Comparison of the Ct-values for genomic and subgenomic SARS-CoV-2 RNA reveals limited predictive value for the presence of replication competent virus.

SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19. In addition to the full length positive-sensed, single-stranded genomic RNA (gRNA), viral subgenomic RNAs (sgRNAs) that are required for expression of the 3' region of the genome are synthesized in virus-infected cells. However, whether these sgRNA-species might be used as a measure of active virus replication and to predict infectivity is still under debate. The commonly used methods to monitor and quantitate SARS-CoV-2 infections are based on RT-qPCR analysis and the detection of gRNA. The infectivity of a sample obtained from nasopharyngeal or throat swabs is associated with the viral load and inversely correlates with Ct-values, however, a cut-off value predicting the infectivity highly depends on the performance of the assay. Furthermore, gRNA derived Ct-values result from nucleic acid detection and do not necessarily correspond to active replicating virus. We established a multiplex RT-qPCR assay on the cobas 6800 omni utility channel concomitantly detecting SARS-CoV-2 gRNAOrf1a/b, sgRNAE,7a,N, and human RNaseP-mRNA used as human input control. We compared the target specific Ct-values with the viral culture frequency and performed ROC curve analysis to determine the assay sensitivity and specificity. We found no advantage in the prediction of viral culture when using sgRNA detection compared to gRNA only, since Ct-values for gRNA and sgRNA were highly correlated and gRNA offered a slightly more reliable predictive value. Single Ct-values alone only provide a very limited prediction for the presence of replication competent virus. Hence, careful consideration of the medical history including symptom onset has to be considered for risk stratification.

A novel isothermal digital amplification system and its application for absolute quantification of respiratory infectious virus

Herein, we establish a novel isothermal digital amplification system termed digital nicking and extension chain reaction system-based amplification (dNESBA) by utilizing the isothermal NESBA technique and the newly developed miniaturized fluorescence monitoring system (mFMS). dNESBA enables parallel isothermal NESBA reactions in more than 10,000 localized droplet microreactors and read the fluorescence signals rapidly in 150 s by mFMS. This system could identify the genomic RNA (gRNA) extracted from target respiratory syncytial virus A (RSV A) as low as 10 copies with remarkable specificity. The practical applicability of dNESBA was also successfully verified by reliably detecting the gRNA in the artificial sputum samples with excellent reproducibility and accuracy. Due to the intrinsic advantages of isothermal amplifying technique including the elimination of the requirement of thermocycling device and the enhanced portability of the miniaturized read-out equipment, the dNESBA technique equipped with mFMS could serve as a promising platform system to achieve point-of-care (POC) digital molecular diagnostics, enabling absolute and ultra-sensitive quantification of various infectious pathogens even in an early stage.Copyright © 2023

Infectious viruses from transfected SARS-CoV-2 genomic RNA.

SARS-CoV-2 emerged at the end of 2019, and like other novel pathogens causing severe symptoms, WHO recommended heightened biosafety measures for laboratories working with the virus. The positive-stranded genomic RNA of coronaviruses has been known to be infectious since the 1970s, and overall, all experiments with the possibility of SARS-CoV-2 propagation are carried out in higher containment level laboratories. However, as SARS-CoV-2 RNA has been routinely handled in BSL-2 laboratories, the question of the true nature of RNA infectiousness has risen along with discussion of appropriate biosafety measures. Here, we studied the ability of native SARS-CoV-2 genomic RNA to produce infectious viruses when transfected into permissive cells and discussed the biosafety control measures related to these assays. In transfection assays large quantities of genomic vRNA of SARS-CoV-2 was required for a successful production of infectious viruses. However, the quantity of vRNA alone was not the only factor, and especially when the transfected RNA was derived from infected cells, even small amounts of genomic vRNA was enough for an infection. Virus replication was found to start rapidly after transfection, and infectious viruses were detected in the cell culture media at 24 h post-transfection. In addition, silica membrane-based kits were shown to be as good as traditional TRI-reagent based methods in extracting high-quality, 30 kb-long genomic vRNA. Taken together, our data indicates that all transfection experiments with samples containing genomic SARS-CoV-2 RNA should be categorized as a propagative work and the work should be conducted only in a higher containment BSL-3 laboratory.

