In silico epitope prioritization shortlisted six HLA I epitopes and six B-cell-derived HLA II epitopes. These high-ranked epitopes were all connected to each other via flexible GPGPG linkers, and at the N-terminus side, the sequence of Cholera Toxin β subunit was attached via an EAAAK linker. Structural modeling of the vaccine was performed, and molecular docking analysis strongly suggested a positive association of a multiepitope vaccine with Toll-like Receptor 3. The structural investigations of the vaccine-TLR3 complex revealed the formation of fifteen interchain hydrogen bonds, thus validating its integrity and stability. Moreover, it was found that this interaction was thermodynamically feasible. In conclusion, our data supports the proposition that a multiepitope vaccine will provide protective immunity against COVID-19. However, further in vivo and in vitro experiments are needed to validate the immunogenicity and safety of the candidate vaccine.The global health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, has resulted in a negative impact on human health and on social and economic activities worldwide. Researchers around the globe need to design and develop successful therapeutics as well as vaccines against the novel COVID-19 disease. In the present study, we conducted comprehensive computer-assisted analysis on the spike glycoprotein of SARS-CoV-2 in order to design a safe and potent multiepitope vaccine.  The unprecedented global health crisis due to the COVID-19 pandemic, with associated economic and social problems, poses a great threat to the world order today . Wuhan cCOVID-19 patients typically show a variety of symptoms—from mild symptoms to severe illness. Common symptoms include dry cough, fever, shortness of breath, fatigue, and upper airway congestion . After vSARS-CoV-2 is implicated in COVID-19 pathology. Its genome organization is thought to be similar to that of other human coronaviruses . The genSome studies explored the recombinant spike protein-based vaccine design against COVID-19. Notably, Keech et al. recently performed Phase 1–2 trials of a SARS-CoV-2 recombinant spike protein-based nanoparticle vaccine and obtained good results . Ren et The spike protein has also been subjected to epitope prediction by numerous researchers. Chen et al. focused on the prediction of B-cell and T-cell epitopes in the SARS-CoV-2 spike and nucleocapsid proteins . FurtherIn silico epitope mapping and prioritization were done on the spike protein of novel coronavirus species to select promising T-cell (CD4+ and CD8+) and B-cell epitopes. All the prioritized epitopes were later incorporated in a single multiepitope vaccine.Currently, there are no effective drugs or vaccines available against COVID-19. Considering the gravity of the situation globally, an effective vaccine or antiviral drug is the need of the hour. Reverse vaccinology is the genome-based mining of pathogenic microorganisms to identify suitable targets to guide the development of vaccines , 22. In We conducted three-dimensional structural modeling of a candidate vaccine using bioinformatic tools and evaluated its effectiveness. The selected model was then used in molecular docking studies with Toll-like Receptor 3 (TLR3) to characterize its ability to trigger specific immunological responses against the novel coronavirus disease. TLR3 acts as a sensor of viral infection and activates downstream innate immunity-related signaling pathways . StudiesThe overall methodology is illustrated in The full-length amino acid sequence (FASTA format) of the surface glycoprotein of the SARS-CoV-2 reference genome (NC_045512.2) with accession number YP_009724390.1 was downloaded from the NCBI GenBank database.The sequence of the full-length surface glycoprotein YP_009724390.1 was fed into the NetCTL 1.2 server to check the binding affinity with twelve specified HLA supertypes: A1, A2, A3, A24, A26, B7, B8, B27, B39, B44, B58, and B62 . BLASTp The sequence of full-length surface glycoprotein (YP_009724390.1) was subjected to HLA II epitope prediction by NetMHCIIpan. Prediction was performed specifically to hunt epitopes binding to the following HLA II supertype alleles: DRB1∗0101, DRB1∗0301, DRB1∗0401, DRB1∗0701, DRB1∗0801, DRB1∗1101, DRB1∗1301, and DRB1∗1501 . NetMHCIAll the epitopes found to interact with at least three supertype alleles  were checked for their antigenic properties using VaxiJen version 2.0, and only those with antigenicity scores greater than 0.4 were retained . Next, tAll the peptides obtained were subjected to BLASTp against the human genome to identify those with homology to any region in the human genome so they could be discarded to avoid the risk of autoimmunity. Meanwhile, the glycoprotein sequence was also submitted to the iBCE-EL web server to predict linear B-cell epitopes . All theβ subunit (CTB) sequence was incorporated at the N-terminal side of the vaccine construct. A linker sequence of EAAAK joined the adjuvant to the first epitope in the designed construct. CTB, a nontoxic component of cholera toxin, exhibits good affinity towards monosialotetrahexosylganglioside on the surface of the gut epithelium as well as standard antigen-presenting cells such as dendritic cells, B-cells, and macrophages [Both the HLA I epitopes and B-cell-derived HLA II epitopes were checked to ensure that there was no overlapping of any kind. Amongst the overlapping peptides, only the most appropriate epitopes were selected to proceed towards multiepitope vaccine design. These finalized epitopes were joined together via flexible GPGPG linkers. This step was essential for effective separation of the epitope domains and for limiting the chances of forming junctional epitopes . Moreoverophages . This isThe multiepitope protein sequence was then submitted to the Protparam tool at ExPASy to calculate physicochemical features such as molecular weight, isoelectric point (pI), half-life, grand average of hydropathicity (GRAVY), aliphatic index, and instability index , 41. ForThe allergenicity of the multiepitope vaccine was assessed through two servers: AllergenFP 1.0 plus AllerTOP 2.0 , 47. AllThe three-dimensional structural model of the polyepitope vaccine was modeled by a 3Dpro tool . It was The three-dimensional structure of the refined model of our polyepitope subunit vaccine was subjected to discontinuous B-cell epitope prediction. For this purpose, two servers, Ellipro and DiscoTope version 2.0, were used with default parameters , 52. The∆G), which is an important thermodynamic parameter to assess whether the binding affinity in a biological complex is energetically feasible or not [The binding affinity of the polyepitope vaccine with TLR3 was analyzed using the PRODIGY web server . PRODIGYe or not . UsuallyB-factors, covariance, and deformability. The deformation of protein chain/s depends on the deformability at the residue level. The eigenvalue associated with each normal mode tells us information about the motion stiffness. This also provides crucial insights into the energy needed to deform the protein(s) structure(s). The easier deformation is clearly indicated by low eigenvalues [Normal mode analysis was conducted in order to confirm the stability of protein-protein complexes after using HADDOCK. The stability of the protein(s) can be gauged by comparing their dynamic behavior with normal modes , 56. It envalues .E. coli K12 strain, the protein sequence of the candidate vaccine was subjected to reverse translation followed by codon optimization. For this purpose, the JCat online server was used [In order to obtain the best codon-optimized sequence for expression in the was used . Care waA total of eighteen epitopes belonging to SARS-CoV-2 spike glycoprotein were projected to have strong binding interactions with at least four HLA I subtypes. These epitopes, as well as the names of their associated HLA supertypes and total number of supertypes, are given in Table BLASTp analysis of the shortlisted peptides against the human genome did not reveal homology of these sequences to any human genomic region. Further, it was confirmed that these epitopes had not been experimentally tested previously and thus represent novel predictions. It was found that ProPred I also classified these peptides as potential HLA I epitopes .A total of fifteen HLA II epitopes corresponding to SARS-CoV-2 spike glycoprotein were predicted to show positive associations with at least four specified HLA DRB alleles using the PREDIVAC tool Table  sheet 1 Meanwhile, a total of fourteen 15-mer putative HLA II epitopes in the SARS-CoV-2 spike glycoprotein were predicted to associate with at least three specified HLA DRB alleles using NetMHCIIpan version 3.2 Table  sheet 1 In total, seven HLA II epitopes were shortlisted using the abovementioned tools (five obtained using PREDIVAC and two using NetMHCIIpan version 3.2). After checking the epitopes in the IEDB database, it was confirmed that none of them had been experimentally tested previously concerning the COVID-19 disease, thus indicating novelty. It was confirmed that all of them shared an overlap with B-cell epitopes predicted by the iBCE-EL tool Table . So, theAs explained MTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANEAAAKFVFLVLLPLGPGPGSTQDLFLPFGPGPGWTAGAAAYYGPGPGYLQPRTFLLGPGPGYQPYRVVVLGPGPGQIITTDNTFGPGPGYFKIYSKHTGPGPGGINITRFQTLLALHRGPGPGFELLHAPATGPGPGYECDIPIGAGPGPGSIIAYTMSLGAENSVGPGPGFGAISSVLN.Escherichia coli (in vivo). An instability index of 28.27 was computed. As the value is less than 40, this projects stability of our vaccine protein. The aliphatic index of 79.08 was calculated for our vaccine, which is a strong indication of thermostability at varying temperatures. Analysis revealed that the grand average of hydropathicity (GRAVY) is -0.081. Since it is a negative value, this suggests that the vaccine is hydrophilic in nature and has the ability to interact favourably with water molecules which is a desired property. Both ANTIGENPro and VaxiJen projected the antigenic nature of the vaccine construct, with scores of 0.764456 and 0.5098, respectively [A physicochemical feature check of the multiepitope revealed that it was composed of 284 amino acids and had an 29832.13 amu molecular weight. Low molecular weight proteins (less than 110 kDa) are thought to be good candidates for developing vaccines . The theectively . The ccSectively . So, altZ-scores  while that of TLR3 was 1905 (Å2).Molecular docking resulted in a good interaction pose between our vaccine and TLR3 chains . A totalHydrogen bonds are enticing powers of the dipole-dipole and are formed when another electronegative atom absorbs a hydrogen atom bound to a strongly electronegative atom such as F, N, and O. The frequency of the hydrogen bond ranges from 4 kJ per mole to 50 kJ. In molecular recognition, hydrogen bonds are considered essential and have rigidity in achieving stable conformation. The vaccine was developed with 15 hydrogen bonds (chain A (vaccine)-chain B (TLR3); 1-214, 4-243, 4-244, 8-240, 82-247, 146-291, 148-319, 186-375, 186-403, 198-272, 200-276, 200-299 (2 H-bonds), 210-325, and 221-377). Upon detailed analysis of protein chains, it was found that Met1 forms a hydrogen bond with Pro214 at 2.85 Å. Similarly, Gln4-Leu243 forms a hydrogen bond at 3.21 Å distance, Gln4-Glu244 at 3.22 Å, Asp8-Lys240 at 2.62 Å, Lys82-Asn247 at 2.77 Å, Tyr146-Asn291 at 2.82 Å, Pro148-His319 at 2.77 Å, Asn186-Phe375 at 3.06 Å, Asn186-Ser403 at 3.16 Å, Tyr198-Lys272 at 2.78 Å, and Lys200-Leu276 at 2.72 Å. Lys200 formed two hydrogen bonds with Gln299 at distances of 2.99 Å and 2.81 Å, respectively. Finally, Asn210 formed one hydrogen bond with Gln325 at a distance of 3.20 Å, while His221 developed a hydrogen bond with Gly377 at a distance of 2.89 Å.After careful observation, we found that most of the hydrogen bond distances between the interacting residues of the