ObjectiveTo investigate the potential mechanisms of Zingiberis Rhizoma in treating inflammatory bowel disease （IBD） by integrating network pharmacology with in vitro and in vivo experiments. MethodsTraditional Chinese Medicine Systems Pharmacology Database And Analysis Platform （TCMSP）， Traditional Chinese Medicine Integrated Database （TCMID） Database were used to obtain the active component targets of Zingiberis Rhizoma. GeneCards was used to obtain the IBD targets. DAVID was used to perform Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analyses on core targets. Cytoscape 3.10.2 was used to establish the "active component-disease target-signaling pathway" interaction network. Mice were randomly assigned to control， model， and Zingiberis Rhizoma （400 mg·kg^-1） groups. An IBD model was induced via dextran sulfate sodium （DSS）. The colonic tissue was collected post-treatment to assess histology， expression of Ly6C⁺ monocytes/macrophages， and mRNA levels of Toll-like receptor 4 （TLR4）， and inflammatory cytokines. The effect of Zingiberis Rhizoma aqueous extract on RAW264.7 cell viability was evaluated. Furthermore， the effects of the extract at 100， 10， and 1 mg·L^-1 on LPS-induced differentiation of RAW264.7 cells into Ly6C^hi monocytes/macrophages， mRNA levels of TLR4 and inflammatory cytokines， and protein levels of factors in the TLR4/mitogen-activated protein kinase （MAPK） signaling pathway. ResultsA total of 241 targets were identified for Zingiberis Rhizoma and 6 787 for IBD， with 122 shared targets among Zingiberis Rhizoma， ulcerative colitis （UC）， and Crohn's disease （CD）. The enrichment analyses yielded 297 GO terms and 88 KEGG pathways. Associations were noted between Zingiberis Rhizoma's active component targets and IBD targets. In vivo experiments： Compared with the control group， the model group showed decreased body weight and disease activity index （DAI）（P<0.01）， shortened colon length， damaged mucosal epithelium with inflammatory cell infiltration， raised pathological scores （P<0.05）， increased Ly6C^hi and Ly6C^lo monocytes/macrophages （P<0.05）， and up-regulated mRNA levels of TLR4， TNF-α， IL-1β， and IL-6 （P<0.05） and protein levels of TLR4， phosphorylated extracellular signal-regulated protein kinase 1/2 （p-ERK1/2）， and phosphorylated p38 MAPK （p-p38 MAPK） （P<0.05）. Zingiberis Rhizoma intervention reversed these changes and reduced Ly6C^hi monocytes/macrophages （P<0.01）. In vitro experiments： compared with the control， LPS increased the proportion and number of Ly6C^hi monocytes/macrophages and mRNA levels of TLR4， TNF-α， IL-1β， and IL-6 （P<0.01） and enhanced the expression of TLR4， p-ERK1/2， and p-p38 MAPK （P<0.05）. Zingiberis Rhizoma reduced Ly6C^hi monocytes/macrophages （P<0.05）， down-regulated the mRNA levels of inflammatory cytokines （P<0.05）， and suppressed the TLR4/MAPK pathway （P<0.05）. ConclusionZingiberis Rhizoma alleviates IBD by suppressing the TLR4/ERK/p38 MAPK signaling pathway and reducing inflammatory cytokine levels in Ly6C^hi monocytes/macrophages.

