Virus-like particles (VLPs) are multimeric nanostructures composed of one or more structural proteins of a virus in the absence of genetic material. Having similar morphology to natural viruses but lacking any pathogenicity or infectivity, VLPs have gradually become a safe substitute for inactivated or attenuated vaccines. VLPs can achieve tissue-specific targeting and complete and effective cell penetration. With highly ordered epitope repeats, VLPs have excellent immunogenicity and can induce strong cellular and humoral immune responses. In addition, as a type of nanocarrier, VLPs can be used to display antigenic epitopes or deliver small molecules. VLPs have thus become powerful tools for vaccinology and biomedical research. This review highlights the versatility of VLPs in antigen presentation, drug delivery, and vaccine technology. Vaccination is one of the most cost-effective ways to control and prevent the spread of infectious disease . PathogeVLPs are non-genetic multimeric nanoparticles formed through the self-assembly of one or more viral structural proteins in vivo or in vitro. Having no viral genetic material, VLPs therefore have no pathogenicity or infectivity . In addiFree of genetic material inside, VLPs have broad potential applications and are a hot topic in many current research areas. In terms of structure architecture, VLPs can be divided into two types: non-enveloped and enveloped . Non-envVLPs have become important tools in the biomedical field. VLPs are capable of self-assembly, they have a repetitive order and, because of the absence of a viral genome, VLPs can confer high immunogenicity and antigenicity . VLPs arNanoparticles are widely used in materials science, medicine, and biology. VLPs are regarded as a type of nanoparticle because they structurally allow for the insertion of foreign genes or peptide fragments for antigen presentation. The repetitive sites on the surface of VLPs can be modified or coupled with foreign antigens of other pathogens by chemical or genetic fusion to form chimeric VLPs, which substantially enhance the stimulation of the immune system by the foreign antigens ,20. ForePlasmodium falciparum infection, and human papillomavirus [Numerous VLPs  have been fused with viral structural proteins for the display of foreign antigens . A frequomavirus . In summThere are significant advantages of using VLPs as delivery platforms, including specific targeting, efficient host cell penetration, biocompatibility, and degradability . SimilarAdenovirus (Ad) is the most commonly used viral vector for drug delivery or gene therapy . The strAt present, 110 viral proteins derived from 35 viral families have been shown to assemble into VLPs . From thHepatitis B virus (HBV), an enveloped DNA virus grouped in the Hepadnaviridae family, is the main causative pathogen of hepatitis B . The infSaccharomyces cerevisiae system to stably express HBsAg, producing particles about 20 nm in size and with a regular octahedral symmetrical structure. These vaccines are regarded as safer and more immunogenic than the first-generation, blood-borne hepatitis B vaccine, and continue to be widely used today [To date, three generations of VLP vaccines against HBV have been developed. The first generation of vaccine was Heptavax-B, a hematogenous HBV vaccine composed of hepatitis B surface antigen particles  isolated from the blood samples of patients with hepatitis B . It was ed today . The thied today . FurtherRecently, a new HBV vaccine, Heplisav-B, has been approved for use in adults in the United States. This vaccine uses the same 20 nm VLP self-assembly from HBsAg as the former three generations of vaccines, but also uses a CpG sequence 1018 as an adjuvant. Inoculation with two dosages within the month leads to better effect than the three-dosage Engerix-B within six months. Moreover, its immunogenicity is higher than that for Engerix-B. Therefore, Heplisav-B is expected to become a new generation of vaccine against HBV infection ,82.In addition to these preventive vaccines, VLP-based therapeutic hepatitis B vaccines are also being developed. In 2019, one group showed that multiple copies of the HBsAg-aa113-135 (SEQ13) polypeptide epitope could be displayed on a novel immuno-enhanced VLP carrier (CR-T3) derived from the roundleaf bat HBV core antigen (RBHBcAg). This core antigen could induce a specific antibody response mediated by HBV/HBsAg clearance to achieve a therapeutic effect, and is currently undergoing further research .HPV is a circular, double-stranded DNA virus, and persistent HPV infection is a major cause of cervical cancer and genital warts . At presEscherichia coli expression system, which could greatly reduce the cost of vaccine production. This system has excellent safety and efficacy in phase 3 clinical trials [E. coli has entered into phase 2 clinical trials [In most developing countries, vaccine production via eukaryotic expression systems renders the vaccine unaffordable. Recently, a recombinant HPV type 16/18 bivalent vaccine (Cecolin) was developed using the l trials ,59, prodl trials .More recently, it was proposed that VLPs could be used to express multiple antigens to offer protection against multiple strains of the same virus type through chimeric design. To this end, an “anti-multiple” single-particle chimeric vaccine was designed. The authors showed that the L1 loop region is closely related to HPV-type specificity, and that homologous substitution of the L1 loop region with that of a closely phylogenetically related loop region can offer cross-protection. A triple-type chimera of HPV33/58/52 VLPs can induce neutralizing titers that are comparable to a mixture of three wild-type VLPs, and this occurs both in mice and non-human primates. Based on this strategy, the authors are at present developing seven anti-20 type-cross vaccines, which are expected to prevent and control more comprehensively HPV-related diseases  .E. coli expression system, this largely reduces the cost associated with vaccine production and thus would be beneficial for vaccination in developing countries. At present, it is the only vaccine that can effectively prevent hepatitis E [E. coli system, is still in phase 1 clinical trials [The hepatitis E virus (HEV) VLP-based vaccine, Hecolin, launched in China in 2011, has brought significant benefits to the prevention of HEV infection . HEV, a atitis E . Besidesatitis E . Meanwhil trials .VLP-based vaccine design has been extrapolated for the treatment of other viruses and pathogens. Many of these vaccines are still in experimental or development stages or still in clinical trials, some proving to be more complicated than others. For example, the development of a universal vaccine to achieve effective prevention against influenza has proven to be difficult. The influenza viruses have high genetic variation and exist as multiple types/subtypes. As such, in recent years, the design of multivalent chimeric VLP-based influenza vaccines has become a hot topic in influenza research. Recently, Novavax described the design of a nano-VLP influenza vaccine, tNIV, which is highly immunogenic and shown to be safe in clinical trials, and is capable of eliciting antibodies that target multiple broadly cross-reactive epitopes on hemagglutinin, covering a variety of conserved antigenic sites, to better circumvent the seasonal antigenic drift ,66. AnotPlasmodium falciparum parasite, which causes malaria in humans via mosquitos. The antigen in the RTS,S/AS01 vaccine consists of a recombinant form of circumsporozoite protein (CSP) linked to HBsAg(S) via an amino acid repeat and T-cell epitope domain. These proteins can spontaneously enter the cells to form VLPs that subsequently serve as a vaccine [Mosquirix  is considered to be the first advanced VLP vaccine targeting the pre-erythrocytic stage of the  vaccine . In phas vaccine . The vac vaccine . Another vaccine .In addition to VLPs containing antigens against a single viral strain, “consensus VLPs” that were designed with multiple antigens from different strains, can be used with the goal to potentially induce cross-protective immunity. The GII.4 consensus norovirus VLP, engineered from sequences of three genetically distinct naturally occurring GII.4 strains, can induce cross-reactive immune responses against different clusters of GII.4 noroviruses when immunization to animals by the intramuscular route with aluminum hydroxide as adjuvant . CurrentFinally, prophylactic and therapeutic vaccines targeting different viruses, such as rotavirus, norovirus, and enterovirus, have evolved with the maturity of VLP vaccine technology . These nThe administration route is a fundamental parameter to be considered because it influences the stability of VLPs and the pharmacological outcome. Current vaccination methods include oral, intramuscular, intradermal, and intranasally, etc. ,104,105.Virosomes, a spherical, unilamellar vesicles with a mean diameter of ~150 nm, and without nucleocapsid and genetic material of the source virus, are also used as important delivery tools in vaccine development ,110. SimVLPs are widely used in drug delivery, gene therapy, and vaccine development. As an immunogen, they are suitable in size and have a morphological structure similar to that of natural viruses, and can therefore induce a stronger immune response. As a nanocarrier, VLPs not only display foreign antigens, but can also be modified to express chimeras through chemical or genetic fusion technology, offering cross-neutralization protection against different viral strains. Finally, the targeted delivery of small molecule drugs and nucleic acids through VLPs improves the bioavailability of the delivered substance. However, it should not be overlooked that, although dozens of VLPs have been successfully prepared in the laboratory stage, there are still many problems to be solved in the development of effective vaccines and carriers. Indeed, it remains to be determined how to improve the expression of VLPs to meet the scale-up production process, how to effectively assemble VLPs, and how to reduce the immune response caused by VLPs when used as a delivery vehicle. Continued advances in VLP vaccine design will provide new possibilities for the treatment of numerous viral infections, and offer powerful tools for biomedical and vaccination research.


