Talimogene laherparepvec (T-VEC) is a modified oncolytic herpes Simplex virus, type 1 (HSV-1) encoding granulocyte-macrophage colony stimulating factor (GM-CSF). T-VEC is adapted for selective replication in melanoma cells and GM-CSF was expressed to augment host anti-tumor immunity. T-VEC is indicated for the local treatment of melanoma recurrent after primary surgery and is the first-in-class oncolytic virus to achieve approval by the FDA in 2015. This review will describe the progress made in advancing T-VEC to the most appropriate melanoma patients, expansion to patients with non-melanoma cancers and clinical trial results of T-VEC combination studies. Further, strategies to identify predictive biomarkers of therapeutic response to T-VEC will be discussed. Finally, a brief outline of high-priority future directions for investigation of T-VEC and other promising oncolytic viruses will set the stage for a best-in-class oncolytic virus to bring the maximum benefit of this emerging class of anti-cancer agents to patients with cancer. Oncolytic viruses (OV) are a new class of anti-cancer therapeutic agents that utilize native or genetically modified viruses to treat cancer. While early reports of tumor regression in patients infected with various viruses has been reports for over a century, advances in molecular genetics and virology have only recently allowed a more directed approach to therapeutic development in this area . Based oViruses are among the most immunogenic agents recognized by the host immune system and the ability of OVs to induce immune responses likely explains the major mechanism involved in OV-mediated anti-cancer activity . The indProgress in molecular biology and cloning technology have also allowed expression of eukaryotic genes by viruses. Genomic stability and expression levels are dependent on the size of the gene or genes expressed, the size of the viral genome, the impact on viral integrity and likely additional epigenetic factors, large viruses have been shown to efficiently encode multiple human genes, which can be used to provide additional anti-tumor activity. In many cases, the genes selected for expression are cytokines to enhance local immune responses against the virally infected cancer cells, other strategies have included expression of suicide genes, apoptosis-inducing genes, and radiosensitizers among others . The con®) is approved in combination with chemotherapy for treatment of head and neck cancers (®) is approved for the treatment of melanoma in several Eastern European countries (®), which was granted U.S. Food and Drug Administration (FDA) approval in 2015 for the treatment of melanoma  34.5 genes, which encodes the neurovirulence factor and deletion improves tumor cell selective replication. In addition, the viral ICP47 gene is deleted and this encodes a viral inhibitor of peptide attachment to major histocompatibility complex (MHC) class I, which the virus uses to prevent immune detection during natural infections. The ICP47 deletion was thought to be important in allowing MHC class I loading of tumor-associated peptides, which would be necessary to promote anti-tumor immunity. Finally, T-VEC is modified by inserting two copies of the human granulocyte-macrophage colony stimulating factor (GM-CSF) genes to promote dendritic cell recruitment and activation following antigen uptake from lysing tumor cells. The virus demonstrated therapeutic activity in the murine A20 lymphoma model and was adapted for clinical translation  that was originally isolated from a fever blister . The virnslation .6 plaque-forming units (pfu) to allow seroconversion in HSV-1-naïve patients, followed by a higher dose of 1 × 108 pfu. This was followed by a multi-institutional phase 2 single-arm study of T-VEC in patients with superficially accessible melanoma ] (p < .0001) was seen (p = .0494). The 5-year survival for patients was 33.4% and the impact on survival was most pronounced for patients with stage IIIB/C and IVM1a melanoma . In the OPTiM trial, 54% of patients exhibited some degree of disease progression based on caliper measurement or imaging prior to achieving an objective response, suggesting that pseudo-progression may be possible with T-VEC treatment. Based on these data T-VEC was approved by the U.S. FDA in 2015 for the local treatment of melanoma that recurs after initial surgery and T-VEC was approved in Europe in 2016 for the local treatment of melanoma patients with stage IIIB-IVM1a disease. Australia, Israel, and Switzerland have also approved T-VEC for the treatment of melanoma. The adverse events in the phase III clinical trial were similar to earlier phase studies establishing a favorable safety profile for T-VEC.Based on the emerging data from the early phase clinical trials, the OPTiM study, was developed as a prospective, multi-institutional randomized phase III clinical trial to determine the clinical benefit of T-VEC in patients with superficially accessible melanoma. In this study, 436 patients with stage IIIB-IV melanoma were randomized in a 2:1 manner to treatment with T-VEC or recombinant GM-CSF. The control arm was selected to allow study participants to receive potentially active therapy and at the time there was interest in single agent GM-CSF for melanoma, although this was not supported by subsequent studies . The stu= .051)] . At a mewas seen . FurtherT-VEC was the first OV approved for cancer treatment and provided a new therapeutic strategy for patients with melanoma. Importantly, the approval in 2015–2016 corresponded to a time with major changes in the therapeutic landscape of melanoma. In 2011 the first BRAF inhibitor was approved for metastatic melanoma patients with BRAF V600E/K mutated tumors, which would be followed by combination BRAF and MEK inhibition therapy . In addiThe launch of T-VEC was challenging for several reasons. First, OV storage and intratumoral delivery of the agent offered unique challenges for local pharmacies and healthcare providers, as outlined in In a retrospective review of T-VEC in patients with stage IIIB-IV1a melanoma at a single German facility, 27 patients were treated with T-VEC between June 2016 and July 2017 . All patA more recent real-world report was published on 127 patients in the national German prescription database . Of the In another single institution retrospective study of T-VEC, 27 patients with a median age of 75 years were treated and results reported at a median follow-up of 8.6 months . In thisIn a small study of 12 melanoma patients with a median age of 83 years, T-VEC was used and resulted in an overall response rate of 58.3%, durable response rate of 41.7% and a complete remission rate of 25% . In thisIn a subset analysis of the OPTiM clinical trial, a higher response rate was noted in patients with head and neck melanomas . Of the Another subset of melanoma patients that were not treated in the OPTiM trial are patients with organ allografts. This represents an important unmet medical need as malignancy is more common in transplant recipients with an increased incidence over time and cutaneous tumors, including melanoma are especially common. Because of the risk of allograft rejection, treatment with potent immunotherapy, such as immune checkpoint blockade, may not be possible. Thus, the potential benefit of OV therapy in this setting has gained recent attention. Indeed, there are two case reports demonstrating complete responses of locally advanced melanoma with T-VEC treatment in transplant recipients, one with a heart transplant and one with a heart and kidney transplant . In bothin situ vaccination at an earlier time prior to extensive immunoediting as occurs in metastatic disease. A randomized phase 2 trial was conducted in 150 patients with resectable stage IIIB-IVM1a melanoma . The reasons for the lack of benefit are not entirely clear as the final data has not yet been published. It is possible that the response rate of pembrolizumab alone in stage IIIB-IVM1a melanoma is higher than in stage IV disease and the study was not adequately powered to detect a narrower response difference between arms. Another difference between the phase I and III trial was that in the phase I study pembrolizumab was started after the second injection of T-VEC to allow seroconversion for HSV-naïve patients and avoid rapid vial clearance by enhanced anti-viral immune responses mediated by pembrolizumab. In the phase III trial, however, no T-VEC lead in was employed and both drugs were given on the first day. Further scrutiny of the data may be needed to better understand why this study was negative.T-VEC infection triggers type 1 interferon production by infected cells, and this in turn can result in expression of immune inhibitory surface receptors, such as PD-1 ligand 1 (PD-L1) on tumor cells . Even whInterestingly, in another phase I study T-VEC was combined with ipilimumab, an anti-CTLA-4 monoclonal antibody in patients with advanced melanoma who were immune checkpoint inhibitor naïve . In thisPredictive biomarkers of immunotherapy response have been important for better identifying patient populations likely to respond to treatment. For immune checkpoint blockade, several biomarkers are now recognized as clinically important, including a high tumor mutation burden, elevated local tumor PD-L1 expression, presence of tumor-infiltrating lymphocytes, and a high interferon gene expression pattern, all of which are associated with improved therapeutic responses . Biomarkin vitro. These data suggest that JAK1 and JAK2 expression may be an important biomarker of OV activity but clinical validation is still required. In our lab we also found that loss of STING expression, a known biomarker for anti-PD-1 resistance, was associated with improved oncolytic activity of T-VEC in vitro  remains a significant clinical challenge and recent evidence that oncolytic HSV-1 (teserpaturev) has activity in glioblastoma suggests that T-VEC could be considered for treating CNS melanoma. Direct access to the CNS for bimonthly injections remains a logistical challenge but further clinical studies may be warranted. Further studies are needed to confirm a role for T-VEC in the neoadjuvant setting and for other cancers. While studies of combination treatment with immune checkpoint inhibitors have been contradictory, other therapeutic combinations await clinical validation, including combinations of T-VEC with radiation therapy, targeted therapy, chemotherapy, and adoptive T cell therapy. Predictive biomarkers are also needed and early work suggests that elements of the intracellular anti-viral machinery may be important predictors of OV sensitivity and merit further evaluation. As techniques for single cell genomic analysis have matured, this will provide assays to interrogate tumor cells Oncolytic viruses represent a new class of cancer therapeutics that have, thus far, resulted in limited approvals for cancer. Newer viruses with more rationally designed transgene payloads, coupled with a better understanding of the underlying biology, should lead to new approvals and best-in-class agents across a range of tumor types and clinical indications. Further studies to explore T-VEC injection of visceral lesions as well as determining the risks and benefits of intravenous delivery are needed. What is established is the tolerable safety profile of T-VEC and other OVs in clinical development with most exhibiting similar low grade and short duration constitutional and local injection site reactions. The safety profile may allow better patient acceptance and expansion of OVs into more immunologically sound combination clinical trials for patients with cancer.


