Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5′-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome. The classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), formerly termed hog cholera. CSFV has been eradicated in most developed countries, but despite longstanding eradication efforts by the OIE , CSF is still an economically important disease. Recent outbreaks of CSFV in the domestic pig population and/or endemic situations in known wild boar reservoirs are reported from Africa, Asia, the Caribbean, Central and South America and Europe ,2,3. ThePestivirus within the Flaviviridae family [rns, E1 and E2) are located in the N-terminal third of the polyprotein downstream of an autoprotease (Npro). The structural proteins are processed by host cell proteases  and are not involved in genome replication [The CSFV is a member of the genus e family . The smae family . This gee family . The IRElication . The nonlication , while mlication ,10,11. Tlication . NS3 matpro (BVDV strain cpPE515), or by the insertion of other cellular processing signals, such as ubiquitin (BVDV strain CP1) [The regulation of NS2-3 cleavage hallmarks the non-cytopathogenic (ncp) biotype of pestiviruses, which is able to establish persistent infections after diaplacental infections and does not cause cytopathic effects in cultured cells. In contrast, efficient maturation of NS3 and high-level replication are observed in the cytopathogenic (cp) biotype of pestiviruses that cannot establish persistent infections in the natural host, is highly attenuated, and induces apoptosis in cell cultures ,14. The ain CP1) . Cp BVDVain CP1) ,30. In contrast to BVDV, helpervirus-independent cp genomes of CSFV have not been found in the field ,32. Thuspro gene. Since the Npro is an intrinsic unstable protein [Reporter CSFVs were used decades ago for the direct detection of viral replication without the use of immunolabeling (reviewed in ). Studyi protein , even sh protein ,47. E2 f protein . Conside2 concentration of 5%. Mutagenesis of CSFV was performed modifying an established reverse genetics system for the CSFV strain Alfort/Tuebingen [SK-6 cells  kindly puebingen . The nuc® D-BVD, CP 4350; Pfizer Animal Health, New York, NY, USA). The cellular insertion was amplified using the oligonucleotides Ubi*_forw (5′-GAGAATGGCAAAATCAGTCGCCTTC-3′) and Ubi*_rev (5′-CCCACCACGAAGTCTCAACACTAG-3′) and the One-Step OneTaq RT-PCR Kit . After sub-cloning in a T-vector , the gene was amplified by extension PCR with suitable nucleotide overhangs for an assembly reaction with oligonucleotides CSFV-353-Ubi*-GA_forw (5′-ATCTCTGCTGTACATGGCACATGGAGGTACATGGCACATGGGAGAATG-3′ and CSFV-5163-Ubi*-GA_rev (5′-CTTGCAAACAGCTGGCCCCCCACCACGAAGTCTCAAC-3′). The CSFV cDNA clone was used for the generation of a replication-competent DI9-like subgenome. Therefore, the pBR322 plasmid backbone with the 5′-UTR and the non-structural protein region of CSFV was amplified using a Phusion reaction together with the oligonucleotides CSFV-378_rev (5′-CTCCATGTGCCATGTACAGCAGAGAT-3′) and CSFV-5140_forw (5′-GGGCCAGCTGTTTGCAAGAAGG-3′). This in vitro plasmid assembly reaction  generated the plasmid pR4 (cp CSFV-DI-Ubi*).A cellular ubiquitin insertion cassette was amplified from the cp BVDV strain CP Rit encoding an N-terminally truncated (3 aa) ubiquitin moiety together with parts of the ribosomal protein S27a . CP Rit E. coli, strain ER2420 (NEB), which has an EcoK r m, McrBC, Mrr Dam and Dcm background. The plasmid pBeloBAC11 carries a gene encoding resistance to chloramphenicol (Cam). After transformation and to retain the plasmid, cells were grown with 17 µg/mL Cam. The cp CSFV strain containing a duplication of the non-structural proteins NS3 to NS4B downstream of the cellular insertion cassette was constructed in a bacterial artificial chromosome (BAC) backbone using a three-piece assembly approach. Therefore, a BAC-backbone  with a SP6 promoter was amplified by a Phusion polymerase reaction using the oligonucleotides BAC-SP6_rev (5′-GTATAGTGTCACCTAAATCGTTACAATTCACTGGCCGTCG-3′) and CSFV-12271-BAC-GA_forw (5′-GACTAAGGTAATTTCCTAACGGCCCTAAATAGCTTGGCGTAATCATGGTC-3′). Then, the CSFV cDNA was amplified with an SP6 promoter from the 5′-end to the desired insertion locus within the N-terminal part of the NS4B gene using the plasmid p447 as a template with the oligonucleotides SP6_forw (5′-ACGATTTAGGTGACACTATAG-3′) and Ubi*-CSFV-7776-GA_rev (5′-GAAGGCGACTGATTTTGCCATTCTCTTGTTGTGTTTCTGTGTCTCCTG-3′). The plasmid pR4 (cp CSFV-DI-Ubi*) was used as a template to amplify the ubiquitin insertion and the upstream non-structural proteins NS3 to NS5b together with the 3′-UTR with the help of the oligonucleotides Ubi*_forw and CSFV-12295_rev (5′-GGGCCGTTAGGAAATTACCTTAGTC-3′). The DNA fragments were assembled, producing the cDNA plasmid pR6 (cp CSFV-Ubi*). The pBeloBAC11 derived plasmid pR6 was transformed in 1512A in the NS2 protein that prevents NS2-3 cleavage by the inactivation of the NS2 autoprotease. Mutagenesis was performed using PCR and DNA assembly. The oligonucleotides CSFV-4891-A1512_forw (5′-GGACCACCAGTGGTCGCCGGTATGACCCTAGCCGATTTC-3′) and CSFV_12295_rev were used for the amplification of one fragment, while CSFV-4932-A1512_rev and CSFV-12271-BAC-GA_forw were used for the other. The resulting plasmid was named pR14 (cp CSFV-Ubi*-C1512A). The cp CSFV-Ubi* was stabilized by mutagenesis, causing the amino acid exchange Cpro, Erns and E1 were amplified from pR14 using the oligonucleotides CSFV-12271-BAC-GA_forw and CSFV-2442_rev (5′-CCGCCCTTGTGCCCCGGTCACCAGCAGCAGCC-3′). The E2 gene and downstream CSFV sequences, including the cellular insertion cassette and duplicated non-structural proteins, were amplified using the oligonucleotides CSFV-2443_forw (5′-CTAGCCTGTAAGGAAGACTACAGGTATGCGATC-3′) and CSFV-12295_rev. A mCherry coding DNA fragment was amplified using extension PCR to provide complementary sequences using the oligonucleotides CSFV-2424-mCherry-GA_forw (5′-GACCGGGGCACAAGGGCGGGTGAGCAAGGGCGAGGAGGATAAC-3′) and CSFV-2465-mCherry-GA_rev (5′-CTGTAGTCTTCCTTACAGGCTAGCTTGTACAGCTCGTCCATGCCG-3′). The PCR products were purified and assembled using a NEBuilder reaction. The resulting plasmid was termed pR31 (cp CSFV-Ubi*-C1512A-mCherryE2). Mutagenesis of the constructs was validated using a commercial Sanger sequencing service . A scheme of the respective constructs is shown in For the insertion of the mCherry gene at the 5′-end of the E2 gene of CSFV, a three-piece assembly reaction was designed. The complete BAC-backbone, the 5´-UTR and the coding region of N7 to 9.5 × 107, according to the manufacturer’s product information. This error rate comes close to the range of the DNA plasmid replication in E. coli, which has an error rate between 1 × 109 and 1 × 1011, depending on genotype and growth conditions [4 [1512A and cp CSFV-Ubi*-C1512A-mCherryE2 to analyze the occurrence of mutations during passaging.PCR products were used as the template for SP6-dependent in vitro RNA synthesis. The DNA templates were amplified from the respective plasmids using Phusion polymerase (NEB) together with the oligonucleotides SP6_forw and CSFV-12295_rev. A total of 250 ng of phenol-chloroform purified PCR products were used as the template for in vitro transcription reactions as previously described [nditions . Howevertions [4 . Therefov/v) Triton-X 100  in PBS, and stained with the monoclonal antibody (Mabs) 8.12.7 anti NS3 [wt/vol) polyacrylamide tricine gels and transferred onto nitrocellulose membranes . The membranes were blocked with 5% (wt/vol) skim milk  in phosphate-buffered saline (PBS) with 0.05% (v/v) Tween-20 . The murine Mabs A18 anti-CSFV E2, 8.12.7 anti NS3, GL5A1 anti NS5A [Indirect immunofluorescence assays were performed as previously described . Brieflyanti NS3  or A18, nti NS5A  and E5D8nti NS5A  , serum toxicity controls (serum dilution 1/5), cell controls and virus back titration controls were included in each assay. Cells were fixed with 4% paraformaldehyde in PBS for 20 min at 4 °C after 48 h, when strong fluorescence signals and cytopathogenic effects were visible in the control wells with the cp CSFV-Ubi*-C1512A-mCherryE2 back titrations. All CSFV infected cells were stained with the E2-specific Mab A18, and a FITC labeled goat anti-mouse IgG for comparison. The SVNA reactions were analyzed using a fluorescence microscope . The 50% neutralization dose (ND50/mL) was calculated for each virus-serum combination using the Spearman–Kaerber method.SVNAs were performed as described previously . Serum dRNA extractions were performed using a volume of 140 µL (cell-free culture supernatant or cell lysates). Total RNA was extracted using the QIAamp Viral RNA Mini Kit  or the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions on a QIAcube Connect device (QIAGEN). RT-qPCRs were performed on a StepOne real-time PCR system  using the Luna Universal Probe One-Step RT-qPCR Kit (NEB). CSFV specific primers and probes were used as previously described  togetherpro that autoproteolytically processed NS3 very efficiently [pro transfected cells. An indirect immunofluorescence test using Mab 8.12.7 anti NS3 showed a strong cytoplasmic fluorescence signal in the dead cells 48 h post-transfection. The bright NS3 dependent staining within the cytoplasm together with the death of the transfected cells is shown in The cp BVDV strain CP Rit has incorporated a cellular mRNA sequence coding parts of the ribosomal protein S27a fused with an N-terminally truncated ubiquitin moiety . This fuiciently . We obse2468 (aa 132 of NS4B) of CSFV. In analogy to the organization of the BVDV CP Rit, the genes from NS3 to NS4B were duplicated and embedded in the complete non-structural protein module (NS3-NS5B) downstream of the Ubi*. The cDNA clone of cp CSFV-Ubi* with a total genome length of 15.2 kb was constructed in a BAC backbone to ensure genetic stability considering the homologous duplication of more than 2.5 kb. Template cDNA was produced by long-range PCR, and synthetic RNA was transcribed via SP6-polymerase. Already 24 h after transfection of the synthetic RNA of cp CSFV-Ubi* into SK-6 cells, the first cytopathic effects were observed, and the cellular monolayer was largely destroyed within 48 h. The high replication and protein expression levels of the cp CSFV-Ubi* were demonstrated by strong fluorescence signals in immunofluorescence staining of the NS3 antigen . Furthermore, RT-PCR analyses with CSFV-RT-qPCR-99_forw and CSFV-5694_rev revealed no evidence for co-passage of subgenomic DIs, as no 5’-end truncated fragment can be observed in Recognizing this apparent instability of our construct, we searched for a way to hinder homologous recombination or the emergence of wild-type-like CSFV phenotypes following deletions of the insertion. Because even complete codon exchange in the duplicated region does not necessarily stabilize such a virus, we decided to exchange the active cysteine residue (aa 1512) within the NS2 protease domain against an alanine. This mutation inactivates the NS2 autoprotease and thus prevents NS2-3 cleavage so that Ceplicate . Accordi1512A strain was genetically labeled using the insertion of a mCherry gene at the 5′-end of the E2 gene as presented earlier [1512A-mCherryE2 was rescued from synthetic RNA by electroporation and analyzed in virus passages. The mCherry-E2 fusion protein in cp CSFV-Ubi*-C1512A-mCherryE2 is