The interaction of a virus with a species-specific receptor is a primary genetic determinant of host tropism and therefore constitutes a major interspecies barrier at the level of viral entry (18–20). CR2 (complement C3d receptor 2) is expressed on the surface of B lymphocytes, and its binding to Epstein-Barr virus is a prerequisite for viral infectivity (21–23). SCR1 and SCR2 are the two most membrane distal CR2 repeats where EBV glycoprotein gp350/220 attaches (24, 25). To ascertain whether the genetic determinants would restrict the viral receptor affinity for tree shrews CR2, we analyzed the orthologs of CR2 and SCR1-SCR2, particularly the residues of the EBV receptor-binding domain (RBD), across a diverse set of species ( Figure 2C ). Phylogenetic analysis revealed that tree shrew was genetically closer to primates than rodents, based on the CR2 ( Figure 2A and Supplementary Table 1 ) and the SCR1-SCR2 protein sequences ( Supplementary Table 1 ). Through molecular modeling for human SCR1-SCR2 complexed with the EBV RBD, we found 32 potential binding sites on the receptor. Out of these sites, we identified 4 key residues that formed hydrogen bonds and provided a substantial amount of energy to stabilize the CR2-gp350 complex ( Figures 2B, C ). Of note, the key residues of tree shrew shared 100% identity with human, which was much higher than Sapajus apella (50%), Oryctolagus cuniculus (50%), and Rattus norvegicus (25%) ( Figure 2C ). Finally, we compared the distribution of CR2 in 9 different tree shrew tissues using RT-qPCR. High expression of CR2 was observed in the spleen, lymph node, and thymus, with limited expression in other tissues ( Figure 2D ). Moreover, results of immunohistochemical analysis indicated that CD21 (CR2)-positive cells were mainly distributed in the germinal center, while CD3 staining was largely absent in the germinal centers of the spleen ( Figure 2E ). Overall, these results indicate that tree shrew may be an ideal animal model for studying EBV infection.

Human peripheral blood mononuclear cells (PBMCs) are susceptible to EBV infection and EBV promotes proliferation and immortalization of infected cells in vitro (26, 27). To determine if tree shrew PBMCs had similar characteristics, we first examined the effect of different virus concentrations on the proliferation of tree shrew PBMCs. As shown in Figure 3A , a significant increase in the number of tree shrew PBMCs was observed (P < 0.01 compared to control group) after 21 days of co-culture with 1x10⁷ copies of EBV. The number of PBMCs in this group showed continuous proliferation throughout the observation period, while the number of PBMCs in the other two groups decreased significantly after day 28. A similar trend was observed using the CCK8 assay indicating that increase in cell proliferation was consistent with the increase in cell numbers ( Figure 3B ). Therefore, 1x10⁷ copies of EBV were used for subsequent assays. Cell morphological analysis clearly showed that PBMCs co-cultured with EBV had a tendency to grow in clusters after 14 days and these clusters gradually formed compact round-like multicellular aggregates over time ( Figure 3C ). However, apoptosis was induced in control cells cultured without EBV. To further verify that tree shrew PBMCs are indeed susceptible to EBV infection, we detected the expression of EBV genes (BLLF1, BMRF1, LMP1, EBNA1, EBNA3A, and EBNA3B/3C) in the tree shrew PBMCs throughout the co-culture period ( Figure 3D ). These results suggest that tree shrew PBMCs were susceptible to EBV infection.