Characterizing SARS-CoV-2 transcription of subgenomic and genomic RNAs during early human infection using multiplexed droplet digital PCR.

BACKGROUND: Control of SARS-CoV-2 (SCV-2) transmission requires understanding SCV-2 replication dynamics. METHODS: We developed a multiplexed droplet digital PCR (ddPCR) assay to quantify SCV-2 subgenomic RNAs (sgRNAs), which are only produced during active viral replication, and discriminate them from genomic RNAs (gRNAs). We applied the assay to specimens from 144 people with single nasopharyngeal samples and 27 people with >1 sample. Results were compared to qPCR and viral culture. RESULTS: sgRNAs were quantifiable across a range of qPCR cycle threshold (Ct) values and correlated with Ct values. The ratio of sgRNA:gRNA was stable across a wide range of Ct values, whereas adjusted amounts of N sgRNA to a human housekeeping gene declined with higher Ct values. Adjusted sgRNA and gRNA amounts were quantifiable in culture-negative samples, although levels were significantly lower than in culture-positive samples. Daily testing of 6 persons revealed that sgRNA is concordant with culture results during the first week of infection but may be discordant with culture later in infection. Further, sgRNA:gRNA is constant during infection despite changes in viral culture. CONCLUSIONS: Ct values from qPCR correlate with active viral replication. More work is needed to understand why some cultures are negative despite presence of sgRNA.

COVID-19 Pandemic: An Overview of its Origin, Current Status, and Ongoing Clinical Trials

The COVID-19 pandemic is raging across the globe, with the total active cases increas-ing each day. Globally over 63 million COVID-19cases and more than 1.4 million deaths have been reported to WHO. Throughout the world, academicians, clinicians and scientists are working tirelessly on developing a treatment to combat this pandemic. The origin of novel SARS-CoV-2 virus still remains foggy but is believed to have originated from a bat coronavirus RaTG13 with which it shares approximately 96% sequence similarity. In the present review, the authors have pro-vided an overview of the COVID-19 pandemic, epidemiology, transmission, developments related to diagnosis, drugs and vaccines, along with the genetic diversity and lifecycle of the SARS-CoV-2 based on the current studies and information available.Copyright © 2022 Bentham Science Publishers.

Immunological inflammatory factors in patients diagnosed with COVID-19

Introduction and Aim: Due to the Coronavirus outbreaks, the SARS-CoV-2, also known as COVID-19, has claimed several lives around the world, with the majority of them being elderly, suffering from underlying chronic illnesses, or living in vulnerable conditions. This study aimed to find the immunological factors CD-79, CD-4, IL-2, and TNF-B in COVID-19 patients utilizing nucleocapsid-(N), a protein structure that interacts with genomic RNA to create complexes. Material(s) and Method(s): SARS-COV-2 infection was detected using RT-PCR. The serum levels of IL-2 and TNF-B, as well as the concentrations of CD4 and CD79, were measured. This study included 100 COVID-19 patients. Result(s): The results showed that the serum concentration of TNF-beta and IL-2 in COVID19 patients was significantly higher than that in the general population (with acute and moderate illness) when compared to normal control groups (p<0.05). COVID-19 patients reported higher levels of CD79 as well as CD4 expression than healthy control groups in a study of activated markers. Conclusion(s): Infection with SARS-COV-2 has a high impact on various immunological and inflammatory markers in patients.Copyright © 2023, Indian Association of Biomedical Scientists. All rights reserved.

Experimental and computational methods for studying the dynamics of RNA-RNA interactions in SARS-COV2 genomes.