fully capable of virion morphogenesis, receptor binding, and host cell infection as demonstrated by its intact infection cycle. As in the cp CSFV-Ubi*, the first cytopathic effects were observed 24 h post-transfection, but viral spread in the infected culture was notably slower in direct comparison. A strong fluorescence of mCherry was observed 24 to 48 h after transfection, and the cellular monolayer was largely destroyed within 48 h post-transfection, as shown in To generate a new and cost-efficient diagnostic tool for indirect CSFV detection, we constructed a dual reporter virus suitable for fluorescence-verified plaque reduction assays. Therefore, the helpervirus-independent stable cp CSFV-Ubi*-C earlier . The vir1512A-mCherryE2, which show no or only very weak mCherry fluorescence signals, can be stained with CSFV specific antibodies. To verify that the observed fluorescence signals arise due to the mCherry-E2 expression, indirect immunofluorescence using Mab 8.12.7 anti-CSFV NS3 and FITC conjugated goat anti-mouse IgG was applied. By overlaying the signals, a close correlation between red and green fluorescence was documented. However, it must be mentioned that signal amplification in immunohistochemical staining results in a very high sensitivity. Therefore, some cells at the margin of the plaques of cp CSFV-Ubi*-C1512A, the cp CSFV-Ubi*-C1512A-mCherryE2 was stable on SK-6 cells for 11 passages. The RT-PCR analyses of cp CSFV-Ubi*-C1512A-mCherryE2 presented in As the parental cp, CSFV-Ubi*-C1512A, and cp CSFV-Ubi*-C1512A-mCherryE2 showed reduced progeny virus titers in direct comparison to the wild-type ncp CSFV Alfort/Tuebingen. In the experiment, ncp CSFV produced a peak titer of 1 × 106 TCID50/mL 48 h post infection, while cp CSFV-Ubi*, cp CSFV-Ubi*-C1512A and cp CSFV-Ubi*-C1512A-mCherryE2 released about 2 log10 lower titers of progeny virus in the cell culture supernatant. No effect of the C1512A mutation on the progeny virus production was observed comparing cp CSFV-Ubi* and cp CSFV-Ubi*-C1512A. The insertion of the mCherry gene additionally reduced infectious virus production by a factor of two in comparison to cp CSFV-Ubi*. We also measured intra- and extracellular viral RNA levels in ncp CSFV Alfort/Tuebingen and cp CSFV-Ubi*-C1512A 12, 24, 48 and 72 h post infection  of 0.01 to study the viral spread and to generate viral growth curves. Progeny virus titers were measured 24, 48 and 72 h after inoculation, as presented in nfection B,C. Vira1512A, cp CSFV-Ubi*-C1512A-mCherryE2 and ncp CSFV Alfort/Tuebingen was compared to investigate their cell culture spread. SK-6 cells were infected with 10 PFU/FFU of the respective virus. After 2 h of infection, the infection medium was removed and replaced with 1% carboxymethyl cellulose medium to prevent the long-distance spread of viral particles. Focus and plaque size was determined by counting the infected cells within single foci or plaques using immunofluorescence 48 h after infection. In 1512A consisted of 55 cells (95% CI +/− 2.4) and the plaques of cp CSFV-Ubi*-C1512A-mCherryE2 contained only 46 cells (95% CI +/− 2.6).The plaque or focus size of cp CSFV-Ubi*-C1512A-mCherryE2 strain, which has not been characterized so far. The elevated NS protein expression levels and the efficient NS protein processing became apparent by the immunodetection of NS5A in 1512A-mCherryE2 strain with a strong band at 110 kDa (E1-mCherryE2 heterodimer) and weaker bands at 80 kDa (unglycosylated mCherryE2) and 160 kDa (mCherryE2-mCherryE2 homodimer). The unglycosylated mCherryE2 with 80 kDa, a strong band at 110 kDa (E1-mCherryE2 heterodimer) and a weak band with 160 kDa (mCherryE2-mCherryE2 homodimer), were also seen in the immunoblot in We also analyzed the protein expression of the different CSFV strains at 24 h post-transfection. The immunoblot against the helicase domain of NS3 in 1512A-mCherryE2 strain was designed as a novel tool for the indirect detection of CSFV infections using a fluorescence-verified plaque reduction assay. For the validation of this potential application, SVNAs with cp CSFV-Ubi*-C1512A-mCherryE2 and ncp CSFV Alfort/Tuebingen were performed in parallel following the standard procedure. Defined positive and negative reference antisera were tested  were transfected in cells pre-infected with an ncp CSVF. The emerging helpervirus-independent cp CSFVs were isolated by subsequent plaque purification. This approach yielded an 18.1 kb long cp CSFV strain (CP G1), in which a complete DI sequence was integrated at the 3′-end of the ORF. However, CP G1 was genetically unstable and reverted completely during passaging into the parental replicative subgenomic DIs .The first genetically stable cp CSFV was presented in 2008 using genomic integration of a complex Jiv insertion of 1539 nucleotides derived from the cp BVDV strain CP8. The resulting cp CSFV-Jiv was tested in an animal experiment. It showed no reversion to wild-type-like genomes, was completely avirulent and induced a strong protective immune response. The cp CSFV-Jiv was proposed as a suitable vaccine candidate . Howeverpro to NS2 protein genes against Ubi*, a ubiquitin fusion protein gene derived from the cp BVDV strain CP Rit. The Ubi* gene has previously been characterized in this well-known temperature-sensitive vaccine strain [1512A, which leads to NS2 autoprotease deficiency, prevented the replication of ncp recombination products and stabilized the cp CSFV. It can be hypothesized that genetically stable cp BVDV strains with duplicated viral sequences known from the field were evolutionarily stabilized in a similar manner. In the selection of the fittest viruses, adaptation processes probably took place compensating the imbalance between mature NS3 and uncleaved NS2-3 by inhibition of the NS2-autoprotease [1512A could potentially be further stabilized and optimized by the introduction of additional mutations within NS2 and/or NS3. Since we did not detect reversion in cp CSFV-Ubi*-C1512A within 11 passages, we assume that the C1512A mutation alone is sufficient to stabilize the cp CSFV-Ubi*. The emergence of mutants carrying larger genomic deletions, which inhibit the growth of helper-independent cp CSFV as DIs, was not observed, probably because the cp CSFV-Ubi*-C1512A already replicated its genome with a very high efficacy. The emergence of such DIs was also never observed in cell culture propagation of cp BVDV field strains containing duplicated viral sequences together with insertions of cellular mRNA sequences.First, we generated a cp CSFV-DI-Ubi* by exchanging the cassette encoding Ne strain . The ence strain ,61. As eprotease . Cp CSFV1512A strain to generate a new valuable tool for veterinary CSFV diagnostics. We inserted an additional red fluorescent protein gene (mCherry) at the 5′-end of the E2 ectodomain to produce a dual reporter virus that destroys the cellular monolayer with clearly visible plaques and allows the verification of the virus-related causation of the cell damage by fluorescence microscopy. The application of the cp CSFV-Ubi*-C1512A-mCherryE2 in fluorescence verified virus neutralization assays was successfully tested in this study.Our new reverse genetics system for a cp CSFV with functionally uncoupled NS2-3 and NS3 proteins allows, for the first time, the study of the independent functions of NS3 and NS2-3 within a single cistron. Future studies will use mutational analyses of separated protease, NTPase and helicase domains as well as of the packaging module to investigate their importance for the different steps in the pestiviral lifecycle. Furthermore, the stabilized cp CSFV showed an enormous antigen expression, which might be beneficial in live attenuated CSFV vaccines. Since this concept can be applied to any CSFV strain, known safe vaccine strains already in use could be modified by inserting stabilized duplications. In this study, we applied the stable cp CSFV-Ubi*-C1512A-mCherryE2, which causes a strong fluorescence signal only in cells killed by the cp CSFV infection, immunofluorescence staining is often inconclusive in such cases because the dead cells are washed away during the staining steps, and a nonspecific background staining occurs in the remnant cells. LED technology has greatly reduced the purchase price and maintenance costs of fluorescence microscopes [1512A-mCherryE2 could reduce costs and improve the speed and reliability of SVNA against CSFV. The stabilized cp CSFV-Ubi*-C1512A still has to be tested in animal experiments to confirm and document its avirulence and immunogenicity, which could be inferred from analogies with cp CSFV-Jiv and various known cp BVDV strains. Given the high cost of such animal studies, the authors of this article cannot perform these tests without industrial collaborators or external funding. Due to the strong antigen expression, the virus may also be suitable as a live vector for the expression of foreign genes in wild boar. In summary, we generated a cytopathogenic CSFV strain rationally stabilized by a point mutation, which has many different potential applications in basic research, prophylaxis, and diagnostics.Currently, various measures are used to control or eradicate CSFV in different regions of the world. The most important measures in countries with successful CSFV eradication programs are molecular biological diagnostics, which are mainly accomplished by RT(q)-PCRs. Unfortunately, there are still many countries where CSFV is endemic and domestic pig herds must be protected from disease by vaccination. In this regard, SVNA is still a common laboratory system for identifying CSFV affected herds in less developed and economically weak countries. It is also still the “gold-standard” test to confirm questionable reactions in antibody ELISA since a definitive diagnosis can be obtained with the high specificity of the SVNA reaction. It is also possible to distinguish the humoral immune response of pigs against CSFV from reactions against other related viruses, such as BVD or BVDV. The SVNA itself is simple to perform, involves comparatively low equipment and consumable costs, and can be performed even in rural areas in any cell culture laboratory with the necessary safety equipment. In addition, the SVNA can be used to check the protection status of pigs after vaccination, which may be relevant in vaccine development and testing as well as during vaccination campaigns. However, SVNAs cause a very high labor burden when they are performed with ncp viruses, as the cell cultures have to be fixed, permeabilized and immune-assayed before evaluation. The dual reporter CSFV presented in this study can significantly reduce the effort and assay cost. It can also improve the assay performance because the cp CSFV causes a clearly visible cytopathogenic effect, which allows a faster and easier evaluation of SVNA after approximately 48 h as a plaque reduction assay under the light microscope. In case of questionable results, the reactions can be validated by the fluorescence of the infected cells under a fluorescence microscope. Such doubtful reactions occur, for example, when the sera to be tested are toxic to the cell culture system at lower dilution levels, which is frequently observed in sera from diseased animals. In contrast to cp CSFV-Ubi*-Croscopes . Due to The author B.L. is the inventor of a patent on pestiviral marker vaccines .