vaccine and TLR3 lie within 2–3 Å, thus indicating significantly high interactions . So, the∆G value of the complex was -19.5 kcal/mol. The negative ∆G value indicates that the molecular association between our designed vaccine and TLR3 structure is thermodynamically possible and could definitely occur in nature. Also, the dissociation constant Kd value was calculated at 25°C and found to be 5.30E−15.Gibbs free energy-based assessment of the refined vaccine-TLR3 complex was performed to assess the binding affinity. Analysis revealed that the B-factor values were considered to be comparable to RMS  of cDNA was 0.876584. As this value is greater than 0.8, it was found to be convenient [The optimized cDNA sequence of the candidate vaccine for the nvenient . The GC nvenient .The development of effective therapeutics and vaccines against COVID-19 is much needed to control this pandemic. Vaccination provides the most medically robust prevention against infectious disease eradication. Pursuing this route, the world has witnessed the end of the deadly smallpox disease, a medical feat that has indeed few parallels in science. Today, the most urgent global problem faced by humans is posed by the highly contagious SARS-CoV-2, the viral pathogen responsible for COVID-19. However, due to a lack of up-to-date immunological knowledge of the novel coronavirus species and the protective immune responses needed to clear the pathogen, vaccine development poses great challenges .Recently, many studies have been performed to envision a COVID-19 vaccine. More than 135 vaccine candidates are undergoing preclinical testing, 15 vaccines have entered Phase I trials, and 11 vaccines are being subjected to Phase II trials . A totalThe spike protein is thought to be a crucial antigenic target for both SARS-CoV-2 and genomically similar SARS-CoV due to its high antigenicity and its functional role in terms of viral attachment and entry into human cells , 80. It β subunit (CTB) which is the nontoxic part of the cholera toxin [The main aim of the present study was to propose a multiepitope vaccine against COVID-19 using computer-aided analysis. The rationale of including a molecular adjuvant in the multiepitope construct was that epitope-based peptide vaccines, although safe and highly specific, are normally poor immunogens . So, we ra toxin . CTB is ra toxin , 84. FurZ-score [A high-quality near-native vaccine structure was obtained after structural modeling and molecular refinements. This was confirmed using the ProSA web server and its Z-score . QualityZ-score . Only a Z-score .It is known that the majority of B-cell epitopes (~90%) are not linear, i.e., they are not present in the primary structure . After pTo check the effectiveness of our multiepitope vaccine, we conducted molecular docking of the vaccine structure with the TLR3 structure. This was necessary as TLR3 acts as a sensor of viral infection and activates downstream innate immunity-related signaling pathways . StudiesE. coli K12 strain as we showed after codon optimization of the candidate vaccine.The guru level interface in HADDOCK provided good results in terms of stability as well as flexibility of protein-protein docked clusters . The HADOne limitation of the designed study is the lack of follow-up wet laboratory studies to validate whether the multiepitope vaccine will provide strong protective immune responses in humans or not. The epitopes incorporated in this vaccine have been strictly identified as per computer-aided analysis. Currently, we cannot find any indication of their experimental testing concerning COVID-19 through the IEDB resource that has the repository of experimentally characterized epitopes associated with both T-cells and B-cells. After scanning the literature, we found some published studies concerning epitope prediction in the spike protein –20. Aftein silico work and should be validated by future studies before conducting trials in humans.Effective vaccines are urgently required worldwide to contain the spread of the COVID-19 pandemic and save human lives. In this study, we carried out a computer-assisted analysis on the spike protein of SARS-CoV-2 to identify highly promising epitopes to guide the development of a safe universal vaccine. These prioritized epitopes were then fused with each other and an adjuvant to form a polyepitope subunit vaccine using computational biology approaches. Three-dimensional structural modeling of the vaccine was carried out and subjected to docking studies with Toll-like Receptor 3 to assess its immunological effectiveness. Our results indicated a strong interaction between our vaccine and the innate receptor, strengthening the proposition that it will lead to the induction of TLR-mediated downstream immune pathways. The present study is entirely


The donor sequences (MG-HR) for homologous repair were designed and cloned by PCR. MG-HR and pGL3-MGP-RNA were transformed into E. coli MG1655 (pCas9). The cpxP gene expression cassette was amplified by PCR and subcloned into pBBR1MCS-2. Then the pBBR-cpxP was independently transformed into E. coli MG1655. The results of motility experiment suggest that cpxP gene had a significant effect on the movement ability of E. coli strain. The CpxP protein had a significant inhibition of bacterial activity. The lastest 81 CpxP proteins sequences were selected and analyzed by multi-sequence alignment and molecular cluster. The CpxP proteins were roughly divided into three categories. Our results suggest that the CpxP protein was involved in bacterial motility, infection and pathogenicity.Based on the analysis of  E. coli MG1655-ΔcpxP were successfully obtained.The cpxP gene had a significant effect on the movement ability of E. coli strain.The The CpxP proteins had a significant inhibition of bacterial activity.Escherichia coli  cpxP gene of MG1655 strain was successfully knocked out by CRISPR-Cas9 system-based gene editing strategy. Likewise, the system can efficiently edit a large plasmid, target genes on the bacterial chromosome, or be adapted to introduce functional gene cargos alongside the gRNA cassette.This study constructed an expression vector pGL3-MGP-RNA including gene-targeting short guide RNA and cloned a donor sequences (MG-HR) for homologous repair. The cell-envelope stress modulator E. coli DH5α by Plasmid extraction kit . The pCas9 Plasmid was transfected into E. coli MG1655  to acquire E. coli MG1655 (pCas9). The transformed E. coli MG1655 was screened using Kanamycin (50 mg/L) that was added to the LB medium. Genomic DNA was extracted from E. coli MG1655 by Bacterial genome extraction kit .The pCas9 and pGL3-U6-SgRNA-PGK-Puromycin Plasmid  was extracted from The MG-HR-S and MG-HR-X were amplified by PCR using the genomic DNA as a template. The primers were designed with prime primer 5.0. The primers were synthesized by the Beijing Invitrogen Biotechnology Company. The primers TCTGGTGTGTCTGGCGAAGT and TGCTAATTCGTGGAGCTTATGCCAGCG TTGAGGCCATG were used to amplify MG-HR-S. The primers TAAGCTCCACGAATTAGCATCAGCAGATGCGAGATCTTAT and CTATGGC AAGGAAAACAGGGT were used to amplify MG-HR-X. MG-HR-S was connected to MG-HR-X by PCR to acquire a MG-HR.The MGP-sgRNA and pGL3 were amplified by PCR using the pGL3-U6-SgRNA-PGK-Puromycin Plasmid as a template.The primers CGGGATCCTTGACAGCTAGCTCAGTCCTAGGTATAATACTAG TTCAGGCGATAACTGGCATCCGTTTTAGAGCTAGAAAT and GGGGTACCGG AACCACGCCCAGAGCAG were used to amplify MGP-sgRNA.The primers GGGGTACCGCTCACTGACTCGCTGCGCT and CGGGATCCGC TTAATGCGCCGCTACAGG were used to amplify pGL3.cpxP gene expression cassette comprising said promoter, a cpxP gene and said terminator was amplified by PCR using the genomic DNA as a template.The cpxP-F: CCCAAGCTTACGCGGTCTAATTCACTGCC 3′cpxP-R: CGCGGATCCAGACAGGGATGGTGTCTATGGC 3′The PCR was carried out by using the Gene Amp PCR system 9700 (Applied Biosystems). PCR products were confirmed on 1.0% agarose gels and recycled by the agarose gel extraction kit .Recycled PCR products of the MGP-sgRNA and pGL3 were digested by KpnI and BamHI ,and then connected by T4 DNA Ligase  to acquire a pGL3-MGP-sgRNA.E. coli MG1655 (pCas9). The transformed E. coli MG1655 (pCas9) were screened using Kanamycin (50 mg/L) and Ampicillin (100 mg/L) that were added to the LB medium.400 μg pGL3-U6-sgRNA-PGK-puromycin, 400 μg pGL3-MGP-RNA, 400 μg pGL3-MGP-RNA and 1.6 μg MG-HR were separately transformed into cpxP gene expression cassette were digested by BamHI and HindIII , purified by the agarose gel extraction kit. The purified PCR products were directly subcloned into pBBR1MCS-2  (No. pBBR-cpxP). Then the pBBR-cpxP was independently transformed into E. coli MG1655 to acquire the overexpression transformants and E. coli MG1655-ΔcpxP to acquire a revertant. The transformed E. coli MG1655 were screened using Kanamycin (50 mg/L).Recycled PCR products of the Escherichia coli MG1655, overexpression transformants, the knocked out mutants and revertants were inoculumned on LB medium for overnight. Then the cultures (2 μl) were added to the LB semi-solid medium for overnight.2O, 200 μl Kan (2.5 mg/ml) and 200 μl CpxP proteins (8 mg/ml) were independently added to the LB solid medium by 6 mm Oxford cup containing 100 μl E. coli MG1655 and E. coli K88 (105 cfu/ml) for 16 h at 4 °C. Then they were kept at 37 °C for bacteriostatic experiment.200 μl ddHhttp://www.ncbi.nlm.nih.gov/) and done multi-sequence alignment and molecular cluster by Clustal X and Treeview  were digested with KpnI and BamHI. According to the electrophoresis, the recombinant plasmids (pGL3-MGP-sgRNA) were successfully obtained  as a template. According to the electrophoresis, the lengths of PCR products were 888 bp without knockout and 782 bp after knockout of cpxP gene , the overexpression transformants and revertant were successfully obtained  . These results suggest that cpxP gene had a significant effect on the movement ability of E. coli strain.Many studies have confirmed that the motility of bacteria plays an important role in the pathogenicity of bacteria during the early interaction with the host. To investigate the effect of the 2O , and there was significant difference compared with Kan   exhibited 100%, 100% and 89.2% homology with the Shigella flexneri (AUU33472), Shigella sonnei (AMG18626) and Salmonella enterica (AMG25549) homologous proteins, respectively. All the bacterial names corresponding to the gi numbers and sequences were showed  gene has been investigated for many years, but there are few studies on its function. The CpxP proteins can inhibit activation of CpxA and are indispensable for the quality control system of P pili.The cell-envelope stress modulator cpxP-overexpression E. coli MG1655, the knocked out mutants E. coli MG1655-ΔcpxP and the revertants E. coli MG1655-ΔcpxP (pBBR-cpxP) were obtained. Deprivation of the cpxP gene resulted in significant enhancement in the mobility of E. coli strains. The overexpression of the cpxP gene also resulted in significant attenuation in the mobility of E. coli strains. The mobility of E. coli revertants strains was lower than E. coli MG1655. The mobility of bacteria had important pathological significance, moreover, and mainly played its role during the early stage of the infection (Mao and He Salmonella enterica serovar Typhimurium was found to depend on the motility of the bacteria (Siitonen and Nurminen cpxP gene resulted in significant enhancement in the mobility and infection of E. coli strains.In this report, the results showed that the Shigella flexneri, Shigella sonnei and Salmonella enteric which are the main pathogenic bacteria in China. Our results suggest that the overexpressions of the cpxP gene may significantly reduce the pathogenicity of these bacteria.Multi-sequence alignment and molecular cluster indicated that the CpxP proteins had a high homology at protein level with Additional file 1. The electrophoregram and sequences of cpxP.