ObjectiveTo clarify the repair effect of Mume Fructus on the intestinal mucosal epithelial barrier in the mouse model of inflammatory bowel disease （IBD） and explore the repair mechanism. MethodsThirty-six male C57BL/6 mice were randomly assigned into six groups： normal， model， low-， medium-， and high-dose （200， 400， and 800 mg·kg^-1） Mume Fructus， and sulfasalazine （300 mg·kg^-1）. Except the normal group， the rest groups had free access to 2% dextran sulfate sodium （DSS） solution for seven days to establish the IBD model， followed by a seven-day drug intervention. The body weight change and disease activity index （DAI） were recorded. After the last administration， spleen and colon tissue samples were collected to analyze the differences in colon length and spleen index. Hematoxylin-eosin staining was used to observe the morphology of the colon tissue. The level of diamine oxidase （DAO） in the serum was measured by the DAO assay kit. Immunohistochemistry was employed to determine the expression of tight junction proteins such as Claudin-1， Occludin， and zonula occludens-1 （ZO-1） in the colon tissue. Real-time PCR was performed to measure the mRNA levels of tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） in the colon tissue. Finally， Western blot was employed to determine the protein levels of mitogen-activated protein kinase kinase （MEK）， extracellular signal-regulated kinase （ERK）， phosphorylated （p）-MEK， and phosphorylated ERK in the colon tissue. ResultsCompared with the normal group， the model group exhibited decreases in body weight and colon length （P<0.01）， increases in DAI， spleen index， and serum DAO level （P<0.01）， damaged colonic epithelium and goblet cells， and obvious infiltration of inflammatory cells. In addition， the model group exhibited higher positive expression of Claudin-1， Occludin， and ZO-1 （P<0.01）， higher mRNA levels of TNF-α and IL-1β （P<0.01）， and higher protein levels of p-MEK and p-ERK （P<0.05， P<0.01） than the normal group. However， sulfasalazine and three doses of Mume Fructus markedly decreased the body weight and DAI （P<0.05）， recovered the colon length and spleen index， alleviated colon tissue damage， lowered the level of DAO in the serum （P<0.01）， and down-regulated the mRNA levels of TNF-α and IL-1β （P<0.01） and the protein levels of p-MEK and p-ERK （P<0.05）. Sulfasalazine and low- and medium-dose Mume Fructus increased the positive expression of Occludin， Claudin-1， and ZO-1 （P<0.05， P<0.01）. Furthermore， high-dose Mume Fructus elevated the protein expression of Occludin （P<0.05）. ConclusionMume Fructus can restore the expression of intestinal epithelial tight junction proteins by inhibiting the phosphorylation of proteins in the MEK/ERK signaling pathway and down-regulating the levels of TNF-α and IL-1β， thus repairing the intestinal mucosal barrier in the mouse model of IBD.

ObjectiveTo investigate the modulating effect of modified Wumeiwan （MWMW） on the ulcerative colitis （UC）-associated intestinal helper T cell 17 （Th17）/regulatory T cell （Treg） balance and intestinal flora by using a human flora-associated model of UC patients with dampness-heat obstruction syndrome， thus providing a new idea for the UC-related research and therapeutic strategies. MethodsThe 24 male C57BL/6J mice were randomized into normal control， model， and MWMW groups （n=8）. Model and MWMW groups were first treated with an antibiotic cocktail （vancomycin， 0.1 g·kg^-1； neomycin sulfate， 0.2 g·kg^-1； ampicillin， 0.2 g·kg^-1； metronidazole， 0.2 g·kg^-1） for 21 days. At the end of antibiotic treatment， the gavage of fecal microbiota suspension from UC patients with dampness-heat obstruction syndrome was started at a dose of 0.2 mL·d^-1 for 19 consecutive days， by which a human flora-associated model of UC was obtained. The MWMW group was administrated daily with MWMW liquid （12.5 g·kg^-1）， while the normal control and model groups were administrated by gavage with an equal amount of sterile water for 7 consecutive days. The symptoms of dampness-heat obstruction were observed. The colon length and spleen index were measured and calculated， and the proportions of Th17 and Treg cells were detected by flow assay. The intestinal flora was analyzed by 16S rRNA high-throughput sequencing. ResultsCompared with the normal control group， the model group showed shortened colon （P<0.05） and increased spleen index （P<0.01）. Compared with the model group， the MWMW group showed prolonged colon （P<0.01） and decreased spleen index （P<0.05）. After the intervention of MWMW， the Th17 proportion and Th17/Treg ratio in the colon decreased （P<0.01）， and the proportion of Treg cells increased （P<0.05）. The number of species and alpha and beta diversity of intestinal flora in mice were regulated by MWMW （P<0.05）. In terms of intestinal flora composition， MWMW increased the relative abundance of several phyla （Firmicutes， Proteobacteria， Fusobacteriota， Actinobacteriota， and Gemmatimonadota）， the genus Bacteroides， and two species （Bacteroides thetaiotaomicron and B. fragilis） in model mice. Moreover， Spearman's correlation analysis showed that the relative abundance of B. thetaiotaomicron and B. fragilis were negatively correlated with the Th17 level （P<0.05）. In addition， the above changes in intestinal flora caused the changes in microbial genes involved in 14 pathways， such as glycolysis， amino acid degradation， inorganic nutrient metabolism， biosynthesis of pyrimidine deoxyribonucleotides， antibiotic resistance， and degradation of polysaccharides. ConclusionsThe human flora-associated model successfully simulated the changes （marked by a decrease in the abundance of Bacteroides） of intestinal flora in UC patients with dampness-heat obstruction syndrome. MWMW can enrich the abundance of beneficial bacteria such as B. thetaiotaomicron and B. fragilis and promote the synergistic intestinal immune modulation with the metabolic functions centered on glycolysis， amino acid metabolism， and nucleotide synthesis through bacterial polysaccharide utilization sites to reduce the Th17/Treg ratio， thereby exerting a protective effect on UC.