Long-range ribonucleic acid (RNA)-RNA interactions (RRI) are prevalent in positive-strand RNA viruses, including Beta-coronaviruses, and these take part in regulatory roles, including the regulation of sub-genomic RNA production rates. Crosslinking of interacting RNAs and short read-based deep sequencing of resulting RNA-RNA hybrids have shown that these long-range structures exist in severe acute respiratory syndrome coronavirus (SARS-CoV)-2 on both genomic and sub-genomic levels and in dynamic topologies. Furthermore, co-evolution of coronaviruses with their hosts is navigated by genetic variations made possible by its large genome, high recombination frequency and a high mutation rate. SARS-CoV-2's mutations are known to occur spontaneously during replication, and thousands of aggregate mutations have been reported since the emergence of the virus. Although many long-range RRIs have been experimentally identified using high-throughput methods for the wild-type SARS-CoV-2 strain, evolutionary trajectory of these RRIs across variants, impact of mutations on RRIs and interaction of SARS-CoV-2 RNAs with the host have been largely open questions in the field. In this review, we summarize recent computational tools and experimental methods that have been enabling the mapping of RRIs in viral genomes, with a specific focus on SARS-CoV-2. We also present available informatics resources to navigate the RRI maps and shed light on the impact of mutations on the RRI space in viral genomes. Investigating the evolution of long-range RNA interactions and that of virus-host interactions can contribute to the understanding of new and emerging variants as well as aid in developing improved RNA therapeutics critical for combating future outbreaks.

A Pilot Study Evaluating Subgenomic RNAs for Detection of Infectious SARS-CoV-2 in Nasopharyngeal Swabs

Background: The prolonged persistence of viral ribonucleic acid (RNA) in coronavirus disease 2019 (COVID-19) patients and the difficulty in differentiating between infectious virus and noninfectious viral RNA have impeded the use of current molecular diag-nostic tests as a decision tool in quarantine termination. The performance of new methods to detect surrogate viability markers, such as subgenomic RNAs (sgRNAs), has been discussed, and numerous important questions are still needed to be addressed before broad implementation.Objectives: This study aimed to primarily evaluate the performance of SYBR green quantitative reverse transcription-polymerase chain reaction (RT-qPCR) targeting N and E sgRNAs as a surrogate of viability markersMethods: This pilot study was carried out to detect genomic RNAs (gRNAs) and sgRNAs using RT-qPCR in cell culture infected with severe acute respiratory syndrome coronavirus 2 and nasopharyngeal swabbing samples from COVID-19 patients, and the results were compared to viral culture as a gold standard method for infectious virus detection. The diagnostic parameters and Cohen's Kappa correlation index were then analyzed.Results: E subgenomic RNA detection was the most reliable predictor for actively replicating the virus as it showed the highest value of all diagnostic parameters with a good correlation with viral cultivation. The lowest cycle threshold value of gRNAs and sgN detection become undetectable by sgE within the range of 23 -26.Conclusion: Using a suitable sgRNA type was important for test accuracy. The findings suggested E sgRNA detection as a promising surrogate approach to indicate a truly active viral infection, and when performed with a low-cost molecular test of SYBR green-based assay, it could support huge demands for routine analysis.

A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis.

The coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously affected public health around the world. In-depth studies on the pathogenic mechanisms of SARS-CoV-2 is urgently necessary for pandemic prevention. However, most laboratory studies on SARS-CoV-2 have to be carried out in bio-safety level 3 (BSL-3) laboratories, greatly restricting the progress of relevant experiments. In this study, we used a bacterial artificial chromosome (BAC) method to assemble a SARS-CoV-2 replication and transcription system in Vero E6 cells without virion envelope formation, thus avoiding the risk of coronavirus exposure. Furthermore, an improved real-time quantitative reverse transcription PCR (RT-qPCR) approach was used to distinguish the replication of full-length replicon RNAs and transcription of subgenomic RNAs (sgRNAs). Using the SARS-CoV-2 replicon, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 facilitates the transcription of sgRNAs in the discontinuous synthesis process. Moreover, two high-frequency mutants of N protein, R203K and S194L, can obviously enhance the transcription level of the replicon, hinting that these mutations likely allow SARS-CoV-2 to spread and reproduce more quickly. In addition, remdesivir and chloroquine, two well-known drugs demonstrated to be effective against coronavirus in previous studies, also inhibited the transcription of our replicon, indicating the potential applications of this system in antiviral drug discovery. Overall, we developed a bio-safe and valuable replicon system of SARS-CoV-2 that is useful to study the mechanisms of viral RNA synthesis and has potential in novel antiviral drug screening.

