In contrast to bone scan and computed tomography (CT), which depend on osteoblastic response to detect bone metastasis, whole-body magnetic resonance imaging (WB-MRI) may be able to directly detect viable tumors. A 75-year-old male who had progressive metastatic prostate cancer during primary androgen deprivation therapy was referred to our hospital. Although bone scan and CT showed multiple bone metastases, WB-MRI suggested nonviable bone metastasis and viable tumor of the primary lesion. Prostate needle biopsy demonstrated viable prostate cancer cells from 10 of 12 cores. In contrast, CT-guided needle biopsy from bone metastasis of the lumbar vertebra revealed no malignant cells. Based on these findings, we reasoned that viable tumor cells inducing disease progression may primarily exist in the primary lesions and not in the metastatic lesions, and combined prostate radiotherapy and systemic hormonal therapy resulted in successful clinical response and disease control. The use of WB-MRI to detect viable disease lesions may enable us to design optimal treatment strategies for patients with metastatic castration-resistant prostate cancer. As the disease condition of metastatic prostate cancer may change over time, it is necessary to evaluate and adjust its corresponding treatment as necessary, according to the given circumstances. Although bone scan and computed tomography (CT) have been the most widely used methods of evaluating metastases of prostate cancer, accurately evaluating bone metastases is often difficult because bone scan and CT depend on the osteoblastic response induced by tumor cells infiltrating the bone. In addition, bone scan and CT cannot evaluate the prostate. In contrast, whole-body magnetic resonance imaging (WB-MRI) allows direct detection of viable tumors not only in the metastatic sites but also in the primary lesion, owing to the method's excellent soft-tissue contrast, high spatial resolution, and lack of ionizing radiation . AlthougA 75-year-old male visited a urological practitioner because of nocturia. An elevated serum prostate specific antigen (PSA) level of 76.2 ng/mL was observed, and digital rectal examination showed diffuse induration of the prostate. Pelvic MRI demonstrated extensive high signal of the prostate in diffusion-weighted imaging (DWI) . FluorodTo evaluate the patient's current disease status, we performed CT, bone scan, and WB-MRI. We observed discrepancies between the WB-MRI, bone scan, and CT. CT showed multiple osteoblastic lesions in the spine and pelvic bone Figures  and shru−3 mm2/s); however, b = 0 and b = 1200 of DWI exhibited no high signal intensity, suggesting a benign osteoblastic change induced by the treatment effect . After this, his elevated PSA immediately declined and was controlled at a level of <0.2 ng/mL.The present case has demonstrated the diagnostic accuracy of WB-MRI in a patient with metastatic CRPC, whose histopathological examination results were consistent with the WB-MRI results but not with the bone scan and CT results. The results of WB-MRI and histopathological examinations enabled us to provide optimal treatment to the patient, including combining prostate RT and systemic hormonal therapy.18F-NaF and 18F-fluorocholine PET/CT, demonstrated a higher diagnostic accuracy of bone metastasis in the past 10 years [18F-NaF PET/CT for bone metastasis were both 100% and those of 18F-fluorocholine PET/CT were 91% and 99%, respectively. Similarly, Beheshti et al. [18F-fluorocholine PET/CT as follows: (a) bone marrow involvement (positive on 18F-fluorocholine PET and negative on CT); (b) typically osteoblastic but less often osteoclastic lesions (positive on 18F-fluorocholine PET and CT); and (c) densely sclerotic lesions (negative on 18F-fluorocholine PET and positive on CT). The densely sclerotic lesions that exhibited no metabolic uptake of 18F-fluorocholine could be attributable to the therapy-induced apoptosis of cancerous cells. More recently, 68Ga-prostate-specific membrane antigen (PSMA) PET/CT has been reported as a promising imaging modality, particularly for the detection of biochemical recurrence. Despite the cut-off value of PSA failure being <1.0 ng/mL, the detection rate of recurrence sites in patients with PSA failure following radical prostatectomy was 50% [68Ga-PSMA PET/CT with WB-MRI for the staging of 36 high-risk patients with prostate cancer. 