Epithin/PRSS14, a type II transmembrane serine protease, is an emerging target of cancer therapy because of its critical roles in tumor progression and metastasis. In many circumstances, the protease, through its ectodomain shedding, exists as a soluble form and performs its proteolytic functions in extracellular environments increasing cellular invasiveness. The seemingly functional integrity of the soluble form raises the question of why the protease is initially made as a membrane-associated protein.In this report, we show that the epithin/PRSS14 intracellular domain (EICD) can be released from the membrane by the action of signal peptide peptidase-like 2b (SPPL2b) after ectodomain shedding. The EICD preferentially localizes in the nucleus and can enhance migration, invasion, and metastasis of epithelial cancer when heterologously expressed. Unbiased RNA-seq analysis and subsequent antibody arrays showed that EICD could control the gene expression of chemokines involved in cell motility, by increasing their promoter activities. Finally, bioinformatics analysis provided evidence for the clinical significance of the intramembrane proteolysis of epithin/PRSS14 by revealing that the poor survival of estrogen receptor (ER)-negative breast cancer patients with high epithin/PRSS14 expression is further worsened by high levels of SPPL2b.These results show that ectodomain shedding of epithin/PRSS14 can initiate a unique and synchronized bidirectional signal for cancer metastasis: extracellularly broadening proteolytic modification of the surrounding environment and intracellularly reprogramming the transcriptome for metastatic conversion. Clinically, this study also suggests that the intracellular function of epithin/PRSS14 should be considered for targeting this protease for anti-cancer treatment. Protease serine 14 (PRSS14), a gene of the 14th serine protease, was initially named as epithin , MT-SP1 Our previous study showed that epithin/PRSS14 is synthesized as a single-chain zymogen and is cleaved between Gly149 and Ser150 in the SEA  domain to produce two fragments, the N-terminal fragment (NTF) and the C-terminal ectodomain containing the protease domain (named as Epi-S), which are non-covalently associated on the membrane . The cleHere, the intracellular function of NTF is presented. Specifically, it was found that NTF of epithin/PRSS14 left on the plasma membrane upon ectodomain shedding can be further processed and liberated from the membrane by intramembrane proteolysis. It was also found that the epithin/PRSS14 intracellular domain (EICD) can promote migration, invasion, and cancer metastasis, possibly through transcriptional changes. The intracellular function of the protease would be locked until ectodomain shedding occurs, and when unlocked, it can potentiate cellular behaviors mediated by the released extracellular protease. Therefore, we suggest that its existence as an integral membrane protein may be required for the cooperative functions of extracellular and intracellular domains in a synchronized manner at the time of ectodomain shedding.When 427.1.86 thymoma cells (427 cells) expressing epithin/PRSS14 endogenously were stained with an antibody generated against the ectodomain of epithin/PRSS14 (anti-C), a membrane staining pattern was observed preferentially in the cell-cell contacts  was generated in which the GAL4-VP16 is inserted into the cytoplasmic tail of the protease  . Becauseion Fig. a. In conion Fig. b and c. ion Fig. d, PMA-inTo establish the existence of EICD, the consequences of its liberation from the membrane were investigated. To this end, 427(SP2bKD) cells were utilized in which shedding-dependent EICD formation is impaired Fig. e. When tp value < 0.05, Additional file Next, the molecular basis of the EICD-mediated increase in cell motility was assessed. Because EICD is found in the nucleus, it was hypothesized that EICD might contain transcription activity as is often observed in other RIP substrates . First, high group showed poorer survival than the SPPL2Blow group , implying an adverse effect of SPPL2B on the survival of patients with risks from epithin/PRSS14. When the expression levels of both SPPL2B and epithin/PRSS14 were considered in patients that were ER-negative, the epithin/PRSS14highSPPL2Bhigh group showed the poorest survival pattern  or full length was amplified by PCR and cloned into NotI and BamHI sites of the p3XFLAG-CMV10 vector. For lentiviral infection of EICD, 3XFLAG-EICD was also cloned into a lentiviral vector, pRRLSIN.cPPT.PGK-IRES-GFP.WPRE, modified from pRRLSIN.cPPT.PGK-GFP.WPRE . Human SPPL2b cDNA was purchased from OriGene. UAS-GFP reporter was generated by cloning EGFP cDNA into pHR_5x Gal4 UAS vector . CCL20-GFP reporter constructs were purchased from GeneCopoeia (MPRM34109-LvPF02). cDNA encoding SPPL2b tagged with C-terminal HA was generated by PCR. Epithin/PRSS14 N-terminal end from Met1 to Ala44 fused with GAL4-VP16, EICD-GAL4-VP16, were generated by PCR and inserted into the pM vector (Clontech). The DNA sequence corresponding to Lys45-Val855 of epithin/PRSS14 was inserted into the 3′ of VP16 in the Epi (1-44)-GAL4-VP16 construct, to generate full-length Epi-GAL4-VP16. Epi-GAL4-VP16-strep construct was generated by cloning of PCR-amplified Epi-GAL4-VP16 into pcDNA3.1 vector containing Strep coding sequence. For transient knockdown of epithin/PRSS14, two epithin/PRSS14-specific siRNAs, Epi-1 (target sequence: GCTTGCTGGTGTGGCACTTCCATTA) and Epi-2 (target sequence: GGTGCGCTTCAAACTCTTC), and a universal control siRNA (target sequence: TTCTCCGAACGTCTCACG) were purchased from GenePharma.427, 427(epiKD), and 4T1KD cells were maintained as described previously . To geneCells grown on gelatin-coated slide covers were fixed with 3.7% paraformaldehyde in PBS for 10 min, permeabilized with 0.1% TritonX-100 in PBS (PBS-T) for 30 min, and incubated in blocking solution  for 30 min. After washing with PBS-T, samples were treated with a primary antibody in blocking solution for 1 h. Cells then were washed with PBS-T and incubated with FITC-conjugated secondary antibodies  for 50 min. The coverslips were mounted with DAPI  and observed under the fluorescence microscope . Images were processed in Photoshop CS6 (Adobe). For quantification of nuclear dots stained by anti-N antibody, a threshold level was set as 1.5 times the mean fluorescence of pixels of which intensities are less than top 60% fluorescence intensity of each nuclear region. If the mean fluorescence of a particular pixel and the 8 pixels surrounding it in the nuclear image is greater than the threshold, the pixel was considered as an EICD-positive pixel. The sum of the fluorescence intensity of EICD-positive pixels in each cell divided by the mean fluorescence of nuclear region of the cell was calculated and represented as normalized EICD nuclear localization. This procedure was automatically performed by a custom Matlab code.To examine the liberation of the intracellular domain of epithin/PRSS14, 427 cells were transfected with Epi (1-44)-GAL4-VP16, Epi-GAL4-VP16, or empty vector with GAL4-UAS-Luciferase and pCMV-β-galactosidase (Promega). At 24 h after transfection, these cells were starved for serum and stimulated with PMA/serum and/or ecotin. Cells were lysed, and the luciferase assay was performed according to the manufacturer’s instructions (Promega). Luciferase and β-galactosidase activities were measured using a MicroLumat Plus LB 96 V (Berthold technologies), and the luciferase activity was normalized by β-galactosidase activity.2ketone  for 16 h. Then, cells were further incubated with 1 μM PMA  for additional 2 h. Whole media were harvested and concentrated using trichloroacetic acid and analyzed for Epi-S′, as described previously [12GSQDFGAGLKYNSR25 was pre-incubated with anti-N antibody.Serum starved 427 cells on a 100-mm dish were treated with 20 μM TAPI-0  for 2 h, DAPT  for 16 h, or (Z-LL)2, 2 mM EDTA, and protease inhibitors) and passed through a 26-G needle on ice. The nuclear pellets were collected after centrifuge at 700g for 10 min at 4 °C. The supernatants were centrifuged at 10,000g for 10 min to discard contamination of nuclear fraction and re-centrifuged at 100,000g for additional 1 h. The supernatants were collected as cytosolic fraction. The nuclear pellets were resuspended with nuclear lysis buffer , incubated for 1 h on rotator at 4 °C, and clarified by centrifugation at 17,000g for 20 min. Resulting cytosolic and nuclear fractions were analyzed by Western blot.Cells were incubated with serum-free media for 2 h and treated 1 μM PMA for additional 2 h. Those cells were harvested in a lysis buffer for subcellular fractionation  according to the manufacturer’s instructions. The 3 × 105 cells from the mixture in 100 μl PBS were injected into the tail vein of 5-week-old BALB/c mice (n = 5). Three weeks after injection, all mice were sacrificed by cervical dislocation and their lungs were removed and frozen. Each lung tumor nodules from the mice were lysed with RIPA buffer and analyzed for EICD expression using western blot.4T1KD cells and 4T1KD/EICD:GFP cells were mixed at approximately 7:3 ratio and analyzed by flow cytometry to confirm the ratio. Approximately 5 × 10FLAG-EICD was co-transfected with pEGFP-c1 (Clontech) into SP2bKD-10 cells. At 24 h after transfection, GFP-positive cells were sorted using FACSAria II (BD Bioscience). The collected GFP-positive cells were immediately lysed with TRIzol , and RNA was extracted. The quality of extracted RNA was verified using an Agilent 2100 BioAnalyzer. RNA-seq was performed at TheragenEtex Bio Institute  using Illumina HiSeq 2500 paired-end RNA sequencing. Reads were aligned with Tophat (v2.1.1) . CuffdifSP2bKD-10 or SP2KD/EICD cells were incubated with 0.5% serum containing DMEM for 24 h. Then, conditioned medium (CM) were concentrated 10 times by centrifugation at 4000 rpm for at 4 °C using ultrafiltration units . The concentrated CMs were analyzed to determine the expression levels of secreted cytokines by Proteome Profiler Mouse XL Cytokine Array (R&D Systems), according to the manufacturer’s instructions. The signals were detected by ImageQuant LAS 4000 Mini , and the pixel density was analyzed by imageJ software.r values between two genes were calculated using the Pearson correlation, and unpaired two-tailed Student’s t test was performed. For the 5 years survival rate, Kaplan-Meier survival analysis was used using TCGA breast cancer data excluded data whose contacts were lost in 5 years. The p values were calculated using a log-rank (Mantel-Cox) test, and the hazard ratio (HR) was determined by the Mantel-Haenszel method.TCGA data were downloaded using the Broad Institute’s Firehose, a web portal site that has been developed aiming to deliver automated analyses of the TCGA data to general users. For expression correlation analysis, expression values of all patients were drawn in scatter plots with linear interpolation curves between the two genes. The correlation coefficient Additional file 1: Figure S1. Surface localization of Epi-GV fusion protein. (A) Diagram of Epi-GV construct with C-terminal Strep tag (Epi-GV-strep). (B) Epi-GV-strep construct (or MOCK) was transfected to 293F cells with a trace amount of EGFP cDNA as a transfection marker, and its surface expression was analyzed by flow cytometry. Representative dot plots are shown. Figure S2. PMA-induced nuclear localization of epithin/PRSS14 intracellular domain. (A) 427 cells were transfected with two siRNAs against epithin/PRSS14, starved for serum, and stimulated with PMA or not. Cells were then stained with anti-N antibody (green) and DAPI (blue). Representative images are shown. Scale bars, 20 μm. (B) The computational method for determining EICD-positive pixels is illustrated. If the mean fluorescence of a particular pixel and the 8 pixels surrounding it in the nuclear image is greater than the threshold described in Methods section, the pixel was considered as an EICD-positive pixel. (C) Representative anti-N antibody-stained original image and binary image with epithin/PRSS14 positive pixels indicated in white are shown. (D) In the experiment described in A, normalized EICD nuclear localization (the sum of the fluorescence intensity of EICD-positive pixels in each cell divided by the mean fluorescence of nuclear region of the cell) was calculated and represented as bar graph. At least 57 cells from three microscopic fields were used for each condition. The error bar indicates SEM. ****p < 0.0001 (unpaired two-tailed Student’s t test). Figure S3. Localization of EICD in the nuclear fraction. (A) 427 cells transfected with specific siRNAs for epithin/PRSS14 and stimulated with PMA as in Fig. Figure S4. Effects of γ-secretase inhibitor and SPPL family inhibitor on intramembrane proteolysis of epithin/PRSS14. The 427 cells were treated with indicated concentration of DAPT or (Z-LL)2ketone for 16 h, and the spontaneous EICD generation was detected by immunoprecipitation and Western blot using anti-N antibody. Arrowhead and arrow indicate NTF and EICD, respectively. Figure S5. Reduced nuclear localization of EICD in SPPL2b-knockdown cell lines. (A) EICD nuclear localization in 427 wild type and SP2bKD-10 cells shown in Fig. p < 0.0001 (unpaired two-tailed Student’s t test). (B) The localization of EICD in cytosolic and nuclear fraction of SP2bKD cells was analyzed as in S3B. Arrowhead and arrow indicate NTF and EICD, respectively. GAPDH (empty arrowhead) and Histone H3 (asterisk) were used for cytosolic and nuclear marker, respectively. Figure S6. Epithin/PRSS14 and EICD-dependent cell motility. (A) 427 cells were transfected with siRNAs against epithin/PRSS14, and their wound healing migration was tested as in Fig. n = 3). (B) 427 cells were transfected with siRNAs against epithin/PRSS14, and their invasion through Matrigel-coated pores of the transwell chamber was analyzed as in Fig. n = 3). (C) Wound healing migration of 427, SP2bKD-10, and EICD-transfected SP2bKD-10 cells was analyzed as in (A). Data are presented as the means of the recovered area . (D) Transwell invasion of 427, SP2bKD-10, and EICD-transfected SP2bKD-10 cells was analyzed as in (B). Numbers of invaded cells to the other side of the chamber for 24 h were counted, normalized against that of 427 cells, and shown as bar graphs . Error bars indicate SEM in all panels. p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (unpaired two-tailed Student’s t test). Figure S7. Western blot analysis of tumor nodules. (A) Twenty tumor nodules from each lung of five mice injected with 4 T1 and 4T1KD/EICD:GFP mixture were isolated, lysed, and analyzed by Western blot to detect GFP expression. Western blot using anti-tubulin antibodies were used as loading controls in the same blot. GFP-positive nodules are indicated with red font. (B) In case GFP expression was not evident due to the small size of the nodules and the subsequent low protein concentration in the sample, e.g., lanes 2, 15, 17 from mouse #4, the Western blot was repeated using negative (4T1KD) and positive control cells (4T1KD/EICD:GFP). Figure S8. Ontology analysis of the EICD-induced gene set. The gene ontology (GO) of total 233 DEGs with more than two-fold increases in EICD-transfected cells was analyzed as in methods. The top ten ranked GO terms were indicated with -log. Figure S9. Cytokine array. Positions of various cytokine antibodies spotted in the array are shown. Figure S10. EICD in human breast cancer patients. The normal (N) and tumor (T) tissues from four ER-negative breast cancer patients were lysed and analyzed by Western blot with anti-human C-terminal (IM1014) and anti-human N antibodies. Arrowhead and arrow indicate NTF and EICD, respectively.Additional file 2: Table S1. Differentially expressed genes (DEG) in the control and EICD-transfected cells. DEGs with p values less than 0.05 from the RNA-seq analysis are listed with gene information, FPKMs in the control and EICD-transfected cells, the binary logarithm of the ratio (M), and the geometric mean (A).


Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Histo-Blood Groups Antigens (HBGAs) have been described as attachment factors, promoting HuNoV infection. However, their role has not yet been elucidated. This study aims to evaluate the ability of HBGAs to protect HuNoVs against various factors naturally found in the human digestive system. The effects of acid pH and proteolytic enzymes  on GII.4 virus-like particles (VLPs) and GII.4 HuNoVs were studied, both during interactions and non-interaction with HBGAs. The results showed that GII.4 VLPs and GII.4 HuNoVs behaved differently following the treatments. GII.4 VLPs were disrupted at a pH of less than 2.0 and in the presence of proteolytic enzymes . VLPs were also partially damaged by lower concentrations of trypsin and chymotrypsin (0.1 mg/mL). Conversely, the capsids of GII.4 HuNoVs were not compromised by such treatments, since their genomes were not accessible to RNase. HBGAs were found to offer GII.4 VLPs no protection against an acid pH or proteolytic enzymes. HuNoVs are transmitted through the faecal-oral route, and are among the main causes of viral foodborne outbreaks in both Europe and the United States of America (USA)3.Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide, with more than 700 million cases reported annuallyCaliciviridae family and can be divided into 10 genogroups (G): GI, GII, and GIV are responsible for most diseases in humans. In the GII genogroup, genotype 4 (GII.4) HuNoVs are the most prevalent type7. The capsid of about 30 nm have a major protein (VP1) and a minor protein (VP2). The external P2 subdomain of VP1 expresses the hypervariable region of the capsid. The capsid includes a single stranded RNA genome (~ 7.5 kilobases). VP1 can spontaneously assemble into virus-like particles (VLPs).Noroviruses belong to the 10. Reverse transcription–polymerase chain reaction (RT-qPCR) techniques are therefore currently used to detect and quantify HuNoVs in humans, foodstuffs, and the environment, in accordance with the ISO 15216 standard12.There is currently no available routine method for the detection of infectious HuNoVs in foodstuffs and the environment, although some innovative approaches to the in vitro replication of a few HuNoV genotypes have been described15, and also with HBGA-like carbohydrates present in food, bacteria, and the environment16. HBGAs are complex carbohydrates resulting from the successive addition of monosaccharides to a precursor. The biosynthesis of HBGAs is governed by the ABO and FUT2 and FUT3 fucosyl-transferase gene families18. They can be found on red blood cells and mucosal epithelial cells. They can also be present as free antigens in biological fluids, such as saliva or milk19. The expression of active FUT2 defines secretor status, while non-active FUT2 defines non-secretor status17. Interactions between HuNoVs and HBGAs are highly specific, with multiple binding patterns and various affinities20. The binding of HuNoVs to HBGAs takes place in the P2 subdomain, which is composed of four or five highly conserved amino acids and a number of more variable amino acids22. Several studies have described HBGAs as attachment factors for several HuNoV strains that promote infection in humans, but it is still unclear whether they act as (co)-receptors or facilitate access to the infection site15.It has been clearly demonstrated that HuNoVs interact with Histo-Blood Group Antigens (HBGAs) in humansUpon entry into the human body via oral ingestion, HuNoVs may (or may not) interact with salivary HBGAs, before passing through the gastrointestinal tract via the stomach. Thus, various factors may alter the structure of virions (i.e. infectious particles), especially the acid pH of the stomach and proteolytic enzymes in the gastrointestinal tract.23. No reduction in HuNoV genomes was observed in Greenshell mussels at pH 3.8 following a 4-week incubation period24. To gain a better understanding of HuNoV survival, many studies have used cultivable infectious HuNoV surrogates, such as feline calicivirus (FCV), murine norovirus (MNV), tulane virus (TV), and VLPs. The combined results showed that a 30 min exposure to an acid pH ranging from 2.0 to 4.0 led to a 0–2 log10 reduction in these surrogates26, with the exception of FCV, which appeared to be more sensitive than the others27. The effect of pH on VLPs has been thoroughly reviewed and differences at the genogroup level have been highlighted, given that GII seemed more resistant to basic pH in the 8–10 range than GI29.There is little data available on the resistance of HuNoVs as a function of pH. It has been observed that HuNoVs treated at pH 2.7 for 3 h were able to cause infection in human volunteers31, while VP1 cleavage could be observed in the same genotypes32. Chymotrypsin and pepsin seemed to promote the infectivity of reovirus, while a reduction below 1 log10 in GI.1 and GII.4 HuNoV genomes could be observed after 42 days in a buffer mimicking the conditions of gastric fluid34. To our knowledge, no data are available regarding the influence of HBGA-binding on the potential protection or degradation of the viral capsid towards these inactivating factors found in the human digestive system.Other factors that can affect the structure of virions include proteolytic enzymes, whose effect is dependent on the nature of each enzyme. Pepsin enzymes, which are active at pH 1.0–3.0, may be present in the stomach and can hydrolyse the C-terminal of specific peptide bonds, such as phenylalanine, leucine, tyrosine, and tryptophan. Trypsin and chymotrypsin enzymes, which are active at pH 7.0–9.0, can be found in the duodenum and jejunum. Trypsin can hydrolyse peptide bonds located after an arginine or lysine residue, while chymotrypsin hydrolyses the C-terminal of peptide bonds, such as tyrosine, tryptophan, leucine, methionine, and phenylalanine. The different positive and negative effects of these enzymes on viruses have been described. Trypsin seems to promote HBGA-binding to the viral capsid of GII.3 and GII.6 VLPs35.The aim of this study was (1) to evaluate the effects of acid pH, pepsin, chymotrypsin, and trypsin on GII.4 VLPs and HuNoVs and, (2) investigate the potential protective effect of HBGA-binding on these viral particles, using the same parameters. The resulting effects on VLPs were analysed by examining capsid integrity as reflected in the overall structure of the particles (using transmission electron microscopy: TEM); measurements of particle size (using dynamic light scattering: DLS); protein mass modifications (using sodium dodecyl sulfate–polyacrylamide denaturing gel electrophoresis: SDS-PAGE); and conformational changes to the capsid (using a fluorescent hydrophobic probe: SYPRO Orange). The resulting effects on HuNoVs were evaluated using RNase treatment followed by RT-qPCR to estimate the number of genome copies (gc) from fully intact capsidsThe effects of acid pH and proteolytic enzyme  treatments at 37 °C for 4 h on the overall structure of GII.4 VLPs and the VP1 protein were evaluated.36. Applying a pH of 3.0 or 2.0 did not change the mean number of particles per field, particle size, or morphology. At pH 1.3, the number of GII.4 VLPs decreased significantly, with no particles observed per field   . With chymotrypsin at pH 8.0, the mean numbers of particles per field were 70, 14, and 1 at 0, 0.1, and 100 mg/mL, respectively . The mean size of particles was not modified after proteolytic enzyme treatments. All these results are summarized in Table t-test, p > 0.3). Interestingly, the mean size of particles either increased or decreased using DLS, depending on test conditions. The mean particle size was less than 6.4 nm after treatments with 100 mg/mL trypsin and chymotrypsin, and greater than 215.4 nm at pH 1.3. It ranged from 41.2 to 59.9 nm under the other conditions tested. This suggests that either GII.4 VLPs or their capsid proteins were unstructured or aggregated following proteolytic digestion (100 mg/mL) and at pH 1.3, respectively. The different mean sizes of isolated VLPs given by TEM and DLS are due to the fact that DLS measures the hydrodynamic diameter of the viral particles in aqueous media while TEM measures the physical diameter of the capsid after drying. On the other hand, when aggregation take place, DLS cannot detect particles over 6 µm and may be more sensitive to detect low numbers of particles between 200 nm and 6 µm compared to TEM. Tm value of the native VLPs at pH 8.0 was measured at 72.1 °C. No difference in the Tm values of the VLPs was observed at pH 2.0 or 3.0. The Tm value strongly decreased after the 0.1 mg/mL enzymatic treatments , which means that proteolytic enzymes impacted the capsid structure, making it more vulnerable to heat. Tm values could not be estimated following the 100 mg/mL enzymatic treatments because of the high protein content caused by the addition of the enzymes.The overall structure of GII.4 VLPs was analysed using TEM, DLS, and SYPRO Orange probe. The study of conformational changes to the capsid using SYPRO Orange probe provided information about the melting temperatures  of the particles. The results obtained by TEM are shown in Fig. 37. Applying a heat treatment of 95 °C to native VLPs for 10 min allowed the two expected VP1 bands to be observed using SDS-PAGE analysis. This suggests that, under denaturing conditions, SDS-PAGE disturbed the migration of VP1 to the expected MWs because of the incomplete dissociation of VP1 multimers and/or the conservation of some secondary structures. We will henceforth refer to such bands as “VP1 multimers.” Thus, the effects of different treatments, on GII.4 VLPs, both separately and in combination with heat treatment  on both the isolated form of VP1 protein and the form assembled into VLPs were investigated. As described above, the effect on isolated VP1 was studied by heating at 95 °C for 10 min prior to proteolytic enzyme treatments  treatments at pH 3.0 had no effect on the structure of VLP/VP1; (ii) treatments with 0.1 mg/mL trypsin or 0.1 mg/mL chymotrypsin or at a pH of less than 2.0 had partial effects; (iii) treatments with 1,500 units/mL pepsin, 100 mg/mL trypsin, or 100 mg/mL chymotrypsin induced VP1 digestion and VLP disruption.None of the methods used on GII.4 VLPs were applicable to GII.4 HuNoVs because the quantity and purity of particles were much greater in VLPs than in HuNoVs. The DLS, SYPRO Orange probe, and SDS-PAGE methods were not sensitive enough to allow us to study the behaviour of HuNoVs when treated with an acid pH and proteolytic enzymes. We therefore drew on the results obtained from GII.4 VLPs, to determine whether proteolytic enzymes and acid pH could disrupt HuNoVs. The difference between disrupted and structured particles was determined based on the number of HuNoV gc from fully intact capsids. Thus, the HuNoV genome was systematically quantified using RNase treatment followed by RT-qPCR before and after proteolytic enzyme and acid pH treatments. However, it cannot be ruled out that degradation takes place at the level of the capsid without making the RNA accessible to RNases. Therefore, the thermal stability of HuNoVs was evaluated using the same approach, to determine whether the viral particles were disrupted by such treatments. Minor degradations may impact thermal stability.p > 0.01).The effects of all these treatments on HuNoVs are shown in Fig. 10 (C/C0) values were determined by dividing the mean GII.4 HuNoV gc values after each proteolytic enzyme or acid pH treatment, following treatment at temperatures of 60–85 °C, by the mean GII.4 HuNoV gc values obtained after the same enzymatic treatment and treatment at 60 °C. The results are shown in Fig. t-test, p < 0.005). Above this temperature, three profiles could be identified: (i) a decay rate similar to that of the native HuNoVs at pH 8.0 following treatment with 0.1 mg/mL chymotrypsin and 100 mg/mL trypsin ; (ii) a decay rate lower than that of the native HuNoVs at pH 8.0 following both pH 1.3 treatment alone and pepsin treatment at pH 1.3 ; and (iii) no decay rate for HuNoVs treated with 100 mg/mL chymotrypsin . It could, therefore, be concluded that the various treatments had no deleterious effects on the HuNoV capsids. Some of the treatments even seemed to play a protective role, which may be explained by their high enzyme content or by the aggregation of the capsid proteins, rendering the viral genome inaccessible to RNase.As previously demonstrated with GII.4 VLPs, the thermal stability of HuNoVs could further explain the partial damage to capsids induced by proteolytic enzymes and their effects on the Tm values needed to disassemble the HuNoV capsid. HuNoV gc from fully intact capsids were therefore quantified at different temperature gradients, to evaluate the effects of acid pH and proteolytic enzyme treatments. The logIn conclusion: none of the proteolytic enzymes or acid pH treatments led to a decrease in the Tm values needed to disassemble the GII.4 HuNoV capsids.