Hydrophobic Alpha-Helical Short Peptides in Overlapping Reading Frames of the Coronavirus Genome.

In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture.

Background: Low frequency intrahost single nucleotide variants (iSNVs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been increasingly recognised as predictive indicators of positive selection. Particularly as growing numbers of SARS-CoV-2 variants of interest (VOI) and concern (VOC) emerge. However, the dynamics of subgenomic RNA (sgRNA) expression and its impact on genomic diversity and infection outcome remain poorly understood. This study aims to investigate and quantify iSNVs and sgRNA expression in single and longitudinally sampled cohorts over the course of mild and severe SARS-CoV-2 infection, benchmarked against an in vitro infection model. Methods: Two clinical cohorts of SARS-CoV-2 positive cases in New South Wales, Australia collected between March 2020 and August 2021 were sequenced. Longitudinal samples from cases hospitalised due to SARS-CoV-2 infection (severe) (n = 16) were analysed and compared with cases that presented with SARS-CoV-2 symptoms but were not hospitalised (mild) (n = 23). SARS-CoV-2 genomic diversity profiles were also examined from daily sampling of culture experiments for three SARS-CoV-2 variants (Lineage A, B.1.351, and B.1.617.2) cultured in VeroE6 C1008 cells (n = 33). Results: Intrahost single nucleotide variants were detected in 83% (19/23) of the mild cohort cases and 100% (16/16) of the severe cohort cases. SNP profiles remained relatively fixed over time, with an average of 1.66 SNPs gained or lost, and an average of 4.2 and 5.9 low frequency variants per patient were detected in severe and mild infection, respectively. sgRNA was detected in 100% (25/25) of the mild genomes and 92% (24/26) of the severe genomes. Total sgRNA expressed across all genes in the mild cohort was significantly higher than that of the severe cohort. Significantly higher expression levels were detected in the spike and the nucleocapsid genes. There was significantly less sgRNA detected in the culture dilutions than the clinical cohorts. Discussion and Conclusion: The positions and frequencies of iSNVs in the severe and mild infection cohorts were dynamic overtime, highlighting the importance of continual monitoring, particularly during community outbreaks where multiple SARS-CoV-2 variants may co-circulate. sgRNA levels can vary across patients and the overall level of sgRNA reads compared to genomic RNA can be less than 1%. The relative contribution of sgRNA to the severity of illness warrants further investigation given the level of variation between genomes. Further monitoring of sgRNAs will improve the understanding of SARS-CoV-2 evolution and the effectiveness of therapeutic and public health containment measures during the pandemic.

Molecular Epidemiology of AY.28 and AY.104 Delta Sub-lineages in Sri Lanka.

Background: The worst SARS-CoV-2 outbreak in Sri Lanka was due to the two Sri Lankan delta sub-lineages AY.28 and AY.104. We proceeded to further characterize the mutations and clinical disease severity of these two sub-lineages. Methods: 705 delta SARS-CoV-2 genomes sequenced by our laboratory from mid-May to November 2021 using Illumina and Oxford Nanopore were included in the analysis. The clinical disease severity of 440/705 individuals were further analyzed to determine if infection with either AY.28 or AY.104 was associated with more severe disease. Sub-genomic RNA (sg-RNA) expression was analyzed using periscope. Results: AY.28 was the dominant variant throughout the outbreak, accounting for 67.7% of infections during the peak of the outbreak. AY.28 had three lineage defining mutations in the spike protein: A222V (92.80%), A701S (88.06%), and A1078S (92.04%) and seven in the ORF1a: R24C, K634N, P1640L, A2994V, A3209V, V3718A, and T3750I. AY.104 was characterized by the high prevalence of T95I (90.81%) and T572L (65.01%) mutations in the spike protein and by the absence of P1640L (94.28%) in ORF1a with the presence of A1918V (98.58%) mutation. The mean sgRNA expression levels of ORF6 in AY.28 were significantly higher compared to AY.104 (p < 0.0001) and B.1.617.2 (p < 0.01). Also, ORF3a showed significantly higher sgRNA expression in AY.28 compared to AY.104 (p < 0.0001). There was no difference in the clinical disease severity or duration of hospitalization in individuals infected with these sub lineages. Conclusions: Therefore, AY.28 and AY.104 appear to have a fitness advantage over the parental delta variant (B.1.617.2), while AY.28 also had a higher expression of sg-RNA compared to other sub-lineages. The clinical implications of these should be further investigated.