68Ga-PSMA-PET/CT detected a higher number of patients with regional and distant lymph nodes  compared with that with WB-MRI . In contrast, the findings of a primary lesion in 68Ga-PSMA-PET/CT corroborated those in WB-MRI only by 52.7%, implying that WB-MRI is better than 68Ga-PSMA-PET/CT in terms of the simultaneous evaluation of prostate and metastatic lesions. These new imaging modalities, including WB-MRI, could contribute to offer precision medicine to patients with metastatic prostate cancer.Several meta-analyses have shown that WB-MRI has greater diagnostic performance for bone metastasis than bone scan and CT. The sensitivity and specificity of WB-MRI in the detection of bone metastasis were found to be ≥95%, whereas the equivalent measures for bone scan were just 78% and 85%, respectively, and for CT, just 77% and 83%, respectively –4. In adi et al.  reportedIt has been reported that WB-MRI can directly detect tumor viability, with the support of DWI . To evalIt is noteworthy that diagnosis using WB-MRI for both primary and bone metastases in a patient with metastatic CRPC was histopathologically proven. Regarding the consistency of MRI and pathological findings, Perez-Lopez et al.  investigRecently, local therapy for metastatic prostate cancer has attracted attention because the median overall survival rate of metastatic prostate cancer is still only 42 months, despite the availability of several new drugs such as docetaxel, enzalutamide, abiraterone, cabazitaxel, and radium-223 . RecentlThere are two hypotheses as to why local therapy for metastatic prostate cancer can improve oncological outcomes. The first hypothesis is the “abscopal effect” of RT. This is a phenomenon where local irradiation causes the shrinking not only of the irradiated tumor but also tumors outside the irradiation field . This maThis case report has several limitations. First, we do not have access to pretreatment prostate biopsy, baseline WB-MRI, and bone scan results because the patient's initial diagnosis and treatment occurred before his referral to our hospital. Second, we could not exclude the possibility of a false-negative result in the bone biopsy, although we performed the CT-guided bone needle biopsy via the osteoblastic lesion, which was diagnosed with bone metastasis by CT and bone scintigraphy, with an orthopedically appropriate technique to detect bone metastasis . FurtherAccurate evaluation of the viability of both primary and metastatic lesions in patients with metastatic CRPC using WB-MRI may enable us to select patients who would benefit from local therapy. Prospective studies are needed to validate the treatment strategy associated with the use of WB-MRI for metastatic CRPC.


Subjects participated in two meal-challenge-tests with high-carbohydrate  and normo-carbohydrate  meal intake. Fasting and 30, 60, 120, and 180 min after meal intake plasma samples were fingerprinted by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In HR-genotype men, the area under the curve (AUC) of acetylcarnitine levels was higher after the HC-meal  and the NC-meal  intake. After the NC-meal, the HR-risk genotype carriers presented lower AUCs of oxidized fatty acids  and higher linoleic acid , while after the HC-meal, they presented lower AUCs of ornithine , sphingosine , linoleamide , and several lysophospholipids . Moreover, lower AUC  of taurocholate after the HC-meal and higher  glycodeoxycholate levels after the NC-meal were observed. Our results revealed differences in postprandial metabolites from inflammatory and oxidative stress pathways, bile acids signaling, and lipid metabolism in PROX1 HR-genotype men. Further investigations of diet–genes interactions by which PROX1 may promote T2DM development are needed.The prospero homeobox 1 (PROX1) gene may show pleiotropic effects on metabolism. We evaluated postprandial metabolic alterations dependently on the rs340874 genotypes, and 28 non-diabetic men were divided into two groups: high-risk (HR)-genotype (CC-genotype carriers,  Type 2 diabetes mellitus (T2DM) is a major public health issue affecting 415 million people worldwide in 2015 .The Genome-Wide Association Studies (GWAS) and other, different scale meta-analyses and studies have indicated that the rs340874 single nucleotide polymorphism (SNP) in the prospero homeobox 1 (PROX1) gene is a strong genetic susceptibility factor for T2DM ,6. It haWe hypothesize that one of the pathways involved in the T2DM development in subjects with the PROX1 rs340874 CC genotype may be a lipid metabolism path, and its further oxidative stress consequences can be modulated by different diets with varying macronutrients content. In our previous studies, we found that some