It is controversial whether B12 deficiency causes dementia or B12 treatment can prevent dementia.To assess associations between low plasma (P-)B12 levels, B12 treatment, and risk of Alzheimer’s disease  and all-cause or vascular dementia (secondary outcomes).We conducted a population-based cohort study using Danish registry data to assess associations between low P-B12 levels, high-dose injection or oral B12 treatment, and risk of dementia (study period 2000–2013). The primary P-B12 cohort included patients with a first-time P-B12 measurement whose subsequent B12 treatment was recorded. The secondary B12 treatment cohort included patients with a first-time B12 prescription and P-B12 measurement within one year before this prescription. For both cohorts, patients with low P-B12 levels (<200 pmol/L) were propensity score-matched 1:1 with patients with normal levels (200–600 pmol/L). We used multivariable Cox regression to compute 0–15-year hazard ratios for dementia.For low P-B12 and normal P-B12 level groups, we included 53,089 patients in the primary P-B12 cohort and 13,656 patients in the secondary B12 treatment cohort. In the P-B12 cohort, hazard ratios for AD centered around one, regardless of follow-up period or treatment during follow-up. In the B12 treatment cohort, risk of AD was unaffected by low pre-treatment P-B12 levels, follow-up period and type of B12 treatment. Findings were similar for all-cause and vascular dementia.We found no associatio1n between low P-B12 levels and dementia. Associations were unaffected by B12 treatment. Results do not support routine screening for B12 deficiency in patients with suspected dementia. Vitamin B12 (B12) is essential in DNA synthesis, and in both fatty acid and amino acid metabolism. It is important in the maturation of hematopoietic cells and in the normal functioning of the nervous system via its role in myelin synthesis [Low plasma B12 (P-B12) is associated with various neurological disorders including dementia, but two systematic reviews concluded that findings were inconsistent . Some inAlthough, high-dose B vitamins that include B12 might reduce cerebral atrophy , meta-anTo this end, we assessed the risk of Alzheimer’s disease and other dementia types in a population-based cohort of Danish patients with various levels of P-B12 and P-MMA and evaluated the potential protective effect of treatment with high-dose B12 on dementia risk, including the specific effects of three different B12 formulations.We conducted a population-based, propensity score-matched cohort study in the Northern and Central administrative regions of Denmark using Danish health registries. These two regions cover a population of about 1.8 million persons, approximately a third of the Danish population. The tax-supported Danish health care system provides free access to general practitioners, specialists and hospital care. The Danish Civil Registration System  assigns The LABKA database  containsThe Aarhus University Prescription Database  (AUPD) cThe Danish National Prescription Registry  holds daThe Danish National Patient Registry  (DNPR) rThe Danish Psychiatric Central Research Register  (DPCRR) The Danish Register on Personal Income  holds daData on education from the Danish Education Register  is colleData on employment status at the individual levels was collected from the Danish Register on Labor Market Affiliation . This reWe selected a primary P-B12 cohort with a first-time P-B12 measurement in LABKA from January 1, 2000 to December 31, 2013, with the date of P-B12 measurement serving as the index date.Propensity Score Matching).We defined low P-B12 as <200 pmol/L (the lower reference limit for the population) and normal P-B12 as 200–600 pmol/L. We used propensity scores to match patients with low P-B12 values in a 1:1 ratio to patients with normal P-B12 values  Age <40 years; 2) P-B12 levels >600 pmol/L, since high values may reflect severe disease, including cancer or liver disease ; 3) DiagThe main outcome was Alzheimer’s disease and the secondary outcomes were all-cause dementia and vascular dementia recorded in the DNPR. All-cause dementia was defined as a code for either Alzheimer’s disease, vascular dementia, or another dementia type .All members of the patient cohorts were followed until the date of an incident dementia diagnosis, emigration, death, or December 31, 2015, whichever came first.The propensity score included diseases that are potential risk factors for dementia and diseases other than dementia that are associated with B12 deficiency. Data on diseases were collected as diagnosis codes recorded in the DNPR or DPCRR and prescriptions contained in the AUPD for drugs used to treat these diseases  for all patients and visually illustrated the propensity score distribution among low and normal P-B12 levels. Using a nearest neighbor matching, we matched each participant with low P-B12 levels with the closest possible normal P-B12 levels group participant. The matching was performed without replacement, within a maximum matching range  in propensity score of 0.2 times the standard deviation of the propensity score.The secondary B12 treatment cohort was further divided into three treatment sub-cohorts and propensity score matching was repeated according to the three types of B12 drugs. The analyses included the covariates describe below:While P-MMA is a more sensitive marker for B12 deficiency than P-B12, it is less widely used than P-B12 due to laboratory costs . TherefoUsing the Danish Register on Personal Income, we categorized personal income in quartiles in the same year as the index dates for the two cohorts. The highest achieved education was obtained from the Danish Education Register and categorized as primary school, youth education, high school or similar education, and higher education (bachelor’s or higher degree). Employment status from the Danish Register on Labor Market Affiliation during the same year as the index date was categorized as employed, unemployed, early retirement, and state pensioner. Patients with missing socioeconomic data were excluded.The Danish National Prescription Registry was used to capture data on the approximately 15% of B12 prescriptions that are redeemed without reimbursement. Due to data restrictions, we were unable to include these data in the phase of propensity score matching or as exclusion criteria in the initial creation of the two study cohorts. Hence, we did not use data on non-reimbursed B12 prescriptions to identify the secondary B12 treatment cohort, but only to capture B12 treatment during follow-up and for exclusion after propensity score matching in a sensitivity analysis in the primary P-B12 cohort.We computed incidence rates (IRs) per 1000 person-years of follow-up with 95% CIs according to the two groups defined by low or normal P-B12 levels.We used a stratified Cox proportional hazards regression model to compute adjusted hazard ratios (HRs) with corresponding 95% CIs, treating patients with normal P-B12 values as reference. We adjusted for age, sex, income level, highest achieved education, employment status, and calendar year of the index date for the two cohorts.Estimates were computed separately for the primary P-B12 cohort and the secondary B12 treatment cohort and stratified according to primary and secondary outcomes and years of follow-up .First, we defined B12 treatment during follow-up in the primary P-B12 cohort as at least one B12 drug prescription in the AUPD after the index date to assess if B12 treatment during follow-up affected the association between low P-B12 and dementia outcomes. We computed IRs and HRs for both treated and untreated patients during follow-up, as described above.Second, to assess any drug-specific effects, we repeated the analyses in the secondary B12 treatment cohort for each of the three B12 drug treatments.2 were excluded. Patients were categorized according to P-MMA levels in three categories: not B12 deficient: ≤0.28μmol/L; possibly B12 deficient: 0.29–0.45μmol/L; and B12 deficient: ≥0.46μmol/L. We computed IRs with 95% CIs for Alzheimer’s disease according to P-MMA categories in both the two cohorts. Using patients with P-MMA values ≤0.28μmol/L as the reference, we performed stratified Cox proportional hazards regression, adjusting for the same covariates as in the main analysis plus the variables used to define the diseases included in the propensity score. Both IRs and HRs were disaggregated according to years of follow-up.Third, we excluded patients with kidney disease from the analysis of P-MMA by using the P-creatinine level to compute estimated glomerular filtration rates based on the modified Modification of Diet in Renal Disease Study equation . Patient20,036 patients were excluded from the secondary B12 treatment cohort due to incomplete propensity score matching. To assess the potential bias created by this exclusion, we analyzed the secondary cohort before propensity-score matching, including the drug-specific subanalysis. We used Cox regression analyses with additional adjustment for the same diseases that were included in the propensity score.We added data on B12 drug prescriptions from the Danish National Prescription Registry to capture data on non-reimbursed B12 prescriptions for exclusion and to assess B12 treatment during follow-up in the primary P-B12 cohort.All statistical analyses were performed using SAS version 9.4 . The study was approved by the Danish Data Protection Agency (record number: 2014-54-0922). Ethics approval is not needed for using registry data for research in Denmark .The primary P-B12 cohort included 53,089 patients with a median age of 63 years in each of the two P-B12 level groups. Median follow-up time was 5.1 years (interquartile range: 2.8–7.6 years) for patients with normal P-B12 values and 5.0 years (interquartile range: 2.7–7.5 years) for patients with low P-B12 values. In total, 6.8% of patients with normal P-B12 levels and 48.6% of patients with low P-B12 levels received a B12 drug prescription during follow-up.The secondary B12 treatment cohort consisted of 13,656 patients in each of the two P-B12 level groups. The median age was 65 years, and median years of follow-up were 4.5 years (interquartile range: 2.5–6.8 years) among patients with normal P-B12 values and 4.8 years (interquartile range: 2.6–7.2 years) among patients with low P-B12. For further characteristics of the two cohorts, see The IRs for the primary outcome, Alzheimer’s disease, were slightly different between the B12 level groups, but only within the first two years of follow-up . This diThe IRs for the primary outcome, Alzheimer’s disease in the secondary B12 treatment cohort were similar among low and normal B12 level groups for all follow-up strata, and all HRs centered around 1 . ResultsWe found no effect of any of the three specific B12 drugs on the primary outcome, Alzheimer’s disease risk according to pre-treatment P-B12 levels, except for injection therapy with 1 mg cyano-B12 in oil suspension, which showed a tendency toward lower IRs and HRs with longer follow-up. However, risk estimates were imprecise with wide CIs .Higher P-MMA levels were associated with a higher IRs of Alzheimer’s disease, mainly within the first two years of follow-up. After adjusting for potential confounders, the HRs centered around 1 and revealed no dose-response association between P-MMA levels and risk of Alzheimer’s disease .Without propensity score matching, results were similar compared to the main analysis, except for higher IRs for patients with P-B12 values <200 pmol/L . The samWhen adding data on non-reimbursed B12 drug prescriptions to the primary cohort, 79 patients were excluded due to B12 treatment before