Identification and functional-structural characterization of the enzymes linked with stable genome size increase of (+) RNA viruses

Nidovirales is an extraordinary order of complex positive-stranded RNA viruses including some of the largest RNA genomes (12-41 kb) among which notable human health burdens: SARS-CoV-1, SARS-CoV-2, MERS-CoV, etc. Recent advance in genome sequencing is slowly filling the gaps between and beyond the classified nidoviral families. Still, the research is lagging behind to understand the evolution of RNA genomes. For example, how are these large genome RNA viruses able to bypass the length and stability constraints of an RNA molecule? Is there any link between increasing length and gaining a functional domain or a special structural feature? To answer these questions, we started with database mining to extract novel nidoviral genomes and annotated different domains in polyproteins of classified and unclassified nidoviruses using HHpred and HHblits tools (Zimmermann L, et al. 2018). We observed a significant variation across the order regarding presence/absence, fold/structure type, co-factor (or enhancer) presence/absence, presence of one motif or the other and genome location of enzymes: Exonuclease (ExoN), N-7 Methyltransferase (MTase), 2'-O-MTase and RNA dependent RNA polymerase (RdRp). A trend seen with this bioinformatic analysis directly implies that stable RNA genome increase as well as maintenance is driven by the synergy of modifying enzymes: MTases, RNA proofreading by ExoNs and fast & processive RdRps (Ferron F, et al. 2021). Next, after their identification, we are trying to characterize these large RNA genome genetic markers: MTase(s) & ExoN, to have a comprehensive understanding of nidoviruses evolution. We have identified, expressed and purified a new nidoviral MTase from a Tobaniviridae family member, White Bream Virus (WBV). This enzyme is unique in terms of its location in ORF1a and not in ORF1b (Ferron F, et al. 2019). Functional and mutational studies show this new MTase to contain N-7 guanine specific, S-adenosyl-methionine (SAM) dependent capping activity (cap-0). Aligning with our predictions, structural characterization confirms that it has a Rossmann fold (RF) SAMdependent RNA-cap N7-guanine MTase. This study answers the missing link of capping activity in these members, which is somewhat only established for coronaviruses in this large genome order. Evaluating such enzymes is a step forward in the direction of fundamental understanding of how these RNA viruses are successfully expanding and maintaining their large genomes as well as coping up to fight against the host innate immunity.

SPECIFIC DETECTION and REPLICATION KINETICS of SARS-CoV-2 USING smRNA-FISH

Background: SARS-CoV-2 is a positive-sense single-stranded RNA virus and its replication begins after the synthesis of virally encoded polymerase complex that is required for replication and transcription of genomic RNA (gRNA) within the infected cells. Despite the global interest in the study of SARS-CoV-2, the kinetics of SARS-CoV2 RNA replication and transcription during the early phase of viral infection is poorly understood. Here, we used the single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) for sensitive detection of SARS-CoV-2 at single molecule level and to determine the replication of genomic RNA (gRNA) and sub-genomic RNA (sgRNA) in the infected cells, at very early stages of infection. Methods: We designed highly specific smRNA-FISH probes targeted to gRNA and Spike gene sgRNA of SARS-CoV-2 virus, using stellaris method and optimized the method to simultaneously visualize these two RNAs at single cell and single molecule level. Because of the high sensitivity of our probes, we applied smRNA-FISH technology to detect SARS-CoV-2 positive cells from autopsy samples obtained from diseased COVID-19 patients. Furthermore, we used high-resolution and high-speed scanning microscopy to detect extent of infection in cell models of SARS-CoV-2 and in COVID-19 patient samples. Results: A time course analysis SARS-CoV-2 replication indicated that single molecules of gRNA could be detected as little as 30 min to 2 hr. post-infection. Distinct "Replication Centers" (RC) began to appear one to two hours post-infection and the sgRNAs began to migrate out of these RCs. Replication after the initial delay appeared to be rapid and gRNA and sgRNAs dispersed throughout the cell within 4-5 hours post infection forming multiple RCs. We found that our RNA-FISH correctly detected the SARS-CoV-2 positive samples from patient autopsy samples that were characterized by qRT-PCR or immunological detection methods. The signals of spike gRNA and sgRNA along with the spike proteins co-localized within the same cells of the SARS-CoV-2 infected patients within the cells of lung, kidney, and heart autopsy samples. Conclusion: We propose that the specific probes and the methodology that we have developed will be highly applicable to the study of SARS-CoV-2 replication in depth and to characterize SARS-CoV-2 infection in COVID-19 patient samples. This study may open a novel direction towards COVID-19 pathophysiology, drug screening and diagnostics.