Suppressive tumor microenvironment is closely related to the progression and poor prognosis of lung adenocarcinoma (LUAD). Novel individual and universal immune-related biomarkers to predict the prognosis and immune landscape of LUAD patients are urgently needed. Two-gene pairing patterns could integrate and utilize various gene expression data.The RNA-seq and relevant clinicopathological data of the LUAD project from the TCGA and well-known immune-related genes list from the ImmPort database were obtained. Co-expression analysis followed by an analysis of variance was performed to identify differentially expressed immune-related lncRNA (irlncRNA) (DEirlncRNA) between tumor and normal tissues. Two arbitrary DEirlncRNAs (DEirlncRNAs pair) in a tumor sample underwent pairwise comparison to generate a score (0 or 1). Next, Univariate analysis, Lasso regression and Multivariate analysis were used to screen survival-related DEirlncRNAs pairs and construct a prognostic model. The Acak information standard (AIC) values of the receiver operating characteristic (ROC) curve for 3 years are calculated to determine the cut-off point for high- or low-risk score. Finally, we evaluated the relationship between the risk score and overall survival, clinicopathological features, immune landscape, and chemotherapy efficacy.Data of 54 normal and 497 tumor samples of LUAD were enrolled. After a strict screening process, 15 survival-independent-related DEirlncRNA pairs were integrated to construct a prognostic model. The AUC value of the 3-year ROC curve was 0.828. Kaplan–Meier analysis showed that patients with low risk lived longer than patients with high risk (p <0.001). Univariate and Multivariate Cox analysis suggested that the risk score was an independent factor of survival. The risk score was negatively associated with most tumor-infiltrating immune cells, immune score, and microenvironment scores. The low-risk group was correlated with increased expression of ICOS. The high-risk group had a connection with lower half inhibitory centration (IC50) of most chemotherapy drugs  and targeted medicine—erlotinib, but with higher IC50 of methotrexate.The established irlncRNA pairs-based model is a promising prognostic signature for LUAD patients. Furthermore, the prognostic signature has great potential in the evaluation of tumor immune landscape and guiding individualized treatment regimens. Globally, lung cancer remains the main cause of cancer death . Lung adLong noncoding RNAs , a crucial class of pervasive genes playing a variety of cellular and physiologic functions, are known to be related to tumorigenesis and metastasis . LncRNAsAn inspiring research developed and validated an individualized immune prognostic signature for lung cancer using a strategy of immune-related gene (irgene) pairing in each sample. This gene-pairing strategy left out the normalization of data from diverse platforms .In the present study, we retrieved irlncRNA data of LUAD patients from the Cancer Genome Atlas (TCGA) to establish and verify an individualized and multiple-data applicable prediction model for LUAD by applying the irlncRNA-pairing strategy. Furthermore, we investigated the value of the prognostic model in evaluating the immune landscape and prediction of effects of chemotherapy and targeted therapy.The RNA-seq and matched clinicopathologic data of LUAD were downloaded from the TCGA database. The mRNAs and lncRNAs were distinguished by annotation files from the Ensembl databank for subsequent analysis. The list of confirmed immune-related genes (irgenes) was downloaded from the ImmPort databank. Immune-related lncRNA (IrlncRNAs) were acquired through co-expression analysis between irgenes and lncRNAs with correlation coefficients >0.4 and the p-value <0.001 as thresholds. The “limma” R package was used to discriminate the differential expressed irlncRNAs (DEirlncRNA) between tumor and normal tissues with the thresholds set as log fold change (logFC) >1 and false discovery rate (FDR) <0.05. Patients lacking clinicopathological data and those with survival data <30 days (who may die of other diseases rather than LUAD) were excluded.The DEirlncRNAs in each tumor sample were randomly paired. The score of DEirlncRNAs pair (DEirlncRNA 1/DEirlncRNA 2) was assigned to be 1 if the expression of DEirlncRNA 1 was more than DEirlncRNA 2; otherwise, the DEirlncRNAs pair score was 0 . TherefoFor screening alternative DEirlncRNA pairs to construct a prognostic model, we first performed a Univariate Cox proportional hazard analysis to screen survival-related DEirlncRNA pairs. Then, a 10-fold cross-validation Lasso regression  was perfFor the validation of the clinical significance of the constructed model, chi-square test was used to explore the relationship between risk score and clinical data. The band diagram was plotted for visualization . We performed Wilcoxon signed-rank test to show the risk score differences among various groups divided by clinical characteristics, which were shown by the box diagram. To confirm whether the risk score can be used as an independent risk-stratification factor, Univariate and Multivariate Cox regression analyses were conducted between the risk score and clinical features. Forest maps were utilized to display the results. These procedures were utilized by the R packages, namely, Survival and pHeatmap.First, to investigate the association between the risk score and immune cells in the tumor microenvironment, we estimated the infiltrating-immune cells among the samples by acknowledged algorithms, namely, CIBERSORT , CIBERSOTo evaluate the value of the signature in the LUAD treatment efficacy prediction, we counted the IC50 of common chemotherapy and molecular targeted drugs for each sample using pRRophetic  . AntitumA total of 457 cancer cases with survival time >30 days and matched clinicopathological features (except for M stage for 26.5% of them were missing or unknown) from the TCGA was included for the following analysis. Using an iteration loop and a 0-or-1 matrix screening among 160 DEirlncRNAs, 9,931 valid DEirlncRNA pairs were identified. After a Univariate Cox proportional hazard analysis, 260 DEirlncRNA pairs were extracted. Then a modified Lasso regression analysis was utilized to prevent overfitting and screened to 45 DEirlncRNA pairs, followed by a Multivariate Cox proportional hazards analysis, and 15 of them were incorporated into the prognostic model based on step-by-step approach. . A breakthrough has been made in immunotherapy (cell adoptive therapy and immune checkpoint blocking therapy). We subsequently explored whether the prognostic signature based on irlncRNAs pairs had a relation to the tumor immune landscape. Results showed that most immune cells in tumor microenvironment including CD8gorithms L calculIn addition to immune checkpoint blockades therapy, we tried to explore whether there were associations between risk score and the sensibility of LUAD patients to the common chemotherapeutics and molecular targeted therapy. Results showed that risk score was negatively related to IC50 of chemotherapy drugs such as etoposide (p = 0.0098), paclitaxel (p <0.0001), vinorelbine (p = 0.017), gemcitabine (p = 0.041), and docetaxel (p <0.0001), whereas it was positively associated with IC50 of methotrexate (p <0.0001), which suggested that the model possessed great potential in predicting chemotherapeutic sensitivity K. In adLung cancer is still the most afflicting cancer in the world and the 5-year survival rate of lung cancer is only 10–20% in many countries  and chemPrognostic signatures associated with the tumor immune landscape possess great potential in recognizing new molecular biomarkers and ameliorating patient management . Our pro+ T cells, CD8+ T cells, B cells, and dendritic cells, which was consistent with previous studies :2019ZX09301001, and the Ministry of Science and Technology of the People’s Republic of China (CN):2020YFC0844300. The research sponsors did not participate in the study design, data collection, analysis and interpretation, and they were not involved in the writing of the manuscript and the decision to submit the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SARS-CoV-2 functionality by explorative interpretation of sequence alignments. Centrality analysis of the Sgp dissects the importance of these residues in the interaction network of the RBD-ACE2 (receptor-binding domain) complex and furin cleavage site. Correspondence of RBD to threonine500 and asparagine501 and furin cleavage site to glutamine675, glutamine677, threonine678, and alanine684 was observed; all residues are exactly located at the interaction interfaces. The harmonious location of residues dictates the RBD binding property and the flexibility, hydrophobicity, and accessibility of the furin cleavage site. These species-specific residues can be assumed as real targets of evolution, while other substitutions tend to support them. Moreover, all these residues are parts of experimentally identified epitopes. Therefore, their substitution may affect vaccine efficacy. Higher rate of RBD maintenance than furin cleavage site was predicted. The accumulation of substitutions reinforces the probability of the multi-host circulation of the virus and emphasizes the enduring evolutionary events.Spike glycoprotein (Sgp) is liable for binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the host receptors. Since Sgp is the main target for vaccine and drug designing, elucidating its mutation pattern could help in this regard. This study is aimed at investigating the correspondence of specific residues to the Sgp It has emerged in Wuhan, China causing the ongoing outbreak of COVID-2019 (coronavirus disease of 2019)2.