P-B12 measurement; 57 among those with normal P-B12 and 22 among those with low P-B12. A total of 2,312 patients were re-classified as B12 treated during follow-up; 354 among those with normal P-B12 and 1,958 among those with low P-B12. Estimates of IRs and HRs were very similar to the results of the primary analysis, with only reimbursed B12 prescriptions (data not shown).In a large population-based cohort study in Northern and Central Denmark, we found that low P-B12 levels and high P-MMA levels were not associated with risk of Alzheimer’s disease, all-cause dementia, or vascular dementia. Moreover, high-dose B12 treatment did not alter these associations regardless of pre-treatment P-B12 or P-MMA levels, and regardless of the specific B12 treatment.We found higher IRs of Alzheimer’s disease among patients with low P-B12 in the primary P-B12 cohort during short-term follow-up and particularly among those treated with high-dose B12. This finding could be due to confounding by indication for initiating B12 treatment, since higher risk was associated with high-dose treatment. We also observed lower IRs with longer follow-up among B12-treated patients with a low P-B12 level, a finding that suggests a possible compensatory deficit for the higher rates found within the first two years. The results from the secondary cohort support this view, showing no effect of pre-treatment P-B12 or P-MMA levels on risk of Alzheimer’s disease during both long and short-term follow-up. Findings were similar for all-cause dementia and vascular dementia. Taken together, these results suggest that B12 deficiency is not an important risk factor for Alzheimer’s disease or other dementias and that B12 treatment is ineffective for prevention, even in B12 deficient patients.A recent meta-analysis, primarily involving people without dementia, did not find an effect of B vitamin treatment in any cognitive domain  and anotEarlier clinical guidelines published by the UK National Institute for Health and Care Excellence recommended routine screening for B12 deficiency among patients suspected of dementia . This reOur study has limitations. We studied patients who had a P-B12 measurement without knowing the clinical indication for the test. This patient group might have different disease risk profiles compared to the general population and also according to P-B12 levels. In this context, potential bias due to patient selection relates closely to the confounding effect of indication, a concern in observational studies. Neurological or psychological symptoms could be an indication to measure P-B12 levels and possibly also to initiate B12 treatment, as recommended in earlier guidelines . HoweverA particular strength of our study is the good comparability of P-B12 measurements . Hence, In conclusion, these robust results in a large patient population suggest that B12 deficiency does not contribute to the risk of Alzheimer’s disease or other dementia types, and that high-dose B12 treatment does not prevent these diseases regardless of P-B12 or P-MMA levels. Our findings from hospital inpatients, hospital outpatients, and general practitioner patients are highly representative of all Danish patients. These findings have high external validity at least for high-income countries. They have public health implications for the assessment of dementia and imply that guidelines suggesting routine screening for B12 deficiency in patients with suspected dementia should be reconsidered.Click here for additional data file.


A conserved mechanism regulates the dominantly inherited double-flower trait, and involves natural allelic variation at the microRNA172 binding site in the novel PETALOSA subclade of TARGET OF EAT (TOE)-type genes. Rosa rugosa, and identified mutant alleles of TARGET OF EAT (TOE)-type genes characterized by a disruption of the miR172 target sequence and of the C-terminal portion of the encoded protein. Despite the phylogenetic distance between these eudicots, which diverged in the early Cretaceous, the orthologous genes carrying these mutations all belong to a single TOE-type subgroup, which we name as PETALOSA (PET). Homology searches allowed us to identify PET sequences in various other species. To confirm the results from naturally occurring mutations, we used CrispR-Cas9 to induce lesions within the miR172 target site of Nicotiana tabacum PET genes, and this resulted in the development of supernumerary petaloid structures. This study describes pet alleles in economically important ornamental species and provides evidence about the possibility of identifying and engineering PET genes to obtain the desirable double-flower trait in different plants.The double-flower phenotype has been selected by humans for its attractiveness in various plant species and it is of great commercial value for the ornamental market. In this study we investigated the genetic determinant of the dominant double-flower trait in carnation, petunia, and  Prunus persica) and the Rosa genus (APETALA2/ETHYLENE RESPONSIVE ELEMENT-BINDING FACTOR (AP2/ERF) family and are divided into two groups, AP2-type and TARGET OF EAT (TOE)-type, both of which are characterized by the presence of a target site for the binding of microRNA172 (miR172), which negatively regulates their activity  is a desirable trait that increases the number of petals and overall flower size . Investiactivity , with APD85 locus; Do1 locus; R. rugosa, and discovered mutant alleles of TOE-type genes—hereafter referred to as PETALOSA (PET) genes—characterized by a disruption of the miR172 target sequence. To further support a conserved role of allelic variability in PET genes, we used a genome-editing approach to induce mutations within the miR172 target site of Nicotiana tabacum PET genes and obtained tobacco lines characterized by additional petals and petaloid stamens.Dominant flower doubleness is widespread amongst angiosperms, and it has been selected as a leading ornamental trait in commercially important plants such as carnation (Dianthus varieties were kindly provided by Floricoltura Billo (https://www.floricolturabillo.it/) and Hybrida srl. Rosa rugosa material was kindly provided by Le Rose di Firenze (https://www.lerosedifirenze.com). Petunia plants were obtained from local garden centres and are identified using their commercial names.Dianthus pet allele), CA-9f/CA-4r , PH-9F/PH-M-3R , or PH-9F/PH-10R . RACE analysis was carried out in a 25-µl reaction using GOTaq from double-flower (DF) petunia cDNA using PH-9F and B25 or from DF Dianthus cDNA using CA-9f and B25.Total RNA was extracted from floral buds using a Quick-RNA Miniprep Kit (Zymo) following the manufacturer’s protocol, with the modification of adding 2% PVP and 4% beta-mercaptoethanol (Sigma-Aldrich) to the tissue lysis buffer just before use. Then, 1 μg total RNA was treated with DNAseI (Invitrogen) and first-strand cDNA was obtained using Goscript Reverse Transcriptase (Promega), using either a standard oligo-dT primer or the B26 primer containing an adaptor sequence (R. rugosa genomic DNA were digested with 2 µl TaqIa (NEB) in Buffer Cutsmart in a 20-µl final reaction . The digestion reaction was purified with PCR clean-up kit (Promega) and the concentration adjusted to 50 ng µl–1. Then, 20 µl of B25 and B25_TaqI_adapter primers (100 pmol µl–1) were mixed, heated for 5 min at 75 °C and allowed to cool down to room temperature to anneal into 5´-CG-3′ overhang adapters. Following this, 200 ng of digested DNA and 100 pmol of B25 adapter were ligated using T4 Ligase (Invitrogen). The ligation was used as template for a two-step nested PCR. A 13-cycle pre-PCR cycle was carried out using RO-8F/B25, and this was used as the template for nested PCR, using RO-8F2/B25. Amplified bands were gel-purified and sequenced.Samples of 2 µg of single-flower and DF JXB online) as follows: CA-9f/CA-4r or CA-9f/CA-1r, ; PH-9F/PH-10R or PH-9F/PH-M-3R ; and RO-8f/RR-3UTRr for both R. rugosa wild-type and pet alleles.Genomic DNA was extracted from 200 μg of leaf tissue using a DNeasy 96 Plant Kit (Qiagen); 10 ng samples of genomic DNA were used in the PCR reactions using GoTaq and the appropriate primer combinations , R. rugosa ‘Hansa’, using the primers CA-F1/CA-4R (Dianthus), PH-F1/PH-UTR-R , PH-F1/PH-M-3R , and RO-1F/RR-UTR-R (R. rugosa). PCR bands were extracted from agarose gels using a Wizard SV Gel and PCR Clean-Up System (Promega) and paired-end sequenced on an Illumina MiSeq instrument, following the manufacturer’s instructions, using a transposome-based Nextera XT kit (Illumina) to generate the libraries. FASTQ files were mapped with BWA-MEM against reference sequences on the Galaxy Platform (Full-length genomic amplicons were obtained with GoTaq Long (Promega) using genomic DNA obtained from P. persica, R. chinensis, P. hybrida, P. axillaris, A. thaliana, and Vitis vinifera were obtained from previously published work , and sequences for D. caryophyllus were obtained from the reference genome website (http://carnation.kazusa.or.jp/). For Dianthus Dca21030 the wild-type allele (Dca21030.2) was used (see Results). Phylogenetic relationships were estimated in MEGAX . To ensure the transcription of specific guide RNA, the oligonucleotides TOB-CRISPR_for and TOB-CRISPR_rev were annealed and ligated into AarI-digested pHAtC. Nicotiana tabacum cv TI 527 ‘Kentucky’ plants were transformed with a c58 Agrobacterium suspension following the method of –1). T0 plants were grown under standard greenhouse conditions until flowering. Mutations in the NtBEN miR172 binding sites were assessed by Sanger sequencing of PCR fragments obtained using either the forward primer NtBEN_016482517_F (XP_016482517) or NtBEN_016499635_F (XP_016499635), and a common reverse primer, NtBEN_SEQR. T1 seedlings were PCR-screened for the presence of the transgene using the primers LBfor/LBrev and RBfor/RBrev, specific for the T-DNA sequence. The presence of off-targets in transgene-free T1 plants was assessed using a high-resolution melting (HRM) analysis-based approach. Primers  that include both single- and double-flower varieties. Homology searches on the carnation (Dianthus caryophyllus L.) genome  that leads to flower doubleness in ‘Francesco’. Allelic comparison of the 3´ region revealed the presence of only the wild-type allele in the single-flower accessions D. superbus ‘Primadonna’ and D. deltoides ‘Flashing lights’  harboured a complete miR172 target site and a further 83 bp of coding sequence  and P. axillaris Peaxi162Scf00472g00069.1, while the second showed an insertion positioned at the level of the 10th exon, 69 bp upstream of the miR172 target site  and BROTHER OF BEN (BOB), two get site , as confxillaris  and P. i domains . The gen domains . Interesarieties . Expressarieties .R. hybrida pet marker  was found in C. papaya, which belongs to the order Brassicales, suggesting that a PET was indeed originally present in the lineage. A survey of the reference allotetraploid N. tabacum genome , and a NtBOB   . Notablye itself  resultede itself .PET genes and the dominant DF trait in different plant species. We therefore investigated whether artificially induced mutations in the miR172 target site of a PET gene would be sufficient to induce the DF phenotype in a different plant species without naturally occurring DF variants. To this end, we used CrispR-Cas9 to specifically create mutations at the PET-miR172 target sequence in tobacco , while a 1-bp insertion in both NtBEN genes resulted in the strongest DF phenotype (line IV).Our results demonstrated a strict association between a range of naturally occurring mutations in orthologous  tobacco . Of seve flowers . Molecul editing . Thus, tobtained . Sequencubleness . A singlThe understanding of the mechanisms governing flower development has been a major goal in plant biology, and knowledge gained from model species does not always apply to other plants. Initial insights into the molecular basis of dominant mutations that confer the double-flower (DF) trait were recently obtained when structural variants that disrupt miR172 target sites within a subclass of TOE-type genes were pinpointed as prime candidates in Rosaceae  and Vitales (V. vinifera) belonging to Group I, as well as in Asterids  and Caryophyllales  belonging to Group II. These two groups are estimated to have diverged around 120 Mya, in agreement with the radiation of the Pentapetalae lineage that recent studies have set in the early Cretaceous  have been reported to act redundantly in various plant developmental processes, such as seedling innate immunity  under BioProject accession number PRJNA600422.The FASTQ files of the amplicon sequencing data have been deposited at the NCBI Sequence Read Archive (SRA;