ORF1 REGION of SARS-C V-2 GENOMIC RNA AS A PROMISING TARGET for siRNA-BASED THERAPY

Background: A promising approach to tackle the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. However, it is unclear so far, which viral replication steps can be efficiently inhibited with siRNAs. Here we report the first-ever in-depth analysis of RNAi-accessible SARS-CoV-2 replication steps. Methods: siRNAs were designed against four genomic regions of SARS-CoV-2. Initial screening of siRNA activity was performed with a dual luciferase reporter assay. Efficacy of siRNAs to terminate various viral replication steps was analyzed by infecting VeroE6 cells with wildtype SARS-CoV-2 or a GFP expressing recombinant SARS-CoV-2 and monitoring viral spread in real-time by time-lapse fluorescence microscopy. Positive and negative sense viral RNA transcripts were distinctly quantified via sense specific cDNA synthesis and reverse-transcriptase quantitative PCR. Finally, the antiviral activity of the siRNAs was primarily evaluated in a highly relevant model, SARS-CoV-2 infected human lung explants. Results: When applied in a prophylactic fashion, siRNAs were able to target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, terminating replication before start of transcription, thereby preventing cytopathic effects. Surprisingly, siRNAs were not active against intermediate negative sense transcripts formed during replication. Targeting sequences that are commonly shared by all viral transcripts indeed allowed a simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. However, siRNAs that targeted ORF1 which is solely part of gRNA, presented an enhanced antiviral activity. We show that the reason for this was that siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs. Based on these findings, we developed a chemically stabilized siRNA, which targets a highly conserved region of ORF1, and which inhibited SARS-CoV-2 replication by >90% ex vivo in explants of the human lung. Conclusion: Our work strongly encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with targeting ORF1, might be key for high antiviral efficacy.

Molecular Methods in the Identification of Pulmonary Pathologic Traits of the SARS-CoV-2 Delta Variant at Autopsy

Background: The highly contagious Delta variant of COVID-19 accounts for more than 80% of SARS-CoV-2 cases in the fall of 2021. Our aim was to determine whether molecular methods for variant and lineage detection could be utilized at autopsy to examine pathologic findings of Delta variant as compared to non-Delta variant cases. Design: We evaluated the lungs from 20 decedents with death due to SARS-CoV-2 confirmed by antemortem nasopharyngeal RTPCR in July and August 2021 (Delta wave), as well as from 40 autopsy cases prior to February 2021 with death due to SARS-CoV- 2. The patient population included males and females, with an age range of 37-67 years in the Delta group, and 44-79 In the non- Delta group. The population demographic was considered at risk for death due to COVID-19, and only one decedent, with immunosuppression, was known to be vaccinated. Lung specimens were examined on H&E and with SARS-CoV-2 nucleocapsid immunostain (IHC). Results: The time from initial symptoms to death averaged 9 days within the Delta wave and 16 days in non-Delta cases. Steroids, anticoagulation, antibiotics, and monoclonal antibody infusion were frequently part of the clinical treatment of Delta wave cases. Notably, SARS-CoV-2 PCR of lung swabs at autopsy were positive in all but one case examined in the Delta variant group, and viral genome RNA sequencing from lung at autopsy confirmed Delta variant lineage. In both groups, gross features of the lungs included edema, while grossly identifiable thrombi were more commonly seen in non-Delta variant cases. Histologic examination revealed diffuse alveolar damage (DAD) in all cases, most commonly early stage DAD in Delta variant cases. SARS-CoV-2 IHC demonstrated patchy to strong positivity in the alveoli of the majority of Delta variant cases - a finding not frequently seen in non-Delta cases. Figure 1 - 15 Conclusions: Our study is the first to incorporate PCR and viral genome sequencing from the lung at autopsy to correlate the Delta variant wave with histopathologic findings - a technique that may be useful in identifying important pathologic features of future variants. While the finding of DAD remains the same across viral variants, the majority of Delta cases showed a significant presence of SARS-CoV-2 in the lung by IHC, with minimal inflammatory infiltrate and reduced thrombotic complication. Whether these findings are the result of a shorter time interval between disease onset and death, therapeutic intervention, or increased viral load remains to be determined.