ObjectiveTo explore the evolution of medication patterns and syndrome-herb associations of traditional Chinese medicine （TCM） in treating inflammatory bowel disease （IBD）， providing a theoretical foundation for precise syndrome differentiation and treatment in clinical practice. MethodsMedical case literature on TCM treatment of IBD from 1960 to 2024 was retrieved to establish a database. Frequency statistics， cluster analysis， change point detection， and association rule mining were employed to comprehensively analyze the syndrome distribution， therapeutic methods， medication patterns， and their temporal variations. ResultsA total of 685 medical cases were included. Common syndromes were dampness-heat （66.42%） and spleen deficiency （56.20%）. Primary therapeutic methods included heat clearing （63.65%）， spleen invigorating （47.45%）， and dampness draining （36.79%）. High-frequency herbs included Coptidis Rhizoma （354）， Paeoniae Radix Alba （303）， Aucklandiae Radix （292）， Codonopsis Radix （253）， and Glycyrrhizae Radix et Rhizoma （244）. Initial prescription clustering revealed three core therapeutic method combinations： heat clearing and detoxifying （represented by Baitouweng Tang）， spleen invigorating and Qi reinforcing （represented by Shenling Baizhusan）， and cold-heat regulation （represented by Wumeiwan combined with Shaoyao tang）. Temporal analysis identified 2008 as a key transition point in TCM treatment of IBD， with significantly increased usage frequency of heat-clearing and dampness-drying herbs such as Fraxini Cortex， Phellodendri Chinensis Cortex， Sophorae Flavescentis Radix， and Scutellariae Radix as well as hemostatic herbs such as carbonized Sanguisorbae Radix， Bletillae Rhizoma， Agrimoniae Herba， and Notoginseng Radix et Rhizoma. Follow-up efficacy analysis showed median improvement rates of 64.0% at the first follow-up， 76.0% at the second follow-up， and 78.7% at the third follow-up. Syndrome-drug association analysis revealed specific herb pairs with significant therapeutic advantages， including Notoginseng Radix et Rhizoma + Coicis Semen， Sanguisorbae Radix + Coptidis Rhizoma， and Codonopsis Radix + Aconii Lateralis Radix Praeparaia. ConclusionTCM medication patterns for treating IBD demonstrate distinct temporal evolution characteristics， with significantly increased usage frequency of herbs such as Fraxini Cortex， Notoginseng Radix et Rhizoma， and Agrimoniae Herba. Significant therapeutic method-herb associations and syndrome-herb association patterns exist， with the formation of specific herb pairs， providing evidence-based support for precise syndrome differentiation and treatment of IBD.

Objective:To analyze the predictive factors associated with late-onset sepsis(LOS) in very low birth weight infants,and to develop a risk prediction score scale applicable to these infants three days postnatal.This will provide valuable insights for early diagnosis and timely intervention.Methods:Very low birth weight infants admitted to the Children's Hospital of Fudan University from January 1,2022,to June 30,2024,were selected as research subjects.These infants were categorized into two groups:the LOS group and the non-LOS group,based on whether they developed LOS.LASSO regression analysis,alongside univariate and multivariate regression analyses,was employed to identify predictive factors for LOS in this population.A Logistic model was constructed using the optimal combination of predictive variables,and a risk assessment scale was subsequently developed.The prediction performance of the model was evaluated using the Hosmer-Lemeshow chi-square test and the receiver operating characteristic curve.Results:A total of 444 cases of very low birth weight infants were included,of which 185 had LOS and 259 did not.After screening the variables,seven independent factors were included into the model:birth weight,gestational age,tracheal intubation,abnormal skin color,abdominal distension,elevated C-reactive protein levels,and right hand perfusion index.A predictive scoring scale was developed based on the regression coefficients of each variable,with corresponding risk scores assigned as follows:1,4,3,2,1,1,and 2; a score of ≥3.5 indicated high-risk groups.The Hosmer-Lemeshow test results demonstrated that χ2 = 7.602( P = 0.473).The area under the receiver operating characteristic curve was 0.792 ( P<0.001),with a sensitivity of 73.5% and specificity of 71.0%. Conclusion:The risk score scale developed in this study exhibits significant predictive capability,providing valuable insights for clinical medical personnel to assess the risk of LOS in very low birth weight infants during the early postnatal period.