Background: Combating viral disease outbreaks has doubtlessly been one of the major public health challenges for the 21st century. (2) Methods: The host entry machinery required for COVID-19 (SARS-CoV-2) infection was examined for the gene expression profiles and polymorphism. (3) Results: Lung, kidney, small intestine, and salivary glands were among the tissues which expressed the entry machinery coding genes Ace2, Tmprss2, CtsB, and CtsL. The genes had no significant expression changes between males and females. The four human population groups of Europeans, Africans, Asians, and Americans had specific and also a common pool of rare variants for the X-linked locus of ACE2 receptor. Several specific and common ACE2 variants including S19P, I21T/V, E23K, A25T, K26R, T27A, E35D/K, E37K, Y50F, N51D/S, M62V, N64K, K68E, F72V, E75G, M82I, T92I, Q102P, G220S, H239Q, G326E, E329G, G352V, D355N, H378R, Q388L, P389H, E467K, H505R, R514G/*, and Y515C were of the utmost importance to the viral entry and infection. The variants of S19P, I21T, K26R, T27A, E37K, N51D, N64K, K68E, F72V, M82I, G326E, H378R, Q388L, and P389H also had significant differences in frequencies among the population groups. Most interestingly, the analyses revealed that more than half of the variants can exist in males, i.e., as hemizygous. (4) Conclusions: The rare variants of human ACE2 seem to be one of the determinant factors associated with fitness in the battle against SARS viruses. The hemizygous viral-entry booster variants of ACE2 describe the higher SARS-CoV-2 mortality rate in males. This is also supported by the lack of gender bias for the gene expression profiles of entry machinery. A personalized medicine strategy is conceived for isolating high-risk individuals in epidemic circumstances.(1)  The early 21st century has been a momentous era in contagious disease history. During the last twenty years, public health has been repeatedly threatened by the pathogenic respiratory beta-coronaviruses (CoV) including the Severe Acute Respiratory Syndrome CoV (SARS-CoV) . These aThe variations of susceptibility to SARS-CoV among different species have been found associated with the inter-species genetic polymorphism of the host machinery for viral entry ,10,11. Ahttps://www.gtexportal.org/home/. Tissue-specific co-expression analysis was performed on TPM measurements by using the BicMix biclustering. The human SNP data was extracted from the GenBank, the database for Single Nucleotide Polymorphisms (dbSNP) including the 1000 genomes project data, the exome aggregation consortium data, and the genome aggregation data. For every variant, data was obtained from the experiments and pooled. Data, i.e., number of individuals carrying the reference or variant alleles, was processed using chi-square statistics. The abundance of the rare variant  and the corresponding reference allele  in every comparison were used to calculate the expected values for the chi-square test.Gene expression data was extracted from the Genotype-Tissue Expression (GTEx) project  publiclyAce2, Tmprss2, CtsB, and CtsL genes were found expressed in different tissues  ,18, the  tissues a. The mi tissues a. Ace2 ahe heart a. Among ith Ace2 b. Furtheith Ace2 b. The adhttps://www.ncbi.nlm.nih.gov/snp/, ∼ 100 to 400 missense SNPs were extracted after filtering for the non-coding regions of Ace2, Tmprss2, CtsB, and CtsL genes  and Africans  specific variants, respectively. Furthermore, another group of 18 SNPs including rs1348114695 [E35K], rs146676783 [E37K], rs1192192618 [Y50F], rs760159085 [N51D], rs1569243690 [N51S], rs1325542104 [M62V], rs755691167 [K68E], rs1256007252 [F72V], rs766996587 [M82I], rs759579097 [G326E], rs143936283 [E329G], rs370610075 [G352V], rs961360700 [D355N], rs751572714 [Q388L], rs762890235 [P389H], rs1016409802 [H505R], rs1352194082 [R514G/*], and rs1263424292 [Y515C] were found as interaction-inhibitor variants. The amino acid substitutions at the positions 505, 514, and 515 had no reported frequency. The variants E37K, N51D, K68E, F72V, M82I, G326E, Q388L, and P389H had significant changes in frequency among the population groups  and Africans  specific variants, respectively. The impact on the viral infectivity was not clear for the Asians specific variants of rs751603885 [R697G], rs763593286 [A532T], rs745514718 [S257N], and rs200180615 [E668K].The global prevalence for rare variants of parisons a. Statisparisons . Interesparisons a. Interen groups a. The Q3Ace2 locus are lethal and have been under strong negative selection or not, the incidence of variants in males, i.e., the hemizygous state, were analyzed using the genome aggregation data (https://gnomad.broadinstitute.org/). Here, the majority (98.3%) of missense variants were as heterozygous in females. In addition, 51% of the variants were also present in males indicating non-lethality in the hemizygous state. Finally, 7 out of 10 interaction-booster variants  and 9 out of 14 interaction-inhibitor variants  were also found in males. Therefore, more than half of the variants have the chance to influence the interaction between the human ACE2 and the viral S1 protein in the hemizygous state, i.e., in the absence of the reference allele, in males. Accordingly, gender bias has been observed towards a higher mortality rate in males accounting for 60%−70% of death despite the similar SARS-CoV-2 infection rates between the genders [As an X-linked phenotype, the efficacy of interaction-booster and interaction-inhibitor rare variants of ACE2 can be stronger in males than females. This is due to the hemizygosity in males. To examine whether the rare variants of the X-linked  genders .−5 without significant difference among the population groups (Weak ACE2-S1 interactions lead to lower levels of SARS-CoV susceptibilities in rats, and also to some extent, in pig and mouse ,10. Thren groups a. As seen groups a, b.Ace2, Tmprss2, CtsB, and CtsL in asexual tissues. For any population group of interest, therefore, it is worth to investigate the enrichment of the rare variants among the SARS-CoV-2 infected cases with severe symptoms. The results have the potential to advance the personalized medicine strategies, e.g., by screening for the high-risk individuals that need isolation against the viral disease outbreaks.Taken together, the human ACE2 has a rich pool of rare variants, which can explain the individual competence in the battle against the SARS viruses. Most interestingly, there are statistically significant variations in the frequencies of the rare variants among the human population groups. These alleles, as 34 introduced in this study, can potentially be decisive in SARS-CoV/CoV-2 recognition and infection. As an X chromosome linked phenotype, more than half of the ACE2 rare variants were also found in males. The impact of the ACE2 rare variants can be more substantial in males than females. Accordingly, the interaction-booster variants of ACE2 can explain the gender bias towards a higher mortality rate caused by SARS-CoV-2 in males; this is in line with the lack of gender bias for the expression of


Virus-like particle (VLP) is a self-assembled nanostructure incorporating key viral structural proteins. VLP resembles molecular and morphological features of authentic viruses but is non-infectious and non-replicating due to lack of genetic materials. Successful applications of VLP has been shown in vaccinological and virological research. As an accessibly safe and relevant substitute of naturally pathogenic viruses, the construction of SARS-CoV-2 VLPs is much in demand in the ongoing fight against 2019 Coronavirus disease (COVID-19) pandemics. In the current study, using mammalian expression system, which is advantageous in maintaining correct protein glycosylation patterns, we efficiently constructed SARS-CoV-2 VLPs. We showed that among four SARS-CoV-2 structural proteins, expression of membrane protein (M) and small envelope protein (E) are essential for efficient formation and release of SARS-CoV-2 VLPs. Moreover, the corona-like structure presented in SARS-CoV-2 VLPs from Vero E6 cells is more stable and unified, as compared to those from HEK-293T cells. Our data demonstrate that SARS-CoV-2 VLPs possess molecular and morphological properties of native virion particles, which endow such VLPs with a promising vaccine candidate and a powerful tool for the research of SARS-CoV-2. Coronaviridae family (The ongoing pandemic of 2019 Coronavirus disease (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a newly emergent member of e family . Though e family , SARS-Coe family . In the e family . Moreovee family . The lage family . Considee family , the intPseudo-viral system, which usually utilizes adenoviral or lentiviral vectors following BSL-2 practices have been used as experimental models to study entry events of enveloped viruses, including SARS-CoV-2 . HoweverAs a vaccine candidate, virus-like particle (VLP) has also been applied in the study of key processes of viral life cycle . Due to SARS-CoV-2 virions consist of four structural proteins, namely S, small envelope (E), membrane (M) and nucleocapsid (N) protein . Since tNheI and NotI  restriction sites were placed at 5′ and 3′ ends, respectively. The four genes were cloned into the double NheI and NotI restriction sites of the expression vector pcDNA3.1. Then the transformation experiments were performed with chemically competent cells DH5α  using the heat shock method in the water bath at 42°C for 1 min, followed by shaking at 37°C for 45 min. After centrifugation at 2,800 × g for 3 min, the transformed cells were plated on LB plates containing 50 μg/ml ampicillin and the plates were inverted and incubated at 37°C overnight. The resistant single colony was picked and amplified in LB medium. The correct orientation of the insertions was examined by restriction enzyme analysis and the open reading frames of recombinant plasmids were verified by DNA sequencing.Human codon optimized sequences of genes encoding S, M, E and N structural proteins of SARS-CoV-2 with C-terminal FLAG tag (peptide sequence: DYKDDDDK) were synthesized by Genscript Biotechnology : the major structural S glycoprotein (Gen Bank: QHD43416.1), E protein (Gen Bank: QHD43418.1), M protein (Gen Bank: QHD43419.1) and N protein (Gen Bank: QHD43423.2). 