Correlation of Clinical and Histopathological Findings with Viral Variant in the Gastrointestinal Tract of COVID-19 Autopsy Cases

Background: Initial evidence has shown the occasional presence of SARS-CoV-2 in enterocytes in the intestines of patients with COVID-19. Our aim is to further assess the clinical and pathologic changes in the gastrointestinal tract caused by the highly contagious Delta (B.1.617.2) variant as compared to viral variants originating earlier in the pandemic. Design: Intestinal samples from 32 patients with death due to COVID-19 were obtained at autopsy. Decedents were males and females, with an age range of 32-73 years. Twenty-one of the decedents self-identified as Black/African American, eight as Caucasian, and three as Hispanic. Two groups were differentiated by viral genome RNA sequencing from autopsy tissue: those with Delta variant (n=16), and those with non-Delta variant (n=16). SARS-CoV-2 expression in the intestine was evaluated by immunohistochemical (IHC) detection of the SARS-CoV-2 nucleocapsid protein (N-protein). Results: Clinically, the Delta group reported diarrhea more frequently (25%) as compared to the non-Delta group (6%). Patients in the Delta group had a shorter time interval between the onset of gastrointestinal symptoms and death (mean = 19 days), as compared to the non-Delta group (mean = 25 days). Histologic examination revealed mostly normal to mild, non-specific chronic inflammation within the epithelium and lamina propria in both groups. Macrophages with positivity for N-protein IHC were present beneath the epithelium, most notably within the Delta group. N-protein positivity occurred most frequently in small submucosal and mesenteric blood vessels. Patchy positivity for N-protein in enterocytes was seen frequently in cases of Delta variant in which the time from initial symptoms to death was short (<14 days). Figure 1 - 11 Conclusions: As in prior studies, intestinal microscopic changes in COVID-19 were minimal, though our findings indicate that SARS-CoV-2 may be detected within enterocytes more frequently in the Delta group. Patients with the Delta variant experienced both a higher rate of diarrhea and a shorter interval between gastrointestinal symptom onset and death. Whether increased Nprotein in enterocytes is a result of the Delta variant itself, or earlier intestinal sampling relative to symptoms in this group, remains to be determined. Autopsy studies can add to our understanding of the effects of COVID-19 on the digestive system, by allowing a greater volume of tissue sampling, as well as temporal sampling relative to disease onset that is not always possible at endoscopy.

Therapeutic approaches and vaccination in fighting COVID-19 infections: A review.

Coronavirus disease 2019 (COVID-19) is a remarkably contagious and pathogenic viral infection arising from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which first appeared in Wuhan, China. For the time being, COVID-19 is not treated with a specific therapy. The Food and Drug Administration (FDA) has approved Remdesivir as the first drug to treat COVID-19. However, many other therapeutic approaches are being investigated as possible treatments for COVID-19. As part of this review, we discussed the development of various drugs, their mechanism of action, and how they might be applied to different cases of COVID-19 patients. Furthermore, this review highlights an update in the emergence of new prophylactic or therapeutic vaccines against COVID-19. In addition to FDA or The World Health Organization (WHO) approved vaccines, we intended to incorporate the latest published data from phase III trials about different COVID-19 vaccines and provide clinical data released on the networks or peer-review journals.

