Despite several decades of intense research focused on understanding function(s) and disease-associated malfunction of p53, there is no sign of any “mid-life crisis” in this rapidly advancing area of biomedicine. Firmly established as the hub of cellular stress responses and tumor suppressor targeted in most malignancies, p53’s many talents continue to surprise us, providing not only fresh insights into cell and organismal biology, but also new avenues to cancer treatment. Among the most fruitful lines of p53 research in recent years have been the discoveries revealing the multifaceted roles of p53-centered pathways in the fundamental processes of DNA replication and ribosome biogenesis (RiBi), along with cellular responses to replication and RiBi stresses, two intertwined areas of cell (patho)physiology that we discuss in this review. Here, we first provide concise introductory notes on the canonical roles of p53, the key interacting proteins, downstream targets and post-translational modifications involved in p53 regulation. We then highlight the emerging involvement of p53 as a key component of the DNA replication Fork Speed Regulatory Network and the mechanistic links of p53 with cellular checkpoint responses to replication stress (RS), the driving force of cancer-associated genomic instability. Next, the tantalizing, yet still rather foggy functional crosstalk between replication and RiBi (nucleolar) stresses is considered, followed by the more defined involvement of p53-mediated monitoring of the multistep process of RiBi, including the latest updates on the RPL5/RPL11/5 S rRNA-MDM2-p53-mediated Impaired Ribosome Biogenesis Checkpoint (IRBC) pathway and its involvement in tumorigenesis. The diverse defects of RiBi and IRBC that predispose and/or contribute to severe human pathologies including developmental syndromes and cancer are then outlined, along with examples of promising small-molecule-based strategies to therapeutically target the RS- and particularly RiBi- stress-tolerance mechanisms to which cancer cells are addicted due to their aberrant DNA replication, repair, and proteo-synthesis demands. As part of this intricate interplay, MDM2 can bind p53, potentially inhibiting MDM2 transactivation. In this regulatory feedback loop, p53 regulates MDM2 at the transcription level and MDM2 regulates the activity of p53 [BCL-2 [NOXA and PUMA [The year 2022 marks the 30th anniversary of one of the milestones in cell biology, discoveries that inspired the concept of p53 as the guardian of the genome . A year n tumors . Biologin tumors . MDM2 coy of p53 , 8. The y of p53 –11. The y of p53 , 13. Eary of p53 . Upon se3 [BCL-2  and up-rand PUMA .The exquisite regulation of p53 comes in many flavors, such as post-translational modifications, which influence p53 stability or specificity of its target genes. Phosphorylation  can leadGenotoxic stressors activate the multifaceted cellular signaling network called the DNA damage response (DDR). In response to oncogene-induced DNA damage, the DDR including p53 provides a biological barrier against tumor progression , 22. DDRp53 is also regulated by acetylation mediated by the acetyltransferases p300, PCAF, and CBP. Acetylation might not be critical for p53 activation, as unacetylated p53 retains its ability to induce the p53-MDM2 feedback loop, nevertheless, the loss of p53 acetylation might impact the p21-mediated stress response . MDM2 prTP53 in human cells [TP63 and TP73 encoding p63 and p73, respectively, share some degree of amino acid sequence identity with the transactivation domain, the DNA-binding domain, and the oligomerization domain of p53. Currently, p53, p63, and p73 constitute the p53 family of transcription factors [TP53−/− hESCs fail to differentiate into neural progenitor cells [To add an extra layer of complexity, at least nine different isoforms of p53 can be expressed from its gene an cells . Two add factors , whereby factors , 40. In  factors . Therefo factors . p53 kno factors . In huma factors , 45. The factors  and ther factors . In mous factors . Furtheror cells . In any It is probable that all stressors impacting cell function lead to post-translational modification(s) and activation of p53. The list of cellular stressors includes, but is not limited to, oncogene activation, DNA damage, telomere shortening, replication stress, dysregulated transcription, altered ribosome biogenesis, hypoxia, nutrient deprivation, mitochondrial stress, mitotic defects, thermal shock, protein misfolding, and ROS accumulation, with more likely to be identified . To limiAny condition that negatively impacts DNA synthesis and compromises replication fork integrity qualifies as replication stress (RS). RS typically causes fork arrest and collapse, however, it can also accelerate the speed of fork progression, activating the DNA damage response –54. AccuThe ATR/CHK1 signaling pathway plays a major role in the response to RS . Perturbp53 avoids RS at the telomeres, specifically, telomeres contain difficult-to-replicate repetitive DNA sequences protected by capping proteins  and RS aAnother example of p53’s versatility in terms of dealing with RS is p53’s role in silencing the Long Interspersed Element 1 (LINE-1). LINE-1 is a family of autonomous retrotransposons that are active in the human genome. LINE-1 contains two open reading frames (ORF1p and ORFp2) that are necessary for this element to spread to new genomic loci. Under physiological conditions, LINE-1 is silenced in somatic cells but cases of derepression and overexpression have been observed in cancer . LINE-1 Our previous work showed that p53 depletion caused reduced fork speed and accumulation of arrested forks. Interestingly, fork defects were alleviated by concomitant double-knockdown of p53 and PARP1 . Upon DNp21 gene repression. Upon DNA damage, PARylated PARP1 gets released from this complex, allowing recruitment of a co-activator p53/p300 and hence p21 transcription [Sensing chromosomal breaks and rearrangements emerging from defective forks or unfinished DNA replication is another major role of p53, with such checkpoint potentially operating directly at the fork level and/or transcriptionally regulating factors involved in preserving fork integrity. Notably, chronic induction of p21 in a p53-independent manner, mimicking p21 expression triggered by deregulated cytokines, hormones, or growth factors in advanced p53-mutant cancers, resulted in RS and genomic instability, reflecting the inability of PCNA to interact with and regulate the degradation of the replication licensing factors CDT1 and CDC6 . p53, Ctcription . Togethecription , the abop21, while PARylated PARP1 also releases p21 from the p21-PARP1 complexes. After prolonged fork arrest, processive DNA polymerases dissociate from modified PCNA [We proposed that any break in front of the replication fork is promptly recognized by PARP1, whose activity is then enhanced Fig. . PARylatied PCNA  and are ied PCNA . Therefoied PCNA . Perhapsied PCNA . RPA sigied PCNA . Defectsied PCNA . Future During DNA replication, forks encounter challenges, including damaged bases, non-histone proteins bound to DNA, transcription, repetitive sequences, and DNA in non-canonical structures such as DNA quadruplexes . RibosomDe novo nucleotide synthesis pathways have been increasingly investigated as potential cancer therapy targets. Inhibition of these metabolic pathways often immediately impairs both rRNA synthesis and DNA replication. Both IRBC  and DDR are involved in p53 activation following RiBi defects. ATR activation and imbalanced nucleotide pools were found in cellular and zebrafish models of ribosomal protein (RP) deficiency, and RP loss elicited DDR that likely contributed to p53 activation [de novo pyrimidine synthesis pathway, blocks cancer cell proliferation through induction of replication and nucleolar stress, activation of p53, and the ATR/CHK1 pathway [de novo GMP synthesis, a hierarchical organization was proposed, whereby IRBC provides the primary “nucleotide sensor”, while in a setting of highly effective IMPDH inhibition, p21 degradation takes place and attenuates the IRBC-mediated G1 arrest, allowing entry into the S phase and subsequent DDR activation [Unrestrained oncogenic activity can trigger enhanced nucleotide usage to sustain a high rate of ribosome biogenesis (RiBi) and DNA replication needed to drive cancer cell growth and proliferation, leading to nucleotide pool imbalances and combined replication and nucleolar stress. tivation . The inh pathway . It is utivation . These rtivation .Dysregulated rDNA transcription can increase R-loop formation, reflecting conflicts of replication and transcription machineries . Conversnucleolar stress [ribosomal stress. A broader hypothesis postulated that the nucleolus senses cellular stress and as soon as nucleolar function is impaired the p53 abundance increases. In parallel, the nucleolar p19Arf (mouse)/p14ARF (human) tumor suppressor was shown to bind and inhibit MDM2 to activate p53 in response to oncogenic signals [Perturbations in RiBi activate a p53-dependent cellular response, and the RiBi machinery is intimately connected to the control of MDM2 and p53. While the hypothesis that nucleolar integrity is linked to p53 turnover  and evidr stress , today a signals . Subsequ signals , 104. Ac signals   signals . In contAlso other members of the p53 family seem to have a role in ribosome biogenesis. It was shown that RPL11 and RPL5 could associate with the N-terminal domain of p73, and enhance the transcriptional activity of p73 by antagonizing p73-MDM2 interaction . InteresRPL5 and RPL11 binding to MDM2 and p53 activation also requires 5S rRNA , 110. 