We previously confirmed that intestinal I/R induces intestinal flora disorders and changes in metabolites, but the role of different metabolites in intestinal I/R injury is currently unclear. Based on targeted metabolic sequencing, pravastatin (PA) was determined to be a metabolite of the gut microbiota. Further, intestinal I/R model mice were established through superior mesenteric artery obstruction. In addition, a co-culture model of small intestinal organoids and type II innate lymphoid cells (ILC2s) was subjected to hypoxia/reoxygenation (H/R) to simulate an intestinal I/R model. Moreover, correlation analysis between the PA level in preoperative feces of patients undergoing cardiopulmonary bypass and the indices of postoperative intestinal I/R injury was carried out. IL-33-deficient mice, ILC2-deleted mice, and anti-IL-13 neutralizing antibodies were also used to explore the potential mechanism through which PA attenuates intestinal I/R injury. We demonstrated that PA levels in the preoperative stool of patients undergoing cardiopulmonary bypass were negatively correlated with the indices of postoperative intestinal I/R injury. Furthermore, PA alleviated intestinal I/R injury and improved the survival of mice. We further showed that PA promotes IL-13 release from ILC2s by activating IL-33/ST2 signaling to attenuate intestinal I/R injury. In addition, IL-13 promoted the self-renewal of intestinal stem cells by activating Notch1 and Wnt signals. Overall, results indicated that the gut microbial metabolite PA can attenuate intestinal I/R injury by promoting the release of IL-13 from ILC2s  Intestinal ischemia/reperfusion (I/R) injury is a severe condition in some critical clinical settings that is commonly associated with serious infection, shock, and some surgical procedures, including cardiopulmonary bypass (CPB), strangulated ileus, and small bowel transplantation, among others . IntestiStreptomyces xanthochromogenes RIA 1098 for enhanced pravastatin production . All experimental procedures were carried out in accordance with the National Institutes of Health guidelines and were approved by the local Animal Care and Use Committee of the Nanfang Hospital of Southern Medical University.Six-to-eight-week-old specific pathogen-free male C57BL/6 mice were purchased from the animal center of Nanfang Hospital of Southern Medical University . IL-33Patients who need cardiopulmonary bypass (CPB) surgery were used as cases for collecting intestinal I/R samples as previously described , 21. TheBlood samples were collected preoperatively (T0) and at 6 h (T1) and 12 h (T2) after surgery for analyses of intestinal fatty-acid binding protein (IFABP) and citrulline, respectively. The level of IFABP in plasma is a confirmed positive marker , while cThe mouse model for intestinal I/R injury was established as in our previous study , 6. Brie2 in a 37°C incubator for 4 h  with 2 mg/kg PA (MedChemExpress) 1 h before inducing intestinal I/R. Organoids were cultured alone or with ILC2s (H/R co-culture model)  were randomly assigned to I/R and I/R + PA groups.To explore the role of IL-33/ST2 signaling in the protective effect of PA against intestinal I/R injury, wild type (WT) mice were randomly divided into the following groups  I/R + PA−/− mice and ILC2s from WT mice were randomly assigned to H/R and H/R + PA groups , an I/R + rmIL-33 group  2 h before intestinal I/R), an I/R + ILC2−/− group  on days −5 and −2 before I/R surgery), and an I/R + rmIL-33 + ILC2−/− group  single cells were harvested as described . For int-Sca-1+KLRG1+ . The cell surface was stained using APC-conjugated Rat Anti-Mouse Ly-6A/E , PE-conjugated Anti-Mouse KLRG1 Antibody , and FITC-conjugated mouse Hematopoietic Lineage Antibody Cocktail . ILC2 were identified as Lin1+KLRG1+ .+ 10 ng/ml IL-25 in 10% (vol/vol) FBS RPMI complete medium to induce ILC2 cell activation and rapid growth. For cell maintenance, we add 10 ng/ml IL-7 to 10% (vol/vol) FBS RPMI complete medium. Change half of the medium every 2 days. The ILC2 and organoids were co-cultured at a ratio of 25:1 in Matrigel.We cultured and expanded ILC2 as described . Briefly−/− mice were administered an anti-CD90.2 antibody  or control rat IgG2b on days −5 and −2 before I/R surgery as described  for further computer analysis. The chromatographic separation was performed on the Thermo Scientific Prelude SPLC system, and detection was performed using a Thermo TSQ Vantage triple quadrupole mass spectrometer. Data collection and processing were performed with TraceFinderTM software version 3.3 sp1 .Targeted metabolomics (PA measurement) was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously . BrieflyThe levels of organoid injury were assessed with a CCK-8 kit  and LDH kit . The detection of CCK-8 and LDH was carried out based on the manufacturers’ protocols.Hematoxylin-eosin (HE) was used to evaluate local pathological damage of the small intestine, all procedures were carried out as our previous . The patImmunofluorescence and immunohistochemistry were performed as previously described . Anti-zo-ΔΔCT method. The target gene primers are shown in RNA was extracted with the TRIzol reagent . Real-time PCR was performed using the ABI Q5 Real-Time PCR System , with the SYBR Green detection protocol . The expression of target genes in mice was normalized against that of the housekeeping gene 18S using the 2P values were corrected for Tukey’s test (for comparisons between multiple treatment groups). In addition, the Spearman method was used for correlation statistical analysis. A value of p < 0.05 was considered significant.Data were analyzed and performed using GraphPad Prism software (version 7.0) by investigators blinded to the group allocation. The results are expressed as the mean ± SEM. Statistical analyses were performed using two-sided Student’s t tests, one-way ANOVA as indicated in the figure legends. p < 0.05 and a variable importance in projection > 1. Untargeted metabolomic results showed that intestinal I/R caused a significant decrease in PA content in the mouse cecum  or IFABP  at T0 (p = 0.029) and T2  but was negatively correlated with the plasma content of IFABP, a positive marker of intestinal I/R injury, at T1  and T2 , and the AGI score at 2 d  after surgery (We then collected stool and plasma samples from patients before (T0) CPB surgery and collected plasma samples from patients at 6 (T1) and 12 h (T2) after CPB surgery, determined acute gastrointestinal injury (AGI) scores at 2 d after surgery, and examined the relationships between PA levels and citrulline, intestinal fatty-acid binding protein (IFABP) concentrations, or AGI scores. Results of correlation analysis did not reveal significant relationships between the PA content in stool and plasma levels of citrulline -based morphological damage and scores  or ST2 (anti-ST2) and IL-33−/− mice (IL-33−/− organoids) to block interactions between IL-33 and ILC2, as well as the subsequent activation of ILC2s . The gut microbiota metabolite capsiate enhances Gpx4 expression and inhibits ferroptosis by activating TRPV1 in intestinal I/R injury. Based on two independent studies using the same research methods, PA and capsiate were both confirmed to be metabolites of intestinal flora, significantly reduce intestinal and organoid H/R injury, and be related to the level of postoperative intestinal I/R injury in clinical patients. The difference is that capsiate directly reduces intestinal I/R injury, but the PA-mediated reduction in intestinal I/R injury requires the interaction among intestinal epithelial cells, intestinal flora/metabolites, and immune cells. Moreover, as different drugs reduce intestinal I/R injury, their protective mechanisms are also completely different.via IL-33/ST2 signaling. Furthermore, data from patients showed that the levels of PA in preoperative feces of patients undergoing CPB were negatively correlated with the degree of postoperative intestinal I/R injury. Taken together, PA might be effective for treating intestinal I/R injury.Some statins have been reported to reduce organ I/R injury. Simvastatin reduces subsequent intestinal tissue damage by suppressing oxidative stress, inflammatory damage, and apoptosis , 38. AtoIL-33 mRNA and intracellular IL-33 protein levels in both human adult cardiac myocytes and fibroblasts  immune responses by interfering with the IL-33 pathway , Key Program of National Natural Science Foundation, Beijing, China (81730058 to K-XL).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We provide a novel single restriction enzyme  digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I “F gene” class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research.IMPORTANCE The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme  digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis. Avulavirus, which belongs to the family Paramyxoviridae in the order Mononegavirales. Avian paramyxoviruses (APMVs) are categorized into 15 serotypes (APMVs 1 to 15); with APMV-1 containing all economically relevant, naturally occurring strains of NDV (1511–Newcastle disease (ND) remains one of the most complex diseases to control in commercial and backyard poultry around the world. Newcastle disease virus (NDV) is a member of the genus  of NDV 14. APMV-1of NDV 158. Class of NDV 15, 9, 10, of NDV 15, 9. The of NDV 15, 11–14. Various fast techniques, such as RT-PCR followed by restriction enzyme (RE) analysis, became available with the development of molecular tools for discriminating between avirulent and virulent NDV isolates –18, and –In the present study, for the first time, we analyzed 4,000 NDV sequences available in NCBI and identified a single RE site in the F gene cleavage site that can be used to differentiate avirulent (including lentogenic and mesogenic vaccine strains) NDV pathotypes from virulent strains. This appears to be the first report of RT-PCR-based single restriction enzyme digestion technique to differentiate avirulent, lentogenic, and mesogenic strains from virulent NDV strains for easy, economic, and specific diagnosis.BsaHI restriction site within the F gene cleavage site. Thirteen amino acid motifs (112G/E/V/K-K/R/Q-Q-E/D/G-R/Q-L-I/V-G-A120) at the F