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the new member of beta coronaviruses3, existing-drug repurposing6, novel drug development9, and utilizing the plant-derived components10, the most promising way out of the pandemic seems to be vaccine development11. Vaccines should induce a long-lasting memory with minimal side effects. Such a vaccine candidate demands a careful selection of epitopes from the existing repertoire of the viral determinants12.Despite many suggestions and efforts such as social distancing13. Spike is a surface glycoprotein (~ 1300 amino acids) with vital roles in the pathogenicity of SARS-CoV-2. Receptor  binding, proteolytic activation of Sgp, and deliverance of the conserved fusion peptide into the host cell membranes are pre-internalization events mediated by the spike. At each step, the congregation of mechanisms and strategies are progressed16. Collectively, the virus entry into the host cells demands a splendid choreography of multifaceted pre-infection events17.The most focal candidate for vaccine design is spike glycoprotein (Spg)19, which is highly pronounced in the RNA viruses due to their unstable genome20. In such situations, harnessing fast and reliable approaches that could predict emerging mutations are highly amenable. Several groups have attempted to distinguish the antigenic determinant of the Sgp. On the other hand, genomic data from all over the world evidenced a clonal and rapid in-human evolution of the SARS-CoV-223. Various substitutions are continuously reported in the spike sequence24. This flexibility of the coronavirus genome warns about a great risk of infection severity and also foretells the possibility of vaccine26 or therapeutics27 failure.One of the main obstacles facing the vaccine design is antigenic drift28 including South African 501Y.V2; B.1.35129 (beta variant), Brazilian 501Y.v3; P.1 (gamma variant)30. The variant is more transmissible and has been estimated to have a growth rate of 40 to 70%31. The N501Y governs an increasing receptor affinity32, which accents the eminence of special mutations at certain positions.Although mutations in any open reading frame of the virus genome could have implications on the severity or transmissibility of SARS-CoV-2, the insertions, deletions, and certain substitutions in the spike sequence could be of major concern. Examples of such substitutions include the dominant variant identified in the United Kingdom, known as B.1.1.7 . This variant holds mutation of N501Y; this mutation is also found in other variants of concern (VOCs)33. This protein’s holistic property is tightly entitled by its amino acid composition, which further dictates the secondary and tertiary structures and subsequently the function of the protein, which is subjected to natural selection. However, a selective constraint on a single site of a given protein can be interpreted in the context of its other building blocks. Since any substitution may affect the rest of the protein, the first changes may be affected again subsequent to the modifications, leading to a complicated web of reaction loops; which is indicative of a tangled bank of amino acid interactions. This issue introduced the phenomenon of evolutionary “stokes shift”; in which a protein as a whole entity, tends to make the resident amino acid(s) gradually stable35. Although the differences between emerging sequences and homologs are obvious and easy to spot through sequence comparisons, it would be appealing to define the corresponding residues and to inspect their substitutions. The corresponding residues make a target sequence odd and have key roles in the sequence function or are likely the main targets of evolution. We hypothesized that these sorts of substitutions are unique characteristics of proteins. Additionally, these residues may play an important role in the web of interactions in the protein; such substitutions might more effectively come into play in the way the SgpSARS-CoV-2 behaves. This dramatically shapes the queries on how these amino acid substitutions are associated with the eccentric behavior of emerging sequences; more importantly, whether these substitutions are going to be stable or tend to be modified.Overall, Sgp -similar to other proteins- is a critical combination of a complex web of ionic interactions, hydrophobic interactions, hydrogen bonds, and many other factors36. The aim of this study was investigating the correspondence of specific residues to the sequence of the SgpSARS-CoV-2 by featuring the corresponding residues in the sets of aligned sequences. These data were complemented by the structural data to better grasp the importance of singled-out residues. The RBD and furin cleavage site were mainly focused here owing to their importance37. The study further discusses how these residual changes shape some critical traits of the SgpSARS-CoV-2.The corresponding residues can be singled out through sequence alignment by principal component analysisSARS-CoV-2, a 1273 amino acid long sequence, is divided into five distinct domains as shown in Supplementary Table Sgp–4. A preliminary review of the libraries showed that the libraries of RBD and N-terminal domain (NTD) were mostly occupied by beta coronaviruses, while other libraries contain more divergent members. Clustering experiments—in the following section—will better assess this issue.To better focus on domains of the protein, the sequences of the divided domains were searched against the databases separately. The search results were used to build the non-redundant libraries of sequences. Each library included sequences of similar length and e-value lower than 10The disparity index test showed a homogenous pattern of substitution for all datasets (data not shown); therefore, all sequences were retained for further evaluations and considered suitable for alignment approaches.SARS-CoV-2.To define the relationship between sequences, each library was clustered based on the strength of their all-against-all pairwise sequence similarities. The network-based clustering approach also identified the closely related sequences and divided them into separate groups. Members of the sequence libraries in this section belong to coronaviruses excluding the SARS-CoV-2 (the limitation strategy of BLAST). The sequence collections are uniform in length and are the result of HMM profiling by querying the SgpAlpha, beta, gamma, delta (if existed), and unclassified (UC) sequences formed completely separate clusters Fig. .Figure 1As illustrated in Fig. SARS-COV-2 and the other homologous sequences. Contrary to the N terminal, the C-terminal segments including CH, CR1, and CR2 involved virtually all groups of coronaviruses suggesting that these are the general determinants of spike , a correspondence analysis was performed. This analysis provides a lower-dimensional representation of the alignment data in a scatterplot. The most striking observation that emerged from correspondence analysis was the dependencies of major domains  to a few residues Table . The majA couple of corresponding sites were identified in the RBD domain viz. Thr500 and Asn501 and occurred in 45.9% and 7% of the MSA, respectively Fig. . These t38. Sequence bundle visualization of MSAs allowed us to extrapolate the harmonious location of the residues in these sites.Herein, as well as two aforesaid positions, two other positions, namely 486 (Gln) and 493 (Phe), were evaluated, because they are all involved in receptor-ligand interaction. These are relatively variable sites . In our dataset, the sequences containing Asn at the same position include A0A023PTS3, A0A023PUW9, A0A2D1PXC0, U5WHZ7, and U5WLJ7. The striking observation is that all these sequences belong to the viruses that are hosted by Rhinolophus affinis (intermediate horseshoe bat).The position of Asn501 in SgpSARS-CoV-2), position 8 (493 SgpSARS-CoV-2), and position 16 (501 SgpSARS-CoV-2)  (Supplementary Data SARS-CoV-2), (493 SgpSARS-CoV-2), and (501 SgpSARS-CoV-2)] with the remaining residues. Additionally, the Mann–Whitney U test was used to investigate the differences between those three focused residues of RBM  and the remaining parts of RBM or the remaining parts of RBD. The results suggest that the distribution of all ten parameters was the same in RBM. Further, for the three focused residues, only similarity and identity scores differed significantly  and the remaining part of RBD. The results showed that the distribution of flexibility, ASA, CX, Average Bfactor, identity scores, and similarity scores were not the same in the two populations . The core region is occupied by positively charged residues, and the flanking regions are more flexible and surface-accessible residues. The furin binding site significantly corresponds to Gln675, Gln677, Thr678, and Ala684  and the proprotein convertase (furin) were docked. The resulted complex was used for centrality analysis. This complementary approach examined whether or not the corresponding residues attain significant centrality Z-score in the complex form. The results .48. Among many substitutions that happen in a protein, few mutations are critical and are actual targets of evolution. A simple and fast method to find these sorts of substitutions might be helpful to understand the nature of emerging diseases, developing novel vaccines, and explaining the behavior of progressive virulence factors. In emerging viruses, the amino acid changes would drastically affect the sensitivity of protein to certain neutralizing antibodies or cause a vaccine or therapeutic failure49. Understanding the rate and positions of the mutations and even predicting the stability of certain parts of the protein is important for vaccine design, planning any therapeutic approach, and studying the nature of the emerging sequence. Furthermore, it is critical to define how these substitutions shape the novel traits of the emerging pathogen. Among pathogens, the genome adaptability of RNA viruses makes them more susceptible to jump to new hosts50. The spillover and transmissibility in these cases usually depend on the existence of virus receptor(s) on the host cells51; the more the receptor is conserved among different target species, the higher the virus is anticipated to spread. The recent examples within the last two decades are members of coronaviruses causing the SARS and the Middle East Respiratory Syndrome (MERS), and more recently SARS-CoV-252.Amino acid changes can range from biochemically similar (conservative substitutions), to dramatically dissimilar amino acids 53. This paper appraised the alignment method along with principal component analyses  to describe the dependencies of small segments of SgpSARS-CoV-2 to specific residues. The work also attempted to predict whether these substitutions would be stable or tend to be modified.The interactions of residues in a protein and