses to COVID-19 infection.

A case of successive development of possible acute necrotizing encephalopathy after COVID-19 pneumonia.

COVID-19 infection often results in an excessive inflammatory response with a spectrum of neurological manifestations. Here, we describe an 81-year-old female with severe COVID-19 pneumonia and subsequent alteration of consciousness after high-dose intravenous dexamethasone and remdesivir. A non-contrast head computed tomography (CT) demonstrated bilateral hypodensities involving bilateral cerebellar hemispheres, thalami, cerebral peduncles and medial parieto-occipital areas. There was no improvement and repeat CT showed progression with findings suggestive of acute necrotizing encephalopathy. Interleukin-6 levels were initially normal; however, subsequent levels were found to be markedly elevated. Acute necrotizing encephalopathy associated with COVID-19 may occur in the setting of severe pneumonia and may represent an immune-mediated process involving inflammatory cytokines such as interleukin-6.

Early detection of neutralizing antibodies against SARS-CoV-2 in COVID-19 patients in Thailand.

BACKGROUND: The presence of neutralizing antibodies (NAbs) is an indicator of protective immunity for most viral infections. A newly developed surrogate viral neutralization assay (sVNT) offers the ability to detect total receptor binding domain-targeting NAbs in an isotype-independent manner, increasing the test sensitivity. Thus, specimens with low IgM/ IgG antibody levels showed strong neutralization activity in sVNT. METHODS: This study aimed to measure the %inhibition of NAbs measured by sVNT in PCR-confirmed COVID-19 patients. The sensitivity of sVNT for the diagnosis of SARS-CoV-2 infection and its kinetics were determined. RESULTS: Ninety-seven patients with PCR-confirmed SARS-CoV-2 infection were included in this study. Majority of the patients were 21-40 years old (67%) and 63% had mild symptoms. The sensitivity of sVNT for the diagnosis of SARS-CoV-2 infection was 99% (95% confidence interval (CI) 94.4-100%) and the specificity was 100% (95% CI 98.3-100%). The negative predictive value of sVNT from the samples collected before and after 7 days of symptom onset was 99.5% (95% CI 97.4-100%) and 100% (95% CI 93.8-100%), respectively. The level of inhibition at days 8-14 were significantly higher than days 0-7 (p<0.001). The median %inhibition values by severity of COVID-19 symptoms were 79.9% (interquartile range (IQR) 49.7-91.8%); 89.0% (IQR 71.2-92.4%); and 86.6% (IQR 69.5-92.8%), for mild, moderate and severe/critical symptoms respectively. The median level of sVNT %inhibition of severe was significantly higher than the mild group (p = 0.05). CONCLUSION: The sVNT is a practical and robust serological test for SARS-CoV-2 infection and does not require specialized biosafety containment. It can be used clinically to aid diagnosis in both early and late infection especially in cases when the real-time RT-PCR results in weakly negative or weakly positive, and to determine the protective immune response from SARS-CoV-2 infection in patients.

Evidence for SARS-CoV-2 related coronaviruses circulating in bats and pangolins in Southeast Asia.

Among the many questions unanswered for the COVID-19 pandemic are the origin of SARS-CoV-2 and the potential role of intermediate animal host(s) in the early animal-to-human transmission. The discovery of RaTG13 bat coronavirus in China suggested a high probability of a bat origin. Here we report molecular and serological evidence of SARS-CoV-2 related coronaviruses (SC2r-CoVs) actively circulating in bats in Southeast Asia. Whole genome sequences were obtained from five independent bats (Rhinolophus acuminatus) in a Thai cave yielding a single isolate (named RacCS203) which is most related to the RmYN02 isolate found in Rhinolophus malayanus in Yunnan, China. SARS-CoV-2 neutralizing antibodies were also detected in bats of the same colony and in a pangolin at a wildlife checkpoint in Southern Thailand. Antisera raised against the receptor binding domain (RBD) of RmYN02 was able to cross-neutralize SARS-CoV-2 despite the fact that the RBD of RacCS203 or RmYN02 failed to bind ACE2. Although the origin of the virus remains unresolved, our study extended the geographic distribution of genetically diverse SC2r-CoVs from Japan and China to Thailand over a 4800-km range. Cross-border surveillance is urgently needed to find the immediate progenitor virus of SARS-CoV-2.

Identification of a Novel Pathogen Using Family-Wide PCR: Initial Confirmation of COVID-19 in Thailand.

In resource-limited countries, early detection of novel pathogens is often challenging, due to financial and technical constraints. This study reports the efficacy of family-wide polymerase chain reaction (PCR) in screening, detecting, and identifying initial cases of the novel SARS-CoV-2 in Thailand. Respiratory secretions were collected from suspected individuals traveling from Wuhan, China to Thailand at the beginning of January 2020. Family-wide PCR assays yielded positive results for coronavirus in one traveler within 12 h on January 8, 2020. Nucleotide sequences (290 bp) showed 100% similarity to SARS-CoV-2. The whole genome sequence was further characterized by Next Generation Sequencing (NGS) for confirmation. Combining family-wide PCR, as a rapid screening tool, with NGS, for full genome characterization, could facilitate early detection and confirmation of a novel pathogen and enable early containment of a disease outbreak.