5SRPL11 mRNA translation resulting in increased 5S RNP-MDM2 complex formation and p53 activation [The 5S RNP-MDM2 interaction is enhanced upon alterations in ribosome production that leads to a 5S RNP surplus, and is needed to elevate p53 in response to for example chemotherapeutics , ribonucleotide depletion or loss of ribosomal proteins   Fig. 4)4). This tivation . Depletitivation . Yet anotivation .Fig. 4ThFurthermore, oncogenes including E2F-1 and Myc , and theRPL5 being frequently altered in human tumors. The IRBC’s anti-cancer role is supported by several findings: (I) RPL5 or RPL11 deficiency impairs p53 activation and may contribute to the increased risk of Diamond Blackfan Anemia (DBA) patients to develop cancer [Rpl11 are prone to radiation-induced lymphomagenesis, and loss of Rpl11 attenuates p53 activation in response to DNA damage in fibroblasts [RPL5 heterozygous mutations or deletions occur in up to 34% of breast cancer, melanoma (28%), glioblastoma (11%), and multiple myeloma (up to 30%) [RPL5 displayed worse overall survival in glioblastoma and multiple myeloma [TP53-intact tumors [The 5S RNP-MDM2-p53 IRBC pathway likely provides a barrier to cancer development, with p cancer ; (II) Miroblasts ; (III) Kroblasts ; (IV) RProblasts ; (V) RPL to 30%) . Further myeloma . RPL5 mu myeloma . Hemizygt tumors . Myc-drit tumors . Consistt tumors , while rt tumors . These sTp53, or pharmacological inhibition of p53 [Rps6 haploinsufficiency caused various limb phenotypes attributable to changes in mRNA translation patterns. Surprisingly most of the differential translational changes were restored upon loss of p53 [Increased p53 activity contributes to pathogenesis of ribosomopathies. In animal models of the human syndromes DBA and Treacher Collins syndrome (TCS) p53 mediates some but not all phenotypes as shown by co-deletion of n of p53 . In DBA,s of p53 . Thus, pEif4a3 haploinsufficiency in mice triggers microcephaly and aberrant neurogenesis through p53 activation [MDM2 and DNA damage, while inactivation of 5S RNP only partially attenuated the p53 response in EIF4A3-depleted cells [MDM2 exon 3 skipping was described in mice deficient in the pre-mRNA splicing factor Eftud2, resulting in p53 activation [MDM2 splicing, impaired RiBi, activation of IRBC, DNA damage, as well as p53 elevation and apoptosis [MDM2 is altered by splicing, yet there is nucleolar stress, DNA damage and IRBC activation, p53 induction cannot in such a complex scenario be solely attributed to only one pathway or mechanism in RCPS. Nevertheless, while EIF4A3 is an essential protein these combined effects, including mis-splicing of MDM2, should be further explored in cancer treatment.DDX21 and EIF4A3 are RNA helicases whose loss triggers multifaceted cellular stress responses converging on p53. The RNA binding exon junction complex (EJC) consists of, among others, the proteins MAGOH, RBM8, and EIF4A3. Deficiency in individual EJC components causes abnormal neural development, exemplified by the Richieri Costa Pereira syndrome (RCPS), an autosomal-recessive acrofacial dysostosis presenting with craniofacial malformation and limb defects due to deregulation of EIF4A3 . Eif4a3 tivation . We havetivation . While led cells . Interestivation . Taken tpoptosis  , is a DEAD-box RNA helicase also involved in RiBi and nucleolar function. DDX21 associates with genes actively transcribed by RNA Pol I and II and unwinds R-loops. In the nucleolus, DDX21 occupies transcribed rDNA genes and promotes rRNA synthesis. Depletion of DDX21 activates the IRBC and p53, but also leads to accumulation of R-loops and DNA damage , 141 Fi. TCS, thAs p53 responds to diverse stress signals, compounds modulating such converging pathways, including replication stress tolerance as a targetable cancer vulnerability –145, mayAmong the fundamental biological processes mechanistically linked to p53, the fields of ribosome biogenesis and replication stresses, including their involvement in human pathologies including cancer, have witnessed striking progress in recent years. In this review, we provide concise background information about the roles of p53 in DNA replication and ribosome biogenesis, with emphasis on deregulation of these mechanisms and the roles of p53 pathways in the maintenance of cell homeostasis, including the emerging functional links between such stress response mechanisms. Apart from providing an overview of current mechanistic understanding of these rapidly evolving areas of biomedicine, we also briefly outline the vulnerability of cancer cells due to their adaptation to chronic ribosome biogenesis stress, with examples of emerging compounds to target such aberrant conditions therapeutically. Finally, throughout the article, we also point out some burning open questions the elucidation of which requires further research, both basic and clinical.


At present, mixed infection with multiple pathogens is an important reason for the complexity of swine diseases in China. Among them, African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are extremely important DNA viruses that cause reproductive abnormalities in sows. Therefore, there is an urgent need for rapid and sensitive detection methods that can diagnose three kinds of DNA viruses in the clinic. Herein, a multiplex real-time PCR (qPCR) assay based on TaqMan probes was developed for the simultaneous determination of ASFV, PCV2, and PRV. To ascertain the use of the multiplex real-time qPCR, 383 field specimens from the pig farms of four provinces in East China were collected. The survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV + PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. The assay established in this study could be used as a differential diagnostic tool for the monitoring and control of ASFV, PCV2, and PRV in the field.1 copies/L of each pathogen, and its repeatability was excellent, with intra- and inter-group variability coefficients of <2%. Applying this assay to detect 383 field specimens collected from 2020 to 2022, the survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV + PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. Overall, the assay established in this study provides an effective tool for quickly distinguishing the viruses causing sow reproductive disorders, suggesting its huge clinical application value in the diagnosis of swine diseases.African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are important DNA viruses that cause reproductive disorders in sows, which result in huge losses in pig husbandry, especially in China. The multiplex qPCR assay could be utilized as a simultaneous diagnostic tool for field-based surveillance and the control of ASFV, PCV2, and PRV. Based on the conserved regions on the p72 gene of ASFV, the Cap gene of PCV2, the gE gene of PRV, and the porcine endogenous β-Actin gene, the appropriate primers and probes for a multiplex TaqMan real-time PCR test effective at concurrently detecting three DNA viruses were developed. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to swine reproductive diseases. In addition, its sensitivity was great, with a detection limit of 10 At present, among these pathogens, the porcine respiratory and reproductive syndrome virus is the most concerning, but three DNA viruses, ASFV, PCV2, and PRV, also cannot be ignored. At the same time, because PCV2 can inhibit type I interferon induction to promote the infections caused by other DNA viruses [In China, the breeding density and scale of pig herds are increasing. The pigs in some large farms are experiencing mixed infection or secondary infection with multiple pathogens, leading to high morbidity and mortality . Severe  viruses , the pro viruses ,5,6. Afr viruses . Since 2 viruses . ASFV is viruses ,10,11. T viruses ,13. PCV2 viruses . ORF1 an viruses . PRV is  viruses . PR is a viruses ,18,19,20These three viruses are difficult to distinguish using clinical symptoms alone. At present, diagnosis mostly depends on molecular diagnosis. Considering the convenience, sensitivity, and rapidity of identifying clinical viral infections in pigs, several technologies, such as PCR, real-time PCR, and digital PCR, are frequently used. There are also some articles about isothermal nucleic acid amplification, such as recombinase polymerase amplification (RPA), recombinase-aided amplification (RAA), and loop-mediated isothermal amplification (LAMP) ,25,26,27Special primers and probes were designed based on the conserved regions of the ASFV (MK333180.1), PCV2 (KT719404.1), and PRV (KT809429.1) for the creation of constructed plasmid standards and the establishment of a real-time qPCR assay. An internal reference, β-Actin, was amplified as a control . The priChlamydia suis, and Toxoplasma gondii were from specimens previously confirmed using PCR and RT-PCR assays. ASFV-positive DNA was a gift from the Lanzhou Veterinary Research Institute . All the positive nucleic acid above of swine pathogens was stored in the lab for specificity experiments. In all, 383 field specimens, consisting of lymphoid tissues, placentas, sera, and blood, were collected from sows with reproductive issues and aborted piglets in the pig farms of four provinces of East China from 2020 to 2022. The information of the specimens was shown in Positive cDNA of JEV and CSFV were from the NJ-2008 strain (GQ918133) and the Shimen strain (AF092448). The positive DNA of PRV was from the SD-2019 strain (GenBank accession No. MW805231). Other positive DNA or cDNA of PCV1, PCV2, PCV3, PCV4, PRRSV, PPV, The conserved regions of ASFV p72, PCV2 Cap, or PRV gE were cloned into a pEasy vector and produced three recombinant plasmids, named pEasy-ASFV, pEasy-PCV2, and pEasy-PRV, and sent to Nanjing GenScript Biotechnology Co., Ltd. , for DNA sequencing. The concentration of plasmid standards was determined using a NanoDrop spectrophotometer . Finally, the copy numbers of the three recombinant plasmids were calculated .7 copies/μL to 3 × 104 copies/μL were amplified as the templates. Firstly, a mixture of three diluted plasmids with a concentration of up to 1 × 104 copies/μL was prepared. Secondly, four mixed pairs of customized primers and probes were added to one reaction condition and set to determine the optimal work concentrations for the development of this assay. The final volume of 20 μL included 10 μL 2 × AceQ qPCR Probe Master Mix , 0.1–1.8 μL of each of the primers, 0.1–0.8 μL of each of the probes, and 2 μL of templates. Thirdly, FAM, HEX, Texas Red, and Cy5 were configured as the fluorescence channels for the real-time qPCR instrument’s four fluorescence channels. Finally, a commercialized real-time PCR instrument  was used to collect the fluorescence signals.The optimal reaction conditions were determined according to a previous report . In brie7 to 101 copies/μL) in distilled water were employed as amplification templates for this assay. The logarithm of the copy number determined the horizontal axis of the standard curve, while the vertical axis was determined by the Ct value. The Prism software was used to draw the standard curves.According to the report described previously , the ampChlamydia suis, and Toxoplasma gondii as detection templates, the plasmid standards as positive controls, and ddH2O as a negative control. All templates and ddH2O were repeated three times under the optimal reaction conditions for specificity of the multiplex PCR.The specificity of the multiplex real-time qPCR assay was determined by utilizing the positive DNA or cDNA of the above-mentioned swine pathogens PCV1, PCV3, PCV4, PRRSV, CSFV, PPV, 7 copies/μL and 1 × 101 copies/μL in nuclease-free water. The diluted standard plasmids were used as templates for multiplex qPCR amplification in the optimal reaction conditions.The detection limit determines the occurence of false negatives during detection. To analyze the sensitivity of the established multiplex real-time qPCR, the linearization standard plasmids, pEasy-ASFV, pEasy-PCV2, and pEasy-PRV, prepared above were diluted 10-fold serially to a final concentration between 1 × 107 copies/μL to 104 copies/μL using nuclease-free water. The diluted standard plasmids were used as templates for multiplex real-time qPCR amplification. Using the optimized amplification