We also assessed the susceptibility of adult tree shrews to EBV infection. Six tree shrews were inoculated with 200ul (1x10⁸ copies/ml) EBV suspensions via the femoral vein injection. The body weight (BW) of each animal was measured using an electronic scale before inoculation (day0) and after infection at various time points (day 3, day 7, day 14, day 21, day 28, day 35, day 42, day 49, day 56, day 70, day 84) to monitor BW changes during the whole experimental period. Figure 4A shows that the BW initially decreased and then increased over time. Specifically, inoculation of tree shrews with EBV caused significant weight loss at 3 days and 7 days post-inoculation (dpi) (p < 0.01 to 0 dpi). However, the lowest BW was recorded at 3 dpi, after which the BW gradually recovered until14 dpi when the weights were indistinguishable from 0 dpi (p > 0.05 to 0 dpi, Figure 4A ). Interestingly, loss of BW was also observed at 49 dpi (p <0.05 to 0 dpi), probably due to viral resurgence in tree shrews which will be described in detail later. The rectal temperature (RT) was also taken simultaneously with the BW measurement, using a mercury thermometer. The results indicated that RT began to rise after inoculation and reached the maximum mean temperature on 7 dpi (39.03 ± 0.62°C, p < 0.01 to 0 dpi), after which it gradually returned to normal (0 dpi, 37.43 ± 0.37°C) ( Figure 4B ). An increase in RT was also observed at 49 dpi, corresponding to the time of BW loss mentioned earlier. Remarkably, there were no mortalities throughout the experiment, although most tree shrews presented with symptoms such as fever and weight loss, while other clinical signs such as reduced activity, increasing weakness, slight diarrhea and wet-tail were observed in a few animals. These features might be a part of Infectious Monocytosis (IM) which is considered to be a self-limited infection caused by primary EBV infection (28, 29).

The WBC (white blood cell) count and the main immune cell subsets (lymphocytes, neutrophils, monocytes) counts and percentages in the peripheral blood of tree shrews were measured at 0 dpi, 3 dpi, 7 dpi, 14 dpi, 21 dpi, 28 dpi, 42 dpi, and 56 dpi. The WBC count was significantly elevated at 3 dpi ( Figure 4C , p < 0.01 to 0 dpi). At the same time, we also observed an increase in total cell counts and percentages of lymphocytes ( Figure 4E , p < 0.01 to 0 dpi) and monocytes ( Figure 4F , p < 0.05 to 0 dpi). Unexpectedly, a decline in neutrophils levels, the first line innate immune defense for the host against microbes and viruses (30, 31), was observed ( Figure 4C , p < 0.05 to 0 dpi). The immuno-suppressive effect of EBV is one of the mechanisms of evading immune surveillance to establish life-long infection in the host (32, 33). Later in the manuscript, we investigated if EBV evades immune surveillance by dampening the functions of neutrophils.

To further characterize the dynamics of EBV infection in vivo, blood, plasma and throat swab samples were collected from tree shrews throughout the experimental period and subjected to virological assays. EBV viremia was readily detected in blood and plasma samples of all the animals at 1-3 dpi and 49 dpi after femoral vein infection ( Figures 4H, J ). The peak viral load of blood samples appeared at 1 dpi and the viral loads ranged from 10^5.19 to 10^5.59 copies/ml ( Figure 4G ), however the peak of plasma samples ranged from 10^2.90 to 10^4.73 copies/mL at 3dpi ( Figure 4I ). In addition, EBV viremia was observed in all animals for the first 3 consecutive days after inoculation, but thereafter viremia was only observed in some animals during the experimental period of 91 days ( Figures 4H, J ). These results were in line with the characteristics of EBV that can lurk in the human body for a long time after infection without any symptoms and thereafter be activated to cause recurrent infections (3, 34). Interestingly, the EBV DNA in throat swab was significantly delayed with respect to the viremia kinetics, and was only detectable at 7 dpi and 49 dpi in most animals (100%). The mean viral loads and duration in throat swabs were appreciably lower than those in blood and plasma ( Figures 4K, L ).

To determine the tissue tropism of EBV in tree shrews, six animals (three untreated and three treated with EBV) were sacrificed at 91 dpi. Quantitative PCR (qPCR) analysis showed the presence of EBV DNA in multiple peripheral organs, including the lung, liver, spleen, lymph nodes, stomach and genitals ( Figure 4N ). Similar to EBV infection in humans, the immune organs (spleen and lymph nodes) had higher loads and positivity rate of the virus ( Figures 4M, N ) (35). No viral DNA was detected in untreated control animals. Interestingly, there was no significant difference between the control animals and EBV infected animals with respect to the organ coefficient of spleen ( Figure 4O ). This suggested that tree shrews could carry long-lasting, asymptomatic EBV infection.