protein–protein interface rule the maintenance of new substitutions, highlighting the existence of great harmony in the whole protein. In a MSA, the single sites include relatively less information than the entire MSA. Therefore, large and diverse datasets are required for detecting critical sites54. This prevailing role has propelled the Spg to the forefront of the coronavirus infection investigations, vaccine design, and arrangement of therapeutic plans. While small modifications on RBD may significantly alter the host selection theme of the virus55, it has dramatically shaped queries on these little differences. Moreover, maintenance or changeable prophecy of the protein segments would allow us to select stable and effective epitopes for vaccine design or drug targets56.The multi-domain Spg is likely the most important determinant of coronaviruses, because it is responsible for the multi-step process of host recognition and tissue tropism36 argument in which the authors attributed the important role of human–human transition or host range determination to this residue. The authors especially highlighted the role of the side chain36. Additionally, significant differences in the Z-scores of centrality between free RBD and RBD-ACE2 complex confirmed the importance of this residue and the context wherein this site is involved. The more obvious evidence involves the alpha variant of SARS-CoV-2, in which the substitution of Asn501 with Tyr501 made the virus dominant14.Two adjacent Thr500 and Asn501 amino acids are the corresponding sites of RBD. Asn501 contains a significant Z-score of centrality, when the molecule is in complex with its ligand (whereas a free molecule does not obtain a significant Z-score), emphasizing the important role of this residue and context dependencies. These two amino acids are in the C-terminal proximity of RBM and are involved in the receptor-binding interface. Ascribing a central role for Asn501 corroborates the Li et al.14. In SgpSARS-CoV-2, six out of fourteen residues are semiconservative compared to SARS-CoV: N439SARS-CoV-2 (R426SARS-CoV), L455SARS-CoV-2 (Y442SARS-CoV), F486SARS-CoV-2 (L472SARS-CoV), Q493SARS-CoV-2 (N479SARS-CoV), Q498SARS-CoV-2 (Y484SARS-CoV), and N501SARS-CoV-2 (T487SARS-CoV)57. The sequence data herein suggests that the RBD  is species-specific. Moreover, it appears that the presence of semi-conservative residues, which are the differed parts among previous beta coronaviruses, could have important roles and most likely has an influence on host range determination, tissue tropism, and the current rapid SARS-CoV-2 transmission in humans. Our focus on three hotspots of RBM, namely F486SARS-CoV-2, Q493SARS-CoV-2, and N501SARS-CoV-2, surmises the overall conserved physicochemical properties of RBM, caused by the collaboration of specific residues, leading to a successful viral attachment and cell entry.RBM is the receptor-binding region, isolated from the edge of RBD, with specific sites entangled in ACE2. Presenting the alignments as sequence bundles, which is a sequence-oriented technique, unveiled some hidden properties of the sequences. Our primary assumption for interpreting the conservancy features of RBD and especially RBM was that the less conserved residues would be more effectively involved in the host range determination of the virus, because conserved residues are present in all strains and may not centrally affect the jumping or the specific nature of the viral infection. Together with the role of mutation at a specific site, the effect of the amino acid coalition should be considered when discussing the protein properties. Fourteen positions in RBM are the key residues for binding of SARS-CoV to human ACE251 and demands maintaining harmony between certain residues of the Spg; it is an indication of a complex tangled bank of amino acid interactions. The full functionality of a protein requires maintaining a balance between the physicochemical properties of major amino acids.The viral entry into susceptible host cells is a complex process58. Based on these observations, Li et al.36 concluded that the side chain of the residue at 487 is a key factor for shaping severity (and likely human-to-human transmission)59. These positions in SgpSARS-CoV-2 are replaced by Q493 and N501, respectively. The coexistence of amino acids with specific physicochemical properties could be a marker of the harmonious interaction of residues in this specific region. Therefore, the binding properties of RBM could be more complicated than has been thought earlier.Viruses extracted from the sporadic SARS cases, during 2003–2004, all had asparagine at position 479 and serine at position 487; each virus was an independent cross-species event without the human-to-human transmission61, packing density, and flexibility64. Surprisingly, regarding the increased levels of surface accessibility and flexibility in association with a decreased level of contact density of RBM in comparison with the remaining parts of RBD, it can be concluded that RBM has a greater evolutionary rate. Therefore, it is evident that the harmonious interaction of residues goes far beyond a small motif. While the evolutionary rate of RBM is higher than the remaining part of RBD, it can be finally concluded that the residues in RBM are targeted by evolution, and other parts tend to preserve these substitutions.Similarity and identity scoring strategies reveal the existence of many substitutions in RBD (mostly conservative). The corresponding residues of RBD were found as parts of RBM in the alignment set. Evolutionary variation among different sites depends on various physicochemical properties of the amino acids including surface accessibility66. The modifications along with increase the affinity of SgpSARS-CoV-2 to the ACE2 receptor, cause failure of S gene targeting by molecular diagnostics; an example includes Thermo Fisher TaqPath COVID-19 assay15.Most VOCs are carrying substitutions in the 501 positions. For example, an emerging UK variant: B.1.1.7 harbors an N501Y mutation which increases the interaction of spike with ACE2 receptorIt is worth mentioning that this position is focused in our study and was defined by correspondence analysis including previous coronavirus sequences, which strongly highlights the usefulness and efficacy of our method.67. The successful completion of viral life spam highly depends on the host elements. In the case of coronaviruses and SARS-CoV-2, the cleavage of the spike by host proteases is important in the infectivity and host range modulation68. For instance, a study on MERS-CoV strengthened the concept that along with the virus receptor, the repertoire of proteases expressed by a given cell type, could significantly affect the infectivity69. Activation of the spike is a crucial step of the infection and depends on the host’s furin activity70. For example, despite the ability of MERS-CoV-related bat coronavirus, HKU4, to recognize the human receptor-dipeptidyl peptidase 4, the activation of this virus does not happen in humans, since the process demands additional exogenous trypsin15. Furthermore, the presence of glycan near the S1/S2 boundary may completely abolish the proteolytic priming of the virus71. Cleavage at different sites can occur in a different lifestyle of the virus during biosynthesis or virus entry; whenever it happens, it can critically affect the cell and tissue tropism as well as host range determination39. SgpSARS-CoV-2 harbors a furin cleavage site at the S1/S2 boundary, which is treated during biosynthesis14.Not only the amino acids of a protein but also hosts and viruses are in a tangled bank of interactions38. Exploring the first release of SgpSARS-CoV-2 sequence data, at the first stages of the COVID-19 pandemic, evidenced a four residue insertion at the S1 and S2 boundary in comparison with other SARS coronaviruses39. Indeed, we examined this region as a broader motif of 20 amino acids.Furin cleavage site is known as a consensus pattern of R-X-[K/R]-R⇓ (where X is any amino acid). However, all furin cleavage sites do not follow this pattern38. Their seminal work also mentioned the conservancy of the physical property of this motif among mammals, bacteria, and viruses14. The motif was defined as a core region (P6–P2′) that fits into the catalytic pocket of furin and two flanking flexible solvent-accessible regions (P7–P14 and P3′–P6′). The core region determines the binding strength of the enzyme and its substrate; while the flanking regions provide the core region accessibility to furin. The alteration of residues in this motif would drastically affect the efficiency of furin cleavage39. It also may affect viral expansion, cell and tissue tropism, transmissibility, and pathogenicity72.An evolutionary conserved 20 amino acid motif could better describe the furin cleavage site as explained by Tian et al.47. Our data showed that all modified properties are in favor of furin cleavage activity. It is worth mentioning that these differences are derived from exchanging the conserved residues  in the SgpSARS-CoV-2 sequence along with the insertion of a short peptide. Similar to RBD, it could be hypothesized that these radical substitutions are targets of evolution, and other sets of substitutions are present for retaining these sites.In the furin cleavage pocket, the balance maintenance between hydrophobicity and hydrophilicity is a fascinating characteristic of viral fusion proteins73. Additionally, organisms with a small effective population size tend to accumulate more radical substitutions than those with larger effective population size and more efficient natural selection74. Although the currently available database did not provide adequate information to trace a positive or a negative selection, it is not possible to predict the fate of the spike protein. Nevertheless, regarding the huge effective population size of the virus, the accumulation of conservative substitutions is expectable. Therefore, the modification of the furin cleavage site is more likely to happen, and the maintenance of RBD in the current composition is presumable. Moreover, different sites of the protein may face diverse environmental contexts, thus might have a dissimilar evolutionary fate. This assumption is consistent with our findings on the