+  approach  cause acute gastroenteritis outbreaks regardless of the Lewis or ABO phenotypes of secretor individuals43 and (iii) infect non-secretor individuals45. To obtain a better understanding of the role of HBGAs in the entire host-cell infection process in humans, the protective role of these complex carbohydrates in the presence of various factors naturally found in the human digestive system was studied. The effects of acid pH and three proteolytic enzymes  on capsid integrity in HuNoVs were investigated because interactions in the stomach and small intestine can occur before the virus reaches the primary target of infection and replication.Over the past decade, HBGAs have only been considered as co-receptors or co-factors promoting infection in host cells because of the hypotheses that HuNoV genotypes could (i) bind to cells  independently of the presence of HBGAs46. This difference could have been due to the absence of VP2 and/or the lack of interactions between the viral genome and VP147. These interactions have been reported to promote capsid stability in other viruses, such as F-specific RNA bacteriophages (MS2 and GA) as well as Triatoma virus (TrV), a picorna-like virus with a single-stranded positive-sense RNA genome and an icosahedral T = 3 structure similar to that of HuNoVs49.First, the effects of acid pH and of the three proteolytic enzymes described above on native GII.4 VLPs and GII.4 HuNoVs were tested. Complete disassembly of GII.4 VLPs—but not GII.4 HuNoVs—into VP1 was observed at an acid pH of less than 2.0. Capsid stability clearly differed between VLPs and HuNoVs belonging to the same genotype. The lesser stability of GII.4 VLPs compared to GII.4 HuNoVs was also demonstrated, following natural ageing at 20 °C50. The optimal conditions for trypsin and chymotrypsin were 0.1 or 100 mg/mL at pH 8.0 in ammonium bicarbonate buffer51. Digestion of VP1 was observed after treatment with the three proteolytic enzymes, indicating the accessibility of cleavage sites. According to the ExPASy PeptideMass tool, 34 peptides, with MWs of 0.5–1.5 kDa should be obtained after pepsin treatment at pH 2.0. With trypsin and chymotrypsin, 28 peptides (of 0.5–5.1 kDa) and 48 peptides (of 0.5–2.1 kDa) respectively are expected following enzymatic digestion. Under the conditions tested, degradation of VP1 was confirmed by SDS-PAGE analyses, following proteolytic enzyme treatments. As expected, the VP1 obtained from native VLPs was totally digested after preheating to 95 °C. Nevertheless, VP1 was not completely cleaved when enzymatic digestion was performed using GII.4 VLPs, particularly at lower doses of proteolytic enzymes (i.e. 0.1 mg/mL). Some intact particles were detected by TEM observations, and some VP1 at a MW of 57 and 60 kDa was detected by SDS-PAGE analysis. Our results demonstrated that VP1 is more sensitive to enzymatic digestion in its isolated form than when assembled into GII.4 VLPs, as previously described in the case of GI.1 VLPs52. One peptide of 32 kDa was found in GI.1 VLPs and two peptides of 31 and 26 kDa in GII.3 and GII.6 VLPs respectively following trypsin treatments of 16–500 µg/mL52. Although complete digestion of VP1 at 62.5 °C and 500 µg/mL trypsin was observed using SDS-PAGE analysis, some authors could still observe VLPs using TEM32. The structure and stability of the HuNoV capsid therefore seems to be genotype dependent. It has recently been demonstrated that GII.4 VLPs have T = 4 icosahedral symmetry, whereas GI.1 and GI.7 VLPs have T = 3 icosahedral symmetry53. Thus, the intra- and inter-VP1 interactions of VP1 assembled into VLPs differ between HuNoV genotypes. This may explain why VP1 could be easily dissociated from VLPs in SDS-PAGE under denaturing conditions in some genotypes (i.e. GII.3 and GII.6) but not completely in GII.4 HuNoVs. The persistence of non-covalent multimers of VP1 at MWs above 100 kDa, which were only dissociated after heating at 95 °C or at an acid pH of less than 2.0 in our study, provides further evidence of the specificity of capsid structure at genotype level. Our results demonstrate that enzymatic cleavage of VP1 does not always reflect disruption of GII.4 VLPs because of the incomplete digestion of VP1 when assembled into VLPs. Complete disruption of GII.4 VLPs was obtained after treatments with trypsin and chymotrypsin concentrations higher than 100 mg/mL and pepsin concentrations higher than 1,500 units/mL.The effects of pepsin, trypsin, and chymotrypsin were studied under optimal conditions. The optimal conditions for pepsin were a concentration of 1,500 units/mL in gastric fluid buffer, at a pH of 2.0 for VLPs and 1.3 for HuNoVs54. Unlike GII.4 VLPs, in which evidence of partial or complete disruption was found following proteolytic enzyme treatments, no complete dissociation of GII.4 HuNoVs was obtained under the same conditions, regardless of the concentration of enzymes used. With the aim of evaluating whether these enzymes really could disrupt GII.4 HuNoV capsids, the thermal stability of the viral particles following gradual increases in temperature after proteolytic enzyme treatments was investigated. This provided several insights. Decay rates were similar in native GII.4 HuNoVs and GII.4 HuNoVs treated with 0.1 mg/mL chymotrypsin and 100 mg/mL trypsin after heating at all temperatures in the range except 75 °C; while in those treated with both a pH of 1.3 alone and with a pH of 1.3 and pepsin combined, less reduction in GII.4 HuNoV genomes was observed after heating to 75 °C and 85 °C. Conversely, no reduction in GII.4 HuNoV genomes was detected following 100 mg/mL chymotrypsin treatment over the whole heat treatment range, indicating the potential stability of the HuNoV capsids. These results suggest that chymotrypsin content was high enough to (i) provide a protective effect against heat degradation; and (ii) prevent HuNoV genomes from being accessible to RNases. The protective effect of a high chymotrypsin concentration could also be produced by treating HuNoVs with an acid pH and pepsin. This could be explained by the aggregation of viral particles which can maintain the integrity of particles47. Be that as it may, our results clearly indicate that GII.4 HuNoVs are more resistant to an acid pH and proteolytic enzyme digestion than GII.4 VLPs.While different degrees of enzymatic digestion were detected in GII.4 VP1 and VLPs, different behaviours were observed in native GII.4 HuNoVs following pepsin, trypsin, and chymotrypsin treatments. VLPs are described as structurally, antigenically, and morphologically similar to intact HuNoV particles+ human saliva provided a protective effect on capsid integrity in GII.4 VLPs against these proteolytic enzymes. Several assumptions can be drawn on the basis of these observations. The first is that the cleavage sites of pepsin, trypsin, and chymotrypsin are not located in the regions surrounding the HBGA-binding site in subdomain P2 of VP1. The second is that the steric hindrance caused by HBGAs was not sufficient to prevent capsid digestion by these proteolytic enzymes. However, it is important to bear in mind some methodological constraints on investigating the potential influence of HBGA-binding on the maintenance of capsid integrity. For example, it was difficult to use SYPRO Orange probe to study the Tm values of VLPs. The abundance of proteins in human saliva and the addition of the proteolytic enzymes increased the background noise and hindered the reliable determination of Tm values. Given that proteolytic enzymes and an acid pH of less than 2.0 can disrupt VLPs, the present study demonstrated, for the first time as far as we know, that human saliva containing HBGAs does not influence the stability of the capsid when exposed to factors naturally found in the human digestive system. However, that bile salts or divalent cations could promote capsid stability has not been ruled out. There is ample evidence that they enhance the replication of HuNoVs using human intestinal enteroids but also potentially improve HBGA-binding to HuNoVs56. The protective effect of human gut microbiota could also be involved because it has recently been shown that some enteric bacteria (e.g. Enterobacter cloacae) bind HuNoVs through the H-like carbohydrates present on the bacterial surface58.Second, the protective effect of HBGA-binding on GII.4 VLP capsids was investigated by using human saliva prior to proteolytic enzyme treatments. No similar investigation was conducted using GII.4 HuNoVs because there was no evidence of complete dissociation of the viral particles, induced by the same enzymes. At low doses of trypsin and chymotrypsin (0.1 mg/mL), SDS-PAGE analysis detected no difference between samples with and without HBGA-binding to VLPs. At higher doses of trypsin or chymotrypsin (100 mg/mL), and at 1,500 units/mL pepsin, VLPs were totally disrupted even in the presence of HBGAs. Thus, we found no evidence that ALeFinally, we demonstrated the differences between the behaviours of GII.4 VLPs and GII.4 HuNoVs when exposed to different treatments (acid pH and proteolytic enzyme treatments). There was no evidence of a protective effect of HBGAs on GII.4 VLPs. Further research is required to elucidate how HBGA-binding contributes to capsid stability and to human infections.59. The quantity of proteins was determined using a NanoDrop 2000 spectrophotometer . GII.4 VLPs were prepared at a final concentration of 1 mg/mL with a TNC buffer . Aliquots were stored at − 80 °C, and extemporaneously thawed as needed.GII.4 VLPs  were obtained using the baculovirus-expressed VP1 system and purified using a discontinuous sucrose gradient, as previously described by De Rougemont60, kindly provided by the University Hospital of Nancy, France. The purification, extraction, and genome quantification of GII.4 HuNoVs were performed using the methods described previously46 (see Supplementary Information Method). The purified HuNoV suspensions were stored in darkness at +4 °C at a final concentration of 107 gc/mL until needed. The total protein content of the HuNoV suspensions was measured using a NanoDrop 2000 spectrophotometer at a final concentration of 350 µg/mL.The GII.4 HuNoVs were genotyped from human stools3, 12 mM KCl, 40 mM NaCl, 6 mM CaCl2)50 at pH values ranging from 1.3 to 3.0 or in ammonium bicarbonate buffer . GII.4 VLPs were prepared at a final concentration of 300 µg/mL. For the acid pH treatments, 4 µL of the simulated gastric fluid buffer were added to 10 µL of GII.4 VLPs at 300 µg/mL. For the proteolytic enzyme treatments, 10 µL of GII.4 VLPs at 300 µg/mL was treated with 4 µL of proteolytic enzymes at 37 °C for 4 h. Pepsin  was used at a concentration of 1,500 units/mL (i.e. 600 µg/mL) in simulated gastric fluid buffer at pH 2.0 for VLPs and pH 1.3 for HuNoVs. Trypsin  and chymotrypsin  were used at concentrations of 0.1 and 100 mg/mL in ammonium bicarbonate buffer.Suspensions of GII.4 VLPs were diluted in simulated gastric fluid buffer  was added and the mixture was incubated at 37 °C for 4 h. RNase was added to achieve a final concentration of 100 μg/mL. The results were considered valid if the RNA genomes of MS2 bacteriophages was not detected by RT-qPCR (see Supplementary Information Method). The number of HuNoV gc in the native suspension was quantified using RT-qPCR at pH 8.0 and 37 °C (C0) and following acid pH or proteolytic enzyme treatments at 37 °C (C). The experiments involving GII.4 VLPs and GII.4 HuNoVs were at minimum duplicated, under the same conditions.The protocol developed for proteolytic enzyme treatments on GII.4 VLPs was used on the purified GII.4 HuNoVs. To estimate the quantity of potentially fully intact capsids, the purified GII.4 HuNoV suspensions were treated with RNase A  as described by Brié et al., with some modifications46. TEM is considered to provide a semi-quantitative estimation of the number of particles. Pictures at a magnification of ×15,000 were obtained, to provide