gene cleavage site  were identified among these sequences. Motif patterns 115ERL117, 115DRL117, 115GRL117, and 115EQL117 were present in 91.32%, 4.76%, 3.64%, and 0.28% of the sequences analyzed, respectively. All patterns had an ‘L’ at 117 positions in the F gene cleavage site. When analyzed, the 858 class II avirulent NDV sequences showed 13 amino acid motifs (112G/E/R-K/R/T-Q-G/K/A/R-R-L-I/L-G-A/F120) with the patterns 115GRL117, 115KRL117, 115ARL117, and 115RRL117 present in 98.94%, 0.82%, and 0.12% of the sequences, respectively. These 13 different amino acid motif patterns have 42 (see Tables S2 and S3) different nucleic acid patterns and at least one BsaHI digestion site. The nucleotide sequence GG(A/G)CG(T/C)CT(T/G/C), corresponding to the amino acid pattern 115G/R-R-L117 at the F gene cleavage site in class II-specific avirulent and lentogenic vaccine strain sequences in APMV-1, has a BsaHI digestion site (GGCGCC). The motif 115K/A-R-L117, present in seven sequences (less than 0.94% of class II avirulent sequences) from India, did not have a BsaHI digestion site at the F gene cleavage site, but a 119GA120 pattern was noticed adjacent to the F gene cleavage site. Out of all the class II-specific avirulent NDVs, 54.19% had BsaHI digestion sites both in and adjacent to the F gene cleavage site, and the remaining 45.81% sequences had BsaHI digestion sites at only the F gene cleavage site. The analysis of 2,584 sequences of class II virulent NDV strains showed 19 different amino acid motifs (112R/K/G-R/R-Q/K/R/-K/R/G-R-F-I/V/L-G/S-A120) with the patterns 115KRF117, 115RRF117, and 115GRF117 in 89.97%, 9.99%, and 0.04% of the sequences analyzed, respectively. The 19 different amino acid motif patterns had 134 different nucleic acid patterns (see Table S4 and S5) with no BsaHI digestion sites.Of the 4,000 NDV, F gene nucleotide, and amino acid sequences analyzed, 357 were class I-specific sequences of APMV-1 and did not possess a 119GA120 adjacent to the cleavage site, but in mesogenic strains, the nucleotide sequence at this region corresponded to the BsaHI digestion site (GGCGCC). In velogenic strains, the nucleotide sequence at this region was found to be GG(C/T) GC(C/T) (see Tables S6 and S7) with at least one T in the degenerate nucleotide position.Notably, the mesogenic strains had the same motif pattern at the F gene cleavage site as the pathogenic/velogenic class-II APMV-I strains. The mesogenic and velogenic strains also had the same amino acid motif The amino acid motif at the F gene cleavage site, the nucleotide pattern at F gene cleavage, and the ICPI were analyzed for 212 sequences/strains. The BsaHI digestion of these sequences showed that, in 200 sequences, the ICPI matched with the availability of RE digestion site, but 12 (12/212) sequences did not match (see Table S8).The 82 NDV isolates/strains sequences with MDT, for which the amino acid motif at the F gene cleavage site and the nucleotide pattern at the F gene cleavage site were accessible, accurately matched when subjected to BsaHI analysis (Table S9).The sequences of all NDV vaccine strains except Mukteswar and the H vaccine strain showed a BsaHI digestion site at the F gene cleavage site (Table S10).in silico analysis results, 45 field isolates from India and 8 commonly used vaccine (lentogenic and mesogenic) strains were used, where all the isolates and vaccine strains were found to be positive for 535-bp, class II-specific APMV-1 RT-PCR. The avirulent, lentogenic, and mesogenic strains, excluding Mukteshwar, provided digested fragments when the class II-specific RT-PCR products of 535 bp were exposed to BsaHI digestion, however, the virulent NDV RT-PCR product remained undigested by this enzyme . ICPI is time-consuming, labor-intensive, and inhumane, and has limited use in surveillance programs. Routine PCR-based diagnosis of NDV is nonconfirmatory and gives false-positive results, as many of the avirulent wild population strains and lentogenic vaccine strains used are re-isolated for several weeks post-administration of vaccine, which may give false-positive results , 26. ForGGTGCC nucleotide sequence at the site corresponding to the BsaHI cutting site within the F cleavage site. This indicates that these strains are velogenic strains adapted by serial egg passages to be mesogenic vaccine strains  cases were found to be mismatched between them. In any cases of mismatching between the amino acid motif at the cleavage site and pathogenicity index (ICPI), the ICPI stands the best chance of determining pathogenicity . Although the amino acid motif at the F gene cleavage site chickens . The virchickens , 31.There is a high similarity between virulent, vaccine, and avirulent NDV strains, which hinders the diagnosis of NDV. Recently, Liu et al.  demonstrPathotyping of NDV using RT-PCR combined with an RE digestive approach is faster and less expensive than ICPI, allowing for large-scale surveillance and detection of virulent, avirulent and vaccine strains of NDV.www.ncbi.nlm.nih.gov.in) and analysis of restriction enzyme sites at F gene cleavage sites was performed using NEBcutter v. 2.0 (http://tools.neb.com/NEBcutter2/) and Webcutter 2.0 (http://rna.lundberg.gu.se/cutter2/) online software.Newcastle disease virus F gene nucleotide and amino acid sequences were retrieved from GenBank NCBI  obtained from 1989 to 2013 and maintained in the Virology Laboratory Repository, Avian Disease Section, Division of Pathology, Indian Veterinary Research Institute  were used for this study. These isolates were reconstituted, and inoculums were prepared as explained by OIE, Terrestrial manual (2021). In brief, 0.2 mL of inoculums was inoculated into 9 to 11-day-old specific-pathogen-free (SPF) embryonated chicken eggs through the allantoic route and the eggs were incubated at 37°C until death, or for a maximum period of 120 h, whichever occurred first. The embryos were candled each day and dead embryos were chilled at 4°C overnight and collected allantoic fluids were tested for hemagglutination (HA) activity. After 120 h, all remaining live embryos were collected and chilled at 4°C overnight. The allantoic fluids found negative for HA activity were passaged further into at least one batch of eggs for confirmation. The harvested NDV was confirmed by hemagglutination inhibition test and RT-PCR.ATGGGCYCCAGACYCTTCTAC and C2-R CTGCCACTGCTAGTTGTGATAATCC  following the manufacturer’s instructions. The extracted RNA was used to synthesize cDNA using a random hexamer primer . Reverse transcription (RT) for the first-strand synthesis was carried out using RevertAid H minus  in a standard 20-μL reaction mixture containing 5 μL total RNA, 1 μL random hexamer primer, 4.0 μL 5× RT buffer, 2.0 μL deoxynucleoside triphosphate mix (10 mM each), 0.5 μL RNase inhibitor and 1.0 μL M-MuLV RT enzyme (200 U/μL). Reverse transcription was carried out at 25°C for 10 min; 42°C for 60 min, and 70°C for 10 min. RT-PCR was done for all three pathotypes  with class II-specific fusion (F) gene primers C2-F GATAATCC . The RT-Class II-specific F gene PCR products (535 bp) were analyzed by electrophoresis. PCR products were purified using the GeneJET PCR Purification kit  according to the manufacturer’s instructions. Purified PCR product was digested with BsaHI RE enzyme , as per the manufacturer’s instructions. The reaction included NEB buffer, 4 to 5.0 μL; BSA, 0.5 μL; PCR product, 10.0 μL (1 μg); BsaHI, 1.0 μL (10 U); and nucleus free water, 33.5 μL. The mixture was incubated at 37°C for 2 to 3 h and the reaction was stopped by storing it at −20°C until further use.https://web.expasy.org/translate/) and the corresponding amino acid motif was identified.Amplified F gene PCR products were purified using a GeneJET PCR purification kit  and sequenced through a commercial sequencing service . The F gene nucleotide sequences were translated by DNASTAR (SeqBuilder Pro-17.2)/Expasy translation tool (We used nucleotide sequences related to the NDV F gene from publicly available NCBI databases in this study. Accession numbers and sequence names are provided in


A179L, a non-structural protein of African swine fever virus (ASFV), is capable of suppressing apoptosis by binding the BH3 domain of the pro-apoptotic Bcl-2 family proteins via a conserved ligand binding groove. Our present study aims to determine if A179L affects necroptosis, the second form of programmed cell death induced by DNA and RNA viruses. Here we report that A179L enhanced TNF-α or TSZ -induced receptor-interacting protein kinase (RIPK1), RIPK3, and mixed lineage kinase domain like peudokinase (MLKL) phosphorylation in L929 cells, a murine fibrosarcoma cell line. Sytox green staining revealed that A179L significantly increased the number of necroptotic cells in TSZ-treated L929 cells. Using human herpes simplex virus 1 (HSV-1) to model DNA virus-induced cell death, we found that A179L blocked the HSV-1-induced cleavage of poly (ADP-ribose) polymerase (PARP), caspase 8, and caspase 3 and decreased the number of apoptotic cells in HSV-1-infected IPEC-DQ cells, a porcine intestinal epithelial cell line. In contrast, A179L transfection of IPEC-DQ cells enhanced HSV-1-induced RIPK1, RIPK3, and MLKL phosphorylation and increased the number of necroptotic cells. Consistently, A179L also suppressed apoptosis but enhanced the necroptosis induced by two RNA viruses, Sendai virus (SeV) and influenza virus (IAV). Our study uncovers a previously unrecognized role of A179L in regulating cell death and suggests that A179L re-directs its anti-apoptotic activity to necroptosis. ASF oute family . Its gene family . These Oe family ,5. Severe family ,10,11,12e family . Apoptosis is considered the first-line defense of host cells to restrict virus replication . Both RNNecroptosis is another important form of programmed cell death. When apoptosis is inhibited, necroptosis becomes the second-line defense against intracellular pathogens . In addiA179L gene and suggests that host cells may use an alternative form of programmed cell death to attenuate virus replication. It is well established that inhibition of caspase 8 activity switches TNF-α-induced apoptosis to necroptosis. Though A179L inhibits apoptosis in multiple settings, whether