Moreover, LMP1 protein was detected in the spleen and lymph nodes using immunohistochemistry ( Figure 5B , positive and negative controls pictures in Supplementary Figure 3 ). To further confirm the presence of EBV, Epstein Barr virus-encoded RNA (EBER) was detected using in situ hybridization studies. Clear EBER-positive puncta were observed in PBMCs and spleen ( Figure 5A , positive and negative controls pictures in Supplementary Figure 3 ). Similar results were observed in all experimental animals after being sacrificed.

To better understand the biological processes of the EBV primary infection in vivo, we performed RNA sequencing (RNA-seq) of blood samples at five distinct time points (0 dpi, 3 dpi, 7 dpi, 14 dpi, 28 dpi, three replicates per time point). First, we used Mfuzz (36) to cluster genes with similar temporal expression profiles. Results of cluster analysis showed that there were six types of expression patterns including; initial increase in gene expression that peaked at 3 dpi (Cluster 1); initial decline in expression that reached its lowest at 3 dpi (Cluster 2); increase in expression from 3 dpi that peaked at 7 dpi (Cluster 3); decline in expression from 3 dpi that reached its lowest at 7 dpi (Cluster 4); increase in expression from 7 dpi that peaked at 14 dpi (Cluster 5); and increase in expression from 14 dpi that peaked at 28 dpi (Cluster 6) ( Figure 6A ). The increase indicated up-regulation, while the decline indicated down-regulation, and the greater the change in gene expression, the more important the genes. Hence, we considered the genes in the six clusters as the characteristic genes for each time point depending on when maximum or minimum expression was observed. Specifically, genes in cluster 1 (C1) were the characteristic up-regulated genes at 3 dpi, genes in cluster 2 (C2) were the characteristic down-regulated genes at 3 dpi, genes in cluster 3 (C3) were the characteristic up-regulated genes at 7 dpi, genes in cluster 4 (C4) were the characteristic down-regulated genes at 7 dpi, genes in cluster 5 (C5) were the characteristic up-regulated genes at 14 dpi, while genes in cluster 6 (C6) were the characteristic up-regulated genes at 28 dpi, as shown in Figure 6B . Then we used DESeq2 (37) to identify the differentially expressed genes (DEGs) between each time point after infection and 0 dpi ( Supplementary Table 3 ). A total of 879 DEGs between 3 dpi and 0 dpi were identified, including 524 up-regulated genes (i.e., C1) and 355 down-regulated genes (i.e., C2) ( Figure 6C ). Figure 6D shows the validation results of the DEGs (TXNL1, PYCARD, COX17) using RT-qPCR. A total of 12 genes (two genes per cluster) are shown in Supplementary Figure 2 .

In order to explore the biological functions of the DEGs, we performed Gene Ontology (GO) enrichment analyses ( Supplementary Table 4 ). After filtering, 32 GO categories were further collapsed into 8 pathways ( Figure 6E ) using the GOSemSim package (38). At 3 dpi, pathways involving antigen processing and presentation (C1, pathway2) as well as nucleic acid, protein and energy metabolism (C1, pathway7) were enriched in response to EBV infection. Interestingly, the immune functions of neutrophils and T cells were suppressed over the same period (C2, pathway 1, 3 and 4). On the one hand, a large number of proteins were processed to maturity and transported to participate in the defense responses (C3, pathway 6) at 7 dpi, but immune suppression still persisted (C4, pathway 3). At 14 dpi and 28 dpi, similar biological processes were observed including active processing and transport of proteins (C5 and C6, pathway 6 and 7). In addition, viral transcription-related pathways were enriched at 7 dpi and 14 dpi (C3 and C5, pathway 8).