a representative field permitting an estimation of the number of particles, and at a magnification of ×27,500 to provide a more precise overview of the morphology and size of particles. When assessing each acid pH, proteolytic enzyme treatment, and magnification factor, at least 4 representative pictures were taken into account. The mean number of particles was determined by visual inspection and the size was measured using ImageJ software (Java 1.8.0).TEM was used to observe GII.4 VLPs both with and without acid pH or proteolytic enzyme treatments, after negative staining with phosphotungstic acid, as previously described2HPO4, 0.18 mM KH2PO4, pH 7.4). Size measurements were made using a Zetasizer Nano ZS instrument , as described by Brié et al.61. Diameter measurements of GII.4 VLPs were obtained using at least two independent experiments and each hydrodynamic diameter measurement was performed in triplicate.Only the GII.4 VLPs treated with 0.1 mg/mL and 100 mg/mL trypsin and chymotrypsin were studied using this method. At the end of the proteolytic enzyme treatments, the reaction was diluted by the addition of 1 mL of 0.1× PBS , and at the different temperatures tested using the same treatment (C). For each sample, at least two analyses of HuNoVs were performed.The method used on GII.4 VLPs could not be applied to GII.4 HuNoVs because the viral content of the purified stools was insufficient. A new procedure was therefore implemented to determine the temperature needed to disassemble the GII.4 HuNoV capsids. Following proteolytic enzyme treatments, the samples were placed in a Veriti Thermal Cycler  apparatus and heated at different temperatures  for 10 min. RNase A treatment was then applied to a final concentration of 100 μg/mL. Finally, the viral genomes of MS2 bacteriophages and GII.4 HuNoVs were extracted and quantified using RT-qPCR as described above and in the Supplementary Information. The quantity of HuNoV gc was determined in the initial viral suspensions, following treatment at 60 °C  at 95 °C for 10 min, then placing them on ice.Following the proteolytic enzyme treatments, GII.4 VLPs were heated for 10 min at 95 °C in order to compare the effects of the treatments on VP1 and VLPs.62. Aliquots of 20 µL of each denatured sample were loaded onto a discontinuous polyacrylamide gel . Standard size markers, protein migration, and gel staining were used under the same conditions as previously described62. Analyses of each sample were performed in triplicate.GII.4 VLPs were then denatured using SDS and dithiothreitol (DTT) conditions, as previously described, with slight modifications59, and informed consent was obtained from all donors. All the experiments using saliva were performed in accordance with the relevant guidelines and regulations. The human saliva was boiled and centrifuged, as previously described46. Supernatants were stored at − 20 °C until use. The presence of A, B, and O blood group antigens and Lewis antigens in saliva samples at a dilution of 1:1000 in 1× PBS was determined by ELISA, as previously described63.Saliva samples from healthy adult volunteers were collected. The saliva samples used in this study were previously described, with the approval of the Nantes University Hospital Review Board (study number BRD02/2-P)46, with slight modifications. The modifications involved a negative control. Briefly, saliva samples were treated with 100 mM sodium periodate (prepared in a 50 mM sodium acetate buffer at pH 5) for 40 min at room temperature, and then incubated for 10 min with 1% glycine in 1× PBS.HBGA-binding ELISA was performed using the method previously described450) and the results were analysed as previously described46. All the samples were analysed in duplicate. Results, expressed in percentages, were calculated by taking the strongest adhesion of GII.4 VLPs as a reference.Optical density values were obtained at 450 nm  were added and the samples were stored at 37 °C for 4 h. All results were analysed in triplicate, using SDS-PAGE.Ten µL of native GII.4 VLPs diluted to 300 µg/mL in a buffer of proteolytic enzyme were incubated with 10 µL of ALe64. A Shapiro–Wilk test with an alpha level of 0.01 was run, to test the normality of the data. Student’s paired and unpaired t-tests were used for dependent and independent data respectively, following a normal distribution. Wilcoxon signed-rank and Mann–Whitney U tests were used for dependent and independent data respectively with non-normal distribution. In all tests, the significance level was set to 0.01.All statistical analyses were performed using R statistical software (Rx64 v.3.5.3)Supplementary Information


Open Biology that were identified as part of a group of eight papers with identical figures and tables. Should, as seems likely, these papers prove to be fraudulent then this strikes at the heart of our endeavour. Science depends absolutely on trust, and the vast majority of scientists honestly seek after the truth in their research; if errors are made these are honest mistakes in preparing figures or interpretation of data. But some errors can also be introduced by being too trusting, notably in the specificity of reagents, and, most regrettably, in the very small minority of studies where data are deliberately fabricated. Disturbingly, it appears that the number of fraudulent papers is on the rise, in part through authors purchasing papers through ‘paper mills' that generate papers by recycling text and figures. Thus, it is necessary for all publishers to increase their vigilance against deliberate fraud. The motto of the Royal Society is ‘Nullis in verba' (on the word of no one) and so it is appropriate that Open Biology should require of its authors that they provide rigorous data to support their conclusions. In this spirit, I would like to set out the standards required before publication, and the measures that we take with every paper to prevent fraud now and in the future.We have recently published Expressions of Concern for two papers in —From now on, all gels and immunoblots will require molecular mass markers and for each figure, the whole gel or blot must be submitted as supporting data. Appropriate positive and or negative controls must be run on the same gel or blot, and the specificity of antibodies used for immunofluorescence should be validated by blotting or siRNA controls.—For siRNA studies, at least three separate siRNAs must be used unless the phenotype can be restored by expressing an RNA resistant to the siRNA.—All figure legends should state how many biological repeats were performed: a minimum of three is expected for most experiments.—Images should not be subjected to any processing that could lead to misinterpretation of the original data, and the original, unprocessed data should be made available upon request.Committee on Publication Ethics.To guard against deliberately fraudulent papers, all submitted papers will be screened using anti-plagiarism software and those provisionally accepted for publication will be examined by a third-party image integrity analyst. Any papers confirmed to contain plagiarized, manipulated or fraudulent data will be rejected and recorded in a database. Any published papers subsequently identified as potentially fraudulent will be treated according to the guidelines of the https://royalsocietypublishing.org/rsob/for-authors#question3.More details can be found here: Open Biology; it is only with this that we can all make progress.I am sure that all our authors will understand that these measures are done to ensure that the community can trust in the validity of the papers published in


Background: We experienced a case of malunion of condylar fracture after miniplate fixation in a patient with a 40 pack-year smoking history who restarted smoking at 5 weeks postoperatively. Case: A 64-year-old man lost consciousness and fell down, hitting his chin on the floor. He had malocclusion and open bite bilaterally. The mouth opening was 1.5-finger breadths. He had a 40 pack-year smoking history. Radiology revealed a bilateral condylar fracture and a fracture of the parasymphysis. Intermaxillary fixation was done using the skeletal anchorage system on the first post-trauma day. On the third post-trauma day, vertical ramus osteotomy, miniplate fixation of the fractured condylar neck, and free grafting were performed. When the wire was changed to a rubber band at 5 weeks postoperatively, he started smoking . At 7 weeks postoperatively, the skeletal anchorage system was removed and some absorption of the condylar head was observed. At 3 months postoperatively, his mouth opening was 24 mm and no malocclusion was present, although the condylar head was distorted and malunion was observed. At 4 months postoperatively, his mouth opening was 30 mm but he complained of pain on do so. Distortion of the condylar head was aggravated. At 5 months postoperatively, his pain continued but was endurable. He continued smoking  since 5 weeks postoperatively. Conclusion: In smokers, a longer period of immobilization is needed in bone grafting of the fractured condylar head. Longer immobilization provides sufficient time for healing and prevents smoking, since the patient cannot smoke easily when the intermaxillary fixation is applied. P = .04).Nicotine negatively impacts bone healing.In high condylar fractures, we have used vertical ramus osteotomy, miniplate fixation of the fractured condylar neck, and free grafting.We experienced a case of malunion of a condylar fracture after miniplate fixation in a patient with a 40 pack-year smoking history who restarted smoking at 5 weeks postoperatively.A 64-year-old man lost consciousness and fell on the ground while working in a warm workplace. Upon falling down, he hit his chin on the floor.a).Upon examination, he had malocclusion and open bite bilaterally. The mouth opening was 1.5-finger breadths. He had a 40 pack-year smoking history. Radiology revealed a bilateral condylar fracture and a fracture of the parasymphysis a.b), which made it difficult for him to smoke.Intermaxillary fixation was done using a skeletal anchorage system (SAS) on the first post-trauma day b, which c and 3(PO4)2 . His course in the hospital was uneventful, and he was discharged on the seventh postoperative day.On the third post-trauma day, vertical ramus osteotomy, miniplate fixation of the fractured condylar neck, and free grafting were performed c and 2. d), he started smoking  against the medical advice. Starting at 6 weeks postoperatively, the rubber band was applied only at night.When the wire was changed to a rubber band at 5 weeks postoperatively d, he stae).At 7 weeks postoperatively, the SAS was removed but some absorption of the condylar head was observed in panoramic imaging e.f).At 3 months postoperatively, his mouth opening was 24 mm and he showed no malocclusion. However, the condylar head was distorted and malunion was observed in a panoramic view f.g).At 4 months postoperatively, his mouth opening was 30 mm and he showed no malocclusion, but he complained of pain on mouth opening. The condylar head was distorted and malunion was aggravated in a panoramic view g.h).At 5 and 7 months postoperatively, his mouth opening was 30 mm and he showed no malocclusion; his pain on mouth opening continued but was endurable. He continued smoking  since 5 weeks postoperatively. At 5 months, the condylar head was distorted and malunion was similar in a panoramic view to the findings at 4 months postoperatively h.The principles outlined in the Declaration of Helsinki were followed in this study.-Our method  is a type of osteotomy-osteosynthesis, as described by previous authors.4When totally removing the fractured condyle and placing some hydroxyapatite between the bony gaps, the following 2 things occur: remove it from its blood supply, and prevent revascularization once replaced.Debates continue regarding the effect of smoking on dental implants. Lindfors et alOn the contrary, Chambrone et al,In the present case, we observed that the condylar head was in the glenoid fossa until 5 weeks postoperatively (the nonsmoking period), but 2 weeks after the patient started to smoke, the grafted condylar head started to be absorbed . It is tSince smoking decreases the alveolar oxygen pressure and subcutaneous wound-tissue oxygen, and nicotine causes vasoconstriction, smokers are more likely to experience flap loss, hematoma, or fat necrosis than nonsmokers (a phenomenon known as smoking flap).