conditions, each diluted standard plasmid was amplified three times repeatedly and tested every other week. The different concentration groups were compared, and their intra-group and inter-group coefficients of variation were determined to confirm their reproducibility.To analyze the repeatability and reproducibility of the established multiplex real-time qPCR, the linearization standard plasmids, pEasy-ASFV, pEasy-PCV2, and pEasy-PRV, were diluted by a factor of 10 from 102O were employed as positive and negative controls, respectively. The extracted DNA from field specimens was measured by the assay established in this study, and then the epidemiological survey data were analyzed.A total of 383 field specimens gathered from four eastern provinces of China were detected using the established real-time qPCR. In brief, all the specimens were resuspended and homogenized in phosphate-buffered saline (PBS). Following the manufacturer’s instructions, the nucleic acid was extracted from a homogenized specimen using a PureLink Viral DNA Mini Kit . The constructed plasmid and ddH2O was 4 μL; and the total volume of the reaction was 20 μL. The qPCR reaction program was as follows: 95 °C pre-denaturation for 3 min; followed by 40 cycles, which consisted of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 20 s. Fluorescence channels 1–4 were arranged to amplify the four genes ASFV p72 (FAM), β-Actin (HEX), PCV2 Cap (CY5), and PRV gE (Texas Red), respectively. Finally, all the fluorescence signals were collected by a real-time PCR instrument.After the repeated tests for optimization of primer and probe concentrations, the optimum reaction conditions for multiplex real-time qPCR were obtained as follows: 10 μL 2 × AceQ qPCR Probe Master Mix  was used; initial primer concentrations of 10 μM of ASFV-p72qF, ASFV-p72qR, PRV-gEqF, PRV-gEqR, PCV2-CapqF, PCV2-CapqR, β-Actin-qF, and β-Actin-qR were 0.6 μL each; initial probe concentrations of 10 μM of ASFV-probe, PRV-probe, PCV2-probe, and β-Actin-probe were 0.3 μL each; the volume of the DNA template and ddH7−101 copies/μL in a 10-fold gradient, pEasy-ASFV, pEasy-PCV2, and pEasy-PRV were amplified by multiplex real-time qPCR using the best reaction conditions and procedure. Three standard curves of gene amplification were established using the obtained Ct values and plasmid concentrations. As shown in 2 = 0.9956 for ASFV, R2 = 0.9965 for PCV2, and R2 = 0.9904 for PRV, which proved an excellent correlation coefficient and amplification efficacy. This suggested that the established TaqMan probe-based multiplex real-time qPCR for each virus was reliable and valid.Diluted to 10Chlamydia suis, and Toxoplasma gondii were tested as templates, and ddH2O was tested as a negative control. As shown in There are numerous pathogens in pig farms that might induce reproductive disorders in sows. To evaluate the specificity of the method established in this experiment, we performed specificity tests with prevalent pathogen specimens. The standard plasmids, pEasy-ASFV, pEasy-PCV2, and pEasy-PRV, were tested as positive controls. Positive nucleic acids of JEV, CSFV, PRRSV, PCV1, PCV3, PCV4, PPV, 7 copies/μL to 1 × 101 copies/μL of three constructed plasmids of ASFV, PCV2, and PRV for the sensitivity test. As shown in 1 copies/μL.Multiplex real-time qPCR was performed utilizing the most appropriate reaction conditions to amplify the target genes from seven concentrations ranging from 1 × 10The resulting data showed that the variation coefficients (CVs) of Ct values in the intra-group reproducibility tests and inter-group reproducibility tests ranged from 0.39% to 1.20% and from 0.06% to 1.96% , respectThe established assay was employed to determine the DNA of 383 field specimens collected from East China. As shown in 1 copies/μL, and the intra-group and inter-group variation coefficients are less than 2%. Meanwhile, the assay established has good reproducibility and sensitivity, providing a sensitive and specific tool for the rapid detection of ASFV, PCV2, and PRV in clinical diagnosis.Sow reproductive disorders still have a serious impact on farms, and the widespread availability of weak strains of ASFV complicates the epidemiology of the disease ,37. Give1 copies/μL using the special primers and probe for the conservative region of the ASFV p72 gene. Recently, the wild-type and gene-deleted ASFV strains were detected simultaneously using a multiplex real-time qPCR assay, which tested for ASFV with a detection limit of 32.1 copies/L for the p72 gene and 3.21 copies/L for the I177L, MGF505-2R, and CD2v genes [1 copies/μL. Currently, few qPCR methods can concurrently detect these three viruses. Remarkably, the approach developed in this study compensated for the shortcomings of clinical detection. Moreover, its detection limit reached 101 copies/μL for each virus.Virus gene analysis and detection are crucial components of epidemic control and prevention. There are numerous approaches to analyzing genes . PCR is 2v genes . In addi2v genes  develope2v genes  establisSince August 2018, the ASF epidemic has occurred in 31 provinces nationwide, seriously harming the Chinese pig-breeding industry and causing huge economic losses . The ASFA TaqMan probe-based multiplex real-time qPCR assay was successfully established to detect ASFV, PCV2, and PRV simultaneously, providing a sensitive and specific tool for the rapid detection of swine viral diseases caused by DNA viruses in the field. The detection results also show that there is no obvious prevalence of mixed infection with ASFV and PCV2 or PRV in East China, indicating that the rate of PRV is declining and that PRV will be potentially eradicated in East China. Of course, the detection of ASFV is still the focus of this work, and we should not neglect the prevention and control of PCV2.


Klebsiella spp. (CRK) owing to the wide usage of veterinary antibiotics. “One Health”-studies observing animals, the environment, and humans are necessary to understand the dissemination of CRK in animal breeding areas. Based on the concept of “One-Health,” 263 samples of animal feces, wastewater, well water, and human feces from 60 livestock and poultry farms in Shandong province, China were screened for CRK. Five carbapenem-resistant Klebsiella pneumoniae (CRKP) and three carbapenem-resistant Klebsiella quasipneumoniae (CRKQ) strains were isolated from animal feces, human feces, and well water. The eight strains were characterized by antimicrobial susceptibility testing, plasmid conjugation assays, whole-genome sequencing, and bioinformatics analysis. All strains carried the carbapenemase-encoding gene blaNDM-5, which was flanked by the same core genetic structure (IS5-blaNDM-5-bleMBL-trpF-dsbD-IS26-ISKox3) and was located on highly related conjugative IncX3 plasmids. The colistin resistance gene mcr-8.1 was carried by three CRKP and located on self-transmissible IncFII(K)/IncFIA(HI1) and IncFII(pKP91)/IncFIA(HI1) plasmids. The genetic context of mcr-8.1 consisted of IS903-orf-mcr-8.1-copR-baeS-dgkA-orf-IS903 in three strains. Single nucleotide polymorphism (SNP) analysis confirmed the clonal spread of CRKP carrying-blaNDM-5 and mcr-8.1 between two human workers in the same chicken farm. Additionally, the SNP analysis showed clonal expansion of CRKP and CRKQ strains from well water in different farms, and the clonal CRKP was clonally related to isolates from animal farms and a wastewater treatment plant collected in other studies in the same province. These findings suggest that CRKP and CRKQ are capable of disseminating via horizontal gene transfer and clonal expansion and may pose a significant threat to public health unless preventative measures are taken.Animal farms have become one of the most important reservoirs of carbapenem-resistant  Klebsiella spp. (CRK) are especially concerning owing to their resistance to carbapenems, one of the last resort antibiotics used against gram-negative bacteria, and ability to cause a variety of community- or hospital-acquired infections such as pneumonia, urinary tract infections, bloodstream infections, and septic shock. Carbapenem-resistant Klebsiella pneumoniae (CRKP), which is widely reported in Europe, Asia, Africa, and other regions  and antibiotic-resistant genes (ARGs) has the potential to cause a global health crisis in the 21st century. Carbapenem-resistant  regions , is list regions . HoweverChina is the largest consumer of veterinary antimicrobials, accounting for 45% of global use in 2017, and is expected to remain the largest consumer in 2030 . Thus, AThus, our study randomly selected 60 animal farms, including 20 chicken, 20 pig, and 20 bovine farms, in a typical region of the animal farming industry with a long breeding time and stable animal breeding patterns in Shandong province in July 2019. Based on the perspective of “One-Health,” animal feces, wastewater (excluding chicken farms), well water, and human feces were collected from each farm to screen for CRK. This study aimed to characterize CRK in terms of antimicrobial susceptibility, antibiotic resistance genes, virulence genes and multilocus sequence typing (MLST), investigate the prevalence of CRK in different types of farms in the same animal breeding area, and explore the transmission of CRK among animals, the environment, and humans.2, and has a long breeding time and stable animal breeding patterns. The primary animal husbandry in this area consists of breeding chickens, pigs, and bovines. We selected 60 livestock and poultry farms (n = 20), chicken farms , and bovine farms . In July 2019, a total of 263 samples were collected, with 75 from chicken farms, 86 from pig farms, and 102 from bovine farms. The following samples types were collected: 66 animal feces, 37 wastewater, 107 worker feces, and 53 well water samples. The number and type of samples are summarized in The study site is in Eastern China, has a population of approximately 1,194,365, covers an area of 2,414 kmry farms , includiblaNDM, blaKPC, blaOXA-48, blaVIM and blaIMP carbapenemase genes using PCR, as previously described  was performed in a constant temperature shaker  at 37°C overnight. Enriched solutions (50 μl) were evenly spread on MacConkey agar  supplemented with 2 mg/l meropenem  to screen for carbapenem-resistant isolates. Colonies were selected based on colony morphotypes and repeatedly streaked on the MacConkey agar to obtain pure isolates . The isoescribed . The PCREscherichia coli ATCC 25922 was used as the quality control strain. Multidrug resistance was defined as non-susceptibility to at least one agent in three or more antimicrobial categories (The minimum inhibitory concentrations (MICs) of the 12 antibiotics were determined for CRK. 12 antibiotics were tested at a series of concentrations, and the maximum concentrations of amoxicillin/clavulanate, piperacillin/tazobactam, cefotaxime, ceftazidime, meropenem, imipenem, amikacin, ciprofloxacin, tetracycline, tigecycline, colistin and