subtle metabolism alterations are detectable only postprandially, and since most of the daytime people spend in the post absorptive state, the postprandial metabolism may play a crucial role in metabolic disorders development and/or progression . We obseStudies carried out so far—as well as our own observations—indicate that the mechanisms by which the PROX1 gene affects the susceptibility to T2DM seem to be more complex. Therefore, for further investigation, we used the metabolomics approach. We used a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) to evaluate postprandial changes in serum metabolites during the high-carbohydrate (HC) and normo-carbohydrate (NC) meal-challenge-tests in non-diabetic men dependent on the PROX1 rs340874 genotypes.www.clinicaltrials.gov as NCT03792685. Only males were enrolled into the meal-challenge-tests because of the possible sexual dimorphism of investigated factors . Variables with VIP >1.0 and absolute p(corr) >0.4 were considered significant.Statistical analysis was performed on each metabolite’s mean AUCs within different strata. Patients with the HR-genotype (CC genotype) were compared to patients with the LR-genotype carrying the protective allele T (CT/TT genotypes) in rs340874 of the PROX1 gene. NC- and HC-meal groups were analyzed independently. Selection of statistically significant metabolites was performed implementing both, uni-, and multivariate analyses. For each significant metabolite, Statistically significant metabolites were annotated by matching the spectral data from public databases  with spectral data obtained through MSMS (tandem MS—mass spectrometry) analysis for metabolites present in plasma samples. Detailed information about identified metabolites is included in the The baseline characteristic of the studied population is presented in Samples were divided into two groups according to the type of meal taken, NC- and HC-meals, and then analyzed in independent analytical batches in both polarity modes. This resulted in four datasets, which were aligned according to their polarity: ESI+ for both meals and ESI- for both meals. After application of the QA procedure, there were 1717 metabolic features in ESI+ and 848 in ESI- past QA procedure from both data sets . Principal component analysis (PCA) was implemented to visualize the results of the QA procedure. For each analytical sequence, the QC samples clustered tightly , which iFinal datasets contained only features presented in ≥80% of the samples in at least one of the two studied groups (CC versus CT/TT). It resulted in 1494 and 843 features for the NC- and HC-meals in ESI+ mode and the NC- and HC-meal in ESI- mode, respectively.To select discriminating metabolites, volcano plots  were buiMetabolites discriminating studied genotypes after both meals are presented in Global overview of all the samples revealed that there was a difference in the metabolic response to the meal between men carrying CC and CT/TT genotypes . InteresWe did not observe any crucial differences between studied genotypes in the fasting plasma metabolites profile, as mentioned above. Postprandially, we noted that the AUCs of the postprandial very long chain unsaturated PC36:5 levels were lower after NC-meals, while the AUCs of PE38:6 levels were significantly higher after both meal intakes in the HR-group. The HR-genotype carriers presented lower AUCs after the HC-meal and higher AUCs after the NC-meal for postprandial levels of polyunsaturated LysoPC and LysoPE with 18-22-carbon chain length. Conversely, the AUCs of the monounsaturated and the saturated LysoPCs postprandial levels (18 and 16 carbons in length) after the NC-meal intake in the HR-genotype men were lower compared to the LR-genotype carriers. We also noted higher AUCs of postprandial linoleic acid (LA) levels, lower AUCs of hydroxyeicosatetraenoic acids (HETE), and hydroxyoctadecenoic acid (HODE) levels after NC-meal intake, and after both meals, we noted lower AUCs of postprandial hydroxydocosahexaenoic acid (HDoHe) levels. After the HC-meal intake in HR-genotype men, we observed higher AUCs of postprandial tetradecanedioic acid. The HR-genotype men presented lower AUCs of postprandial leukotriene A4 (LTA4) and sphingosines levels and higher AUCs of postprandial acylcarnitines levels after both meal intakes. Lower AUCs of postprandial linoleamide levels after the HC-meal, and for dodecanamide levels, after both meal intakes, were observed. The AUCs of postprandial taurocholic acid levels were lower after the HC-meal intake, while the AUCs