2 at 37°C.HEK-293T human embryonic kidney cell line and Vero E6 African green Monkey kidney cell line were purchased from the American Type Culture Collection  and cultured in Dulbecco’s modified essential medium (DMEM) from Gibco . The cell culture medium contained 10% fetal bovine serum , penicillin (100 U/ml) and streptomycin (100 μg/ml) and cells were maintained with 5% COD-lysine  12 h before transfection. The plasmid constructs were transfected or co-transfected with lipofectamine 3000 (Invitrogen) and Opti-MEM reduced serum medium (Gibco) into HEK-293T cells and Vero E6 cells, respectively. For transfection of single plasmids, a total of 2.5 μg of each plasmid was applied individually. Co-transfection of double and triple plasmids was conducted with equal molar of each plasmids. Construction of SARS-CoV-2 VLPs was performed by co-transfecting cells with S, M, E, and N with molar ratio at 8:6:8:3 as shown in g for 10 min at 4°C. Afterward, the collected supernatant was filtered through a 0.45 μm filter membrane , and then the filtrates were centrifuged with 20% sucrose at 21,000 × g for 7 h at 4°C. The final pelleted particles were recovered in ddH2O. Meanwhile, attached cells were washed with cold PBS, and then harvested and lysed with prechilled lysis buffer . After centrifuge at 13,000 rpm for 15 min at 4°C, cell lysate was collected. Alternatively, VLP-containing pellets from 20% sucrose centrifugation were resuspended in TNE buffer , loaded on top of 20–60% discontinuous sucrose gradients and ultra-centrifuged for 3.5 h at 26,700 rpm. Nineteen fractions were then collected and analyzed by immunoblotting.Cells were seeded into 6-well plate coated with Poly-The collected samples were applied to 4–20% SDS-PAGE. After SDS-PAGE separation and transfer, membrane blocking was performed with 3% BSA (Sigma) for 1 hr at room temperature. Primary antibody against flag , against SARS-CoV-2 spike glycoprotein , against SARS-CoV-2 nucleoprotein/NP , and secondary anti-Rabbit IgG  were applied before image capture. The expression of VLP proteins was developed by high sensitivity western chemiluminescent HRP substrate (Millipore) and visualized under ChemiDoc MP Imaging System .For negative staining, collected VLPs were placed onto carbon-coated grids for 1 min and stained with phosphotungstic acid (Sigma) for 45 s. After air-dry overnight, the thin sections were observed and imaged with a transmission electron microscope  at 100 kV.t-test. The level of statistical significance was set for p < 0.05. Data and graphs were generated by GraphPad Prism 7.0, and figures were performed using Illustrator CC 2018 (Adobe).All experiments were repeated at least three times independently. All data are presented as mean ± standard deviations (SD) with Student’s To get better understanding of the secretory features of four SARS-CoV-2 structural proteins, we first transfected HEK-293T and Vero E6 cells with vectors expressing S, M, E, or N, respectively. As shown in To gain further insight into how S, M, E, and N may mutually be regulated in terms of protein egress, we then co-transfected HEK-293T and Vero E6 cells with equal molar of 2 out of 4 vectors . Our datAiming to verify the correlation of proteins in double-transfection systems, we next explored the efficiency of protein release by combination of three expressing vectors. Consistent with pairwise relationships, addition of E to S-harboring systems (S + M and S + N) dramatically increased the amount of egressed proteins, confirming the supportive function of E. Moreover, incorporations of S into M-presenting conditions (M + E and M + N) resulted in restrained release of M, highlighting a limiting effect of S on M. In addition, supplementing M or N had no further effects on cells expressing any other two constituents .By using 20–60% discontinuous sucrose gradient , we wereAltogether, these data establish for the first time that M is the most critical protein that drives SARS-CoV-2 VLP egress. Presence of E may substantially potentiate the release efficiency of both M and S. Considering that viral infectivity depends highly on S, the minimal molecular requirement for an efficient assembly and egress of SARS-CoV-2 VLP is both M and E proteins.Though the potency of protein egress is stronger in HEK-293T cells compared to Vero E6 systems , the morcoronaviridae family, M protein is the most abundant envelope protein that drives other structural components to be packed into VLPs. Though E protein has also been implicated beneficial in viral morphogenesis and release, the mechanistic action of E remains unclarified. The S protein, which is responsible for receptor binding, membrane fusion and as targets of drug and vaccine development, and N protein which encapsidates viral genome into virions, do not seem to have indispensable roles in SARS-CoV-2 VLPs assembly. Our morphological evaluation of SARS-CoV-2 VLPs derived from Vero E6 cells further confirmed a high incorporation of S glycoprotein on the surface of VLPs. To the best of our knowledge, most current studies focus on pseudo-virus-based platform of SARS-CoV-2, a successful construction of SARS-CoV-2 VLPs via mammanlian expression system has not yet been reported. The approach in our study suggests that SARS-CoV-2 VLPs molecularly and morphologically resemble the native virion particles, which not only hold promise for virological research, but also present a potential vaccination for SARS-CoV-2.In the current study, we described the efficient construction of SARS-CoV-2 VLPs by plasmid-driven transfection of viral structural proteins in mammalian cells. By comparing expression induced by single, double or triple expressing vectors, we are able to demonstrate that M and E are basically required for efficient assembly and release of SARS-CoV-2 VLPs. Similar to other members in All datasets presented in this study are included in the article/supplementary material.NL, RX, and JL conceived and designed the experiments. RX and NL conducted the experiments and analyzed the data. NL, RX, MS, and PS wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


Human enteroviruses (EVs) are highly prevalent in sewage and have been associated with human diseases with complications leading to severe neurological syndromes. We have used a recently developed molecular method to investigate the presence of EVs in eight samples collected in 2017–2018 from water streams contaminated by drainage channels in three different locations in Nigeria. A total of 93 human EV strains belonging to 45 different serotypes were identified, far exceeding the number of strains and serotypes found in similar samples in previous studies. Next generation sequencing analysis retrieved whole-capsid genomic nucleotide sequences of EV strains belonging to all four A, B, C, and D species. Our results further demonstrate the value of environmental surveillance for the detection of EV transmission of both serotypes commonly associated with clinical syndromes, such as EV-A71, and those that appear to circulate silently but could eventually cause outbreaks and disease. Several uncommon serotypes, rarely reported elsewhere, were detected such as EV-A119, EV-B87, EV-C116, and EV-D111. Ten EV serotypes were detected in Nigeria for the first time and two of them, CV-A12 and EV-B86, firstly described in Africa. This method can be expanded to generate whole-genome EV sequences as we show here for one EV-D111 strain. Our data revealed phylogenetic relationships of Nigerian sewage strains with EV strains reported elsewhere, mostly from African origin, and provided new insights into the whole-genome structure of emerging serotype EV-D111 and recombination events among EV-D serotypes. Water is an essential resource used for critical human activities such as drinking, cooking, washing, recreational events, crop irrigations, and industrial use. While developed countries have made significant progress in providing adequate quality water and sanitation to most households, many of the developing countries are still deficient in these areas, leading to many deaths related to the ingestion of virus-contaminated water and other waterborne pathogens. It is understood that an estimated 663 million people globally regularly consume untreated water obtained from different sources including groundwater and surface water . ContamiEVs are common pathogens that have co-evolved with humans for many years transmitting mainly through the fecal-oral or respiratory routes with use of poor-quality water supply and sanitation being associated with high virus transmission. The EV genome consist of a single RNA positive strand molecule approximately 7400 nucleotides in length that contains a region coding for structural and non-structural proteins flanked by 5′ and 3′ short non-coding regions . A new aAs an alternative to clinical diagnosis, environmental surveillance (ES) has revealed as a very sensitive method for EV detection, particularly for poliovirus, which can be readily identified in sewage and water systems contaminated with sewage even in the absence of paralytic disease ,10,11,12With a view to investigate the prevalence and diversity of EVs in samples from water streams often contaminated by drainage channels in Nigeria, we have used a recently developed NGS method to sequence PCR products synthesized directly from water concentrates retrieving whole-capsid sequences of EV strains from all four A to D species ,17. EV nOne liter grab water samples were collected at random from water bodies such as streams and rivers, presumably contaminated by drainage channels, at three different locations in Nigeria during 2017 and 2018 . CommuniSamples that did not contain poliovirus were specifically selected in order to focus on non-polio EV serotypes and avoid biosafety containment issues. Although, it cannot be completely ruled out, we do not expect bias in EV serotype distribution due to selecting poliovirus-negative samples in our study. First, the workflow used for all samples was identical, following WHO Guidelines for environmental surveillance . Second,Primers 5′NCR (5′-TGGCGGAACCGACTACTTTGGGTG-3′) and CRE-R (5′-TCAATACGGTGTTTGCTCTTGAACTG-3′)Primers MM_EV_F2 (5′-CAGCGGAACCGACTACTTT-3′) and MM_EV_R1 (5′-AATACGGCATTTGGACTTGAACTGT-3′)We followed a method designed to amplify the entire capsid sequences of EV strains from all four A, B, C, and D described before , which iReaction conditions were: 50 °C for 30 min followed by 94 °C for 2 min plus 42 cycles of 94 °C for 15 s, 55 °C for 30 s, and 68 °C for 8 min with a final extension step of 68 °C for 5 min. Amplified products from both reactions were purified using AMPure XP magnetic beads  and pooled (1:1) before being analyzed by NGS. The expected amplicon size for both RT-PCR reactions is approximately 3950 nucleotides .Sequencing libraries were prepared using Nextera XT reagents and sequenced on a MiSeq using a 2 × 301-mer paired-end v3 Flow Cell and the manufacturer’s protocol . NGS sequencing data were processed using Geneious 10.2.3 software package  as described before . BrieflyThe filtered NGS reads were then assembled de novo using stringent assembly conditions as described before : minimumhttp://tree.bio.ed.ac.uk/software/figtree/).EV sequences obtained in this study were compared to those of other EVs available in the NCBI database. The closest virus relatives to the Nigerian EV environmental strains were identified using the RIVM and BLAST online sequence analysis tools ,28 and EThe nearly complete genome of EV-D111 strain from KAT-17-1263 sample was determined by NGS analysis of overlapping PCR products. Primers D111-WGF (5′-CAGGAAACAGCTATGACCTTAAAACAGCYYKDGGGTTG-3′) and D111-IR (5′-GCACATAAACGGTATGGTCATTCTAGCTGG-3′) were used to generate a PCR product covering the 5′-end half of the genome and primers D111-IF (5′-CCAGCTAGAATGACCATACCGTTTATGTGC-3′) and D111-WGR (5′-TTCCCAAKTRACCAAAATTTACCTC-3′) were used to generate a PCR product covering the 3′-end of the genome. Amplification conditions and NGS analysis to obtain consensus sequences for each RT-PCR product were the same as those described in