As mentioned in the preceding sections, a decrease in the number and function of neutrophils was observed in tree shrews at 3 dpi. To further confirm this phenomenon, we performed a series of experiments. First, the serum levels of Neutrophil peptide 1-3 (NP1-3) and Neutrophil activating protein-2 (NAP-2) were determined using ELISA assays in the tree shrews at 3 dpi, 7 dpi, 14 dpi, 21 dpi, and 28 dpi. The results showed a significant decrease in proteins-related to neutrophil functions at 3 dpi ( Figures 7A, B ; p < 0.05 to 0 dpi). Moreover, peripheral blood smears revealed morphological changes in neutrophils at 0 dpi and 3 dpi. As shown in Figures 7C, D , EBV infection induced an increase in the percentage of immature neutrophils (rod-shaped nucleus) and neutrophils which contained specific intoxication particles in the cytoplasm (p < 0.05). Two publicly available RNA-seq data (GSE85599 (39) and GSE45918 (40)) of IM from the Gene Expression Omnibus (GEO) database were used to validate these results in humans. IM is considered to be a symptom of EBV primary infection (41). Downregulated genes from the RNA-seq data were subjected to gene set enrichment analyses for GO-biological process. Supplementary Table 5 and Figures 7E, F show the results of pathways enrichment analysis, clearly displaying that neutrophil activation pathways, neutrophil-mediated immune pathways, neutrophil chemotaxis pathways and neutrophil migration pathways were significantly enriched. Additionally, to further understand the relationship between neutropenia and percentage of rod nuclear neutrophils and clinical symptoms, the data of 138 inpatients with IM were retrospectively analyzed ( Supplementary Table 6 ). Neutropenia (neutrophil count ≤ 2x10⁹/ml) was recorded in 44.93% (62/138) of the patients ( Figure 7G ). Among the patients with neutropenia, 74.19% (46/62) had records of peripheral blood smear examination, with 45.65% (21/46) of the samples showing increase in the percentage of rod nuclear neutrophils ( Figure 7H , percentage ≥ 5%). This suggests that decreased neutrophil count and the increase in the percentage of rod nuclear neutrophils are commonly associated with EBV infection. Moreover, results of regression analysis revealed that there was a moderate positive correlation among percentage of rod nuclear neutrophils, days of fever ( Figure 7I , R² = 0.5478, P = 0.0092) and copies of EBV DNA in blood ( Figure 7J , R² = 0.5800, P = 0.0040). Thus, a decrease in neutrophil count as well as the increase in the percentage of rod nuclear neutrophils are potential immune escape strategies for EBV.

To uncover the critical regulatory genes, we first extracted genes from the neutrophils function related pathways and constructed a PPI network through STRING database (https://string-db.org/). Genes with the top10 DMNC scores were identified as hub genes using Cytoscape plug-in cytoHubba ( Supplementary Table 7 ). As shown in Figure 7K , the darker the red color, the higher the scores. We further calculated the weight value of top10 genes according to GO semantic similarity using GOSemSim package ( Figure 7M ). Finally, critical genes (PYCARD and NCSTN) were selected by taking the intersection of the top 5 genes of DMNC scores and top 5 genes of weight value ( Figure 7L ). Moreover, it was interesting that the FPKM values of PYCARD and NCSTN in the GSE45918 data set had a strong negative correlation with the corresponding copies of EBV DNA in blood ( Figures 7N, O , r = -0.8503, p = 0.0115).

The reference Epstein-Barr Virus Receptor (CR2, complement C3d receptor 2) protein sequence used for constructing the distinct phylogenetic branches and identity analysis, were obtained from the GenBank sequence database of NCBI under the following accession numbers: Homo sapiens (NP_001006659.1), Macaca mulatta (XP_014973177.2), Sapajus apella (XP_032144963.1), Tupaia chinensis (XP_014447439.1), Oryctolagus cuniculus (XP_017203311.1), Rattus norvegicus (NP_001099459.2) and Sus scrofa (XP_020918993.1). Phylogenetic analyses of the CR2 protein sequence were performed with the MEGA (version: 7.0.26). Phylogenetic trees were assembled via the Neighbor-Joining method of the Maximum Composite Likelihood model (74). Statistical evaluation of the branching pattern was performed by bootstrap analysis of 1,000 replicates. The EMBOSS NEEDLE online program (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) was applied to reveal the identity scores among protein sequences.