of deoxycholic acid glycine conjugated were higher after the NC-meal intake. Moreover, we noticed lower AUCs of postprandial ornithine levels after the HC-meal intake in the HR-genotype men.We evaluated the metabolomics analyses at fasting and postprandial states to explore the impacts of the rs340874 SNP in the PROX1 gene on the human metabolism. At the fasting state, we did not observe any crucial differences in metabolites levels between studied genotypes, but the meal-challenge-tests uncovered several postprandial alterations. We noted some differences in the postprandial phospholipid levels. Altered PCs and LPCs plasma profiles were associated with T2DM . LPCs weIn HR-genotype men after the NC-meal intake, we also noticed higher AUCs of postprandial LA and lower AUCs of postprandial levels of long-chain fatty acids (LCFA) esters derived from arachidonic acid (AA), HETE, and from docosahexaenoic acid (DHA), the HDoHe, after both meal intakes. Some of the oxidized fatty acids had a biological activity and could signal through their own receptors to evoke a variety of physiological changes. It has been shown that reduced glucose-induced 20-HETE formations and release contribute to inefficient glucose-stimulated insulin secretion in islets isolated from T2DM humans and mice . MoreoveThe HR-genotype men also presented higher AUCs of postprandial tetradecanedioic acid levels after the HC meal intake, which may suggest an altered peroxisomal beta-oxidation since the oxidation of tetradecanedioic acid has been found to be reduced by more than 75% in peroxisome deficient hepatocytes .Our metabolomics analysis showed lower AUCs of postprandial leukotriene A4 (LTA4) plasma levels in the HR-genotype carriers after both meal intakes. The LTA4 could be further metabolized to form LTB4, which plays an important role in metabolic disruptions . TherefoAfter both meal intakes, the HR-risk genotype carriers presented higher AUCs of postprandial acylcarnitine levels. Increased plasma acylcarnitines levels have been reported in insulin-resistant and T2DM subjects as products of incomplete or inefficient β-oxidation, and tissue accumulation of acylcarnitine molecules may lead to activation of proinflammatory pathways, which are implicated in insulin resistance and T2DM development .Furthermore, in the HR-genotype men, we also noticed lower AUCs of postprandial levels of two fatty acid amides (FAAs)—dodecanamide after both meals and linoleamide after the HC-meal intake. The FAAs may play roles as endogenous brain cannabinoid receptor ligands and may be involved in T2DM pathogenesis . MoreoveWe also observed lower AUCs of postprandial sphingosine levels after both meals in people in the HR-genotype group. Sphingosine is a breakdown product of ceramide degradation, and free sphingosine can be trapped for ceramide regeneration or for sphingosine-1-phosphate (S1P) formation, both of which have been associated with obesity, insulin resistance, and T2DM ,46,47. LThe very interesting finding of our study is that the PROX1 gene may be involved in the postprandial bile acids signaling. After the HC-meal intake in the HR-genotype men, we noticed lower AUCs of postprandial taurocholic acid and higher AUCs of postprandial deoxycholic acid glycine conjugate levels after the NC-meal. It has been found that PROX1 suppress the transcription of the CYP7A1 gene, which codes the key enzyme of bile acid synthesis and may negatively modulate the bile acids synthesis . Bile acThe carriers of the PROX1 HR-genotype also presented lower AUCs of postprandial ornithine levels after HC-meal intake. Increased ornithine levels as a product of arginine catabolism are associated with hyperglycemia and can be involved in the pathogenesis of T2DM ,54, but The PROX1 gene has been shown, thus far, to confer the susceptibility to T2DM mostly by its associations with fasting and glucose-stimulated insulin secretion  as well In conclusion, our results showed an altered postprandial metabolite profile in the PROX1 HR-genotype carriers. Our observations indicate that one of the pathways involved in the T2DM development in subjects with the PROX1 CC genotype may be postprandial alterations, but further functional studies are required to extrapolate implications from our findings for the biochemical pathways associated with PROX1 SNPs and T2DM development. It may allow identifying the pathways and factors that interact with some dietary nutrients, leading to particular metabolic responses that are associated with the development of metabolic diseases.