Surgery that were published during the Kim Jong-un era (2012–2018). The framework for categorization was primarily based on disease entities, surgical specialty, and research methodology. A total of 1792 articles in 28 issues were included in the current study. The frequency of detailed surgical cases and their associated characteristics based on their specialty were investigated. The types of medical imaging techniques and anesthetics that were commonly utilized in clinical fields in North Korea were also evaluated. A large proportion of essential surgeries were covered, with the exception of those for congenital diseases; however, a lack of surgical techniques and infrastructure was revealed. Going forward, further evaluation of the surgical system and a greater focus on non-communicable diseases in North Korea are needed.Information regarding surgical diseases in North Korea is not fully reported despite their clear clinical significance. The current study aimed to indirectly assess the contemporary research trends and medical infrastructure related to surgical diseases in North Korea. We analyzed and categorized articles from the journal  In general, information on the health and disease status of 24 million North Koreans is largely unknown due to limited access to the country itself . Only a Some limited facts are known regarding the health of individuals in North Korea, including the average life expectancy of 71.69 years as of 2016 and the number one cause of death, which is cardiovascular disease . HoweverIn an attempt to gather additional data, there have been efforts to investigate defectors from North Korea rather than current residents . FurtherNotably, there are few reports regarding the status of surgical diseases and surgical environments in North Korea . RegardlAccording to the Institute for Health Metrics and Evaluation, the top 11 causes of death in North Korea are stroke, chronic obstructive pulmonary disease (COPD), ischemic heart disease, liver cancer, lung cancer, Alzheimer’s disease, stomach cancer, road injuries, lower respiratory infection, hypertensive heart disease, and cirrhosis . Among tSurgery. A secondary aim of the present study was to indirectly assess the nature of surgical diseases and the status of infrastructures in North Korea for surgeries, including anesthesia and radiological imaging.In the current study, medical research pertaining to surgical diseases in North Korea was investigated through the North Korean medical journal Surgery [Oegwa] from 2012 to 2018 were reviewed. The selection of articles began in 2012 to correspond to the year when Kim Jong-un became the Chairman of the Workers’ Party of North Korea. The journal Surgery was selected as a representative medical journal to understand surgical diseases in North Korea out of nine known medical journals in North Korea: Internal Medicine; Surgery; Basic Medicine; Korean Medicine; Preventive Medicine; Pediatrics, Obstetrics, and Gynecology; Dentistry, Ophthalmology, Otorhinolaryngology; Korean Pharmacy; Medical Science , between 2012 and 2018, an issue was published every quarter with an average of 64 articles per issue. A total of 1792 articles in 28 issues were published from 2012 to 2018. All of the articles were written in Korean and could be classified into seven different types: editorial, original article, discussion, experience, review article, case report or series, or creative invention. The distribution of article types is reported in In the North Korean journal The majority of original articles reviewed were organized in sections composed of a gyo-si , study population, study method, study results, conclusion, references, and keywords. “Discussion” articles had similar structures but did not include references. Articles categorized as “experiences” similarly had a structure that consisted of sections including population, methods, results, and conclusion, without any references. Out of the 1792 articles, approximately 36% had teachings from Kim Jong-il or Kim Jong-un as a gyo-si see . TeachinThe results of the analysis by disease category are shown in The distribution of articles by specialty is demonstrated in General surgery included those on the lower and upper gastrointestinal (GI) tract, hepatobiliary system, vascular system, thyroid, and breast. Trauma: There were a total of six articles categorized as trauma, which included a splenic injury (2 articles), liver injury (2 articles), and traumatic hemorrhagic shock (2 articles).Pediatric: In terms of pediatric surgery, between 2012 and 2018, the articles covered intussusception (5 articles), hernia (4 articles), biliary ectasia (3 articles), megacolon (2 articles), hypertrophic pyloric stenosis (1 article), and Meckel’s diverticulum (1 article).Helicobacter pylori. A paper on cholangiocarcinoma included 118 cases, 84 of which were male, and reported that cholangiocarcinoma is most common in individuals in their 50’s in the Democratic People’s Republic of Korea (DPRK). Reports of other cancer-related surgeries included breast cancer (6 cases), which included two on oncoplastic techniques. In terms of rectal cancer, one paper reported the mortality from 1996–2005 as 1.97% for men and 1.34% for women.Cancer: Papers reported the use of chemotherapy for upper GI cases of advanced gastric cancer. The use of endoscopic surgery for gastric cancer was reported as well. One paper included a population sample of 2483 cases that specifically covered the topic of mucosa-associated lymphoid tissue (MALT) lymphoma, which is highly related to Non-communicable diseases: For lower GI cases, there were 29 papers on hemorrhoids, anal fistula, or anal fissure that included anal surgeries. Between 2012 and 2018, 19 appendicitis/appendectomy cases were reported. The surgical treatment of vascular diseases including thromboangiitis obliterans (TAO), arteriosclerosis obliterans (ASO), and deep vein thrombosis (DVT) was notable.Others: There were 21 papers that included the use of laparoscopy. Laparoscopic surgeries were used for appendectomy, cholecystectomy, hernia repair (both pediatric and adult), early gastric cancer, and colectomy surgeries. No cases of laparoscopic lobectomy of the liver or laparoscopic surgeries for the pancreato-biliary system were reported. There were no papers reporting thyroid surgery for either benign or malignant cases.Trauma: A total of 46.7% (248 out of 531) of orthopedic papers were trauma cases, which included fractures.Pediatric: For pediatric cases, there were five papers on developmental dysplasia of the hip (DDH) and three on club foot. In 2015, there was one paper with a study population of 2250 for DDH.Cancer: A total of five papers reported on tumors, which included chondroma (1 article), leiomyoma (1 article), osteoma (1 article), and sarcoma (1 article). The additional report included animal models of sarcoma.Non-communicable diseases: A total of 27 papers dealt with back pain, including herniated intervertebral disc (HIVD), spinal stenosis, and spondylosis, and an additional nine papers reported on arthropathy or arthritis. Twenty-six papers were on avascular necrosis (AVN), containing both radiological and clinical factors. Seven papers provided cases on synovitis, bursitis, or tendinitis.Others: There were six papers that used the 3D finite element method (FEM), six on bone substitutes, and two reporting cases with arthroscopy. Of all the orthopedic papers, 24.7% (131 out of 531) utilized X-ray imaging in their studies, 4.5% (24 out of 531) used CT imaging, and 3.2% (17 out of 531) used MRI (with and without contrast). Novocaine (procaine) was used in 19 papers and the use of lidocaine was found in 11 papers.Trauma: There were six papers on trauma. Traumatic hemothorax, pyopneumothorax, and mitral valve injuries were included, as well as cases of electric injury.Pediatric: For pediatric cases, there were three papers on Tetralogy of Fallot, one case report on Marfan syndrome, six papers on ventricular septal defect (VSD), and four on patent ductus arteriosus (PDA).Cancer: There were 12 papers on lung cancer and one on a mediastinal tumor. One author (S.R. Ahn) was included in 25% of the papers on lung cancer (3 out of 12). While lung adenocarcinoma was distinguished into tubular, papillary, mucinous, and poorly cohesive type pathologically according to the WHO 2010 classification, the 2015 paper that studied the survival rate of lung adenocarcinoma classified it into mucinous and non-mucinous types.Non-communicable diseases: A total of 13 papers addressed problems with valves, of which 76.9% (10 out of 13) were on the mitral valve. Two papers were on pulmonary valves, and the other on aortic valvular disease. There were three papers that examined the treatment of pleuritis, one on pericarditis, and two on pleural effusion. One study reported a case of cardiac tamponade. There were three papers on pneumothorax, two on thoracic sympathectomy, and four on coronary artery bypass grafting (CABG).Others: Of all the cardiothoracic papers, 24.7% (38 out of 154) used X-ray imaging, 4.5% (7 out of 154) used CT imaging, and 23.4% (36 out of 154) utilized ultrasonography. There were six papers that used dimedrol, five that used morphine, and four that used novocaine (procaine) for anesthesia.Trauma: For cases of epidural hemorrhage (EDH), subdural hemorrhage (SDH), and intracerebral hemorrhage (ICH), except for those involving skull fracture, incidences were higher in men. There were two papers on EDH, nine on SDH, and three on ICH. There was one report of treatment for Parkinsonism caused by CO poisoning.Pediatric: There were a total of four congenital disease papers, which included three on moyamoya disease and one on cerebral palsy. There were three papers on adult hydrocephalus, but none on pediatric cases. All hydrocephalus papers used endoscopic third ventriculostomy (ETV) as the surgical method, except for one paper that described the characteristics of normal-pressure hydrocephalus (NPH).Cancer: There were 11 papers on brain or spine tumors from 2012 to 2018. For papers addressing brain tumors, two were on CPA tumors, two were on meningioma, and two were on pituitary tumors. In these papers, pituitary tumors were approached using a trans-sphenoidal approach (TSA).Non-communicable diseases: Two papers were on myelitis and one paper reviewed the features of ischemic stroke.Others: Of the neurological papers, 25.2% (28 out of 111) used CT imaging and 9.9% (11 out of 111) used MRI. Five papers reported using novocaine (procaine).Coptis chinensis, Cirsium japonicum var. maackii, and Lonicera japonica. Additionally, there was one case report for the treatment of an electric injury. Burn and wound treatments were common topics. There were 51 articles (35.2%) covering the topic of burn treatment