A179L can inhibit virus-induced caspase activation to enhance necroptosis remains unexplored. Here we report that A179L suppresses apoptosis induced by a DNA virus (HSV-1) and two RNA viruses (IAV and Sendai virus). In contrast, A179L enhanced necroptosis induced by TSZ and by these three types of viruses. Our study reveals a novel function of the ® Green Nucleic Acid Stain) (#S7020) was purchased from Invitrogen Inc. . FITC Annexin V Apoptosis Detection Kit (Cat# 556547) was purchased from BD Pharmingen . Antibodies against human phospho-RIPK1S166 (#65746), murine phospho-RIPK1S166 (#31122), RIPK1 (#3493), human phospho-RIPK3S227 (#93654), murine RIPK3 (#95702), human RIPK3 (#13526), murine MLKL (#37705), human MLKL (#14993), caspase-3 (#14220), cleaved caspase-3 (#9664), caspase-8 (#4790), cleaved human caspase-8 (#9496S), cleaved mouse caspase-8 (#8592), PARP (#9532S), and anti-FLAG antibody (#8146) were purchased from Cell Signaling Technology, Inc. . Antibodies against β-actin (#sc-47778) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology Inc. . Antibodies against murine phospho-MLKL (#ab196436), human phospho-MLKLS358 (#ab187091), and murine phospho-RIPK3T231/S232 (#ab222320) were purchased from Abcam Inc. . The anti-GFP (Code#HT801 Lot#O21209) mouse monoclonal antibody was purchased from TransGen Biotech . Recombinant murine TNF-α (#410-MT-010) was purchased from R and D Systems, Inc. .SMAC (AT-406) (#HY-15454) and z-VAD(OMe)-FMK (#HY-16658) were purchased from MedChemExpress LLC . Sytox green (SYTOXNhe I) and 5′-TTTGTAGTCGGATCCTATCAAATTGCAGTTTCTTAATAACTG-3 (Bam HI). The primers for amplifying DP71L were 5′-CCCAAGCTGGCTAGCGCAATGGGGAGGCGGCGCAAAAAAC-3′ (Nhe I) and 5′-TTTGTAGTCGGATCCCTGCTGCTCCAGTAGCTT -3 (Bam HI). The primers used for amplifying A238L were 5′-CCCAAGCTGGCTAGCGCAATGTTTCCAGAAAGGGAGATAG-3′ and 5′-TTTGTAGTCGGATCCCACACAGGAACTTTGGAATTCTG-3′. The PCR fragments were purified and cloned into the Nhe I- and Bam HI-cleaved pcDNA3.1-FLAG vector by homologous recombination. The expression vectors encoding these three ASFV genes were validated by sequencing. The nonstructural genes of ASFV were amplified by using the homogenized sample of ASFV-infected spleen tissue as the template. The primers used in the PCR reaction for amplifying A179L were 5′-CCCAAGCTGGCTAGCGCAATGGAGGGAGAA-GAGTTAATATATC-3′  in Vero and MDCK cells, respectively. The 50% tissue culture infection dose (TCID50/100 μL) values were determined by using the standard Reed and Muench method.L929 (a murine fibrosarcoma cell line), NL20 , and Vero (CCL-81) cells were purchased from the American Tissue Culture Collection . The L929 cells were grown in DMEM containing 10% fetal bovine serum (FBS). The NL20 cells were grown in F12 containing 4% fetal bovine serum (FBS). The Vero cells were grown in complete αMEM medium containing 10% fetal bovine serum, streptomycin, and penicillin (100 U/mL), sodium pyruvate (1 mM), and L-glutamine (2 mM). The IPEC-DQ cells, a subclone of the porcine intestinal epithelial cell line IPEC-J2, were kindly provided by Dongwan Yoo, College of Veterinary Medicine, the University of Illinois at Urbana-Champaign, Urbana, IL, USA. The HSV-1 (KOS strain) and A/PR8/34 (H1N1) virus were kindly provided by Liqian Zhu . The GFP-tagged HSV-1 (GFP is fused with the VP26 protein of the KOS strain) was kindly provided by Zengfan Jiang . The Sendai virus was kindly provided by Chan Ding . The GPF-tagged Sendai virus was kindly provided by Feng Ma . The HSV-1 was propagated and expanded in the Vero cells. The H1N1 and Sendai viruses were prepared by inoculating 10-day-old specific-pathogen-free embryonic chicken eggs. The Sendai virus and IAV titers were determined by a 10-fold serial dilution  and normalized by the arbitrary units of their corresponding total proteins or β-actin, as indicated. Quantified results were presented as the mean ± standard deviation (SD) from three experiments in the bar graphs. The original Western blot images can be found in Cells grown in 6-well plates were harvested and lysed in NP-40 lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 1 mM phenylmethylsulphonyl fluoride). After incubation on ice for 30 min, the cell lysates were prepared by spinning down at 4 °C, 15,000 rpm for 15 min. The cell lysates were analyzed by Western blot with antibodies against the proteins of interest, followed by horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG and SuperSignal Western Pico enhanced chemiluminescence substrate . The density of the bands was analyzed using NIH Image-J software  (t test. The data in bar graphs are the mean ± SD of three independent experiments.The L929 cells seeded in 6-well plates were infected with HSV-1 or Sendai virus (1 MOI each). After incubation for 24 h, single-cell suspensions were prepared and stained for necroptosis by Sytox green or apoptosis with propidium iodide (PI) and Annexin V by using an FITC Annexin V Apoptosis Detection kit following the manufacturer’s instructions. The NL20 cells infected with IAV were similarly analyzed for apoptosis and necroptosis. Single-cell suspensions were run in a Beckman Coulter flow cytometer (Model CyAn ADP). The fluorescence intensity was analyzed using FlowJo software. The Sytox green- or Annexin-positive cells were gated. The percentage of Sytox green- or Annexin V-positive cells from three independent experiments were calculated and statistically analyzed using the unpaired Student’s t test. A p value of <0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism .The differences among the Western blot band densities and the percent of apoptotic or necroptotic cells were statistically analyzed using an unpaired Student DP71L, A238L, and A179L, and then stimulated with TSZ for 2 h. As shown in We first investigated the effect of A179L, DP71L, and A238L, three nonstructural proteins of ASFV, on TSZ-induced necroptosis in L929 cells. DP71L recruits protein phosphatase 1 (PP1) to dephosphorylate elF2a and inhibit the activation of proapoptotic CHOP ,35. A238We next conducted a flow cytometric analysis of Sytox green-stained cells to verify the stimulatory effect of A179L on TSZ-induced necroptosis. As shown in We further investigated if A179L was able to sensitize L929 cells to TNF-α-induced necroptosis. L929 cells transfected with pcDNA3.1 vector or A179L were treated with TNF-α and/or Smac. As shown in It has been well established that many DNA viruses can induce both apoptosis and necroptosis ,39. The HSV-1-induced necroptosis in murine cells is independent of ZBP1 . Insteadparamyxoviridae, readily induces both apoptosis and necroptosis [Having shown that A179L suppressed apoptosis but facilitated necroptosis induced by HSV-1, we next determined if A179L had similar effects on RNA virus-induced cell death. Sendai virus, a single-stranded RNA virus belonging to the family of roptosis . We firsWe next determined if A179L also enhanced Sendai virus-induced necroptosis. As shown in Both HSV-1 and Sendai virus strongly induce apoptosis and necroptosis . We finaRecent studies have shown that ZBP1 plays an important role in mediating influenza virus-induced necroptosis ,40,41. WtBid gene [Programmed cell death is considered the first-line defense of host cells to halt the growth of invading microbes . The ASFBid gene , in K562Bid gene , and in Bid gene . Our preIt is well established that inhibition of apoptosis facilitates virus replication in the early stage of virus infection ,28. HoweA179L is expressed as an 18-kDa protein at both the early and late post-infection times in macrophages . A179L, A179L is well conserved among different ASFV isolates and shares 94-99% amino acid homology across 179 amino acids encoded by the entire protein ,24. The Several questions remain unanswered. First, whether A179L-deleted ASFV affects cell death in porcine macrophages remains to be investigated. 3D4 cells, a porcine macrophage cell line, do not express RIPK3 and cannot be used to investigate the effect of A179L on cell death (data not shown). Primary porcine macrophages could be used to investigate the role of A179L in cell death. Our ongoing investigation showed that ASFV infection increased MLKL phosphorylation in porcine alveolar macrophages. This preliminary evidence suggests that ASFV is capable of inducing necroptosis in relevant cell types. Second, the ability of A179L to enhance TNF-α and virus-induced necroptosis has not been verified in a virus infection setting. A179L by itself has been shown to block autophagosome formation ,31. Howe


Marek’s disease virus (MDV) is a member of alphaherpesviruses associated with Marek’s disease, a highly contagious neoplastic disease in chickens. The availability of the complete sequence of the viral genome allowed for the identification of major genes associated with pathogenicity using different techniques, such as bacterial artificial chromosome (BAC) mutagenesis and the recent powerful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based editing system. Thus far, most studies on MDV genome editing using the CRISPR/Cas9 system have focused on gene deletion. However, analysis of the expression and interactions of the viral proteins during virus replication in infected cells and tumor cells is also important for studying its role in MDV pathogenesis. The unavailability of antibodies against most of the MDV proteins has hindered the progress in such studies. This prompted us to develop pipelines to tag MDV genes as an alternative method for this purpose. Here we describe the application of CRISPR/Cas9 gene-editing approaches to tag the phosphoprotein 38 (pp38) gene of the MDV vaccine strain CVI988 with both V5 and green fluorescent protein (GFP). This rapid and efficient viral-gene-tagging technique can overcome the shortage of specific antibodies and speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis. Mardivirus. MDV is highly contagious, and chicks get infected via the respiratory route through inhalation of poultry dust from contaminated poultry houses. Once infected, MDV undergoes a cytolytic infection involving different cell populations, including B and T lymphocytes, when high levels of expression of most viral genes are observed [Marek’s disease