This study investigates preexisting and vaccine-induced vector immunity in 30 participants of a Phase-1 VSV-EBOV Ebola vaccine trial. No preexisting immunity was detected, however humoral and cell-mediated immunity against internal VSV proteins was observed in up to 36% of vaccines. In response to the Ebola virus (EBOV) crisis of 2013–2016, a recombinant vesicular stomatitis virus (VSV)–based EBOV vaccine was clinically tested (NCT02283099). A single-dose regimen of VSV-EBOV revealed a safe and immunogenic profile and demonstrated clinical efficacy. While EBOV-specific immune responses to this candidate vaccine have previously been investigated, limited human data on immunity to the VSV vector are available. Within the scope of a phase 1 study, we performed a comprehensive longitudinal analysis of adaptive immune responses to internal VSV proteins following VSV-EBOV immunization. While no preexisting immunity to the vector was observed, more than one-third of subjects developed VSV-specific cytotoxic T-lymphocyte responses and antibodies. Between 2013 and 2016, Africa experienced the largest epidemic of Ebola virus disease (EVD) in history. The unprecedented spread of EVD led to acceleration of vaccine trials. In late 2014, the Ebola virus (EBOV) vaccine candidate VSV-EBOV (V920) entered phase 1 clinical testing , 3 × 106 PFU, and 2 × 107 PFU) [We performed a comprehensive investigation of preexisting and induced vector immunity against VSV in 30 healthy subjects immunized with 3 different doses (3 × 10107 PFU) . PreexisOverall, up to 36% of vaccinees generated vector-directed immune responses. However, the magnitude of responses was highly variable between trial participants. Considering the expanding use of VSV vaccine vectors in emergency vaccine efforts, such as in the recent EVD outbreaks in the Democratic Republic of the Congo (DRC) , the invNCT02283099 was a phase 1 trial of escalating doses of VSV-EBOV in healthy adults. Details about the trial and study protocol have been reported previously [Plasma was analyzed for the presence of VSV matrix–specific (VSV-M) antibodies using the Recombivirus Human Anti-VSV Indiana M Protein ELISA Kit (Alpha Diagnostic). ELISA to EBOV-GP was performed as previously described . VSV neuVSV-specific T cells were analyzed using cryopreserved peripheral blood mononuclear cells (PBMCs) and overlapping peptide pools (OLPs) spanning the VSV nucleoprotein (VSV-N) . FollowiStatistical analysis was performed using GraphPad Prism software (version 7.02.). Intergroup differences were analyzed using Mann–Whitney–Wilcoxon test.Given the very recent introduction of VSV vectors to human study populations , 5, 10, 5 PFU cohort, in 1 of 10 subjects of the 3 × 106 PFU cohort, and in 2 of 9 subjects of the 2 × 107 PFU cohort. The low-dose group showed the highest magnitude of antibody titers.In the current study, we focused on adaptive immunity against VSV-M and VSV-N. First, vector-specific antibody responses against VSV-M were analyzed by ELISA using longitudinal collected plasma samples. None of the subjects showed preexisting VSV-M–specific antibodies (day 0) ; howeverr = 0.7; P < .0001), indicating that vaccine responders generated antibodies not exclusively to the target antigen EBOV-GP, but also to the viral vector itself.We next sought to investigate the association of vector- and EBOV-specific antibody responses. The induction of EBOV-GP antibodies in vaccinees has previously been described , 8. A coSubsequently, the function of VSV-specific antibodies was further analyzed by evaluating the capacity to inhibit VSVwt replication. We analyzed a subset of vaccinees based on their generation of VSV-M–specific antibodies . While i+ T cells stimulated with VSVwt were detectable, but were of low magnitude. While the 2 higher-dose cohorts revealed a peak of cytokine-producing CD8+ T cells at day 28, the low-dose group showed an increase of total cytokine responses at day 56.T-cell responses against the vector may eliminate VSV-infected cells and thereby modulate vaccine efficacy. We first evaluated T-cell responses against the whole VSVwt particle . Total c+ . Note that the induction of IFN-γ expression following VSV-N stimulation was validated in a subset using ELISpot  responses were investigated by CD107a staining. After stimulation with VSV-N OLPs, 75% of subjects of the high-dose cohort showed at least a 2-fold induction of CD107a expression in CD8t day 28 .Taken together, no preexisting humoral or cell-mediated VSV-specific immune responses were detected in this German study population. However, one-third of vaccinees developed nonneutralizing antibodies and T-cell responses against VSV proteins