Homo sapiens SCR1- SCR2 protein sequence was obtained from RSCB Protein Data Bank (PDB; http://www.rcsb.org/pdb/), PDB id: 1LY2. A global-local alignment (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) between the Homo sapiens SCR1- SCR2 protein sequence and the other species CR2 segment was performed to identify the SCR1- SCR2 homologous protein segment across species ( Supplementary Data 1 ). The EMBOSS NEEDLE online program was employed to determine the identity scores among protein sequences. The 3D spatial structure of the SCR1- SCR2 protein (PDB ID: SCR1-21ly2) and receptor-binding domain (RBD) of EBV (gp350) (PDB ID: SGP3502h6o) were obtained from RCSB Protein Data Bank. Molecular modeling and visualization of the protein-protein (SCR-gp350) interactions were performed using the Chimera software (http://www.cgl.ucsf.edu/chimera, in the public domain). Two residues were defined to be in contact if the distance between any of the atom pairs was less than 5Å. Homo sapiens SCR1-2 residues having contact with gp350 were screened by executing the command “select:.#1 &:.#0 zr < 5” and “select:.#0 &:.#1 zr < 5”. Subsequently, using the EMBOSS NEEDLE online program, the Homo sapiens residues were compared to those of corresponding residues in other species. Lastly, water molecules were removed and hydrogen-bond interactions for these residues were explored using Chimera software.

EBV viral supernatant was obtained from the B95-8 strain (NCBI: txid 10377) known to release high titers of transforming EBV. All experiments involving infectious EBV were conducted in biosafety level 2 (BSL2) facilities. B95–8 strain was cultured in 1640 Medium supplemented with 10% FBS and 1×antibiotics (100 kU/l penicillin, 100 mg/l streptomycin) at 37°C and 5% CO₂ in a humidified incubator for 14 days. Next, the whole culture was transferred to a 50 mL centrifuge tube and freeze-thawed thrice to lyse cells and release the virus. The culture was centrifuged at a low speed (1200xg) to remove the cell debris. The supernatant was filtered via a 0.45-μm centrifugal filter device. The filtrate fluid was centrifuged at high speed (16,000 × g for 90 min at 4 °C) and resuspended in 1ml of 1640 Medium to acquire the viral supernatant for analysis. The viral supernatant was stored at −80°C until tree shrew infection (75). DNA was extracted from 200µl viral supernatant to determine viral genome copy numbers before use.

PBMCs were isolated from whole blood by Ficoll (Solarbio, Beijing, China) gradient centrifugation following manufacturer guidelines. PBMCs (1×10⁶ cells) were inoculated on 24-well plates and cultured under the same conditions as the B95-8 strain. PBMCs were assigned to three experimental groups and one control group, with three replicates per group. Subsequently, 1×10⁵ copies, 1×10⁷ copies, and 1×10⁹ copies of EBV were added in the three experimental groups, respectively, and to the control group with the same amount of 1640 medium. Following EBV infection, cell proliferation on Day 7, Day 14, Day 21, Day 28, Day 35, and Day 45 was evaluated using the CCK-8 kit (Solarbio, Beijing, China). Next, 10 µl of CCK8 solution was added to each well, and cells were incubated at 37°C for another 5 hours. The optical density was read at 480 nm by a microplate reader. The number of viable cells was manually counted under a microscope using a hemocytometer at the same time points as CCK8. According to the optimal EBV infection concentration determined above, vitro cultures were further prepared, and cell morphology was examined under a light microscope on Day 7, Day 14, Day 28, and Day 45. Human PBMCs were acquired from healthy volunteers [EBV DNA-negative, from another clinic study by our team, Ethical Approval No: 2020 (KY-E-135)] served as positive controls for better assessment of morphological changes. Total RNAs of PBMCs were extracted with the TRNzol Universal Reagent Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. RNA quantity and purity were determined using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, USA).

Six tree shrews (F1 generation, Production approval number: SCXK (Dian) 2020–0004, 8 ± 2 months, male and female unlimited, 138.1 ± 7.2 g) were acquired from the Kunming Institute of Zoology, Chinese Academy of Sciences. The protocols used in this study were approved by the Animal Ethics Review Committee of Guangxi Medical University (approval number: 202005015). The experimental protocols strictly followed the ‘Guiding Principles for the Use and Care of Experimental Animals issued by the Ministry of Science and Technology of China. The total number of animals and their suffering was minimized according to the 3R principle.