nitrofurantoin were 128/64 mg/l, 128/4 mg/l, 256 mg/l, 256 mg/l, 256 mg/l, 256 mg/l, 256 mg/l, 64 mg/l, 128 mg/l, 32 mg/l, 64 mg/l, 128 mg/l, respectively; the minimum concentrations were 0.0625 mg/l for 12 antibiotics. The MICs of tigecycline and colistin were determined using the broth microdilution method, and others were determined using the agar dilution method according to the Clinical Laboratory Standards Institute (CLSI). The results were interpreted according to the CLSI (document M100-S31), excluding tigecycline, for which MICs were interpreted following the ecological cut-off (ECOFF) of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) SOP 10.2. tegories .E. coli J53 was used as the recipient strain, and CRK was used as the donor strain to evaluate the horizontal transferability of the plasmids mediating carbapenem resistance, colistin resistance, and tigecycline resistance by filter mating. The donor strain and recipient strain were mixed at a ratio of 2:1 and incubated at 37°C for 12 h. Transconjugants carrying carbapenemase gene, colistin resistance gene, and tigecycline resistance gene were selected by cultivation on LB agar supplemented with 100 mg/l sodium azide and 2 mg/l meropenem, 100 mg/l sodium azide and 2 mg/l colistin, 100 mg/l sodium azide and 4 mg/l tigecycline for 18 h at 37°C, respectively. Transconjugants carrying resistance genes were verified using PCR. Moreover, plasmids of tigecycline-resistant strain (KA2) were extracted using the M5 BAC/PAC large plasmid extraction kit  according to the manufacturer’s instructions. XL10-Gold chemically competent E. coli cells  were used as the recipient strain to evaluate the transformation of plasmids that mediate tigecycline resistance using the heat shock method, as previously described (Sodium azide-resistant escribed . TransfogyrA and gyrB) and topoisomerase IV (parC and parE) was performed using Clustal Omega  combined with the PacBio Sequel platform (Berry Genomics Co. Ltd.). Hybrid genome assembly with both short and long reads was performed using Unicycler v0.5.0 . The seqal Omega . Genetical Omega . A distaal Omega . Visualior BRIG; . All genK. pneumoniae and three were K. quasipneumoniae. Five CRK  were isolated from chicken farms , two (KW1 and KW2) from pig farms  and one (KW3) from bovine farms . KH1 and KH2 were isolated from the same chicken farm, whereas the other strains were obtained from different farms. In terms of sample types, four CRK (KW1-KW4) were isolated from well water, two (KA1 and KA2) from animal feces, two (KH1 and KH2) from human feces, and no CRK was isolated from wastewater  and imipenem (≥64 mg/l). In addition to the carbapenemase gene, CKR carried various classes of resistance genes and the most prevalent ARGs , including oqxAB (n = 8), fosA (n = 8), tet (A) (n = 6), aadA2 (n = 5), floR (n = 5), qnrS1 (n = 4), and aac(6′)-Ib-cr , whereas resistance to colistin (50%), amikacin (37.5%), nitrofurantoin (37.5%), and tigecycline (12.5%) was observed to a lesser degree . Eight Cr n = 4; . Three sgyrA and gyrB) and topoisomerase IV (parC and parE) between the CRK isolated in this study and the reference sequence in the NCBI database showed that the amino acid substitutions of quinolone resistance-determining regions (QRDRs) were observed at position 83 (Ser → IIe) of gyrA and position 80 (Ser → IIe) of parC in KA2, KH1, and KH2 , qnrS , qnrB , and aac(6′)-Ib-cr -Ib-cr, and oqxAB, and another strain carrying qnrS1 and oqxAB showed non-susceptibility to ciprofloxacin. The qnr genes encode proteins belonging to the pentapeptide repeat protein family that play a role in binding and protecting topoisomerase IV and DNA gyrase from repression by ciprofloxacin (aac(6′)-Ib-cr gene encodes a variant aminoglycoside acetyltransferase that reduces the activity of ciprofloxacin by N-acetylation at the amino nitrogen on its piperazinyl substituent  to ciprofloxacin, the resistance mechanisms of ciprofloxacin were analyzed. Comparison of the amino acid sequence of DNA gyrase /IncFIA(HI1) plasmid carrying mcr-8.1 was detected on KA2, and IncFII(K)/IncFIA(HI1) plasmids carrying mcr-8.1 were detected on KH1 and KH2.The silico analysis of the general molecular characteristics of the isolates are summarized in typeable . The genblaNDM-5-carrying IncX3 plasmids were successfully transferred from all strains to the recipient strain E. coli J53 via conjugation assays. blaNDM-5 was located on the 46,161 bp IncX3 plasmid among strains KA1, KW2, and KW3, designated pKA1-3-NDM5, pKW2-1-NDM5, and pKW3-1-NDM5, respectively. Plasmid sequence comparison showed that the three IncX3 plasmids shared 100% nucleotide identity and 100% coverage with each other, and were identical to several other IncX3 plasmids found in Enterobacteriaceae isolates in China, such as the plasmid pNDM5_IncX3 (GenBank: KU761328.1) from K. pneumoniae isolated from a patient in Suzhou city, pNDM-SCCRK18-72 (GenBank: MN565271.1) from an E. coli isolated from chicken in Sichuan province, and pEC24-NDM-5 (GenBank: CP060888.1) from an E. coli isolated from a patient in Zhejiang province. blaNDM-5 was flanked by the same genetic structure as that of Tn3-IS3000-ISAba125-IS5-blaNDM-5-bleMBL-trpF-dsbD-IS26-ISKox3  and IncFIA(HI1) replicons. In addition to mcr-8.1, resistance genes blaTEM-1B, blaCTX-M-1, and Oble were identified on the plasmid. The plasmid BLAST query in the GenBank database showed that the plasmid pKA2-3-mcr8.1 exhibited the highest degree of sequence homology with plasmids pKP19-3,138-3 (GenBank: CP090619.1) from K. pneumoniae and pKP46-mcr8 (GenBank: CP088125.1) from K. pneumoniae, with over 99% identity and 81% coverage  and IncFIA(HI1) replicons. The two plasmids shared over 99% nucleotide identity and 94% coverage with the plasmids pKP3 (GenBank: OL804387.1) and pKP91 (GenBank: MG736312.1), which were isolated from chicken anal swab and pig feces, respectively, in Shandong province, China /IncFIA(HI1) and IncFII(K)/IncFIA(HI1) plasmids carried two genes coding the response regulator transcription factor CopR and HAMP domain-containing histidine kinase Baes. The combination of these two protein families usually constitutes a two-component system involved in colistin resistance in Enterobacteriaceae.The coverage . In strae, China , indicatSNP-based phylogenetic analysis revealed that the eight CRK were grouped into four clusters. The first cluster included KW2 and KW3, isolated from the well water of farms P2 and B1, respectively, with four SNPs differences. The second cluster consisted of KA1, KW1, and KW4, among which KW1 and KW4 differed by only six SNPs and were isolated from the well water of farms P1 and LB1. The third cluster included only KA2. The fourth cluster included KH1 and KH2 isolated from two human fecal samples in farm BC3, with two SNPs differences .Klebsiella quasipneumoniae (CRKQ) stains from the GenBank database were characterized  from well water in two animal breeding areas (differed by 4–6 SNPs). KW1 and KW4 were similar to K. quasipneumoniae strain KE1661 from a human rectal swab in Germany  and K. quasipneumoniae strain SDWK24 from the abovementioned WWTT  detection rate in animal fecal samples was higher in chicken farms compared to bovine and pig farms in northern China  of blaNDM-5 among different Enterobacteriaceae species was mainly mediated by IncX3 plasmids in a pediatric hospital in Shanghai  have been identified in different bacteria isolated from animals, the environment, and humans worldwide /IncFIA(HI1) and IncFII(K)/IncFIA(HI1), supporting the notion that plasmids are the main factor in disseminating mcr-8.1.KH1 and KH2 from two fecal samples from workers at farm BC3 and KA2 from animal feces at farm BC2 carried the colistin resistance gene arly CRE . Howeverin China . In the orldwide . Since W in 2018 , K. pneueriaceae . In thisK. pneumoniae carrying blaNDM-5 and mcr-8.1 between human workers in the same chicken farm. However, because the types of samples were limited, the route of dissemination could not be determined in this study, and further research should clarify the route of clonal spread and provide effective control strategies for the dissemination of colistin-resistant CRKP in animal farms. Additionally, two pairs of clonally related CRKP and CRKQ were isolated from well water in four different farms located at a distance of more than 20 km, revealing that clonal spread also has significant implications for the dissemination of blaNDM-5 in the animal breeding area. Unexpectedly, the pair of clonal CRKP was clonally related to six strains from animal farms and a WWTT collected in other studies in the same province, suggesting that blaNDM-5-carrying K. pneumoniae are capable of disseminating within or between farms in the same county, even in different counties within a province, via clonal expansion. Although the exact route of long-distance transmission of CRKP is uncertain, population movement and travel / IncFIA(HI1) and IncFII(pKP91)/IncFIA(HI1) plasmids. Two pairs of clonal CRK from well water and one pair of clonal colistin-resistant CRK from human workers were identified. Thus, both HGT via plasmids and clonal expansion play a significant role in disseminating blaNDM-5 and mcr-8.1 among animals, the environment, and humans in the animal breeding area. The results highlight that systematical monitoring and large-scale investigation of the prevalence and spread of CRK with a “One Health”-perspective in animal breeding areas in Eastern China are urgently needed to enable effective measures for controlling the further spread of CRK.In this study, we investigated the prevalence of CRK by randomly selecting 20 chicken, 20 pig, and 20 bovine farms from a typical region of the animal farming industry in China. Based on the “One-Health” concept, the transmission of CRK among animals, the environment and humans were discussed. All CRK carried The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Ethical approval to conduct this study was granted by the Ethics Committee for Shandong University [Ethics permission number: 20191202]. The patients/participants provided their written informed consent to participate in this study. Ethical review and approval was not required for the animal study because We only collected animal feces from the animal farms and did not contact animals.CY: sampling, experiments conducting, data formal analysis, and writing—original draft preparation. BB and JH: writing—reviewing and editing. HZo, CG, LZ, CM, and HZh sampling. XM and XL: conceptualization, supervision, and writing-reviewing. All authors contributed to the article and approved the submitted version.This work was supported by the National Key Research and Development Program of China [2020YFC1806904] and the National Natural Science Foundation of China (grant nos. 8197120700 and 41771499).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.https://www.frontiersin.org/articles/10.3389/fmicb.2022.1030490/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.