following immunization with VSV-EBOV.http://www.who.int/blueprint/en/).VSV-EBOV represents a promising vaccine candidate and has only recently entered human clinical trials and is now administered in compassionate use programs. The VSV platform is currently being considered for immunization strategies for several World Health Organization priority diseases , the effect of preexisting immunity to the vaccine vector requires careful attention. Given the lack of data on VSV-directed immune responses in humans, we here addressed if natural immunity against VSV is detectable and if VSV-EBOV elicits adaptive immunity against VSV proteins following immunization.In the study population, no preexisting immune responses were detected, possibly related to the fact that all vaccinees originated from and reside in Europe. VSV is endemic in North, Central, and South America, and generally infects cattle. Humans with a high risk of VSVwt exposure are individuals living in these regions in close contact to livestock .While preexisting immunity may be a minor problem for current vaccine trials, acquired vector immunity could emerge as a relevant factor given the increasing number of clinical vaccine trials applying the VSV platform  . In the The effect of acquired preexisting immune responses against a viral vaccine vector has been described in several studies , 7. PossBeyond VSV-N–specific T cells, vector-directed antibodies may also be implicated in reducing vaccine efficacy. In this context, one phase 1 study using homologous prime-boost administration with VSV-EBOV failed to demonstrate efficient antibody induction and showed no significantly increased neutralization titers following a homologous boost on day 28 , possiblWhile the exact impact of induced vector-directed antibodies remains unclear to date, the transiently detected binding VSV-M antibodies potentially contribute to dampening of vaccine responses upon second encounter and may need to be taken into consideration for future prime-boost implementations or further VSV-associated vaccinations. However, it is noteworthy that all tested samples in this study neutralized EBOV and none VSVwt; therefore, the neutralization capacity seems to be specific to and mediated by EBOV-GP , 8 Figu.While concern for an impact of vector-directed antibodies on vaccine efficacy remains, a recent study in nonhuman primates assessed the effect of vaccine vector–induced preexisting immunity to VSV in a preclinical study using Lassa virus (LASV) and EBOV vaccine constructs (boost: day 90) . CynomolThere may also be options to minimize vector immunity. One strategy might be the optimization of prime-boost intervals. One volunteer immunized with VSV-EBOV received a homologous boost 6 months later. Here, a strong boosting effect on antibody titers was demonstrated . Alternatively, genetic modification of the viral vector, as shown for the HPIV-3-platform, may also be explored in this context .We observed distinct magnitudes of humoral responses among participants immunized with different vaccine doses. Interestingly, low-dose subjects showed the greatest number of assay responders and the highest antibody titers. This may be related to the fact that the low number of virus particles administered to subjects immunized with the low dose potentially induces weaker innate immune responses, protracting viral replication compared to subjects, who received higher doses. Therefore, the vaccine may replicate for prolonged periods, resulting in stronger adaptive immune responses, as previously described for the live 17D yellow fever vaccine . AnotherOur study detected vector-specific immune responses in more than one-third of subjects following VSV-EBOV immunization and emphasizes the need to explore vector immunity as part of vaccine evaluations with this new vaccine vector. Homologous boosting may be less effective due to vector-directed antibody responses. Furthermore, cell-mediated responses to internal VSV proteins might inhibit efficient vector replication by limiting virus spread. As humoral and cell-mediated responses peaked at days 28 and 56, VSV-based vector immunity could potentially be minimized by readministration of the vaccine at later time points.In conclusion, our results highlight that immune responses against VSV are elicited after a single vaccine administration of VSV-EBOV in human subjects. Future implementations using the VSV platform require careful consideration of vector immunity with respect to prime-boost or other immunization strategies.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Table 1Click here for additional data file.Supplementary FiguresClick here for additional data file.