The nearly complete genome of EV-D94 was determined in a different project characterizing EVs present in infected cell cultures from clinical stool samples from AFP surveillance. As it is expected that such cell culture samples contain high concentration of virus, we opted for sequencing PCR products generated by random primers as opposed to specific primers required to amplify low concentrations of viral RNA present in environmental samples. PCR fragments for sequencing were generated by sequence-independent single-primer amplification (SISPA) of purified RNAs from infected cells and NGS analysis was performed as described before ,31. RT-Phttps://sray.med.som.jhmi.edu/SCRoftware/simplot/) [p-value cutoff was chosen as 0.05 and the best signals for recombination are associated with the lowest p-values, which indicates the approximate likelihood for the occurrence of exchange of sequences between genomes (recombination) rather than the probability of convergent evolution of the sequences.The whole-genome sequences of EV-D11-KAT-17-1263 and EV-D94-14-534 strains were aligned to those from representatives of Species D EV serotypes D68, D70, D94, and D111 using the program ClustalW (within Geneious 10.2.3) and subjected to recombination analysis using Simplot version 3.5.1 software (implot/)  and the implot/) . Defaultimplot/) , Bootscaimplot/) , Chimaerimplot/) , MaxChi implot/) , SiScan implot/) , and 3Seimplot/)  were useNGS data obtained from Pan-EV RT-PCR products amplified from viral RNAs that were extracted from water concentrates were processed and analyzed as described in Materials and Methods. A high proportion of the total filtered NGS reads from all samples mapped to EV reference sequences (range: 84.07–99.22%) which shows the high specificity of the primers used for EV sequences present in RT-PCR products. A total of 93 contig sequences, corresponding to 93 different EV strains were identified in the 8 water concentrates from Nigeria. In most cases, nucleotide consensus sequences for each EV strain were approximately 4000 nucleotides in length, between nucleotides 553–4459 (numbering as in PV1 Sabin AY184219 reference strain) of the EV genome, covering part of the 5′-end non-coding region, the whole-capsid coding region and part of the P2 non-structural coding region. Nucleotide consensus sequences are available from NCBI sequence database with GenBank numbers MW373870 to MW373962. The results are shown in n = 43), followed by species C (n = 28), A (n = 20), and D (n = 2). We detected EV strains from 10 of 21 (47.62%), 26 of 62 (41.93%), 8 of 23 (34.78%), and 1 of 5 (20.00%), known serotypes for species A, B, C, and D, respectively. Ten EV serotypes were detected in Nigeria for the first time: CV-A2, CVA5, CV-A12, CV-A14, EV-B74, EV-B81, EV-B85, EV-B86, EV-B87, and EV-B106, two of them, CV-A12 and EV-B86, were first described in Africa. E-15 strains were the most widespread, present in five samples, whereas serotypes EV-A119, CV-A13, and CV-B4 were found in four samples each. E-15, EV-A119, and CV-A13 strains were found in all three locations. Some environmental EV strains identified corresponded to uncommon serotypes defined as those rarely associated with clinical cases and/or hardly ever reported elsewhere in the world from which very few nucleotide sequences are available, EV-A119, EV-A120, E-26, EV-B81, EV-B86, EV-B87, EV-C116, and EV-D111. Uncommon serotypes included A119 and D111 mostly seen in Central and South Africa. Several negative control samples were sequenced including RNA extraction, RT-PCR reaction, and water controls. No EV contig sequences were identified in any of the negative control samples following our analysis pipeline.Samples contained EV strains belonging to 45 different serotypes and all four A, B, C, and D species with between 2 and 21 different serotypes identified in each sample. Species B strains were the most abundant (n = 19) and neighboring/nearby countries such as Republic of Niger, Chad, Cameroon, Ivory Coast, Ghana, Senegal or DRC (n = 53). Although little sequencing data are available from recent EV clinical strains from Nigeria, the highest sequence similarity of the Nigerian strains from water samples was found to be with Nigerian clinical strains with few exceptions. In several cases, the observed VP1 sequence drift was compatible with EV strains of various serotypes having circulated in the country for long periods of time. We found sequence similarity between the EV strains from Nigeria and non-African EV strains; a CV-B4 strain from USA (96.0–96.4% VP1 sequence identity), an EV-A71 strain from Denmark (97.4% VP1 sequence identity), and a CV-A5 strain from France (96.6% VP1 sequence identity). Interestingly, the EV strain genetically closest to any of the Nigerian EV environmental strains (98.7% VP1 sequence identity) was found to be an EV-A76 isolate from a healthy chimpanzee found in 2016 in Nigeria. The closest E-15 strains from the water samples were also found to be distantly related to strains from non-human primates, gorillas and chimpanzees, found in Cameron in 2014. Nigerian EV strains from serotypes EV-A119 and EV-D111 were also found to be distantly related to viral sequences from non-human primates. Twelve different CV-A13 strains were identified in total; sample KAT-17-1263 contained 6 different CV-A13 strains and sample ADA-18-059 contained 3. Six different EV-A119 strains, 6 E-15, 4 E-14 and 4 CV-B4 strains were found in total. Phylogenetic trees based on VP1 nucleotide sequence comparisons of species A, B, C, and D EV strains are shown in n = 6), B (n = 39) and C (n = 7)  . As specThe nearly complete genomic sequence of EV-D111, from nt 33 to nt 7353 (based on numbering of EV-D111 strain with GenBank number MT081371) was obtained by NGS analysis of overlapping RT-PCR products as described in Materials and Methods. The NGS analysis produced a main contig generating a consensus sequence of 7315 nucleotides in length. This contig contained 119,853 sequence reads producing high sequence coverage throughout the genome (mean coverage of 3028 reads per site) as shown in The nearly complete genomic sequence of EV-D94 strain 14-534, from nt 1 to nt 7335 (based on numbering of strain with GenBank number MT081370) was obtained by NGS analysis of randomly synthesized PCR products as described in Materials and Methods. The NGS analysis produced a main contig generating a consensus sequence of 7293 nucleotides in length. This contig contained 44,105 reads with high sequence coverage throughout the genome (mean coverage of 1091 reads per site) as shown in p-values of 3.52 × 10−47, 8.84 × 10−55, 1.05 × 10−32, 8.24 × 10−15, 1.08 × 10−30, and 6.35 × 10−14 for methods RDP, Bootscan, Maxchi, Chimaera, SiSscan, PhylPro, LARD, and 3Seq, respectively). EV-D94-14-534 was identified as a recombinant strain with parental sequences EV-D94 in the capsid region and EV-D111-KAT-17-1263 in the non-structural region and breakpoint identified at nucleotide 3802 of the sequence alignment. The whole-genome sequences of EV-D111-KAT-17-1263 and EV-D94-14-534 strains were aligned to those from representatives of Species D EV serotypes to investigate the occurrence of recombination events among them. A main recombination event supported by high statistical significance values using six of the seven RDP algorithms was identified (average This analysis confirmed that EV-D94-14-534 showed closer sequence similarity in the non-structural coding region to EV-D111 strains than to EV strains from other EV-D serotypes including EV-D94. These results strongly suggest that recombination events likely occur between EV-94 and EV-D111 serotypes. Both EV-D111-KAT-17-1263 and EV-D94-14-534 strains showed higher sequence similarity between themselves in the non-structural coding region than they did to EV strains from their respective serotypes while they showed closer sequence similarity to homotypic EV strains in the capsid coding region as expected. However, only a few whole-genome sequences from all EV-D serotypes are available so further research will be needed to better determine and understand these potential recombination events.High prevalence and diversity of human EVs were found in water samples from Nigeria. A total of 93 different EV strains from 45 different serotypes belonging to all four A, B, C, and D EV species were identified in eight water samples collected from water streams presumably contaminated by drainage channels situated in three locations in Nigeria, confirming the high risk associated with domestic use of such water supplies due to sewage contamination. We were able to identify ten EV serotypes detected in Nigeria for the first time, two of them, CV-A12 and EV-B86, firstly described in Africa. EV strains were identified from both serotypes commonly associated with clinical syndromes and those that appear to circulate silently but could eventually cause outbreaks and disease. Among them, several emerging serotypes, rarely reported elsewhere, were detected such as EV-A119, EV-B87, EV-C116, and EV-D111. E-15 strains were the most prevalent, present in five samples, whereas serotypes EV-A119, CV-A13, and CV-B4 were found in four samples each. Our data revealed close phylogenetic relationships of the Nigerian strains with EV strains reported elsewhere, mostly from Central West Africa, indicating that many of these serotypes are endemically circulating in the region. High sequence divergence from the closest relative in some cases suggests long-term silent transmission of specific EV serotypes and possible gaps in surveillance. We found sequence similarity between the EV strains from Nigeria and non-African EVs from serotypes CV-B4, EV-A71, and CV-A5 which could represent recent importation events. EV-A71, in particular, has been associated with neurological disease . Our datOur study further illustrates the power of using next generation sequencing analysis allowing the identification of multiple EV strains in a single sample ,17,18,19Although any amplification-based method such as the whole-capsid RT-PCR assay described here may contribute to bias toward specific strains or serotypes and may risk missing some strains due to mismatches in primer binding sequence regions, we have so far been able to sequence in different studies EV strains from 102 of the 110 different EV serotypes that have been described ,17,19. TNucleotide sequence information at the 3′-end of the capsid coding region can also be used to design EV strain-specific internal primers to synthesize overlapping PCR products and obtain whole-genome sequences of selected EV strains as we have shown here. Analysis of the whole-genome sequences of an EV-D111 strain from a water sample in Nigeria and an EV-D94 strain isolated from a paralytic case in the Republic of Niger as well as representative sequences of all known EV-D serotypes revealed the occurrence of recombination events among species D EVs, a common feature of species A, B, and C EVs. This is not unexpected as frequent EV transmissions are known to occur across the border between the Republic of Niger and Nigeria , and espWe found high prevalence and diversity of human EV serotypes in water samples from Nigeria highlighting the high risk of infection associated with domestic use of contaminated water supplies. Our results show that setting up ES systems in combination with high resolution molecular analysis will help determining the incidence of EV transmission in different populations, being able to monitor the resolution of outbreaks, evaluate the impact of vaccines on virus circulation, and establish alert systems for the early detection of specific EV serotypes known to be associated with human disease.