virus (MDV), the causative agent of MD, is an acutely transforming, cell-associated avian herpesvirus belonging to the genus observed ,2,3. Theobserved ,5. Extenobserved ,7,8,9, Mobserved , the virobserved , and virobserved . While tMeq [Previous studies demonstrated the important role of MDV genes involved in MD pathogenesis, including the genes located in the long repeat regions of the genome, such as Meq ,13,14, pMeq ,16, viraMeq ,17, viraMeq ,19,20,21Meq , as wellMeq  and VP22Meq . All of Meq , pseudorMeq ,27,28,29Meq , Kaposi’Meq , human cMeq ,33, EpstMeq , guinea Meq , duck enMeq , herpesvMeq ,38, and Meq ,40,41.CRISPR/Cas9-based gene editing on the MDV genome has given opportunities to speed up the studies of gene function and identification of pathogenic determinants. The first study to demonstrate effective use of the CRISPR/Cas9 system on the MDV genome involved the generation of a Meq and pp38 deletion mutant of the MDV serotype 1 vaccine strain CVI988/Rispens in MDV-infected CEF using the double-gRNA transfection/virus infection strategy . Using tAlthough CRISPR/Cas9 editing has been increasingly used in MDV gene function and host–virus interaction studies, thus far, only gene disruption/deletion has been applied in MDV gene editing using one or more gRNAs. The physical detection of viral gene expression and details of cellular localization of the viral proteins are valuable for host–virus interaction studies during virus replication and the progression of the disease development. However, the unavailability of antibodies against most of the viral genes has significantly hampered the progress of such studies. Targeted gene editing using CRISPR/Cas9 facilitates the introduction of donor DNA at specific loci in the genome, allowing for tagging proteins of interest with commonly used protein tags ,46. MDV-Primary chick embryo fibroblasts (CEF) were prepared from 10-day-old specific-pathogen-free (SPF) embryos and maintained in M199 medium  supplemented with 5% fetal bovine serum , 100 units/mL of penicillin and streptomycin (Thermo Fisher Scientific), and 10% tryptose phosphate broth (Sigma). Commercial CVI988 vaccine stock Cryomarex Rispens was obtained from Boehringer Ingelheim .http://crispor.tefor.net/, accessed on 24 October 2019) and cloned into the CRISPR/Cas9 vector pX330A-1×2 by introducing the synthesized oligo-DNA primers pp38-gRNA-F and pp38-gRNA-R corresponding to the target sequence into BbsI restriction sites. A single-stranded donor DNA of TTGTCGTTGGGGGAGTGGATTCTGGGGAGGTGGAATCTGGAGAAACAAAATCTGAATCAAATGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGTAAATTTAATACAGTGTAGCCGTACCCGACGTTGAAGGCGGAGATTAAGCGAATTCTCACCTTTACGAATATTGGTGCAGACAAAGACCAAAAAAT harboring Protospacer adjacent motif (PAM)-blocking mutations and V5 tag coding sequence flanked by a 62 bp left homology arm (LHA) and 96 bp right homology arm (RHA) for V5 tagging was synthesized using integrated DNA technologies . Double-stranded donor DNA harboring PAM-blocking mutations and a GFP coding sequence (amplified with primers pp38-GFP-B-F and pp38-GFP-B-R), flanked with 517 bp LHA (amplified with primers pp38-GFP-A-F and pp38-GFP-A-R) and 515 bp RHA (amplified with primers pp38-GFP-C-F and pp38-GFP-C-R) for GFP tagging, was generated by overlapping PCR with primers pp38-GFP-A-F and pp38-GFP-C-R for joining the LHA, GFP, and RHA sequences together. The oligonucleotides used are listed in The gRNA-targeting C-terminal of pp38 of CVI988 genome was designed using CRISPR guide RNA-designing software  and mixed with 2.5 μg of gRNA plasmid DNA, 2.5 μg of donor DNA, and 5 μg of CVI988 genomic DNA, and then electroporated with an optimized condition at voltage 250 V and a pulse width 1.5 ms of poring pulse. At 96 h post electroporation, single plaques from the V5-tagging were moved to a 24-well plate pre-seeded with CEFs and analyzed after 4 days of incubation. For GFP tagging, single GFP plaques were moved at 96 h post electroporation to a 24-well plate pre-seeded with CEFs followed by single live GFP cell sorting and analysis using PCR of the formed plaques following incubation. The harvested cells for PCR analysis were lysed in 1× Proteinase-K-based DNA extraction buffer  at 65 °C for 30 min. A total of 2 µL of the extracted DNA was used as a template for the PCR with primers pp38-tag-F and pp38-tag-R  outside TM 568 goat anti-mouse IgG2a antibody for pp38 expression and primary mouse anti-V5 antibody (BIO-RAD) and secondary Alexa FluorTM 488 goat anti-mouse IgG antibody for V5/pp38 expression. Images were taken using IncuCyte S3 scanning microscope with 10× objective. For a stability check, images were taken from 36 separate regions per well covering approximately 70% of the well in 24-well plates. Only one out of 36 regions for each virus is shown in the results.The expression of pp38 tagged with V5 in the mutant-virus-infected cell was evaluated using IFA. The CEF cells grown in 24-well plates were infected with the mutant and parental CVI988 viruses for 48–72 h. After fixing with 4% paraformaldehyde and permeabilizing with 0.1% Triton X-100, the cells were stained with primary mAb BD1 and secondary Alexa FluorInfected CEF cells on the coverslips in 24 well plates were fixed with 4% chilled paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were then labeled with anti-heat shock protein 60 (HSP60) polyclonal antibody followed by goat anti-rabbit IgG labeled with Alexa Fluor 568 for HSP60 expression, anti-pp38 mAb BD1 followed by goat anti-mouse IgG labeled with Alexa Fluor 633 for pp38 expression, and anti-V5 antibody followed by rabbit anti-mouse IgG labeled with Alexa Fluor 488 for V5 expression. Coverslips were mounted in Vectashield onto microscope slides after cell nuclei staining with 4′,6-diamidino-2-phenylindole (DAPI). Images were taken using a Leica Stellaris 5 confocal laser scanning microscope .g for 5 min at room temperature. The cell pellets were resuspended in cold PBS/5% FBS and single live GFP cells were sorted into a flat-bottom 96-well plate with growth medium by fluorescence-activated cell sorting (FACS) using FACSAria II . For single-cell cloning, the cells were washed twice with PBS containing 1% FBS and centrifuged at 450× 6 infected cells were collected and boiled with TruPAGETM LDS sample buffer (Sigma) for 10 min. The samples were separated on a 4–12% TruPAGETM Precast Gel, and the resolved proteins were transferred onto PVDF membranes. Immunoblots were blocked with 5% skimmed milk and then incubated with anti-pp38 antibody or anti-V5 antibody. After probing with primary antibodies, the blots were incubated with secondary antibody IRDye®680RD goat anti-mouse IgG  and visualized using Odyssey Clx (LI-COR).The expression of pp38 in CVI988-pp38-V5 virus was determined via Western blotting analysis using anti-pp38 mAb BD1 or anti-V5 antibody as the primary antibody. Briefly, 1 × 105 CEFs were seeded in a 24-well plate and infected with the CVI988-pp38-GFP virus (MOI 0.01). Images were captured every 4 h for 112 h from 36 separate regions per well using a 10× objective lens. Images of one region from all time points were exported to generate a movie.The growth of CVI988-pp38-GFP was monitored using IncuCyte S3 live-cell imaging . Briefly, 1.6 × 10The recombinant viruses CVI988-pp38-V5 and CVI988-pp38-GFP were passaged in CEF cells 15 times. The stability of each inserted tag was monitored using PCR and IFA. PCR was performed with primer pairs located at the flanking region of the insertion site (pp38-F and pp38-R) using DNA extracted from every 5th passage. IFA was performed to check the pp38 and V5 expressions every 5th passage following the method mentioned in Previously, we successfully inserted the foreign sequences into both HVT and MDV genomes using the non-homologous end-joining (NHEJ) repair pathway. We now explored a strategy to tag the protein of interest with either the small peptide-coding sequence V5 or the fluoresent protein-coding sequence of GFP using a homology-directed repair (HDR) pathway with pp38 as an example for gene function studies. The gRNAs targeting the pp38 C-terminal were designed using the CRISPR guide RNA designing software. The gRNA closest to the stop codon of pp38 and with high scoring was chosen and cloned into the CRISPR/Cas9 vector pX330A. For V5 tagging, a synthesized single-stranded DNA donor harboring PAM-blocking mutations was used. For the GFP tagging, double-stranded DNA donor with a PAM-blocking mutation and GFP coding sequence flanked with 517 bp LHR and 515 bp RHA was generated using overlapping PCR. Previously, we developed the pipeline for efficient MDV/HVT genome editing, including deletion, insertion, and disruption with the NHEJ pathway. The pipeline involves transfection of the components required for editing of the target sequences, such as gRNA, Cas9, and the donor template, followed by infection of the viruses to be edited and subsequent plaque purification and characterization of the edited viruses. Considering the lower efficiency of the HDR pathway compared with the high efficiency of the NHEJ pathway, we explored the new strategy via the cotransfection of components required for editing and genomic DNA of the virus-infected cells to avoid the high background of the unedited viruses caused by virus infection. The detailed procedure for the generation and characterization of the pp38 fusion protein of the MDV vaccine strain CVI988 with either a V5 or GFP tag is described in 4 cells were lysed for DNA extraction and subjected to direct PCR using primers pp38-tag-F and pp38-tag-R located at the flanking region of the insertion site. As shown in Eleven isolated plaques were analyzed for insertion of the V5 tag. To confirm the V5 sequence was inserted at the C-terminus of pp38 in frame with pp38, 5 × 10We analyzed 12 GFP-positive plaques obtained after sorting. To examine the purity of these plaques, DNA was extracted and PCR was performed using the primers pp38-tag-F and pp38-tag-R. Again, a 240 bp PCR product was generated from the parental CVI988 virus-infected cells, as expected a. SimilaIn addition to imaging the GFP-tagged infected cells directly using the fluorescence microscope, the introduction of the GFP tag allowed for