Six tree shrews were injected with 200ul EBV suspensions (1x10⁸ copies/ml) via the femoral vein as described previously. Samples were isolated the day before inoculation as 0 dpi. Blood and throat swab samples were collected on the 0 dpi, 1 dpi,2 dpi,3 dpi,7 dpi, 14 dpi, 21 dpi, 28 dpi, 35 dpi,42 dpi, 49 dpi, 56 dpi, 63 dpi, 70 dpi, 77 dpi, 84 dpi, and 91 dpi (femoral vein blood, 1 ml each time). Serum and plasma were immediately separated through centrifugation (3,000 g at 4°C) and stored at −80°C. No EBV was detected in any samples at 0 dpi. Genomic DNA was extracted from 150 µl of blood samples using the QIAamp DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) and eluted in 30 µl of nuclease-free water. Total RNA was extracted from 200 µl of blood samples with the RNAprep Pure Hi-Blood Kit (Tiangen, Beijing, China). Viral nucleic acid was extracted from 150 µl of tree shrew plasma with the QIAamp MinElute Virus Spin Kit and eluted in 20 µl of nuclease-free water. Genomic DNA of throat swab samples was extracted using TIANamp Swab DNA Kit (Tiangen, Beijing, China). The quantity and purity of the extracted nucleic acid were determined by a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, USA). Serum samples were used to detect NP1-3 and NAP-2 levels using an ELISA kit (Cusabio, Wuhan, China) following the manufacturer’s guidelines.

Three tree shrews were sacrificed at 91 dpi with an overdose of sodium pentobarbital. Subsequently, multiple tissues, including the lung, liver, spleen, lymph nodes, stomach, and genitals were harvested. Genomic DNA was extracted from 20mg of each tissue using the QIAamp DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany). The remaining tissue was fixed, dehydrated, paraffin-embedded, and sectioned. For the control experiment, tissues were obtained from three healthy tree shrews. Total RNA was extracted from 20mg of the control tissues (lung, liver, spleen, lymph nodes, thymus, stomach, kidney, and brain) for CR2 detection with the RNeasy Protect Mini Kit (Qiagen, Hilden, Germany) and eluted in 60 µl of nuclease-free water.

EBV DNA copies of viral supernatant, blood, plasm, throat swab, and tissue samples were quantified by qPCR using primers (forward primer, 5′- TCT TAG GAG CTG TCC GAG GG -3′; reverse primer 5′- CCC AAC ACT CCA CCA CAC C -3′) and a probe that targets the EBV BamHI W region (probe, 5′- CAC ACA CTA CAC ACA CCC ACC CGT CTC -3′) (76). Standard curves were generated from genomic DNA of Namalwa cell line (containing two integrated viral genomes/cell) (77, 78), purchased from the Chinese Academy of Sciences Institute of Cell Resource Center (Shanghai, China). The culture conditions for the Namalwa cells and the DNA extraction protocol were as previously described. For PCR, Takara Premix ExTaq™ (Probe qPCR) was used with manufacturer-recommended reaction conditions. All experiments were repeated 3 times. The virus load of samples was determined according to the appropriate standard curve (PCR amplification efficiency (E) of 95–105% and correlation coefficient (R²) > 0.995) by the Bio-Rad CFX96 detection system.

RNA (800 ng) was reverse transcribed to cDNA using the PrimeScript™ RT reagent Kit with gDNA (Takara, Dalian, China). RT-PCR was performed with TaKaRa Ex Taq^® (Takara, Dalian, China) according to the manufacturer’s protocol. RT-PCR detection of the expression of BLLF1, BMRF1, LMP1, EBNA1, EBNA3A, EBNA3B/3C was determined using primer sequences presented in Supplemental Table 2 . The “housekeeping gene” β-actin was amplified as an internal control. cDNA from B95–8 cells served as a positive control. Lastly, 3% agarose gel electrophoresis (120 V for 32 min) was performed to visualize the bands under UV light. All the experiments were repeated three times.