Reporter genes are used to visualize intracellular biological phenomena, including viral infection. Here we demonstrate bioluminescent imaging of viral infection using the NanoBiT system in combination with intraperitoneal injection of a furimazine analogue, hydrofurimazine. This recently developed substrate has enhanced aqueous solubility allowing delivery of higher doses for in vivo imaging. The small high-affinity peptide tag (HiBiT), which is only 11 amino-acids in length, was engineered into a clinically used oncolytic adenovirus, and the complementary large protein (LgBiT) was constitutively expressed in tumor cells. Infection of the LgBiT expressing cells with the HiBiT oncolytic virus will reconstitute NanoLuc in the cytosol of the cell, providing strong bioluminescence upon treatment with substrate. This new bioluminescent system served as an early stage quantitative viral transduction reporter in vitro and also in vivo in mice, for longitudinal monitoring of oncolytic viral persistence in infected tumor cells. This platform provides novel opportunities for studying the biology of viruses in animal models. The development of novel strategies for pre-clinical and clinical imaging of viral replication and dissemination is of crucial importance . An impoA recombinant virus genetically engineered to express a luciferase produces light from infected cells after substrate (luciferin) administration, which can be detected using a sensitive, charged-coupled device (CCD) camera. This approach has been used in animal models for several viruses, e.g., Dengue virus, Herpes simplex virus 1 (HSV-1), Sindbis virus, Vaccina virus, Influenza virus and oncolytic viruses (OVs) ,6,7,8,9.Generating recombinant reporter viruses remains a challenge. The smaller the inserted reporter gene, the more stable the viral genome remains, making small reporter tags applicable to different viral strains that do not tolerate large genome alterations such as RNA viruses, or being near the limit of their packaging capacity  . Our proIn our system the very small HiBiT tag (33 base pairs/11 amino-acids) is inserted into the viral genome and interacts with the complementary large part (LgBiT) expressed in the infected cells upon infection of those cells with the tagged virus, reconstituting the full Nluc luciferase. Recently, a Flavivirus reporter has been generated using the NanoBiT complementation approach . TomokazOVs are gaining momentum as an emerging therapy for cancer treatment using engineered viruses to selectively kill tumor cells. Improving in vivo imaging of oncolytic viruses would improve safety and efficacy assessments and localization of viral biotherapeutics, with the potential to translate those findings towards the clinic . A new gHere we demonstrate the power of our system for viral tracking over the course of 6 weeks post infection using an oncolytic adenovirus as model virus. This resulted in the HAdV-5-DELTA-24-RGD-GFP-T2A-HiBiT oncolytic adenovirus carrying the small HiBiT tag. The adenoviral infection and further replication were monitored via expression of HiBiT tag from the E3 promoter which is activated by the E1A proteins and expressed early after infection  and lateThe approaches established within this study will have a direct application to various studies where sensitive detection is needed for a better understanding of infection dynamics in vivo, making it a preferable detection method especially for viruses not tolerating accommodation of large inserts or are at the limit of their packaging ability.We performed a lentiviral transduction of prostate cancer cell lines PC-3, PC346C and PC339C to induce expression of LgBiT. PC-3 and PC346C are adherent cell lines while PC339C grows in suspension. In order to determine the functionality and the expression stability of the HiBiT tag from the NanoBiT system, a series of cell lines were infected with the oncolytic reporter virus (HAdV-5-DELTA-24-RGD-GFP-T2A-HiBiT). After the NanoBiT complex was reconstituted successfully, it formed a functional NanoLuc enzyme after infection with the virus A over sen = 8) or with Phosphate Buffered Saline (PBS) (n = 4) (control). The HFz substrate in its novel formulation proved particularly well-suited for these experiments, where a broad imaging time window was required. In fact, the substrate showed a stable signal 15 min after intraperitoneal administration when formulated in P-407 in mice carrying NanoLuc expressing cells  (Promega) using the following primers; forward primer with a NotI restriction site 5′- TTTA self-inactivating lentivirus pCDH-EF1-LgBiT was produced by transfection of HEK293T packaging cells by transient transfection of HEK293T cells with three packaging plasmids pCMV-VSVG, pMDLg-RRE, pRSV-REV  and PEI transfection reagent 1 mg/mL per μg DNA. Procedures were previously described in detail . Lentivi5 cells and transduced with either EF1-NanoLuc-T2A-copGFP lentivirus plus polybrene (hexametridine bromide)  at a final concentration of 8 μg/mL. Cells were sorted for GFP expression using FACS . Transgene expression was confirmed by the presence of the green fluorescent protein copGFP from (excitation/emission maximum = 475/509 nm).HEK293T cells were seeded in a T-25 flask at a density of 5 × 10TGTACAAGGCTGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCTAATGTACA) and was subsequently cloned in the plasmid pShuttle-ΔE3-Fib-RGD-ADP-EGFP-Kana. The GFP-2A-HiBiT containing fragment (after PacI+AatII+ScaI digestion) was recombined with SpeI-linearized pAdEasy-1 resulting in the plasmid pAE-RGD-GFP-2A-HiBiT. Using a standard recombination procedure [9 PFU/mL.The oncolytic HAdV-5-DELTA-24-RGD-GFP-T2A-HiBiT virus was constructed and produced according to previously described protocols . The aderocedure  this plarocedure . To prevTransduced PC-3 cells transfected with pBIT1.2-N [CMV LgBiT were plated with an equal seeding density  in a black 96-well plate  in 100 µL of RPMI medium . Transduced Xenograft Human Prostate Cancer cells, the PC339C, expressing LgBiT were plated with an equal seeding density  in 1 mL of growth medium in a black six-well plate with a clear bottom (Sigma-Corning 3506) and the PC346C cells expressing LgBiT were plated with an equal seeding in 100 µL of growth media in a black 96-well plate (Greiner-Bio-One).Oncolytic adenoviral infection was performed with several dilutions of the initial HAdV-5-DELTA-24-RGD-GFP-T2A-HiBiT viral stock (ranging from 4 to 0.03 MOI). Twenty-four hours after infection the cells were washed with PBS and the bioluminescence signal from wells was measured with IVIS spectrum system  1 min after substrate addition from the Nano-Glo Luciferase Assay System (Promega) with a final concentration of 0.01 mm. The photon flux (ph/s) was collected using open filter binning = medium, field of view = 12.9 × 12.9 cm, f/stop = 1 and either a 30 s or 60 s exposure time. The experiments were performed in triplicates and were repeated three times. Data were analyzed using Living Image 4.3 software (Perkin Elmer) by drawing the appropriate ROI and then plotted using GraphPad Prism 8. Cells were visualized at the microscopy for checking transfection rate using the GFP signal and viability.The intravenous immunoglobulin G (IvIg)  with a final concentration of 50 mg/mL has been incubated with the oncolytic reporter virus for 1 h and then inoculated to the cells. NanoLuc activity was measured 24 h post infection at the IVIS imager.Animal experiments were approved by the Bioethics Committee of Erasmus MC, Rotterdam, The Netherlands under the approved work protocol 17-867-42, covered by the national project license CCD number 2017867. The experiments are performed in accordance with national guidelines and regulations established by the Dutch Experiments on Animal Act (WoD) and by the European Directive on the Protection of Animals used for scientific purpose (2010/63/EU).BALB/C nude  were obtained from Charles River Laboratory (The Netherlands). All mice were provided access to food and water ad libitum and were hosted in the animal facility at the Erasmus MC, Rotterdam, The Netherlands.6 PC-3-LgBiT expressing cells prepared in PBS and matrigel (Sigma-Corning) solution (50:50 ratio). After tumor cell implantation (PC-3-LgBiT) and tumor formation, mice were intratumorally (i.t.) injected, from three different sites, with 10 µL  of HAdV-5-DELTA-24-RGD-GFP-T2A-HiBiT  or PBS. Animals were monitored daily and were euthanized in case of unusual behavior e.g., weight loss > 20% of baseline, rapid tumor growth > 0.5 cm etc.For the subcutaneous skin model experiments, eight-week-old nude BALB/C nude  were anesthetized using isoflurane and were injected with 2 × 10n = 8) and 20 days for control mice (n = 4) by non-invasive imaging.Mice were monitored over 6 weeks (43 dpi) for virus-treated mice  with open filter binning = medium, field of view = 12.9 × 12.9 cm, f/stop = 1 and a 3-min exposure time for the imaging of viral infection. At the peak of the bioluminescence signal, regions of interests (ROIs) were used as a tool to analyze the signals.To detect the adenovirus hexon proteins, paraffin-embedded sections of the mouse tumors  for the treated ones and 20 dpi for the control) were deparaffinized and rehydrated with xylene and ethanol according to standard procedures . The secp < 0.05.Analysis of the bioluminescence output, where more than two groups were compared, was performed using a one-way ANOVA, followed by Tukey’s t-test. All statistics were calculated using GraphPad Prism version 8 for Windows. Data from each animal were presented as means ± SD. The results were statistically significant when The authors confirm that all relevant data are included in the paper and/or its