imaging of the virus replication dynamics in living cells. To demonstrate this directly, we performed live-cell imaging of GFP-tagged virus infection in CEF using the IncuCyte S3 Live-Cell Imaging system . The vidPreviously, we showed that three foreign gene expression cassettes can be stably integrated into the HVT genome during repeated passages . To detePreviously, we observed the cytoplasmic distribution of the pp38-mCherry fusion protein in CVI988-infected CEF. Further studies using confocal microscopy suggested pp38 expression close to the mitochondria. To obtain a detailed understanding of pp38 subcellular localization and the potential interaction with the mitochondrial structures, we stained the CVI988-infected CEF with the anti-HSP60 antibody, an available chicken mitochondria marker antibody, pp38-specific mAb BD1 (purple), and V5 antibodies (green) . The parThe MDV genome is approximately 180 kb long and encodes more than 100 genes that are involved in various processes of the viral lifecycle. The role of a majority of MDV-encoded proteins and host–virus interactions contributing to the MDV-induced oncogenesis are still to be explored. Following the successful application of the CRISPR/Cas9 system on HVT and MDV genome editing for gene function and virus–host interaction studies ,42,50,51Two general repair pathways, namely, NHEJ and HDR ,54, are Previously, a transfection/infection methodology that involves transfection of Cas9/gRNA expression plasmids into CEF followed by infection of the parental virus, plaque purification, and characterization of the mutant viruses was used for gene deletion of replicating MDV/HVT in CEF cells with NHEJ repair ,42,51. CTo test whether the HDR sequence modification was stably maintained in the MDV genome, both tagged viruses were passaged 15 times in CEF and tested using both IFA and PCR to observe the appearance of the wild-type viruses. The results showed that both V5 and GFP were stably integrated into the CVI988 genome, as there was no untagged virus after 15 passages. Although this is not surprising considering the relatively small size of the insert, as we showed previously that the HVT recombinant can accommodate three foreign gene expression cassettes , this isThe QT35 is a quail fibroblast cell line latently infected with MDV . The potOverall, we present an efficient, precise, and broadly applicable strategy for MDV gene tagging for gene function studies using the CRISPR/Cas9-based homology-directed repair system. By knocking in an affinity/fluorescence tag at the gene of interest in the viral genome, the tagged gene can be studied by immunostaining (affinity tag) and live-cell imaging (fluorescence tag) to investigate the expression, subcellular distribution, host–virus interactions, and dynamic distribution of the tagged protein during virus replication. The current approach can be rapidly developed and expanded to any protein of interest in the MDV genome of both a lytic replicating virus and in MDV-1-transformed cell lines to study MDV gene function, pathogenesis, host–virus interaction, latency, reactivation, and molecular determinants of MDV oncogenicity.


Background: The MSI/MSS status does not fully explain cancer immunotherapy response in colorectal cancer. Thus, we developed a colorectal cancer-specific method that predicts cancer immunotherapy response.Methods: We used gene expression data of 454 samples  and developed a TMEPRE method that models signatures of CD8+ T-cell infiltration and CD8+ T-cell exhaustion states in the tumor microenvironment of colorectal cancer. TMEPRE model was validated on three RNAseq datasets of melanoma patients who received pembrolizumab or nivolumab and one RNAseq dataset of purified CD8+ T cells in different exhaustion states.Results: TMEPRE showed predictive power in three datasets of anti-PD1-treated patients . CD8+ T-cell exhaustion component of TMEPRE model correlates with anti-PD1 responding progenitor exhausted CD8+ T cells in both tumor and viral infection . The global pattern of TMEPRE on 454 colorectal cancer samples indicated that 10.6% of MSS patients and 67.2% of MSI patients show biological characteristics that can potentially benefit from anti-PD1 treatment. Within MSI nonresponders, approximately 50% showed insufficient tumor-infiltrating CD8+ T cells and 50% showed terminal exhaustion of CD8+ T cells. These terminally exhausted CD8+ T cells coexisted with signatures of myeloid-derived suppressor cells in colorectal cancer.Conclusion: TMEPRE is a colorectal cancer-specific method. It captures characteristics of CD8+ T-cell infiltration and CD8+ T-cell exhaustion state and predicts cancer immunotherapy response. A subset of MSS patients could potentially benefit from anti-PD1 treatment. Anti-PD1 resistance MSI patients with insufficient infiltration of CD8+ T cells or terminal exhaustion of CD8+ T cells need different treatment strategies. However, it is already evident from the studies of anti-PD1 response in lung cancer and melanoma that the number of tumor-infiltrating CD8+ T cells is not the only requirement of response to anti-PD1 treatment; the characteristics of exhaustion state of tumor-infiltrating CD8+ T cells is also required  colorectal cancer patients. However, still, approximately 60% of MSI colorectal cancer patients do not respond to single immune checkpoint inhibitor treatment such as anti-PD1, and approximately 40% of MSI colorectal patients do not respond to combinations of immune checkpoint inhibitor treatment . The mecrequired . Therefo+ T-cell infiltration and CD8+ T-cell dysfunction ; MSS tumors with a CD8A level lower than the cutoff are classified as tumors without high CD8+ T-cell infiltration (n = 211). 200 rounds of 10-fold cross-validation between these two groups were performed. In each cross-validation round, a t-test for each gene was performed and p-values of genes were ranked. CD8A gene itself was excluded from the cross-validation procedure. Genes with p-values in the top 60 ranked genes in at least 80% of 200 rounds of cross-validations were selected as the signature of . The selected genes were used to construct the nearest centroid method. The inputs are expression values of these selected genes and the output is a 1) The score function of the TMEPRE model is comprised of two components: + T cells do not display a terminal exhaustion pattern and can still respond to checkpoint inhibitors. To define the terminal exhaustion pattern of tumor-infiltrating CD8+ T cells, we use the co-expression pattern of multiple inhibitory receptors of PD1 and TIM3 because TIM3 is an early acquired co-expressed inhibitor receptor among all co-expressed inhibitory receptors and using an early co-inhibitory receptor, TIM3, could sensitively capture more tumor samples with terminal exhausted CD8+ T cells (+ T-cell infiltration defined in the previous step (n = 78), the median of PD1 expression level is used as the cutoff of PD1 and the median of TIM3 expression level is used as the cutoff of TIM3. MSI tumors with high CD8+ T-cell infiltration and both PD1 and TIM3 higher than cutoffs are defined as a tumor microenvironment of co-expression of multiple early inhibitory receptors. CD8+ T cells in this type of tumor microenvironment become terminally exhausted and resist anti-PD1 treatment (n = 21). MSI tumors with high CD8+ T-cell infiltration but both PD1 and TIM3 lower than cutoffs are defined as a tumor microenvironment in which CD8+ T cells can still respond to anti-PD1 treatment (n = 21). 200 rounds of 10-fold cross-validation between these two groups were performed. In each cross-validation round, a t-test for each gene was performed and p-values of genes were ranked. Genes with p-values in the top 60 ranked genes in at least 80% of 200 rounds of cross-validations were selected as the signature of . The selected genes were used to construct the nearest centroid method. The inputs are expression values of these selected genes and outputs are a 2)  T cells . Within The relative range coverage n = 16, GSE78220)   . The secSE91061) . The plasurvival . The hazsurvcomp .+ T cells, a dataset including normalized RNAseq data from progenitor exhausted and terminally exhausted CD8+ T cells isolated from tumors and chronic viral infection  was used   . Taken t,r = 0.82, r. msi = 0.81) and TIDE cytotoxic T lymphocytes   . The rel,+ T cells can respond to anti-PD1 treatment. To test whether + T cells, we read out the scores of + T cells isolated from tumors and chronic viral infection: terminally exhausted tumor-infiltrating CD8+ T cells that can no longer respond to anti-PD-1 therapy and progenitor exhausted tumor-infiltrating CD8+ T cells that can still respond to anti-PD-1 therapy (GSE122713)  in + T cells as CD8+ T cell is among the top two immune cell types expressing them in the BloodSpot HemaExporer human hematopoiesis database . These nine genes were used to read out + T cells dataset. In both tumors and chronic viral infection, the scores of + T cells (+ T cells that can still respond to anti-PD1.The second component of the TMEPRE modelE122713) . Only gedatabase . Two gen+ T-cell infiltration but no pattern of CD8+ T-cell terminal exhaustion are considered as potential responders to anti-PD1 therapy . A splitted heatmap was plotted . Tumors  therapy .