sequence diversity of SgpSARS-CoV-2. Since the C-terminal of SgpSARS-CoV-2 is located in a relatively constant microenvironment , a low diversity level can be observed in these segments.Radical substitutions are more probable to be chosen against conservative substitutions75, the mutations are reduced in this family including SARS-CoV-2, relative to other RNA viruses. However, as many research groups are continuously monitoring the genomic diversity of SARS-CoV-210, many mutations are indeed reported. The emergence of mutations in the Spg as the most antigenic determinant of coronaviruses, would cause antigenic drift and subsequently vaccine or drug stagnation. Previous research has demonstrated the altered capacity of some neutralizing antibodies against SgpSARS-CoV-2 due to the recent mutations. None of the discussed residues in the present study was included in the set of evaluated mutations76. Furthermore, surveying the genomic database (https://www.gisaid.org/epiflu-applications/phylodynamics/) of SARS-CoV-2 revealed that more mutations are accumulated in the furin cleavage site (and its vicinity) than RBD, which confirms our assumption of maintenance and modification probability of RBD and furin cleavage site, respectively.Due to the proof-reading properties of RNA polymerases of coronaviruses77 for inhibiting critical enzymes of the virus78 or other non-structural components through molecular docking or molecular dynamic simulations80.This paper shows how sequence-based computational approaches could be applied solely to extrapolate important features of an emerging sequence prior to availability of more complex costly structural information. The most prominent feature of this study is the data presentation, especially the visualization of the MSAs as sequence bundles. Dissemination of sequence data and coupling these observations with structural information manifests the usefulness of in silico tools to delve into important features of emerging virulence agents. Moreover, in silico tools hold great potential of screening bioactive componentsSARS-CoV-2 could be a signature of long-lasting evolution. Given these enduring events, it could be hypothesized that the coronavirus has been confronted with different environmental contexts and thus, faced different evolutionary pressures. It is plausible for further studies to be focused on this assumption.A wide research ground is provided here for future studies to describe the dynamic and energetic features of sequence modifications and manifest the role of other nearby residues and their implications in the protein architecture as a whole. In this regard, the accumulation of various substitutions that occurred in SgpThe slight differences of SARS-CoV-2 with its close relatives, shape its distinguished characteristics that are responsible for the easy spread of the virus and its spillover. Within many residue substitutions, a few belonging to RBM and furin cleavage motif, were shown to be correlated with the corresponding domains. Our results implicated that singled-out residues may be the real targets of evolution and other substitutions tend to maintain these resident amino acids at certain positions. Residues in the consortium are responsible for explicit features of RBD and furin cleavage motif. The location of amino acids in certain positions revealed a tangled bank of amino acid interaction web. The compensatory role of amino acids may explain this harmonic localization. While the identified residues are parts of experimentally identified epitopes, it should be pinpointed that antibodies or vaccines that target the mentioned residues would remain effective.While the initial molecular information on emerging pathogens mostly includes the sequence data, the methods that rely on sequences could be the most helpful approaches. This paper illustrates how sequence-oriented techniques and visualization approaches together can be drastically helpful for the interpretation of existing facts prior to the release of structural information obtained through more complicated and costly experiments. Many human pandemics have been rooted in host shifting. Introducing a fast and reliable approach to describe the emerging sequences will help us to tackle them and to discover effective medications and vaccinations.SARS-CoV-2 and its homologous sequences were obtained from the Uniprot Knowledge Base (UniprotKB)79 at www.uniprot.org .All sequences including the Sgp81 was surveyed to extract the experimentally defined and validated linear and conformational epitopes of SgpSARS-CoV-2.The Immune Epitope Database (IEDB)www.ebi.ac.uk. HMMER profiling simultaneously defines the domains on the protein sequence and collects homologous sequences from several optional databases. The database used for building the profile was the UniprotKB82. The data on the domains of SgpSARS-CoV-2 were retrospectively collected from the available literature82 and automatic annotation of UniProtKB (www.uniprot.org). Following this procedure, major domains were defined and were separately searched against UniprotKB (www.uniprot.org) for collecting sequences similar to each domain.Similar sequences were collected by hidden Markov model profiling by HMMER software tool as provided by 83 was performed on all datasets to estimate the probability of rejecting the null hypothesis of substitution pattern heterogeneity. The judgment was stemmed from the extent of composition biases between the sequences. A Monte Carlo test with 500 replicates was employed for estimating the p-values84. The p-values lower than 0.05 were considered significant. The disparity index test was performed by the MEGAX software tool85 for each dataset separately.The disparity index testhttps://toolkit.tuebingen.mpg.de/tools/clans)84, at a p-value of 10–3 and at least 1000 repulsions to avoid collapsing the nodes. The pairwise similarities were visualized in a graph by the CLANS stand-alone java application. The resulting CLANS files were further clustered by the network-based clustering function of the CLANS application. The network-based similarity clustering put similar sequences in separate groups, thereby making it easier to differentiate similar and dissimilar sequences in a complicated network of similarities.Sequence clusters were built for all datasets by an all-against-all BLAST approach at MPI Bioinformatics toolkit by CLANS  34, as provided by the MPI bioinformatics toolkit at https://toolkit.tuebingen.mpg.de88. The alignments were then visualized and dissected by the Alvis alignment visualizer tool89. This alignment visualization as a sequence bundle by Alvis, has several useful features, including the precise definition of each position in the alignment, probing the harmonious location of certain amino acids in certain positions of any sequence in the MSA, and correspondence analysis, which are explained in the next section. The arrangement of letter-coded amino acids by physiochemical properties in the Y-axis of MSA vision makes the MSA presentations more informative. This physicochemical arrangement facilitates the sequence comparison and observation of the residual substitution effect(s).The MSAs were generated for all datasets by the Tcoffee algorithm90 were computed for each MSA. The selected substitution matrix was BLASUM62. As numerical embedding, the Fisher scores of the emission probabilities35 were calculated by Alvis (ver. 0.1) after training a hidden Markov model35 on the MSAs. Then, the correspondence test91 was performed by Alvis (ver. 0.1). The correspondence test is an unsupervised (versus supervised) ordination method to detect dependencies between the sequences, sequence groups, and sites responsible for grouping in the alignment (for details see Ref.92).The explorative interpretation of MSAs was done in a series of numerical experiments. The alignment kernelsSARS-CoV-2 was analyzed for locating the secondary structure elements and disordered regions. The sequence was analyzed by the RaptorX server (http://raptorx.uchicago.edu)91 and PSIpred (http://bioinf.cs.ucl.ac.uk/psipred)93 to reach a consensus position of the structural elements. SARS-CoV-2 related structures were obtained from the Protein Data Bank at www.rcsb.org, including 6VW1: 2019-nCoV chimeric receptor-binding domain complexed with its receptor, human ACE2, and 6VXX: Spg at its closed state94.In addition to the characterization of homologous domains by HMMER, the sequence of SgpSARS-CoV-2 was built by Galaxyhomomer95 at http://galaxy.seoklab.org/. The built structure was automatically refined based on the Cryo-electron microscopy structure of a coronavirus Spg trimer96 (PDB entry: 3JCL).A homology modeling approach was also included to achieve a complete structure to avoid missing residues. The homotrimeric structure of Sgp97. The RINanalyzer enabled the connection of Cytoscape (ver. 3.7.2)97 with Chimera (ver. 1.13)98. The interaction networks were visualized and interpreted by Cytoscape (ver. 3.7.2)99. The hydrogen bonds, contacts, and distances (distance threshold < 5 Angstrom) were considered in the RINAnalyzer setting for extracting the network. Before network mining, the residues of interest were selected in Chimera (ver. 1.13). The network of interaction between the selected residues and neighboring residues was then extracted and visualized by Cytoscape (ver. 3.7.2).The molecular interactions of residues in protein structures  were directly extracted from the tertiary structures by RINalyzer (ver. 2.0.0)98. The centrality measurement is based on the modification of the average shortest path length under the removal of individual residues100. This shortest path within two nodes (residues in the structure) is the path in the network that is required for connecting the first node to the second one with the minimum number of edges. This minimum number of edges is known as the shortest path length and the average shortest path length of all possible pairs of nodes identifies the average shortest path. The specific central residues were determined by calculating the Z-score; based on the alteration of an average shortest path length compared to the primary one. By increasing the average shortest path length upon removing a node, a Z-score would be increased. The Z-scores of greater than 2 were considered