and 32 (22.1%) on the topic of wounds. In the cases of burn treatment, papers reporting the usage of oriental/herbal medicines (Koryo medicines) were noted. Examples of these treatments included Koryo Burn Ointment, arsenic sulfide, extraction from lilac leaves, egg white–rifampicin mix, Skin flaps were also highlighted in 26 articles (17.9%). A total of four papers were on amputation, three on frostbite, one on scalp laceration, and three papers on pressure sores. There were two congenital disease papers, which included case reports of syndactyly and microtia. Only two papers utilized X-ray imaging, while a single article examined the clinical usage of pre-operational Doppler sonography on skin flaps. Nine papers reported the usage of novocaine (procaine).Trauma: There were eight trauma papers, among which, five were on traumatic urethral stricture.Pediatric: There were six pediatric papers, which included reports on congenital adrenal hyperplasia (CAH), circumcision, phimosis, cryptorchidism, Wilms’ tumor, and hypospadias.Cancer: There were 12 papers on urologic cancers. Bladder cancer accounted for half of the articles, while the remaining articles included prostate cancer, cystic cancer, and renal cancer.Non-communicable diseases: There were five papers on benign prostate hyperplasia (BPH), one on sparganosis, and 10 papers covering stones in the urinary tract.Others: Ten papers utilized ultrasonography and a single paper reported the usage of cystoscopy. Three papers used novocaine (procaine), including a case report of treatment for idiopathic priapism with epidural anesthesia.The analysis of papers using oriental medicine for surgical diseases is shown in In addition to research on the living human population, studies using cadavers and animals were reported. The proportion of the studies using cadavers or animals is listed in The distribution and the characteristics of references included in the articles are shown in Details of the usage of medical devices from 2012 to 2018 are shown in For imaging devices, some articles introduced them with specific model names. For example, the EDR-750b, Bulgeungi 66 (Russian product) and F-30III, Tamjeong were used. For endoscopy, the Olympus gif-d2, Intravision-8745, and Model MGA-III were used. For ultrasound, articles reported the use of the Aloka SSD-650 (marketed in 1986 and discontinued in 1997), Aloka-SSD 620 (marketed in 1988 and discontinued in 1996), HP SONOS 5500 2.5MHz (marketed in 1988), ATL Sono-CT HDI-5000 (1996–2006), Medison 128BW, Medison SonoAce 8000, and Sonoace 8800. Additional detail regarding ultrasound machines cannot be specified as these models were manufactured for a long period with different editions . In artiThe papers reviewed listed a total of 56 various kinds of injection materials used for anesthesia. The commonly used anesthetics and supplementary medications are listed in The trends of specific research methodologies are shown in The current study revealed the trends of research conducted in North Korea in the surgical field during the Kim Jong-un era from 2012–2018. An analysis by specialty and disease category reflected the status and characteristics of surgical diseases in North Korea. Trauma had been a constant focus of research, where 25% of articles dealt with traumatic cases that required surgical treatment. Non-communicable diseases were the most common topic, which comprised 35% of journal articles. Significant changes have been made in the sector of health and medicine since the beginning of the Kim Jong-un era . The estSurgery (35.2%) supports the idea that the demand for surgery related to NCDs is high in North Korea. NCDs, including tumors, account for eight of the top 10 causes of death in North Korea, among which, stroke, ischemic heart disease, lung cancer, stomach cancer, and cirrhosis may need surgical interventions to reduce mortality and morbidity [Naegwa [Internal Medicine], which were not included in the analysis of this study.The high proportion of topics related to non-communicable diseases (NCDs) in the journal orbidity . Notablyorbidity . Furtherorbidity ,28. The The overall trends in South Korea do not differ much from the statistics of North Korea, as lung cancer, liver cancer, stomach cancer, and colorectal cancer are reported to be the third, seventh, ninth, and tenth most common causes of death, respectively .The percentage of articles on the topic of neoplasm among non-communicable diseases were relatively low compared to the actual prevalence. Some types of cancers, such as thyroid cancer, which are reported to be frequent in North Korea, were not reported in any articles between 2012 and 2018 . The steCertain surgical treatments, such as liver and kidney transplantation, were not covered in any publications. From this, it can be assumed that organ transplantation is rarely, if ever, performed in North Korea. This could be the result of a lack of surgical techniques or pre-/post-op care, which requires intensive care facilities. A high percentage  of articles covered trauma/injury cases ranging from traumatic brain injury accompanied by hemorrhages to fractures and liver or splenic injuries. This may reflect the high number of injuries in North Korea, where road injuries are the fifth most common cause of death . The higAs the authors’ affiliations were not fully written in the paper, and there was no location information, it is difficult to speculate where these cases were collected. Considering the substandard conditions of provincial hospitals or local clinics in North Korea, it is reasonable to assume that these papers were based on data gathered from tertiary hospitals in Pyongyang .The proportion of papers using oriental medicine (3.3%) was interestingly similar to the proportion of oriental medical doctors (Koryo doctors) among all doctors in North Korea  . ResearcOne-third of the articles (600 out of 1792) did not include any references, while the proportion of papers with references published in the last five years were below 20%. Furthermore, it was notable that articles from China written in Chinese were frequently cited given that in South Korea, articles written in Chinese are rarely cited in medical journals. A few textbooks or journals were preferred and repeatedly cited; further studies with network analysis on the groups of the references may provide visible relationships. In terms of imaging, there were 78 papers (4.4%) that mentioned CT imaging in the study method and 33 (1.8%) that reported the use of MRI as an imaging device. Considering these proportions, CT and MRI may not be widely used imaging modalities in North Korea. This also could be a significant hurdle in setting precise surgical plans. Due to sanctions and ongoing political situations, the DPRK has been attempting to develop their medical equipment, including essential spinal implants , spiral Surgery. In 2017, in issue 1 of the Surgery journal, it was stated that the 23rd National Cardiothoracic Conference, 34th National Abdominal Surgery Conference, and 4th Resuscitation Conference were held. From this issue, it can be assumed that regular medical conferences based on specialties are being held in the DPRK. The list of the committees in the North Korean medical association is provided in The types of medical conferences held in North Korea are not precisely known, but related comments were made in the journal Surgery (In 2015, the World Health Organization suggested 44 essential surgeries that are necessary to reduce the burden of surgically avertable deaths worldwide ,34,35. OSurgery . Eight sThe current study has several limitations. First, this review was conducted using an indirect approach to estimate the epidemiology of DPRK’s surgical needs based on published journal articles as a source of analysis. The research papers reflect North Korean doctors’ research interests and not the full spectrum of disease characteristics or burdens in North Korea. The reviewed research papers did not report where their study populations resided, which could lead to a disproportionate geographical distribution of the study population. A direct survey of the North Korean population, which is almost impossible in the current political climate, is necessary to collect unbiased and reliable data. Second, the scope of the analysis only included data from the Kim Jong-un era. A database of previous periods could have given a more complete picture to identify changes in surgical trends. However, the focus on recent periods has its advantages in recognizing the latest efforts in research on surgical diseases and the medical infrastructure. Third, articles published in the journal were relatively short in length, with an average of 0.5–1 page per paper. It is possible that the papers included in the journal are not full papers but abstracts.Surgery; therefore, obstetrical, gynecological, dental, ophthalmological, and otorhinolaryngological surgeries were not mainly covered. Further investigation is needed to grasp the status of these kinds of surgeries. Fourth, this study only analyzed papers published in the journal Recent research outcomes in North Korea have been published in overseas, peer-reviewed journals under the instruction of Kim Jong-un, which may provide additional information to interpret the current medical situations in one of the most mysterious countries in the world . These sDespite these limitations, the insights gained into surgical and medical fields in North Korea from the current study are considerable. The prevalence of surgical diseases may be the key to understanding unmet needs and priorities, and the analysis can be used as guidance for policymakers with a focus on North Korea. The reports of medical imaging techniques and anesthesia use provide a unique perspective to approach and map out future strategies on the utilization of limited medical resources. Further studies on surgical interventions in North Korea may help to identify deficiencies and guide the subsequent necessary steps to rebuild severed relationships. The findings present a preliminary vision of the surgical diseases of North Korea, from the current focus in each surgical specialty to the status of surgical infrastructures. The characteristics of the research trends in North Korea were unique in the Kim Jong-un era; follow-up studies on the betterment in surgical fields may provide fundamental references for policymakers and researchers. Meaningful discussions on North Korean healthcare have, however, been dwindling in recent years; even the remaining part of the attention is disproportionately concentrated on communicable diseases and nutritional issues. Further attention on non-communicable diseases, including surgical diseases, is needed considering the mortality and morbidity burdens.