+ T cells and 2.8% (n = 8) showed sufficient tumor-infiltrating CD8+ T cells; however, those CD8+ T cells display patterns of terminally exhausted CD8+ T cells. As expected, the anti-PD1 resistance mechanism of the majority of MSS tumors is an insufficient amount of tumor-infiltrating CD8+ T cells.Within 284 MSS tumor samples, 10.6% (n = 30) are classified as responders and 89.4% (n = 254) as nonresponders. This predicted percentage of responders, 10.6% by TMEPRE, is consistent with the reported disease control rate, 11%, in pembrolizumab-treated metastatic MSS colorectal cancers . Among M+ T cells and 16.8% (n = 22) showed sufficient tumor-infiltrating CD8+ T cells; however, those CD8+ T cells display patterns of terminally exhausted CD8+ T cells. Therefore, approximately 50% of MSI nonresponders are caused by terminal exhaustion of CD8+ T cells in the tumor microenvironment, and the rest 50% of MSI nonresponders are caused by an insufficient amount of tumor-infiltrating CD8+ T cells. Treatments of MSI nonresponders with insufficient infiltration of CD8+ T cells and MSI nonresponders with terminal exhaustion of CD8+ T cells need to be designed separately.Within 131 MSI tumor samples, 67.2% (n = 88) are classified as responders and 32.8% (n = 43) as nonresponders. This predicted percentage of responders, 67.2% by TMEPRE, is between the range of the reported immune-related objective response rate, 40%, and the reported disease control rate, 78%, in pembrolizumab-treated metastatic MSI colorectal cancers . Among tThe TMEPRE model identified 10.6% of MSS and 67.2% of MSI colorectal cancer patients whose tumors show biological characteristics that can potentially benefit from anti-PD1 treatment. These predicted percentages of responders in MSS tumors and MSI tumors are consistent with the reported benefits of immune-related disease control rate at 20 weeks of a cohort of colorectal cancer patients treated with pembrolizumab .+ T-cell infiltration. The quantity of tumor-infiltrating CD8+ T cells needs to be supplemented by characteristics of tumor-infiltrating CD8+ T cells to predict anti-PD1 response. In this report, we developed the computational method TMEPRE for colorectal cancer patients, which measures two factors of the tumor microenvironment that contribute to anti-PD1 resistance: CD8+ T-cell infiltration  and seven genes  have high expression levels in purified CD8+ T cells. The expression values of these nine genes are higher in nonresponders, which correlates with the terminally exhausted type of CD8+ T cells. Among the other genes in + T cells failed to respond to anti-PD1 therapy might be not only the co-expression of multiple inhibitors on CD8+ T cells themselves but also the tumor microenvironment of colorectal tumor in which those terminally exhausted CD8+ T cells may be infiltrated with myeloid-derived suppressor cells. Approximately 50% of the MSI nonresponders have a high The first component of TMEPRE, nsidered . The secor cells . This pansidered .By only assessing the MSI/MSS status, it is not recommended that MSS colorectal cancer patients be treated with anti-PD1. However, data from clinical trials showed that the disease control rate in pembrolizumab-treated metastatic MSS colorectal cancers was 11%, and further, in a more recent clinical trial of neoadjuvant setting, the pathological response rate of ipilimumab + nivolumab–treated early-stage MSS colorectal cancers is 27% . These r+ T-cell or tumor microenvironment of terminal exhaustion of CD8+ T cells, and treatment strategies need to be different. TMEPRE will aid personalized medicine options of cancer immunotherapy for colorectal cancer patients.To conclude, we develop a colorectal cancer-specific method, TMEPRE, that predicts cancer immunotherapy response. The global patterns of TMEPRE in colorectal cancer patients explained the mechanism underlying the response of anti-PD1 in MSS patients and the resistance of anti-PD1 in MSI patients. A subset of MSS patients could potentially benefit from anti-PD1 treatment. Anti-PD1-resistant MSI patients could result from tumor microenvironment of insufficient infiltration of CD8


In recent years, there are many studies on the improvement of I/R injury by H2S through regulating autophagy, but the related mechanisms are not completely clear. Therefore, we summarize the related research in the above aspects to provide theoretical reference for future in-depth research.Ischemia/reperfusion (I/R) injury is characterized by limiting blood supply to organs, then restoring blood flow and reoxygenation. It leads to many diseases, including acute kidney injury, myocardial infarction, circulatory arrest, ischemic stroke, trauma, and sickle cell disease. Autophagy is an important and conserved cellular pathway, in which cells transfer the cytoplasmic contents to lysosomes for degradation. It plays an important role in maintaining the balance of cell synthesis, decomposition and reuse, and participates in a variety of physiological and pathological processes. Hydrogen sulfide (H Ischemia/reperfusion (I/R) injury is a term used to represent the functional and structural changes that become obvious during the recovery of blood flow after a period of ischemia. In addition to ischemia, the recovery of blood flow can also lead to potentially very harmful effects, including significant cell swelling, irreversible cell necrosis and uneven blood flow recovery in all parts of the tissue . I/R injAutophagy is an evolutionarily conserved process in which the cytoplasm and organelles are isolated in double membrane vesicles, then transported to lysosomes/vacuoles for degradation and recycling of eukaryotic cytoplasmic components . Accordi2S) has always been considered as a toxic pollutant, but recently it, together with nitric oxide (NO) and carbon monoxide (CO), is regarded as a biological signal molecule, namely gas transmitter , cystathionine-γ-lyase (CSE), as well as 3-mercaptopyruvate sulfurtransferase (3-MST) , thus alleviating I/R injury  . H2S hasammatory , anti-apammatory , relaxinammatory  and antiammatory . It has R injury . In rece2S has been reported to play a protective role against hepatic I/R injury , aspartate transaminase (AST) and inflammatory cytokines, improving pathological changes of liver and reducing hepatocyte apoptosis. The mechanism research revealed that autophagy was enhanced in I/R-induced hepatitis, which could be reversed by exogenous H2S. Moreover, exogenous H2S inhibited c-Jun NH2-terminal kinases (JNK) pathway induced by I/R injury through decreasing JNK1 and extracellular signal-regulated kinase (ERK) phosphorylation, while SP600125, a JNK1 inhibitor, strengthened H2S hepatoprotective effects, indicating that autophagy and JNK pathway are involved in the hepatoprotective effect of exogenous H2S in hepatic I/R injury  is one of the main receptors involved in macrophage-mediated inflammatory response, which plays an important role in cerebral ischemia injury . The mechanism research showed that exogenous H2S promoted the autophagy of peritoneal macrophages by increasing the concentration of LC3B particles and the ratio of LC3-II/LC3-I in fatty liver I/R injury. Exogenous H2S also inhibited inflammation, apoptosis and oxidative stress, and decreased the protein expression of SRA in fatty liver I/R injury, suggesting that exogenous H2S alleviated hepatic I/R injury by enhancing autophagy through suppressing SRA pathway, which needed to be further studied by using the related inhibitors  with LY294002, a PI3K inhibitor, or knocking down Serum- and glucocorticoid-induced kinase 1 (SGK1) with SGK1 siRNA could enhance autophagy and weakened the anti-autophagy and cardioprotection effects of exogenous H2S, while the inhibition of glycogen synthase kinase-3 beta (GSK3β) with tws119, a GSK3β inhibitor, had the opposite effects. Collectively, exogenous H2S plays a cardioprotective role in neonatal rat cardiomyocytes exposed to H/R by inhibiting autophagy through PI3K/SGK1/GSK3β signaling pathway  mice, while autophagy activator rapamycin alleviated the protective effects of H2S, and the autophagy inhibitor 3-MA had the similar results to exogenous H2S, suggesting that autophagy was involved in the protective effects of exogenous H2S. Exogenous H2S suppressed the elevation of LC3-II and the decrease of p62, but had no notably effect on Beclin-1 complex in cerebral I/R injury model mice, indicating that exogenous H2S inhibited autophagy through decreasing autophagosome accumulation. From the above, it can be deduced that exogenous H2S reduced cerebral cortex I/R injury through inhibiting autophagy . Moreover, exogenous H2S had no influences on the -induced upregulation of the ULK1 self-association and reduction of the ATG13 phosphorylation induced by OGD/R, which are vital for the initiation of autophagosome formation. Taken together, it can be inferred that exogenous H2S played neuroprotection against cerebral I/R injury through increasing autophagosome degradation , and subsequent tissue injury. In the process of renal transplantation, the transient stop of renal blood flow leads to acute ischemic injury, and the subsequent reperfusion further deepens the functional and structural damage of human kidney . At presnous H2S , Moreove pathway . Therefo2S ameliorated motor function and infarct area, promoted autophagy by upregulating the expression levels of beclin-1 and LC3II, and downregulating miR-30c expression in rat spinal cord I/R injury model, indicating that autophagy and miR-30c were involved in exogenous H2S improvement of spinal cord I/R injury. Exogenous H2S also improved oxygen glucose deprivation (OGD)-induced ischemic injury in SY-SH-5Y cells  Exogenous H2S improve hepatic I/R injury by inhibit autophagy through suppressing JNK pathway; (2) Exogenous H2S alleviated hepatic I/R injury by enhancing autophagy through suppressing SRA pathway; (3) Exogenous H2S improved myocardial I/R injury by promoting AMPK-mediated autophagy or by inhibiting autophagy through PI3K/SGK1/GSK3β pathway; (4) Exogenous H2S reduced cerebral I/R injury through inhibiting autophagy via promoting autophagosome degradation; (5) Exogenous H2S improved renal I/R injury by inhibiting autophagy through suppressing SRA pathway; and (6) Exogenous H2S ameliorated spinal cord I/R injury by promoting autophagy via suppressing miR-30c (2S can improve ischemia-reperfusion injury by inhibiting autophagy or promoting autophagy. This difference may be due to the different basic levels of autophagy in different tissues, which leads to different responses of autophagy to ischemia-reperfusion injury. In addition, the mechanism of the effects of H2S on autophagy and the response mechanism of autophagy to ischemia-reperfusion injury need to be further elucidated.In this review, the effects of exogenous H miR-30c . It can 2S on autophagy may provide a new strategy for the prevention and treatment of ischemia-reperfusion injury.With the development of research, the effect of exogenous HHW revised, wrote, and funded the manuscript. SL wrote and funded the manuscript. ZW and JW wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.