relevant101. The centrality analysis was done on the structures of RBD both as a free molecule and in complex with ACE2 (PDB entry: 6W41101). Similarly, centrality analysis was performed on the structure of the furin cleavage motif both in the free state and in complex with furin. The structure of the furin cleavage motif nestled in the furin active cleft was obtained by docking the predicted structure of the furin cleavage motif to unbounded furin (PDB ID: 5JXG103). The docking approach was based on the ZDOCK algorithm104 as provided by http://zdock.umassmed.edu.The key residues in the interaction networks were determined by the centrality analysis approach in the RINSpector software (ver. 1.1.0)www.expasy.ch105. The contact map of RBD was predicted using its sequence by RaptorX contact predict106 as provided by http://raptorx.uchicago.edu/. The contact map is indicative of interaction density; this interaction density here is inferred from the sequence data.The physicochemical properties of the RBD sequence were based on amino acid scales of flexibility, surface accessibility, and buried area as calculated by the Protscale at SARS-CoV-2, from MSA, were mapped onto the structure of RBD . The mapping approach was based on the ProtSkin algorithm106 at http://www.mcgnmr.mcgill.ca/ProtSkin/. The conservation property of each site in RBD alignment was calculated even as the percentage of identity to the query sequence, or the average similarity score to the query sequence. The scores were calculated using the BLOSUM62 Block Substitution Matrix by the ProtSkin algorithm107. The obtained scores from the MSA file then were mapped onto the structure by a color gradient.The identity and similarity scores to the RBD of Sgp108 at http://pongor.itk.ppke.hu/. These data along with B-factor were extracted from the PDB structure of RBD: 6W41109.Protrusion (or convexity) index (CX), the depth of each atom in a protein structure (DPX), and the contact number of each residue were calculated by protein core/surface visualization workbench (PCW)SARS-CoV-2 sequences, a collection of all available RBDSARS-CoV-2 sequences was built by a BLAST search against the available SARS-CoV-2 sequences. The best model with the lowest Bayesian Information Criterion scores was identified to describe the substitution pattern of this dataset. All positions containing gaps and missing data were completely deleted. The null hypothesis of dN = dS in favor of dN > dS or dN < dS was tested for tracing the positive or negative selections, respectively. These analyzes were performed in the MEGAX software (ver. 10.1.7).To evaluate the positive or negative selections in RBDSARS-CoV-2 was obtained from the GISAID database110 at https://www.gisaid.org/. The replacement frequency of each position was examined to find any significant differences.Additionally, to monitor the mutation in each certain position , the mutation record of Sgp97.Distance difference for each pair of amino acids was also evaluated based on Grantham’s distancesThe nonparametric Mann–Whitney U test was performed to investigate the significant differences between certain positions and others. The selected positions were those that have been focused on in previous sections.111 and Cytoscape (3.7.2)106. The conservancy of amino acids of RBD was visualized by the PyMol graphic system (ver. 2.3.4)106 using the coloring macro generated by the ProtSkin100 based on similarity or identity scores.Images were prepared by the CLANS Java application and graphical reporting tools of Chimera (1.13)Supplementary Figure S1.Supplementary Legends.Dataset S1.Dataset S2.Dataset S3.Dataset S4.Dataset S5.Supplementary Tables.


A total of 36 studies reported the use of at least prophylactic antithrombotic treatment in the majority of their patients. Meta-regression analysis showed that the prevalence of VTE was higher across studies with a higher percentage of ICU patients and higher study population mean D-dimer values, and lower in studies with mixed dosing of anticoagulation in ⩾ 50% of the population compared to studies with standard prophylactic dosing of anticoagulation in < 50% of the population. The pooled odds ratio for death in patients with COVID-19 and VTE versus those without VTE  was 2.1 . Hospitalized patients with severe COVID-19 are at high VTE risk despite prophylactic anticoagulation. Further research should investigate the individualized VTE risk of patients with COVID-19 and the optimal preventive antithrombotic therapy. PROSPERO Registration No.: CRD42020185543.Severe coronavirus disease 2019 (COVID-19) is associated with increased risk of venous thromboembolism events (VTE). This study performed a systematic review in PubMed/EMBASE of studies reporting the prevalence of VTE in patients with COVID-19 who were totally screened/assessed for deep vein thrombosis (DVT) and/or for pulmonary embolism (PE). Among 47 candidate studies ( Emerging evidence shows that severe COVID-19 is often complicated with coagulopathy, which has prothrombotic effects resulting in high risk of venous thromboembolism events (VTE) and mortality.5 This issue is being increasingly recognized by international societies that strongly recommend the use of thromboprophylaxis in all hospitalized patients.610Moreover, preliminary evidence suggests that anticoagulant therapy might provide a survival benefit in patients with severe COVID-19.This study aimed to review the current evidence regarding the prevalence of VTE in patients with COVID-19 screened/assessed with lower limb ultrasonography or computed tomography pulmonary angiography.This study protocol was registered in PROSPERO; No.: CRD42020185543.www.prisma-statement.org) independently by three investigators  using the following search keywords: (‘coronavirus 2019’ OR ‘2019-nCoV’ OR ‘SARS-CoV-2’ OR ‘COVID-19’) AND (thrombotic OR thrombosis OR ‘deep vein’ OR ‘pulmonary embolism’ OR thromboemboli*) until September 30, 2020. Articles were also selected from references of relevant articles, by searching in journals’ websites and by hand search. Disagreements were resolved by consensus with a senior author (AK).A systematic literature search of PubMed and EMBASE databases was performed in line with the PRISMA recommendations (Eligible studies were full-text articles in English that: (i) reported the prevalence of PE and/or DVT in patients with COVID-19; and (ii) performed screening/assessment in the total sample for DVT (lower limb ultrasonography) or were focused on patients with suspicion for PE (whole study population subjected to computed tomography pulmonary angiography). Case reports and case series studies with ⩽ 10 patients were excluded. The primary endpoint of this analysis was the pooled estimate of PE and DVT prevalence. The secondary endpoint included the pooled estimate of odds ratio for death in patients with COVID-19 with VTE versus non-VTE.11Three investigators extracted independently data concerning study design, main characteristics of included populations and data regarding primary and secondary endpoints from included studies where available. The risk of bias was assessed using the Joanna Briggs Institute’s ‘Critical Appraisal Checklist for Analytical Cross Sectional Studies’.12 with the metaan command in Stata.13 Heterogeneity in the meta-analyzed estimates was quantified using the I2 statistic.14 A random effects model was used, and we opted for a nonparametric bootstrapped DerSimonian–Laird approach.16 The pooled estimates were back-transformed to percentages and are reported as such in forest plots. Poisson regression models were used to examine associations and potential determinants of high heterogeneity in the primary outcome, in a meta-regression setting. The covariates of interest in these analyses were: age, percentage of male patients, percentage of patients in an intensive care unit (ICU), antithrombotic treatment characteristics , mean D-dimer values of the examined sample, and quality of included studies. Meta-regression bubble plots were obtained to further examine the association between VTE prevalence and displaying the Poisson model regression line over study observations. Since this was a meta-analysis of prevalence values, publication bias could not be assessed through tests or funnel plots. Odds ratios for death in VTE versus non-VTE patients were calculated using appropriate formulas.17 Odds ratios and 95% CI values were logarithmically transformed and SEs were calculated from these values and used in the analysis. Mean values of subgroups were combined where feasible .18 Median values were converted to mean values.19 Missing information about study population characteristics  was retrieved through personal communication with the corresponding authors where possible. An alpha level of 5% was used.A pooled prevalence estimate was calculated for each outcome, using the numerators and denominators reported and a Freeman–Tukey arcsine transformationAnalyses were performed using Stata Statistical Software, Release 16 .2066Among 3399 initially identified articles, 47 studies fulfilled the inclusion criteria and were included in the systematic review . The main = 6459; 33 in Europe) reported the prevalence of VTE in totally screened/assessed patients with COVID-19.2066 Among them, 17 studies  reported the prevalence of PE with a pooled estimate of 32%   reported the prevalence of DVT with a pooled estimate of 27%  .A total of 47 studies  ,20,25–2721, 34%) .21–24,28patients .21–25,28p = 0.002) and higher percentage of ICU patients . In addition, prevalence of DVT was higher across studies with higher mean D-dimer values  and lower in studies with mixed dosing of anticoagulation in ⩾ 50% of the population compared to studies with standard prophylactic dosing of anticoagulation in < 50% of the population . Meta-regression bubble plots for noncategorical variables are shown in online Supplementary Figure S3. The above-mentioned estimates regarding the associations of PE prevalence were almost identical when a small study outlier was removed .Meta-regression analysis did not reveal any significant associations between mean age, percentage of males, or quality of the included studies and the prevalence of PE/DVT. However, the prevalence of PE was higher across studies with higher mean D-dimer values 5866 was 