Sequences of Epstein-Barr virus-encoded small RNA (EBER1 and EBER2) probes were acquired from the literature (79, 80). Highly sensitive digoxigenin-labeled oligonucleotide probes were synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China). Briefly, tissue sections (thick 5 um) were dewaxed regularly and digested for 30 min by pepsin (2 min of PBMCs smear) to expose RNA. Pre-hybridization was performed for 2-4 hours in a wet box with an aqueous solution of 20% glycerin to reduce the nonspecific reaction. The working concentration of the probe was 4μg/ml. A denaturation step was performed at 95°C for 15min. Subsequently, a hybridization reaction was performed using the Enhanced Sensitive ISH Detection kit (BOSTER, Wuhan, China). Nuclear fast red (Solaibao, Beijing, China) served as a chromogen. B95-8 cells were used as a positive control.

Antibodies used for immunohistochemical experiments are listed in Supplemental Table 8 . Blood was drawn from the femoral vein and cardiac of the animals before they were sacrificed and PBMCs isolation was performed as previously described. PBMCs were used to make a cell smear using slides treated with 0.1% gelatin/0.1% chromium potassium sulfate for improved cell attachment Tissue samples of spleen and lymph nodes were prepared and subjected to immunohistochemical analysis using SP-HRP kits (SP-9000, ZSGB Biotechnology Co. Ltd., Beijing, China) following the manufacturer’s instructions.

Blood samples harvested at 0, 3, 7, 14, and 28 dpi (three replicates per group) were used for global transcriptome analysis. Oxford Nanopore Technologies (ONT) sequencing is emerging as a new generation sequencing technology based on nanopore single-molecule real-time electrical signal (81). In most cases, the NGS RNA-Seq technology cannot assemble the entire transcripts and recognize an Isoform (transcript of homologous gene, superfamily gene, and allele expression), making it challenging for us to understand life processes. Full-length transcriptome sequencing based on ONT does not require RNA breakage; rather, RNA is directly reverse transcribed to obtain full-length cDNA. The experimental workflow is shown in web extra (https://nanoporetech.com/). The original data format of the Nanopore sequencing output data is the second-generation fast5 format containing all original sequencing signals. Base-calling was performed with the Guppy software in the MinKNOW2.2 package and data in fast5 format was converted to fastq format for subsequent quality control analysis. Short sequences (length<500bp) and low-quality reads (Qscore<6) of raw fastq data were filtered. Notably, if both ends of reads were identified as primers, they were determined as full-length sequences according to the cDNA sequencing principle. The full-length sequences statistics are shown in Supplemental Table 9 . Consistent sequences of each sample were aligned with the reference genome (http://www.treeshrewdb.org/) by minimap2. Then, we filtered out sequences with identity lower than 0.9 and coverage lower than 0.85 to remove redundant for alignment result. CPM (counts per million) (82) served as an indicator to measure gene expression level. Differential expression analysis was performed by DESeq2 (37) with a fold change cutoff of 1 and a p-value cutoff of 0.05.

The Ethics Committee of the First Affiliated Hospital of Guangxi Medical University approved this study [Approval number: 2021(KY-E-112)]. A total of 138 cases of IM which recorded the count of neutrophils were identified following a search of the First Affiliated Hospital of Guangxi Medical University case archives from 2015 to 2020.

All statistical data were analyzed with GraphPad Prism Software version 9.0.0 (GraphPad Software Inc., La Jolla, CA). Outliers were first detected and removed using the “ROUT” method to clean the data. The Shapiro-Wilk normality test was performed on clean data to assess for normality. For normally distributed data, comparison of two groups was performed via Unpaired Student t-test, whereas the Mann–Whitney U-test was applied when data did not conform to normal distribution. Comparison of more than two groups was executed using an ordinary ANOVA test with Tukey’s post hoc test when data were normally distributed with equal variance. However, for data that did not conform to normal distribution or variance was significantly different between groups, non-parametric tests were applied. P < 0.05 denoted statistical significance.