Vitamin D has been implicated in the pathogenesis of skeletal disorders and various autoimmune disorders. Vitamin D can be consumed from the diet or synthesized in the skin upon ultraviolet exposure and hydroxylation in the liver and kidneys. In its bioactive form, vitamin D exerts a potent immunomodulatory effect and is important for bone health. Juvenile idiopathic arthritis (JIA) is a collection of inflammatory joint diseases in children that share the manifestation of inflamed synovium, which can result in growth arrest, articular deformity, bone density loss, and disability. To evaluate the potential effect of vitamin D on JIA disease manifestations and outcomes, we review the role of vitamin D in bone metabolism, discuss the mechanism of vitamin D in modulating the innate and adaptive immune systems, evaluate the clinical significance of vitamin D in patients with JIA, and summarize the supplementation studies. Juvenile idiopathic arthritis (JIA) is the most prevalent debilitating rheumatic disease of childhood, with a prevalence rate of 1/1000 worldwide . The immVitamin D was first known as an essential element in modulating bone health and altering calcium homeostasis ,9. Vitam2  and vitamin D3  are perhaps the most discussed vitamin D compounds D (FGF23) . Howevernd INF-α ,25. Circnd INF-α ,26.2D binds to the intracellular VDR, a transcription factor, to exert its genomic effects. Upon the binding of 1,25[OH]2D, VDR dimerizes with other nuclear receptors and binds to vitamin D response elements (VDREs) in the promotor regions to regulate the expression of genes mediating various biological activities [2D with membrane-bound VDR [2D-induced 24-hydroxylase (CYP24A1) further catabolizes 25[OH]D and 1,25[OH]2D to 24,25[OH]2D and 1,24,25[OH]3D in the kidneys tivities ,25. Moreound VDR . To avoi2D is approximately 5–8 h in humans n humans . Due to  reserve ,30,31. A reserve . Nonethe reserve ,30,31. S reserve ,30.2D can directly affect bone homeostasis in response to various inflammatory mediators in patients with JIA D into 1,25[OH]2D lization . When th25[OH]2D ,34. As atabolism ,34.2D and 24,25[OH]2D are both important for proper endochondral bone formation ormation . VDR sigormation . VDR sigormation . By enhalization ,34. In aogenesis ,34.2D-VDR signaling in osteogenic cells, intestines, and kidneys mainly contributes to the balance of serum calcium by reducing mineralized bone mass when intestinal calcium absorption is decreased ntiation ,56,57,58revealed ,65. Specrevealed ,65. 1,25oduction ,65,66. Fpression . Recent pression .2D has been shown to interfere with the production of antibodies and induce apoptosis among activated B cells  B cells . Additioturation . Due to turation ,71.2D have been shown to inhibit the invasion of synovial fibroblasts and suppress the production of both IL-1β and matrix metalloprotease-1 D was reported to be deficient or insufficient in a high number of JIA patients across numerous studies ,80,81,822D concentration was shown to accurately differentiate 79.6% of the patients into either low or normal-BMD groups D level with bone mineral density (BMD) in patients with JIA has also been investigated ,89,90,91D groups . A margiD groups ,89, it iD groups . NonetheD groups ,93, the Interest in how vitamin D levels alter JIA disease activity has grown along with our understanding of its ability to modulate inflammation ,81,85,94ApaI, BsmI, Cdx2, FokI, and TaqI are the most widely studied polymorphisms. While vitamin D status was independent of VDR genotypes, polymorphisms of ApaI and Cdx2 were significantly different between JIA patients and unaffected controls D D . As PTPNk of JIA .Hypovitaminosis D is currently a global issue for children worldwide and an intake of 400 IU and 600 IU vitamin D daily is generally accepted to prevent nutritional rickets in infants and children ,102. WhiConsidering the high prevalence of hypovitaminosis and the usage of steroids in patients with JIA, special recommendations on the optimal vitamin D requirements for children with chronic inflammatory rheumatic conditions have been discussed in the past decade ,106. Von2D, JIA disease activity, and BMD remained the same [2D [3 at a dose of 2000 IU/day for 24 weeks did not significantly reduce disease activity or improve BMD patients with JIA despite the rise of serum 25[OH]D D . A summaSeveral studies calculated the estimated amount of daily vitamin D intake and reported the supplementation of vitamin D among JIA patients in real-world practice. Many of the vitamin D dosing, however, did not meet the recommended minimal amount of 400 IU daily. Utilizing a three-day diet recall method, Dey et al. calculated daily calcium and vitamin D intake among 35 patients with JIA and their age/gender-matched controls. Lower daily vitamin D intake (123 ± 53.6 IU) was reported among the patients as compared to the controls (309 ± 62.38 IU) . While PJIA is the leading rheumatic disorder driven by a dysregulated innate and adaptive immune system in children. While current treatments provide notable improvements in the control of systemic inflammation and the relieve of symptoms in JIA, issues in long-term bone health and concerns for medication-related side effects, infection, and malignancy, for example, have led to the search for a cost-effective therapy or nutrient supplementation with limited side effects. Vitamin D is an essential hormone in altering calcium homeostasis and modulating bone health. Its deficiency can result in osteomalacia and rickets in humans ,9. BeyonClinical significance of vitamin D in patients with JIA has been documented. Data from limited randomized controlled trials, however, did not support the therapeutic effect of vitamin D supplementation in suppressing disease activity or improving bone mineralization. Heterogeneity between studies in terms of the study population, vitamin D supplementation regimen, outcome measurements, and definitions of vitamin D deficiency is perhaps the most obvious problem. Moreover, as trials were conducted across 30 years in different geographic locations, with various ethnic backgrounds, under diverse qualities of diet, sun exposure, and clothing style, it is hard to draw a conclusion based on the available information at present.Further studies are needed to better evaluate the potential efficacy of vitamin D treatment in JIA. First, larger controlled trials are recommended as sample sizes from existing JIA supplementation studies were small. These trials do not allow for stratification based on baseline vitamin D concentration, JIA subtypes, disease activity, medication, diet quality, sun exposure, and other factors. Next, given the high prevalence of vitamin D deficiency and because vitamin D is noted to be safe for adults in doses of up to 4000 IU/day , trials In conclusion, mechanistic and epidemiologic data support a role for vitamin D in promoting bone health and modulating disease status in JIA. However, additional evidence is needed to clarify the efficacy of vitamin D supplementation in patients with JIA and determine the optimal level of vitamin D for these patients.