2.1   .The main findings of this analysis were the following: (i) the overall prevalence of PE/DVT in hospitalized patients with COVID-19 subjected to assessment was about 30% but with considerable observed heterogeneity; (ii) VTE prevalence was high, even in patients receiving thromboprophylaxis, and appeared to be higher in studies with < 50% of patients anticoagulated; and (iii) patients with COVID-19 and VTE compared to those without VTE had higher risk for death.67 Thus, all hospitalized patients, and especially those in ICUs, are routinely assessed for VTE risk and often administered thromboprophylaxis.It is well recognized that all hospitalized patients with acute medical illness are at high VTE risk. Critically ill patients admitted to ICUs are at very high VTE risk because of ICU-specific risk factors , but also individual patient-related risk factors .2068 It has been suggested that SARS-Cov-2 in severe forms of the disease induces an excessive inflammatory state via cytokine storm combined with endothelial injury and pulmonary vascular microthrombosis, which could considerably increase the risk for VTE, mainly PE.110 In recent autopsy studies, it has been found that the lungs of the infected patients are characterized by a widespread thrombosis with microangiopathy, whereas a high incidence of DVT has been recognized with PE being identified as a direct cause of death.70At present, whether COVID-19 is associated with a higher VTE risk than other infections remains unclear. Initial case reports of VTE events in patients with COVID-19 were followed by case series studies, mainly conducted in an ICU setting, and showed high VTE prevalence, particularly in patients with severe COVID-19.72 These data might support the notion that COVID-19 is associated with a higher risk of thrombosis than other diseases requiring ICU admission but future, well-designed studies should confirm this finding.This review of the current evidence indicates that the course of hospitalized patients with COVID-19 is complicated with DVT/PE in about 30% of cases, irrespective that most of them have received thromboprophylaxis. Unfortunately, studies providing direct head-to-head comparison and using the same assessment methodology between patients with COVID-19 and patients hospitalized for other reasons in terms of VTE prevalence are lacking. However, data from two studies that compared patients with COVID-19 and other (non-COVID-19) patients hospitalized in the same ICU but at different time points, showed higher prevalence of VTE in COVID-19.49Another important finding was that patients with COVID-19 and VTE had a higher risk for death compared to those without VTE. Unfortunately, the exact cause of death  in these patients has not been reported in the included studies. On the other hand, data on the bleeding complications were scarce. However, in three of these studies reporting such information, bleeding complications were uncommon and minor.74 to 20%.76 However, these have included studies with a large methodological heterogeneity, with the results being dependent on the percentage of the study sample assessed for VTE. Shi et al. showed that the pooled prevalence of PE was increased from 8% to 28% when the assessment was performed in the total population.77 In addition, in line with the meta-regression analysis of this study showing that hospitalization in ICU determines a higher prevalence of PE, previous reports have shown a higher pooled prevalence in studies including patients in ICU versus those hospitalized in general wards.78 Thus, the current analysis reported higher prevalence of VTE compared to the existing literature and this was driven by the methodology of the included studies . The clinical relevance of this methodology is highlighted by the fact that most cases of DVT were reported as asymptomatic in many studies.The findings of the current meta-analysis showed a relatively high prevalence of DVT and PE in the range of about 30%. Previous relevant meta-analyses have shown a pooled prevalence ranging from about 13%5 Current recommendations strongly support the use of thromboprophylaxis in all hospitalized patients with COVID-19, although this is based mainly on expert opinion and less so on high-quality evidence.610 Furthermore, important details such as the optimal dose-intensity of the anticoagulation therapy are lacking.It should be noted that the high prevalence of VTE among patients with COVID-19 was observed despite using thromboprophylaxis in the majority of the included studies. In the meta-regression analysis, the prevalence of DVT was lower in studies with mixed dosing of anticoagulation in ⩾ 50% of the population compared to studies with standard prophylactic dosing of anticoagulation in < 50% of the population. A retrospective study in more than 4000 hospitalized patients with COVID-19 showed anticoagulation therapy to be associated with lower mortality and intubation events.The findings of this review should be interpreted by considering several limitations. Most important is the heterogeneity among these studies and the lack of information regarding (i) the patients’ individual VTE risk and (ii) details on the anticoagulant therapy , which might have influenced the outcome. In a sensitivity analysis for identification of publication bias, there was a trend for higher PE prevalence in smaller studies, but there was no apparent trend in DVT prevalence. Furthermore, in a significant proportion of the included studies, exclusive polymerase chain reaction-based diagnosis of COVID-19 was unclear or not reported. Other criteria for diagnosis, such as imaging or other laboratory tests, might have been used but these probably regarded only a minority of patients and not the whole study sample. Thus, the exposure might not have been measured in a strictly reliable way in a minority of patients in some of the studies but this also reflects real clinical practice. However, the outcome was measured in a valid and reliable way in most of the included studies, although, in some of these, adjustment for confounders would be needed for accurate assessment. In addition, meta-regression analysis examined the associations between outcome and several characteristics which were aggregate and summarized at the level of the study, which in turn introduces ecological bias. Last, most of the studies did not provide information on hemorrhagic complications.Since the screening/assessment process for VTE diagnosis represents a significant source of heterogeneity among such studies, we included only studies that screened/assessed the total population. Limb ultrasonography is an easy test that can be performed massively in the context of a research protocol and can identify asymptomatic patients, which is not an uncommon finding. However, computed tomography pulmonary angiography is performed in selected patients upon clinical suspicion combined with the D-dimer value. By selecting studies that performed these assessments in the whole study population, a more realistic estimate of the DVT/PE prevalence among these patients can be calculated, which in turn determines the pre-test probability in such patients. The latter estimate is a major determinant in a Bayesian approach where the diagnostic strategy depends on the pre-test probability. Thus, the findings of this meta-analysis might provide answers regarding the prevalence of DVT in hospitalized patients with COVID-19, including both symptomatic and asymptomatic cases (the latter being quite common), as well as regarding the prevalence of PE in hospitalized patients with COVID-19 and high suspicion based on clinical characteristics and D-dimer values.This systematic review of the evidence suggests that hospitalized patients with COVID-19, who are screened or assessed for VTE, present a pooled prevalence of DVT and PE at about 30% each, and despite thromboprophylaxis in most cases. The VTE risk appears to be considerably higher than in patients without COVID-19 admitted in the same ICUs. Further research is necessary to investigate the individualized VTE risk of patients with COVID-19, the underlying pathogenetic mechanisms, and the optimal preventive anticoagulant therapy.Click here for additional data file.Supplemental material, sj-pdf-1-vmj-10.1177_1358863X21995566 for Venous thromboembolism in COVID-19: A systematic review and meta-analysis by Anastasios Kollias, Konstantinos G Kyriakoulis, Styliani Lagou, Evangelos Kontopantelis, George S Stergiou and Konstantinos Syrigos in Vascular Medicine


The evolving nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has necessitated periodic revisions of COVID-19 patient treatment and discharge guidelines. Since the identification of the first COVID-19 cases in November 2019, the World Health Organization (WHO) has played a crucial role in tackling the country-level pandemic preparedness and patient management protocols. Among others, the WHO provided a guideline on the clinical management of COVID-19 patients according to which patients can be released from isolation centers on the 10th day following clinical symptom manifestation, with a minimum of 72 additional hours following the resolution of symptoms. However, emerging direct evidence indicating the possibility of viral shedding 14 days after the onset of symptoms called for evaluation of the current WHO discharge recommendations. In this review article, we carried out comprehensive literature analysis of viral shedding with specific focus on the duration of viral shedding and infectivity in asymptomatic and symptomatic  COVID-19 patients. Our literature search indicates that even though, there are specific instances where the current protocols may not be applicable ( such as in immune-compromised patients there is no strong evidence to contradict the current WHO discharge criteria. On the 27th of May 2020, the World Health Organization (WHO) provided an update to guide the clinical management of COVID-19, wherein criteria for discharging patients were recommended  Are RT-PCR tests reliable enough? (2) From which site do we take specimens? (3) Is there a correlation between viral load and disease severity & patient status (symptomatic and asymptomatic)? (4) Does the duration of viral shedding correlate with infectivity? Empirical evidence