Older Veterans represent a unique population at high risk for mobility limitations. They are also more likely to be overweight or obese when compared to the general population. We sought to compare changes in mobility function across the obesity spectrum in older Gerofit participants at six different sites. Two-hundred and seventy Veterans (mean age: 74 years) completed six-months of Gerofit participation and mobility assessments at baseline, three and six months. Our assessments included gait-speed, six-minute walk distance, 30-second chair stands, and the eight foot up and go. When comparing weight groups, we found no significant interaction of weight and time, however we found clinically significant (P<0.02) improvements of 7-20% across all mobility measures. Six-months of Gerofit participation appears to be one way to improve mobility function in older Veterans across the weight spectrum.


Spike gene (S_NTD224), which is analogous to the N-terminal domain of porcine respiratory coronavirus. S_NTD224 notably affected the TGEV growth kinetics in PK-15 cells but was not essential for recombinant virus survival. In animal experiments with 13 two-day-old piglets, the TGEV recombinant viruses with/without S_NTD224 deletion induced obvious clinical signs and mortality. Together, our results directly demonstrated that S_NTD224 of TGEV mildly influenced TGEV virulence but was not the enteric tropism determinant and provide new insights for the development of a new attenuated vaccine against TGEV. Importantly, the optimized reverse genetics platform used in this study will simplify the construction of mutant infectious clones and help accelerate progress in coronavirus research.Transmissible gastroenteritis virus (TGEV) is the etiologic agent of transmissible gastroenteritis in pigs, and the N-terminal domain of TGEV spike protein is generally recognized as both the virulence determinant and enteric tropism determinant. Here, we assembled a full-length infectious cDNA clone of TGEV in a bacterial artificial chromosome. Using a novel approach, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems efficiently and rapidly rescued another recombinant virus with a 224-amino-acid deletion in the N-terminal domain of the TGEV  Coronaviridae family, which consists of the Alpha-, Beta-, Gamma-and Deltacoronavirus genera ..50].The animal experiments were performed according to the protocols approved by The Scientific Ethics Committee of Huazhong Agricultural University (Permit number: HZAUSW-2017-007). The animal care and maintenance protocols complied with the recommendations detailed in the Regulations for the Administration of Affairs Concerning Experimental Animals made by the Ministry of Science and Technology of China.ORF3 on TGEV enteric tropism and virulence, the gene encoding ORF3 at the genome position from 24,826 to 28,580 was replaced by the EGFP gene  using total RNA extracted from PK-15 cells infected with TGEV WH-1 A,B. FragGFP gene A,B. ThroGFP gene B,C. The AC clone C,D. In oTo rescue the recombinant CoV of TGEV-GFP (corresponding to pTGEV-GFP BAC), the pTGEV-GFP BAC was transfected into PK-15 cells using Lipofectamine 2000. Sporadic green fluorescence was observed 24 h post-transfection, as depicted in Spike with N-terminal 224 aa deletion in TGEV WH1 is analogous to the spike of a reported natural PRCV strain A. To verTo construct the mutant infectious clone of the S_NTD224 deletion (designated pTGEV-GFP-ΔS_NTD BAC) from the pTGEV-GFP BAC, we produced a 672-nucleotide deletion-specific mutation PCR product using two pairs of primers (rec-672SF/δS-NTDR and rec-672SR/δS-NTDF) . The mutWe rescued the recombinant virus TGEV-GFP-ΔS_NTD from PK-15 cells as previously described, and