Objective:To explore the mediating effect of accompanied childbirth experience among spouses of primipara on dynamic adjustment after marriage and paternal role adaptation, with the aim of providing reference for further improving accompanying childbirth services.Methods:Spouses of primipara who participated in accompanying childbirth at Nanjing Drum Tower Hospital Group Suqian Hospital from January 2023 to June 2024 were selected as the research subjects through convenience sampling. The general information questionnaire, the Chinese version of Dynamic Adjustment Scale, the Parents' Experiences of Childbirth and the Newborn Father's Role Adaptation Questionnaire were used to conduct a cross-sectional survey, and establish the intermediary model between the dynamic adjustment after marriage and the paternal role adaptation.Results:A total of 244 spouses of primipara were included, with an age of (28.86 ± 5.77) years. The score for dynamic adjustment in spouses of primipara was (110.82 ± 16.33) points, the score for experiences of childbirth was (124.27 ± 13.89) points, and the score for paternal role adaptation was (71.66 ± 9.22) points. The accompanied childbirth experience among spouses of primipara was positively correlated with the dynamic adjustment after marriage and the paternal role adaptation ( r=0.197, 0.511, both P<0.01). The dynamic adjustment after marriage was positively correlated with the paternal role adaptation ( r=0.403, P<0.01). The accompanied childbirth experience among spouses had a partial mediating effect between the dynamic adjustment after marriage and the parental role adaptation, which the mediating effect was 0.182, accounting for 32.79% of the total effect. Conclusions:The dynamic adjustment after marriage can directly predict the paternal role adaptation, and the experience of accompanying childbirth has a partial mediating effect between the two. The role adaptation level of the spouses of the primiparas should be improved by further improving the delivery service to meet the needs of the primiparas and their spouses.

Objective To explore the possible mechanism underlying the role of forkhead box A1 circular RNA(circ-FAT1)regulating pericyte pyroptosis in diabetic retinopathy(DR).Methods The experiment was divided into two parts.Firstly,thirty male C57BL/6J mice(totaling 60 eyes)were randomly divided into normal,DR and circFAT1 overex-pression groups,with 10 mice in each group.Fasting plasma glucose(FPG)was detected by a glucometer.The serum lev-els of total cholesterol(TC)and triacylglycerol(TG)were measured by enzyme-linked immunosorbent assay(ELISA)kits.Hematoxylin and eosin(HE)staining was used to examine the pathological structure of mouse retina.Reverse tran-scription-quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression of circFAT1 in retinal tissues.The relative protein expression levels of NOD-like receptor protein 3(NLRP3),Caspase-1 and porin D(GSDMD)in the retina of mice were detected by Western blot.Secondly,retinal perivascular cells were extracted from 5 C57BL/6J mice(totaling 10 eyes)and divided into control,high glucose and circFAT1 overexpression+high glucose groups.The protein expression levels of NLRP3,Caspase-1 and GSDMD in pericytes were detected by Western blot.ELISA kits were used to measure the content of interleukin-18(IL-18)and interleukin-1β(IL-1β)in pericytes.Results(1)Compared with those in the normal group,the levels of FPG,serum TC and TG were increased while the relative expression level of circFAT1 mRNAs in the retinal tissue was decreased in the DR group(all P＜0.05).Compared with the DR group,the circ-FAT1 overexpression group showed decreased levels of FPG,serum TC and TG(all P＜0.05),and increased relative ex-pression levels of circFAT1 mRNAs in the retinal tissue(P＜0.05).The relative expression levels of NLRP3,Caspase-1 and GSDMD proteins in the retinal tissue of the DR Group were higher than those in the normal group(all P＜0.05).The rela-tive expression levels of NLRP3,Caspase-1 and GSDMD proteins in the retinal tissue of the circFAT1 overexpression group were lower than those in the DR group(all P＜0.05).(2)Compared with those in the control group,the relative expres-sion levels of NLRP3,Caspase-1,and GSDMD proteins and the content of IL-18 and IL-1 β in the pericyte of the high glucose group were increased(all P＜0.05).The relative expression of NLRP3,Caspase-1 and GSDMD proteins and the content of IL-18 and IL-1 β in the pericyte of the circFAT1 overexpression+high glucose group were lower than those in the high glu-cose group(all P＜0.05).Conclusion Overexpression of circFAT1 can improve DR by inhibiting pericyte pyroptosis.