on the above questions will help further guide and improve discharge protocols that can shorten patient isolation and minimize transmission risks of convalescing patients. This review critically analyses existing literature on SARS-CoV-2 viral shedding dynamics and infectivity in convalescing patients.Authors attempted to assess in-depth a multitude of COVID-19 reports in PubMed, Google Scholar and Scopus. The following keywords were used as search terms: viral shedding, COVID-19, SARS-CoV-2, transmission, infectivity, viral load, dynamics, viral viability, and WHO discharge guidelines. The date of last search was April 2021. Concerned about the delays between completion of studies and their publication and taking into consideration the high turnover of publications on COVID-19 pandemic, manuscripts on preprint servers were also considered (Research Square medRxiv dimensions and BioRxiv). Articles identified by the searches were reviewed along with any relevant references cited within them. Articles were excluded if the full texts were not available or if they were not published in English.Understanding the dynamics and duration of infectivity of SARS-CoV-2 is critical in the setting up of protocols guiding public health for quarantine, isolation and contact tracing. The period of infectivity is the space of time within which a naive individual may acquire the infection from an infected person . The cliViral loads are generally quantified via RT-qPCR. In most studies, viral loads from upper respiratory tract samples peaked at the time of symptom onset and lasted for a few days, but gradually decreased over the following 1–3 weeks –8. Viraln = 76) was higher than in mild cases. Consequently, patients with severe COVID-19 infection had significantly higher viral loads in respiratory samples compared to those presenting with mild symptoms. In addition, Zheng et al.  starts to clear the infection. Variation in the time of activation of the adaptive immune response in different individuals implies that the SARS-CoV-2 virus can continue replicating and remains viable . In a reIn a case series conducted by Vetter et al. , the autA seroprevalence study was conducted within selected locations in Ghana to estimate the level of exposure of SARS-CoV-2 in individuals across a wide socioeconomic range. Using a strip-in-cassette lateral flow type RDT which separately detects IgM and IgG antibodies against the nucleocapsid protein of the virus, 252/1,305 persons tested positive for either of the antibodies or both . ExposurCheng et al.  reportedAccording to Kissler et al. , the trap = 0.0261) and density (p = 0.0094) of the 3′ end of SARS-CoV-2 genome compared to the 5′ end even if the patients did not display GI symptoms. Moreover, the gut microbiota of COVID-19 patients with GI infection showed increased amounts of opportunistic pathogens, as well as reduced salutary bacterial content. This important study showed the persistence of transcriptional activity of viral infection and replication in the gut even after clearance of SARS-CoV-2 from respiratory samples. Furthermore, the authors deciphered for the first time, the signature of active gut viral infection in COVID-19 patients even in the absence of GI symptoms, suggesting “quiescent” GI infection of SARS-CoV-2 following recovery from COVID-19.Zuo et al.  assessedSeveral studies have been conducted regarding viral shedding in nasopharyngeal swabs, tears, urine and excreta of severe, mild, and asymptomatic COVID-19 cases. Nonetheless, there are still knowledge gaps regarding the viral shedding dynamics during the convalescent phase in COVID-19 patients with mild and severe symptoms. Recent studies suggest that asymptomatic COVID-19 carriers may be as contagious as symptomatic (mild and severe) patients, and therefore may play an important role in transmission through viral shedding –41. HoweCOVID-19 patients displaying mild symptoms were assessed for any unusual signs and their symptoms were correlated with the viral shedding period. RT-PCR was performed every 2–7 days, with the duration of viral shedding ranging from the day of first diagnosis to the day before the first negative test. On average, viral shedding lasted for 24.5 days. According to this study, it was evident that all patients presenting with chest pain and sputum shed the virus significantly longer than patients without these symptoms. Zhou et al.  studied Yan et al.  assessedLi et al.  observedLiu et al.  successf9/L shed SARS-CoV-2 virus for longer time. Lu et al. (Children with mild COVID-19 were clinically assessed at Wuhan Children's Hospital, Wuhan, China and their laboratory test results were also investigated and correlated with viral shedding . On averu et al.  suggestePark et al.  determinLi et al.  assessedLee et al.  reportedIn a recent study conducted on 20 immunocompromised COVID-19 patients, it was found that viral shedding occurred for up to 78 days after symptom onset . InteresKim et al. showed that the time from disease onset to viral clearance in culture with the time to clearance in real-time RT-PCR tests was not in agreement with each other . In factEven though evidence showed that SARS-CoV-2 viral shedding occurs both in patients with and without symptoms, the correlation between transmissibility/infectivity and detectable viral RNA remains unclear . This isOnly few studies have correlated RT-PCR test results with viral cultures and infectivity. Arons et al.  could suThe extent and length of infectious virus replication should be considered when evaluating the possibility of viral transmission which will in turn guide decisions regarding patient discharge and isolation. Qualitative or quantitative viral RNA tests have been used extensively as a potential marker for infectious coronavirus since RNA detection is more sensitive than virus isolation. But this can be problematic because, shedding viral RNA may not necessarily be synonymous to shedding whole viruses. Even though assessing the potential infectivity is a labor-intensive process, it is not possible to estimate the average time frame of viral RNA shedding by investigating the presence of SARS-CoV-2 viral RNA in patient samples as the presence of nucleic acid alone does not necessarily imply infectivity . Severalin vitro infectiousness on cell lines by replication competent virus is regarded as a more informative surrogate of viral transmission (The critical question in the ensuing discussion so far in this review, is how long a person continues to shed infectious virus? In order to answer this question, it is important to note that the determinant of infectious virus is not the detection of sub-genomic RNA which is widely used in testing centers, but smission –66. A nusmission , 67. Thismission , 49 repoVan Kampen et al.  studied Economic challenges and prolonged turn-around times of rRT-PCR-based testing makes it inadequate for post-discharge screening of potentially infectious individuals. For these reasons, antigen-based screening for SARS-CoV-2 is being considered as a complement to rRT-PCR-based testing . StudiesThe most common mode of human to human transmission is via body fluid droplets, infected hands and surfaces . The SAREnvironmental conditions including temperature, humidity, wind speed, water, sewage, air, insects, and surface of objects influence the viral transmission process. The prevalence of COVID-19 with maximum air humidity and wind speed was found to be negligible  while pr2 for a long time had higher death probability (2 hotspots in Europe. Similarly, Zhu et al. (2, and O3 led to increased risk of COVID-19 infection. In summary, most studies indicate that patients developed more severe forms of COVID-19 when they were exposed to air pollutants for long time and also took longer to recover and had increased mortality rates.Few studies have tried to investigate the correlation between air pollution and COVID-19 cases. Wu et al.  observedbability . Furtherbability  correspou et al.  found thCDC has not identified food, food packages, and food handlers as a major mode of viral transmission. However, washing and disinfection of surfaces is highly recommended based on evidence suggesting the persistence of the virus for hours/days . Most imIn addition to environmental factors, human population density and movement, social interactions, climate change also impacts on viral transmission.The WHO played an important role in issuing guidelines and measures to control the worldwide COVID-19 pandemic. In line with this, the latter provided a first set of recommendations for the discharge of COVID-19 patients. Due to challenging conditions and poor infrastructure in many countries, these recommendations were reviewed. Following a critical assessment of follow-up studies involving convalescing COVID-19 patients, we posit that there is no strong evidence which counters the current WHO recommendations. However, we propose the use of a test-based discharge strategy in immunocompromised individuals. RT-PCR tests are less reliable. Indeed, false-negative have been reported due to poor quality specimen collection, testing too early in the incubation period, and other processing errors. LRT specimens were shown to be more sensitive compared to URT ones. Viral loads could be correlated with disease severity with higher viral loads noted for severe forms of COVID-19. However, viral load and viral shedding cannot be directly correlated with infectivity because RT-PCR based assays do not distinguish between degraded viral genetic remains or viable virus. In patients with critical COVID-19, infectious virus may be shed for longer periods in contrast to the WHO guidelines. It has been observed that in the presence of serum neutralizing antibodies, infectious virus shedding drops to undetectable levels below a viral RNA load threshold. This suggests that quantitative viral RNA load and immunological assays are better alternatives in test-based approaches when deciding to relax infection prevention and control precautions. Van Kampen et al.  observedAll authors conceived the literature review concept, conducted a literature search, compiled and interpreted all data, and wrote the entire manuscript. All authors have read and approved the entire manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.