CD47 performs a vital function in cancer therapy by binding to different SIRPα, thrombospondin 1, and integrin. However, its role in tumor immunity and its correlation with prognosis among many cancer types remain unknown. The raw mRNA expression data of CD47 in cancer patients was downloaded from TCGA and GTEx datasets. The protein expression of CD47 was detected using a microarray. Kaplan Meier analysis and forest plot were performed to compare the effects of high and low expression of CD47 on overall survival in different cancers. In addition, the correlations between CD47 expression and immune cell infiltration, stromal components, immune checkpoint genes, tumor mutational burden (TMB), and microsatellite instability (MSI) were analyzed from the public database. The gene function was determined by Gene Set Enrichment Analysis (GSEA). The expressions of CD47 in CHOL, COAD, ESCA, HNSC, KIRC, STAD, and THCA were higher compared with normal tissues. Elevated expression of CD47 predicted poor prognosis in ACC, KICH, KIRP, LGG, PAAD and UCEC. CD47 expression was strongly associated with immune infiltrating cells among KICH, KIRP, LGG, and PAAD. In addition, significant positive correlations with most immune checkpoint genes including PDCD 1 (PD-1), CD274 (PD-L1), CTLA4 in BLCA, DLBC, KICH, KIRC, LUAD, LUSC, PAAD, PCPG, SKCM, STAD, UCEC, and UVM was noted for the expression of CD47. GSEA analysis demonstrated that CD47 was a key regulator in metabolism-related pathways. These findings provide novel evidence that CD47 could be utilized as a promising prognostic biomarker and combination treatment target in various cancers. CD47 is a highly glycated protein distributed on the surface membrane of several cells, including red blood cells and non-hematopoietic cells, but its expression levels varied by cell types5. It is reported to mediate immune homeostasis and cell proliferation, migration, phagocytosis, and apoptosis. CD47 exerts these functions by binding to different ligands, including SIRPα, thrombospondin 1 , and integrin8. SIRPα, also known as protein tyrosine phosphatase substrate 1 (SHPS-1), is mainly expressed on myeloid cell membranes such as macrophages, myeloid dendritic cells, and monocytes; TSP-1 is a glycoprotein of extracellular matrix, composed of binding extracellular matrix components and cell surface receptors, secreted by macrophages, monocytes, platelets and a variety of non-hematopoietic cells10. Usually, CD47 appears as a "don't eat me" signal on healthy cells and could prevent phagocytosis by macrophages, whereas it is down-regulated on old and redundant cells which promotes the macrophages to remove them. CD47 on the cell surface is overexpressed in various cancer cells to help avoid the killing by innate immune cells.CD47 is a 47 kDa transmembrane protein, also known as integrin-related protein (IAP), composed of a short C-terminal intracellular variable spliced tail, five transmembrane helices, and an N-terminal extracellular IgV domain11 first discovered the expression of CD47 on ovarian cancers, subsequently, a series of studies have confirmed that CD47 is overexpressed on tumor cells compared with healthy cells, including acute lymphoblastic leukemia cells12, lymphoma cells7, non-Hodgkin’s lymphoma (NHL) cells13, glioblastoma14, myeloma cells15, bladder tumor cells16, head and neck squamous cell carcinoma cells17, breast18, pancreas19, liver20, lung21, and prostate cancer cells22. Additionally, a few studies found that poor prognosis correlated with increased expression of CD47 in oral squamous cell carcinoma23, nasopharyngeal carcinoma24, triple-negative breast cancers25, ovarian carcinoma26, and non-small cell lung cancer27. These data demonstrated CD47 is of vital importance in cancer progression. Brightwell et al. found that epithelial ovarian cancer patients with low expression of CD47 had a better treatment response than standard therapy as well as extended OS28. Reseachers have also demonstrated that a higher CD47 expression was associated with poor prognosis in clear cell renal cell carcinoma29, non-small-cell lung cancer27, triple negative breast cancer25, oral squamous cell carcinoma30, and hepatocellular carcinoma20. Consequently, CD47 predicted poor prognosis in a couple of cancers, however, its prognosis value in many other cancer types need to be investigated.In the 1980s, Knauf et al.9. CD47 is a novel potent immunotherapy target in malignancies, targeting CD47 may be a novel strategy for cancer immunotherapy. A variety of studies have verified the synergistic effect of anti-CD47 combinated with target therapy31, chemotherapy32, and radiotherapy33. One of the most exciting and promising combination is targeting CD47 and other immune checkpoints. Liu et al.34 found that dual blockade of both CD47 and PD-L1 improved therapeutic efficacy versus monotherapy by increasing DNA sensing, DC cross-presentation, and anti-tumor T cell response. Bispecific antibody targeting CD47 and PD-L1 could maximize antitumor immunity by activating type I interferon pathways, increasing antigen presentation in dendritic cells, macrophage, and Tcf7+ stem-like progenitor CD8 T cell populations35. Targeting CD47 in combination with PD-1 may potentially improve the outcomes of patients with anaplastic thyroid carcinoma36. Similarly, Dual targeting CD47 and CTLA-4 promotes immunity against colon cancers37. Anti-CD47 targeting in pancreatic cancer could remodel tumor- infiltrating immune cells38 and enhance CD19/CD3-bispecific T cell engager antibody-mediated lysis of B cell malignancies39. However, limited is known about the combination treatments of anti-CD47 and other immune checkpoint blockers except for PD1/PDL1/CTLA4.Mounting evidence demonstrates that inhibition of the SIRPa–CD47 innate immune checkpoint is an effective and novel way for cancer therapyIn this study, we performed a pan-cancer analysis between CD47 expression and patients’ prognosis. We further investigated the correlation of CD47 expression with the level of immune infiltration across a large variety of cancers, and explored whether immune checkpoint genes correlated with CD47 expression in 33 cancer types. Lastly, we enriched the functional pathways CD47 which may be involved in cancers. Our findings provide new insights linking CD47 with numerous tumor prognoses, effects and mechanisms of targeting CD47 in anti-tumor immunity. Of note, combination treatment of anti-CD47 and immune checkpoint inhibitors may provide new approaches and improvements for anti-cancer strategies.We downloaded transcriptome RNA-seq data of 33 cancers over 10,000 patients from TCGA (The Cancer Genome Atlas) database. The cancer types were listed in Supplement Table P < 0.05 was considered as a statistical difference.The data of CD47 expressions in both tumor and matched normal tissue were extracted from Genotype Tissue Expression (GTEx) and TCGA datasets. We used log2 (TPM+1) transformed expression data for plotting. Kaplan Meier analysis by log-rank test was applied to compare the overall survival (OS) for patients stratified based on the median gene expression level of CD47. Univariate Cox model was used to investigate the relationship between gene expression levels and patient survival in various cancers. 40. Furthermore, we explored the relationship between CD47 expression and immune checkpoint genes, Tumor mutation burden (TMB) as well as Microsatellite instability (MSI) analysis regarding the relationships were investigated by Sangerbox.To better understand the relation between CD47 and immunity, we analyzed the correlation between the proportion of the tumor infiltration cells and the expression of CD47 by TCGA datasets, such as B cells, CD8+ T cells, CD4+ T cells, dendritic cells (DCs), macrophages, and neutrophils. Additionally, Stroma Score and ESTIMATE Score were utilized to assess tumor purity and stroma cell proportionThe data was divided into two groups according to the level of CD47 expression, then enrichment analysis was performed by KEGG and HALLMARK depending on expression levels of CD47. Gene sets with |NES| > 1, NOM p-val < 0.05, and FDR q-val < 0.25 were determined as significantly enriched.P < 0.05 was considered as a statistical difference.The Student’s t-test was utilized to investigate the gene expression. Spearman’s correlation analysis was used to evaluate the CD47 expression with stroma cell proportion score or immune cell infiltration score or immune checkpoint genes score. All analyses were performed with the R software (version 3.6.1). The study was approved by the ethics committee of the Xiangya Hosptial, Central South University. All the methods and protocol with human data were performed in accordance with ethical standards of the ethics committee and the Helsinki Declaration of 1975 and its later amendments.Informed consent was obtained in all cases for the tissue microarray.To understand the relationship between CD47 and various cancers, we explored the RNA expression level of CD47 in different cancers and matched normal tissues. Initially, we evaluated CD47 expression level between the tumor and matched normal tissues across twenty cancer types via the TCGA database Fig. A, and thP < 0.001), KICH , KIRP , LGG , PAAD , UCEC  , KICH , KIRP , LGG , PAAD , UCEC ; CD4+ cells in one cancer type that is KIRP ; CD8+ T cells in four cancer types including KICH , KIRP , LGG , PAAD ; neutrophils in four cancer types types including KIRP , LGG , PAAD , UCEC ; macrophages in four cancer types including KICH , KIRP , LGG , PAAD ; and dendritic cells in six cancer types including ACC , KICH , KIRP , LGG , PAAD , UCEC   and UCEC . However, it was positively correlated with tumor purity in KIRP  and PAAD  . However, it was positively related to tumor purity in KIRP , PAAD   T cells, immune checkpoint inhibitors, cancer vaccines, therapeutic antibodies, and cell therapy47. As a novel immunotherapy target, to date, there are four major strategies of anti-CD47 treatment: (1) CD47 antibody can enhance cancer cell phagocytosis of macrophages by blocking the binding of CD47-SIRPα; (2) CD47 antibody or SIRPα-Fc fusion protein can also exert cytotoxic effects through the Fc segment; (3) CD47 antibody could directly induce tumor cells apoptosis via a caspase-independent mechanism; (4) CD47 antibody could work with prophagocytic molecules to make DC phagocytose tumor cells, and then present the antigen to T cells, thereby activating the adaptive immune response. Previous studies have also revealed that CD47 inhibition could promote CD8+ T cells for tumor cell killing and loss of CD47 in the tumor stroma increases the sensitivity of some tumors to standard therapy48. Anti-CD47 increased the pro-inflammatory macrophages and reduced the anti-inflammatory macrophages38. In the present study, we found CD47 was closely related to immune cells in various cancers. CD47 expression was positively associated with CD4+ T cell, CD8+ T cell, B cell, neutrophil, dendritic cell, and macrophage infiltration of numerous cancers. However, CD47 could play a different role among different cancers. Although loss or inhibitory of CD47 usually enhanced eliminating