SARS-CoV-2 Infection in Iranian People Living with Human Immunodeficiency Virus-1 Infection

Background: A novel Coronavirus first emerging in Wuhan, China, was named severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). The disease caused by SARS-CoV-2 is known as Coronavirus disease 2019 (COVID-19). HIV-1 infected individuals may be at risk of COVID-19. Objectives: This cross-sectional study evaluated the SARS-CoV-2 infection rate and COVID-19 prevalence among Iranian HIV-1-infected people. Methods: The study was conducted on 155 HIV-1-infected patients from June 2020 to October 2020. COVID-19 Ab (IgG) was detected using an enzyme immunoassay in serum specimens. Furthermore, nasopharyngeal and oropharyngeal specimens were collected. Then, the genomic RNA of SARS-CoV-2 was detected using a real-time polymerase chain reaction (RT-PCR). Clinical symptoms of the studied participants with and without COVID-19 were examined. Results: Of 155 HIV-1-infected individuals, 12 (7.7%) had positive real-time PCR results for SARS-CoV-2. Out of 12 (7.7%) patients with COVID-19, four (33.3%) were males. Anti-COVID Ab (IgG) was detected in 10 (6.5%) participants, of whom eight (80.0%) were males. The most common COVID-19 clinical symptoms, including dry cough, fever, runny nose, anosmia, and hypogeusia, were observed in seven (58.3%), five (41.7%), five (41.7%), five (41.7%), and five (41.7%) patients with COVID-19, respectively. Conclusions: A recent study has shown that the risk of SARS-CoV-2 infection in HIV-infected individuals is similar to that in the general population.

Molnupiravir Maintains Antiviral Activity Against SARS-CoV-2 Variants in Vitro and in Early Clinical Studies

Background. Molnupiravir (MOV, MK-4482, EIDD-2801) is an orally administered prodrug of N-hydroxycytidine (NHC, EIDD-1931), a nucleoside with broad antiviral activity against a range of RNA viruses. MOV acts by driving viral error catastrophe following its incorporation by the viral RdRp into the viral genome. Given its mechanism of action, MOV activity should not be affected by substitutions in the spike protein present in SARS-CoV-2 variants of concern which impact efficacy of therapeutic neutralizing antibodies and vaccine induced immunity. We characterized MOV activity against variants by assessing antiviral activity in vitro and virologic response from the Phase 2/3 clinical trials (MOVe-In, MOVe-Out) for treatment of COVID-19. Methods. MOV activity against several SARS-CoV-2 variants, was evaluated in an in vitro infection assay. Antiviral potency of NHC (IC50) was determined in Vero E6 cells infected with virus at MOI ~0.1 by monitoring CPE. Longitudinal SARSCoV-2 RNA viral load measures in participants enrolled in MOVe-In and MOVe-Out were analyzed based on SARS-CoV-2 genotype. Sequences of SARS-CoV-2 from study participants were amplified from nasal swabs by PCR and NGS was performed on samples with viral genome RNA of >22,000 copies/ml amplified by primers covering full length genome with Ion Torrent sequencing to identify clades represented in trial participants. SARS-CoV-2 clades were assigned using clade.nextstrain.org. Results. In vitro, NHC was equally effective against SARS-CoV-2 variants B.1.1.7 (20I), B.1351 (20H), and P1 (20J), compared with the original WA1 (19B) isolate. In clinical trials, no discernable difference was observed in magnitude of viral response measured by change from baseline in RNA titer over time across all clades represented including 20A through 20E and 20G to 20I. No participants at the time of the study presented with 20F, 20J, or 21A. Conclusion. Distribution of clades in participants in MOVe-In and MOVe-Out was representative of those circulating globally at the time of collection (Oct 2020 -Jan 2021). Both in vitro and clinical data suggest that spike protein substitutions do not impact antiviral activity of MOV and suggest its potential use for the treatment of SARS-CoV-2 variants.