Ebolavirus (EBOV) infection in humans causes severe hemorrhagic fevers with high mortality rates that range from 30 to 80% as shown in different outbreaks. Thus the development of safe and efficacious EBOV vaccines remains an important goal for biomedical research. We have shown in early studies that immunization with insect cell-produced EBOV virus-like particles (VLPs) is able to induce protect vaccinated mice against lethal EBOV challenge. In the present study, we investigated immune responses induced by Ebola VLPs via two different routes, intramuscular and intradermal immunizations, in guinea pigs. Analyses of antibody responses revealed that similar levels of total IgG antibodies against the EBOV glycoprotein (GP) were induced by the two different immunization methods. However, further characterization showed that the EBOV GP-specific antibodies induced by intramuscular immunization were mainly of the IgG2 subtype whereas both IgG1 and IgG2 antibodies against EBOV GP were induced by intradermal immunization. In contrast, antibody responses against the EBOV matrix protein VP40 induced by intramuscular or intradermal immunizations exhibited similar IgG1 and IgG2 profiles. More interestingly, we found that the sites that the IgG1 antibodies induced by intradermal immunizations bind to in GP are different from those that bind to the IgG2 antibodies induced by intramuscular immunization. Further analyses revealed that sera from all vaccinated guinea pigs exhibited neutralizing activity against Ebola GP-mediated HIV pseudovirion infection at high levels. Moreover, all EBOV VLP-vaccinated guinea pigs survived the challenge by a high dose (1000 pfu) of guinea pig-adapted EBOV, while all control guinea pigs immunized with irrelevant VLPs succumbed to the challenge. The induction of both IgG1 and IgG2 antibody responses that recognized broader sites in GP by intradermal immunization of EBOV VLPs indicates that this approach may represent a more advantageous route of vaccination against virus infection. Ebolavirus is a member of the filoviridae family, and infection by ebolavirus in humans and non-human primates (NHPs) results in onset of severe hemorrhagic fevers with high mortality rates . Since tThe high mortality rate of EBOV infection underscores the urgent need for an effective EBOV vaccine. A number of EBOV vaccine approaches have been explored in past studies, many of these vaccine strategies have been shown to be able to protect vaccinated animals against lethal EBOV challenge in small laboratory animal models with various efficacies . MoreoveAmong the different EBOV vaccine strategies, VLPs are a subunit vaccine platform that has been shown to protect EBOV infection in NHPs. In early studies, both EBOV and Marburg virus VLPs have been evaluated in different animal models and shown to be effective in eliciting protecting vaccinated animals against lethal challenge , demonstThe stock for guinea pig-adapted EBOV was produced and titered in Vero E6 cells. All experiments with live EBOV were conducted in the BSL-4 facility at the Texas Biomedical Research Institute (TxBiomed), San Antonio, TX, United States.Spodoptera frugiperda Sf9 cells were maintained in SF-900 II serum-free medium with penicillin/streptomycin. The polyclonal rabbit serum against EBOV was a gift from Dr. P. Rollin (CDC). JC53BL cells . HeLa cells and 293T cells were obtained from ATCC and maintained in DMEM plus 10% FCS.BL cells , which eConstruction and production of recombinant baculoviruses (rBVs) that express EBOV GP and VP40 (designated as rBV-GP and rBV-VP40 respectively) has been described previously . For VLPn = 4) of the same dose as an irrelevant VLP control. For ID injection, VLPs were delivered by using the Mantoux method to four sites (25 to 50 ul per site based on vaccine dose) of the shaved posterior-abdomen skin of guinea pigs under kentamine/xylazine anaesthetization. Blood samples were collected from cranial vena cava under anesthesia at 1 week prior to the first immunization and 2 weeks after the second and third immunizations, and stored at −80°C until being used in analyses.Female Hartley guinea pigs (∼250 g in body weight) were obtained from the Charles River Laboratory and housed at the Division of Animal Research of Emory University. Twelve guinea pigs were divided into three groups with four animals per group. Two groups of guinea pigs were vaccinated by IM or ID injection first with 50 ug of EBOV VLPs two times and then boosted again with 200 ug EBOV VLPs at 4-week intervals. The control group guinea pigs were vaccinated with SIVgag VLPs  has been pre-determined to saturate binding of the amount of coated His-tagged GP antigens. The plates were then washed again and then incubated with sera from individual guinea pigs that had been vaccinated by ID injection of EBOV VLPs or SIVgag VLPs (Control). After incubation for 2 h at room temperature, the plates were washed and the bound IgG1 antibodies were detected with horseradish peroxidase-labeled goat against guinea pig IgG1 secondary antibodies . The wells were added with 50 ul per well of TMB (Sigma) to develop color and the color reaction was stopped with addition of 50 ul per well of hydrochloric acid (0.2N), and afterward the absorbance value at 450 nm of each well was read in an ELISA reader .tat-activated promoter. Neutralization assays were then carried out as described in our previous studies . Guinea pigs were challenged at 22 weeks after the third vaccination by intraperitoneal injection with 1000 plaque-forming units (pfu) of guinea pig-adapted EBOV. After challenge, guinea pigs were monitored at least twice daily for weight loss and disease symptoms, and animals that exhibited severe disease signs and lost significant levels of body weight (over 25%) were sacrificed in compliance with IACUC guidelines.In our previous studies, we showed that mice were effectively protected by two intramuscular immunizations with 50 ug EBOV VLPs produced in insect cells . To invet-test showed that the differences in neutralizing activity between serum samples from guinea pigs in the IM and ID immunization groups at all four serum dilutions  are not statistically significant (p > 0.05).Based on these results, we decided to give one additional boosting immunization using 200 ug EBOV VLPs or SIVgag VLPs for the control group by IM or ID injection respectively as outlined in p > 0.05). On the other hand, ID immunization by EBOV VLPs induced significantly higher levels of IgG1 antibodies against GP than IM immunization (p < 0.05). To investigate whether such differences might also be observed for other vaccine antigens in EBOV VLPs, we further determined the levels of IgG1 and IgG2 antibodies against EBOV matrix protein VP40 in EBOV VLPs for comparison. As shown in p > 0.05). Further, it is also notable that the levels of IgG1 antibodies against GP are only about one-third of the levels of IgG2 antibodies by ID immunization and about one-tenth of the levels of IgG2 antibodies by IM injection. In contrast, the levels of IgG1 and IgG2 antibodies against VP40 are similar in both IM and ID immunization groups. Taken together, these results show that the antibody profiles induced by IM and ID immunization of EBOV VLPs in guinea pigs are different only for the GP antigen but not for VP40.We further characterized the levels of IgG1 and IgG2 subclass antibodies against GP induced by IM or ID immunization with EBOV VLPs for comparison. As shown in Based on the different levels of IgG1 antibodies against GP induced by ID and IM injection, we carried out blocking ELISA to determine whether the IgG1 and IgG2 antibodies against GP may target similar or different epitopes. Briefly, microtiter plates were coated with a limited amount of purified His-tagged GP protein at 0.05 ug per well. After blocking, the wells were treated with sera from guinea pigs vaccinated by IM injection of EBOV VLPs at 1:400 dilutions, a dilution that has been pre-determined to saturate binding of the amount of coated GP antigen. The controls were added with the same dilution of sera from control group guinea pigs or no sera as indicated. After incubating with the first antibody, the plate was washed and then added with sera from guinea pigs vaccinated by ID injection of EBOV VLPs or from control group guinea pigs at 1:400 dilutions as indicated, followed by addition of HRP-conjugated secondary antibody against guinea pig IgG1 to determine the levels of IgG1 antibodies bound to GP for comparison. The results in After the third immunization, the guinea pigs were transferred to the Texas Biomedical Research Institute to evaluate the protective efficacy of EBOV VLP vaccination against lethal EBOV challenge in the ABSL-4 facility. As shown in In the present study, we compared the effect of two different immunization routes, IM and ID injections respectively, on the immunogenicity and protective efficacy of insect cell-derived EBOV VLPs in the guinea pig model. Our results showed that EBOV VLPs administered via both IM and ID routes induced similar levels of antibody responses against EBOV GP and vaccinated were completely protected against a high dose lethal challenge by guinea pig-adapted EBOV. Of particular interest, we observed that while immunization with EBOV VLPs by IM or ID injection induced similar levels of IgG2 subclass antibodies against GP, the levels of IgG1 subclass antibodies against EBOV GP induced by ID immunization were significantly higher than the levels induced by IM immunizations. In contrast, immunization by both IM and ID routes induced similar levels of IgG1 and IgG2 antibody responses against VP40, the EBOV matrix protein in VLPs. Moreover, by further analyses, we observed that binding of IgG1 antibodies induced by ID injection of EBOV VLPs was not blocked by sera from IM-vaccinated animals, indicating that such IgG1 antibodies may react to different epitopes in GP from those recognized by antibodies induced by IM immunization, which are predominantly of the IgG2 subclass.In addition to serving as a protective barrier, skin has also long been known to be an important part of the immune system . The firIn contrast to antibody responses against GP, we found that the immunization by IM or ID injection of EBOV VLPs induced similar levels of both IgG1 and IgG2 antibodies against VP40, the EBOV matrix protein that directs the formation of VLPs. Further, the levels of IgG1 and IgG2 antibody subclasses against VP40 are also similar in animals vaccinated by EBOV VLPs through either IM or ID injection, whereas antibodies against GP are predominantly of the IgG2 subclass regardless of the immunization routes. These results indicate that induction of enhanced IgG1 antibody responses by ID immunization of EBOV VLPs is specific for the GP antigen but not the VP40 antigen, even when these antigens are delivered in the same VLP vaccine complex. The functional properties of guinea pig IgG1 and IgG2 antibodies are still poorly understood. However, in an early study, it was found that the IgG2 antibodies activate complement through the classical pathway whereas the IgG1 antibodies activate complement through the alternative pathway . In thisWe show in this study that the EBOV VLPs produced by the recombinant baculoviruses from insect cells are able to provide effective protection against lethal challenge in the guinea pig model by both IM and ID immunizations. In comparison to IM immunization, ID immunization of EBOV VLPs was able to elicit enhanced IgG1 antibodies against epitopes in EBOV GP that were not recognized by the IgG2 antibodies induced by IM immunization. However, additional studies are needed to understand the underlying mechanism for this observation and to identify the targets recognized by the IgG1 antibodies induced by ID immunizations. Moreover, studies to determine the targets of the antibodies of different IgG subclasses induced by vaccination through different routes will further advance our understanding of the protection against EBOV infection by vaccine-induced immune responses. It will also be of interest to determine the effect of different adjuvant on the epitope breadth as well as subclass switching of antibody responses induced by ID immunizations in comparison with the conventional IM immunization approach. In particular, it will be of interest to determine if the use of an adjuvant will enhance or suppress induction of antibody responses to the subdominant epitopes in a vaccine antigen, and if subclass switching for dominant and subdominant epitopes will be similarly or differently affected by the use of an adjuvant. Moreover, optimization of EBOV VLPs or other subunit vaccines by ID immunization using novel microneedle vaccine delivery technologies will further reveal the potential of this vaccination approach for protection against infection by EBOV as well as other viral pathogens.All datasets generated for this study are included in the article/supplementary material.The animal study was reviewed and approved by the Institutional Animal Care and Usage Committee (IACUC) of Emory University and the Institutional Animal Care and Usage Committee (IACUC) of TxBiomed Research Institute.YL, LY, and ZW conducted production and characterization of EBOV VLP vaccines, immunization studies, and analyses of immune responses. RC, JN, HS, AT, and JP conducted EBOV challenge studies. LY, RWC, and CY contributed to experimental design and data analysis. YL, LY, and CY contributed to manuscript preparation.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.