sporadic and more obvious green fluorescence was observed at 24 and 48 h post-transfection, respectively A. We theTo further evaluate the role of S_NTD224 in TGEV, we measured the growth kinetics of the wild-type virus, TGEV-GFP and TGEV-GFP-ΔS_NTD. The replication kinetics of the TGEV-GFP and wild-type viruses were comparable to each other and considerably different from those of TGEV-GFP-ΔS_NTD E. Twelve5 50% tissue culture infective dose (TCID50) with the respective chimeric virus, and the mock-infected control piglets were inoculated with Dulbecco’s modified Eagle’s medium (DMEM). All the piglets in the TGEV-GFP group exhibited severe clinical symptoms and weight loss, and those in the TGEV-GFP-ΔS_NTD group showed ameliorated but still obvious clinical symptoms (To validate whether S_NTD224 determines the enteric tropism of TGEV, 13 two-day-old piglets were randomly divided into three groups, with five piglets in each virus-infected group and three piglets in the mock-infected control group. The piglets in the two virus-infected groups were inoculated intranasally and orally at a dose of 1 × 10symptoms A,B. In asymptoms C. In addsymptoms A,C. To bsymptoms . TGEV-GFsymptoms D, but nosymptoms D.The postmortem of the moribund piglets in the TGEV-GFP and TGEV-GFP-ΔS_NTD groups revealed that the small intestines were filled with watery contents. In particular, the intestinal walls in the jejunal section of the intestines of these piglets were clearly thinner and more transparent compared with those of the mock group A. HE staThe N-terminal domain of Spike protein is recognized as the TGEV enteric tropism determinant in piglets, as demonstrated through comparisons of the sequences of natural TGEV isolates or those obtained after continuous passage in cell culture ,51,52, bBecause CoVs are the viruses with the largest RNA genomes, the construction of a CoV infectious clone is hampered by two main challenges: large full-length cDNA and toxic sequences in the bacterial clone ,53. AlthNotably, numerous mutation fragments can be inserted efficiently into the linearized BAC at the same time D, which Spike gene of TGEV has been shown to alter TGEV virulence or enteric tropism. However, recombinant TGEV with S protein N-terminal amino-acid deletion was constructed through targeted recombination and passaged several times, which might cause other locus mutations in addition to the S protein deletion [Spike gene changes the TGEV virulence or enteric tropism [The deletion . In partdeletion . No othe tropism . Here, w tropism ,59,60,61 tropism .Using reverse genetics, we confirmed that changes in S_NTD224 alone altered, albeit not completely, the virulence of TGEV. One reason explaining no detection of TGEV-GFP-ΔS_NTD in the two piglets euthanized at 7 days post-inoculation might be related to immunity of recovering piglets. The role of S_NTD224 might be analogous to that of the 197-amino-acid region in the N-terminus of the PEDV S gene when used as a viral virulence marker. Consistent with previous reports, our experiments also detected TGEV-GFP and TGEV-GFP-ΔS_NTD in the jejunal contents tissue by nested RT-PCR ,64, whicIn summary, using the reverse genetics method, we have provided direct evidence showing that the N-terminal domain of Spike protein is not the determinant of TGEV enteric tropism in piglets, although S_NTD224 exerts a mild influence on TGEV virulence. These results provide new insights into the development of a new attenuated vaccine against TGEV. Furthermore, the method developed in this study allows the efficient and rapid editing of the full-length CoV genome in vitro and can theoretically be applied to all viruses with large RNA genomes once the full-length cDNA is obtained.