cancer cells, CD47 could positively and negatively regulate NK cell function in human melanomas, which may explain CD47 was associated with improved survival in melanomas. In addition, increased expression of CD47 was positively associated with TMB in BLCA, ESCA, SKCM, STAD, while negatively correlated with GBM, HNSC, KIRC, KIRP, LGG, PRAD, THCA, UCEC, and UVM. As far as MSI was conferred, elevated expression of CD47 was positively related to COAD, READ, STAD, while negatively associated with DLBC, HNSC, LUAD, LUSC, PAAD, PARD, SKCM, TGCT, and UCS. TMB and MSI analysis also supported its different roles in different cancers. It seems high expression of CD47 was negative with TMB/MSI in several poor prognosis cancers, while positively with TMB/MSI in a few favorable cancers. Taken together, CD47 expression was extensively related to cancer immunity.The tumor immune microenvironment is of great importance for cancer cell proliferation, metastasis, survival, and treatment resistance38. Combination blockade of CD47 and PD1, CTLA4 also achieved better anti-tumor effect37. Furthermore, a couple of ongoing clinical trials are targeting dual immune checkpoints such as CD47/PD-1(HX009), CD47/CD137 (DSP107), CD47/PD-L1 , CD47/CD20 (IMM0306), CD47/CD19 (TG-1801)49. The present analysis found elevated expression of CD47 not only positively correlated with PD1, PD-L1, CTLA4, but also with multiple other immune checkpoints such as IDO1, CD40, CD160, CD86, CD44 across various cancer types. Consequently, the novel combination of CD47 and other immune molecules bcould provide new strategies for cancer treatment.Furthermore, the level of CD47 was positively correlated with most immune checkpoint genes while also negatively with a few genes in pan-cancer analysis. Of note, previous studies have identified combination inhibition of CD47 and PD-L1 could lead to a synergistic inhibition effect on tumor growth in some pancreatic cancer cell lines50, which supported the effect of CD47 in phosphorylation. Other researchers found that CD47 deficiency could increas TGF-β1 expression by phosphorylation of the TGF-β1 receptor-regulated transcription factors SMAD-2 and SMAD-351, which supported the effect of CD47 in TGF-β pathway. Additionally, researchers also demonsrated that CD47 blockade or deficiency broadly prevents CD47 regulating metabolic pathways required to control glucose metabolism, oxidative stress, DNA repair, and energetics after exposure to ionizing radiation52 and combination blockade of CD47 and HER2 achieved an antitumor immune response in radioresistant breast cancer18, which provide evidence for the combination of CD47 and ERBB signaling. Those studies together with our analysis demonstrated CD47 closely correlated with metabolism pathways like TGF-β, phosphorylation and so on. Therefore, the combination of anti-CD47 with other immune checkpoint blockades or a combination of anti-CD47 with metabolism regulators could shed light on novel cancer treatment.Additionally, biofunctional analysis by KEGG and HALLMARK analysis on CD47 through GSEA in the current study suggested high CD47 expression was mainly associated with metabolism-related pathways. Similary, Previous studies have found that activation of CD47 from both neutrophils and epithelial cells caused a shift of specific signaling elements from a tyrosine-phosphorylated to a tyrosine-dephosphorylated status by phosphatidylinositol 3-kinase and tyrosine kinases, which facilitated neutrophil migration across endothelial and epithelial monolayersOur study has some limitations. Firstly, the mRNA level of CDC47 is assessed in our study, its correlation with immune checkpoint inhibitors or metabolism regulators needed to be validate in future experiments. Secondly, we analyzed the possible pathways associated with CD47, the results of bioinformatic analysis needed to be verified in biological experiments for further validation. Further mechanistic studies will be beneficial to undermine the value of CD47 as a potential target of immunotherapy at the molecular and cellular levels.In sum, all the data in the current study suggested the CD47 as an effective prognostic biomarker in various types of human cancers. Anti-CD47 treatment could be utiliezd as a promising strategy in a variety of cancers. Besides, our results shed light on the significant role of CD47 in tumor immunity and metabolic activity. However, more data are needed to clarify the mechanisms of CD47 in those activities. Future prospective studies will be focusing on the combination of anti-CD47 and immune checkpoint inhibitors or metabolism regulators that could provide an immuno-based strategy for cancer therapy.Supplementary Information.


Dmrt (Doublesex and Mab-3 related transcription factor) gene family is a class of crucial transcription factors characterized by a conserved DM domain related to sex determination and differentiation, which has been systematically described in various teleost fish, but less in channel catfish , an important global aquaculture species in the US and China. In this study, seven Dmrt genes from channel catfish genome were identified and analyzed using bioinformatics methods. Seven IpDmrt genes were distributed unevenly across five chromosomes. Synteny analysis revealed that Dmrt1, Dmrt2a, Dmrt2b, Dmrt3, Dmrt4, and Dmrt5 were relatively conserved in teleost fish. Tissue distribution analysis showed that IpDmrt1, IpDmrt2b, IpDmrt5, and IpDmrt6 exhibited sexually dimorphic expression patterns and, among them, IpDmrt1 and IpDmrt6 had high expression levels in the testes, while IpDmrt2b and IpDmrt5 had more significant expression levels in the ovaries than in other tissues. After 17β-estradiol treatment, IpDmrt2b and IpDmrt5 were significantly up regulated, while the expression of IpDmrt1 and IpDmrt6 was significantly repressed in XY channel catfish ovaries compared with XX channel catfish ovaries. The present study provides a comprehensive insight into the Dmrt gene family of channel catfish. The results suggest that IpDmrt1 and IpDmrt6 may play an important role in testis differentiation/development, while IpDmrt2b and IpDmrt5 are critical in ovary development in this species.The  Sex determination (SD) in vertebrates is a complicated physiological process, affected by both environmental and genetic factors . A diverDmy from Japanese medaka (Oryzias latipes) (Gsdf from Philippine medaka (Oryzias luzonensis) (Amh from Patagonian pejerrey (Odontesthes hatcheri) (Amhr2 from Japanese pufferfish (Fugu rubripes) (Sox3 from brackish medaka (Oryzias dancena)  (The reproductive modes of teleosts are divided into gonochorism and hermaphroditism . In natulatipes) , Gsdf fronensis) , Amh froatcheri) , Amhr2 fubripes) , Sox3 frdancena) , and Dmrilaevis) .Dmrt (Doublesex and Mab-3 related transcription factor) gene family is thought to be associated with sex determination and differentiation, embryo and gonadal development, as well as muscle growth, in vertebrates ranging from fish to tetrapods. This gene family belongs to the transforming growth factor-beta superfamily, which contains one or several classical DNA-binding domains. There are at least five Dmrt genes  in teleost fish, among which Dmrt1 is the major male-biased expression gene  is one of the most important freshwater aquaculture fish in the US and China  was selected as a vector to induce the sexual reversal of male fish by feeding catfish larvae continuously for 27 days. The dosage of 17β-E2 in the compound feed was 60 μg/g. The gonads of sex-reversed and normal female catfish at 60 dahs were identified by histological section and sex-linkage molecular marker . Artificial compound feed containing 17β-estradiol  and were stored at -20°C until RNA extraction. During sampling, the experimental fish were anesthetized using 0.1% tricaine methanesulfonate (MS-222) . All animal handling was carried out in accordance with the ethical guidelines and protocols of the Freshwater Fisheries Research Institute of Jiangsu Province.IpDmrt family genes were identified on the channel catfish genome using the BLAST search program based on the conserved DM domain  with amino acids. The identified candidates and their amino acid sequences were downloaded from the channel catfish genome database stored in the NCBI database (https://www.ncbi.nlm.nih.gov/). The genes were further confirmed to contain the conserved DM domains by scanning their sequences through the Pfam (http://pfam.sanger.ac.uk/search), SMART (http://smart.embl-heidelberg.de/), and InterPro (http://www.ebi.ac.uk/interpro/) online databases.Candidate Dmrt genes, we designed seven pairs of primers . The cDNA was subjected to reverse transcription using the PrimeScript™ first Strand cDNA Synthesis Kit  according to the manufacturer’s instructions. According to the DNA sequences of channel catfish  primers  to cloneIpDmrt genes, including the number of introns and exons, coding sequence length, and chromosome location, were extracted from the channel catfish genome database. The physicochemical characteristics of IpDmrt proteins, including the theoretical molecular weight (kDa), lengths, isoelectric points (pI), and grand average of hydropathicity (GRAVY), were evaluated using the online ExPASY tool (https://web.expasy.org/protparam/). The intron and exon positions, and untranslated regions were visualized using the Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/).Basic information related to the https://espript.ibcp.fr/ESPript/ESPript) online software, respectively. The amino acid sequences of Dmrt proteins from human (Homo sapiens), chicken , African clawed frog (Xenopus laevis), zebrafish (Danio rerio), large yellow croaker (Larimichthys crocea), Nile tilapia (Oreochromis niloticus), Japanese medaka, Japanese pufferfish, and channel catfish were selected to construct the phylogenetic tree in MEGA7 using the neighbor-joining method. The Jones-Taylor-Thornton (JTT) algorithm + Nearest-Neighbor-Interchange (NNI) distance were selected to automatically generate the initial tree (Default - NJ/BioNJ) model and evaluate the phylogenetic tree. The Dmrt genes were named using an “Ip” prefix and were numbered in ascending order based on the results of the phylogenetic analysis. The name of Dmrt protein sequences showed in Multiple sequence alignments of the IpDmrt proteins were performed and visualized using the T-COFFEE (tcoffee.crg.cat/apps/tcoffee/index.html) and EsPript 3.0 , Nile tilapia, zebrafish, and Atlantic cod (Gadus morhua), the Synteny Database (http://teleost.cs.uoregon.edu/synteny_db/) and Genomicus Browser (http://www.dyogen.ens.fr/genomicus-63.01/cgi-bin/search.pl) were used as the main visualization tools. To perform the synteny analysis, the channel catfish orthologs of the Dmrt genes neighboring other teleosts’ genes were firstly examined at the chromosome level through the Synteny Database, and at the local neighborhood level using the Genomicus Browser, which provides detailed gene information related to each teleost gene’s orthologs on an individual chromosome. In the synteny analyses of Dmrt genes, the channel catfish neighboring genes as follows: Fbrsl1, Ap1b1, Fbxo21, Fbxw8, Abca2, Adamts3, Dmrt2a, Dmrt3, Dmrt1, and Adgrv1 on Chr22; Abl2, Bend5, Dmrt5, Calr, Abhd17 ab, Tmem47, Tab3, Nexn, Armh1, Multh, and Acbd6 on Chr5; Irrc40, Irrc7, Insl5a, Ak4, Jak1, Foxd3, Kank4, Cyldl, Adcyaplr1b, and Dmrt2b on Chr11; and Ndrg2, Arhgef40, Casq1b, Ipcat4, Khnyn, Drap1, Rela, Elavl2, Dmrt4, Rbpms, and Alpk1 on Chr29.In order to examine the extent of preservation of genomic neighborhoods between IpDmrt gene family were constructed using the STRING online software (https://www.string-db.org).The purpose of protein interaction network analysis was to examine the relationship between Dmrt and other genes and explore their potential functions. The protein interaction networks of the α-tubulin  were identified in the channel catfish genome, and were named based on the nomenclature of teleost Dmrt genes and the results of phylogenetic analyses. These gene numbers were relatively consistent compared to that previously reported in other fish species, such as large yellow croaker (Ctenopharyngodon idella) (Oncorhynchus mykiss)   and rain mykiss) , which en nature .Each IpDmrt protein had a characteristic conserved DM domain , and only protein IpDmrt1 had an additional Dmrt1 domain, while a DMA domain was detected in IpDmrt3, IpDmrt4, and IpDmrt5 .The multiple alignment of IpDmrt protein sequences revealed the presence of a conserved DM domain at the N-terminal , while oIpDmrt gene family members, a phylogenetic tree was generated using 57 Dmrt protein sequences from various vertebrate species, ranging from fish to mammals, based on the maximum likelihood method. As shown in Dmrt1, Dmrt2a, Dmrt3, and Dmrt5 were found in all five teleost species. As expected, Dmrt genes were clustered into six subfamilies, which is consistent with previous studies  were observed in teleosts. However, Dmrt7 and Dmrt8 were lost in teleosts, and are only present in the mammal genome .Gene duplication is an important process in species evolution in terms of gene expansion and functional diversity. The aim of synteny analysis is to show whether genes are conserved during evolution in teleosts. Conservative regions can be clearly identified by observing the distribution and permutation of genes on chromosomes. In this study, in order to investigate the preservation extent of the Dmrt1-Dmrt2a-Dmrt3 syntenic block was highly conserved in teleosts  (Scatophagus argus)  , and spos argus) , to dateal years . In thisDmrt genes range from five (zebrafish) to twelve (common carp), and this is correlated not only with genome size but also with genomic duplication rounds. For example, only five Dmrt genes were identified in Atlantic cod, which has a genome of 686 megabases (Mb) (Dmrt genes. Both zebrafish and common carp (Cyprinus carpio), which belong to the order Cypriniformes, have relatively large genomes   are found in most 3R teleosts, which probably separated during the basal vertebrate genome duplication (2R).Previous reports have shown that the number of ses (Mb) , while Ases (Mb) , has ten,400 Mb) , but, asrt genes , while tDmrt genes have a close relationship with Wnt4, Foxl, Amh, and Cyp19a1. Amh (anti-Müllerian hormone) is considered necessary for female differentiation, because it regulates FSH to induce aromatase activity (Amh is closely related to the development of spermatogonia by interacting with its Amhr2 (anti Müllerian hormone receptor type 2) receptor in gonads (Foxl gene family members have different functions, and Foxl2 in particular is associated with ovary formation, as confirmed by numerous study (Wnt4, which regulates the development of the Müllerian duct by controlling the secretion of sterol in mammals (Cyp19a1 is a key gene for the balancing of the secretion levels of endogenous steroid hormones, and regulated by several upstream genes, including Dmrt1, Foxl2, Amh, and Wnt4.The protein interaction network analysis showed that activity . Moreoven gonads . The Foxus study . Another mammals . Even if mammals . It has  mammals . Cyp19a1IpDmrt genes were detected in several tissues based on the qRT-PCR assay. Among these genes, IpDmrt1 and IpDmrt6 had a higher expression in the testes, while IpDmrt2b and IpDmrt5 had a higher expression in the ovaries, indicating that IpDmrt1/IpDmrt6 and IpDmrt2b/IpDmrt5 may have important regulatory roles in male and female sex differentiation/development, respectively. The expression levels of IpDmrt1, IpDmrt2b, IpDmrt5, and IpDmrt6 in the ovaries of normal and XY channel catfish treated with 17β-estradiol also confirmed our hypothesis; in particular, IpDmrt2b and IpDmrt5 were significantly up-regulated in XY channel catfish ovary, while IpDmrt1 and IpDmrt6 were significantly repressed. Numerous studies have proved that Dmrt1 plays a dominant role in male sex differentiation in avians, turtles, frogs, and teleosts (Dmrt1 participates to antagonistic regulatory networks in association with Foxl2 to maintain the male fate in mammals. Dmrt6 is absent in most teleosts, such as zebrafish, medaka, fugu, and Atlantic salmon (Dmrt6 knockout in tilapia resulted in fewer spermatocytes and produced a lower level of serum 11-ketotestosterone (Dmrt2 gene, while the majority of teleosts present at least two orthologous Dmrt2 genes, Dmrt2a and Dmrt2b, of which the former is expressed in various tissues and promotes the transition of endochondral bone formation by linking Sox9 and Runx2 (Dmrt2b is different from Dmrt2a, Dmrt2b is significantly expressed in teleosts’ ovaries and is critical for their development (Dmrt5 is required for the terminal differentiation of corticotropes and gonadotropes; this gene regulates corticotrope differentiation in the pituitary gland in a cell-autonomous manner, thereby determining gonadotrope numbers (In the present study, the expression levels of teleosts . Howeverc salmon . This gec salmon . It seemosterone . Tetrapond Runx2 . Dmrt2b elopment . Dmrt5 i numbers .


We report a case of vertical transmission of Coxsackievirus (CV)-A6 with severe congenital pneumonia/sepsis. A male infant presented with severe respiratory symptoms at birth and was treated with full cardiopulmonary support, including inhaled nitric oxide. Three days before delivery, his older brother was diagnosed with hand, foot, and mouth disease (HFMD). His mother developed transient fever 1 day before delivery and presented a blister on her thumb 2 days after delivery. A multiplex polymerase chain reaction test on day 2 was positive for human rhinovirus/enterovirus. CV-A6 was later detected in the serum, tracheal aspirate, and stool of the patient sampled on day 6, and in the maternal serum sampled on the day of delivery. He was diagnosed with congenital CV-A6 pneumonia/sepsis caused by vertical transmission, based on VP1 consensus sequences used for typing of the virus that demonstrated a 100% match between the mother and infant. Further, the strain was closely related to the lethal CV-A6-Changchun strains in the phylogenetic analysis of the P2 region, which contributes to the pathogenicity. In conclusion, congenital CV-A6 infection should be considered if a woman exhibits HFMD symptoms during the perinatal period. Detailed virologic examination is useful for understanding its pathogenesis. Coxsackievirus (CV)-A6, a human enterovirus (EV) , has beeIn addition, through detailed virological studies, we demonstrate that the causative virus has a genetic background similar to that of the CV-A6 strains isolated from patients with HFMD in northeast China in 2013, which were highly virulent .2, measured by pulse oximetry) levels persisted under 60% on pulse oximeter; thus, oxygen supplementation was initiated 3 min after birth. Continuous positive airway pressure with 100% oxygen was initiated because his SpO2 levels did not increase with oxygen administration alone; however, the levels persisted below 95%.A 34-year-old woman, gravida 2, para 1, who had been treated for gestational diabetes with insulin therapy, delivered a male infant  at 40 and 5/7 weeks of gestation. Three days before delivery, her older son was diagnosed with HFMD, with a fever and typical rash. She developed a transient fever 1 day before delivery and presented a 2 mm diameter blister on her thumb 2 days after delivery. At delivery, her laboratory data demonstrated an increased C-reactive protein  level, normal white blood cell  count, and negative polymerase chain reaction (PCR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The infant cried and breathed spontaneously immediately after birth, and early skin-to-skin contact was initiated. He began grunting and his oxygen saturation  and in the maternal serum collected on the day of delivery by CODEHOP VP1 reverse transcription-seminested PCR [He was diagnosed with congenital CV-A6 pneumonia/sepsis caused by vertical transmission, based on the full length of the VP1 region that demonstrated a 100% match between the mother (Hyogo14341: GenBank accession no. LC743961) and infant (Hyogo14314: GenBank accession no. LC743960). Phylogenetic analysis of the VP1 region indicated that these strains were closely related to the strains detected in Hyogo Prefecture and China from 2017 to 2019 . In addiFurthermore, all eight amino acid substitutions that differed between the lethal and nonlethal groups were identical to those of Changchun097 and Changchun099, including three substitutions  in the 2C region that matched the three lethal strains  .The serum cytokine profiles on day 1 were consistent with those of a viral infection (increased interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte-colony stimulating factor (G-CSF), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) levels; CV-A6 is a recognized causative agent of herpangina; however, it has been reported as a new causative agent of HFMD in Asia and Europe since 2008 . In JapaWe considered two factors as the causes of severity. First, the viral infection (the onset of HFMD) occurred immediately before delivery; therefore, insufficient maternal neutralizing antibodies were produced against CV-A6, leading to vertical infection in the absence of transferred antibodies in the fetus and the development of fulminant symptoms. Based on the findings of a SARS-CoV-2 study, the production of neutralizing antibodies requires at least 2 weeks; thus, in our case, the fetus most likely did not have adequate immunity against CV-A6 . Second,Moreover, in this case, the serum cytokine profile was measured in residual sera at 1, 4, and 10 days of age, and the IL-6, IL-8, G-CSF, IFN-γ, and TNF-α levels significantly increased at 1 day of age. In a review, Zhang et al. reported on elevated IL-6, IL-8, G-CSF, IFN-γ, and TNF-α in severe EV 71 infection; thus, our results were consistent with the findings of severe EV infection .This case highlighted two aspects. First, CV-A6 can cause vertical infection, severe congenital pneumonia, and PPHN. Second, multiplex PCR can assist in the diagnosis  of congenital pneumonia of an unknown cause.In conclusion, congenital CV-A6 infection should be considered if a woman exhibits symptoms of HFMD during the perinatal period. Detailed virologic examination is useful for understanding the pathogenesis.