Objective To investigate the effects of miR-34a on proliferation and osteogenic differentiation of human periodontal stem cells.Methods Twenty healthy teeth that needed to be extracted for orthodontic treatment were collected.Human periodontal stem cells(hPDLSCs)were isolated and cultured in vitro,and miR-34a mimetics were constructed and transfected into hPDLSCs.The experimental groups were subsequently categorized into the mimics group(miR-34a overexpression group)and the mimics-NC group(control group without load).The transfection efficiency was assessed using real-time fluorescence quantitative PCR(qRT-PCR),while CCK-8 assays were used to evaluate the proliferation capacity of hPDLSCs post-transfection.Osteogenic differentiation of miR-34a-transfected hPDLSCs was induced,with samples being collected at day 0 and day 14 after the osteogenic induction.The expression level of Runx2-associated transcription factor 2(Runx2)was quantified via qRT-PCR,protein levels of Runx2-associated proteins were analyzed through Western blot,and mineralized nodule formation was examined using alizarin red staining.Results The expression level of miR-34a in the mimics group was significantly higher than that in the mimics-NC group(P<0.05).There was no significant difference in the value-added rate between the mimics group and the mimics-NC group on days 1～5(P>0.05),and the value-added rate between the mimics group and the mimics-NC group was significantly lower than that between the mimics-NC group and the mimics-NC group on days 5～11,and the difference was statistically significant.After the osteogenic induction,the mRNA expression level of Runx2 in the mimics group was higher than that in the mimics-NC group(P<0.05),and the expression level of Runx2 protein in the mimics group was also higher than that in the mimics-NC group(P<0.05),and there were more mineralized nodules in the mimics group than in the mimics-NC group after 14 days of osteogenic induction.Conclusion Under in vitro conditions,miR-34a inhibits the proliferative activity of hPDLSCs and promotes the osteogenic differentiation of human periodontal ligament stem cells.

Objective:To investigate the impact of minocycline on myocardial injury in diabetes cardiomyopathy(DCM)rats by regulating 5'-AMP activated protein kinase(AMPK)/sirtuin 1(SIRT1)/peroxisome proliferator activator receptor gamma coactiva-tor 1α(PGC-1α)signal pathway.Methods:SD rats were fed with high-fat diet for 4 weeks,and then a single intraperitoneal injection of 35 mg/kg streptozotocin was used to induce the DCM model.They were randomly grouped into model group,low-dose minocycline(20 mg/kg)group,high-dose minocycline(40 mg/kg)group,high-dose minocycline+Dorsomorphin(AMPK inhibitor,0.2 mg/kg)group,with 12 rats in each group,another 12 normal rats were fed with normal feed for 4 weeks,and then were given a single intraper-itoneal injection of the same dose of citric acid buffer,which was set as a sham operation group,after the intervention with minocy-cline and Dorsomorphin,and the fasting blood glucose(FBG),total cholesterol(TC)and triglyceride(TG)were measured;left ven-tricular ejection fraction(LVEF)and left ventricular short axis shortening(FS)were measured by ultrasound;HE staining and Mas-son staining were applied to detect the pathological morphology of myocardial tissue of rats in each group;the levels of serum and myo-cardial tissue inflammation IL-6,IL-18,and myocardial tissue oxidative stress indicators-superoxide dismutase(SOD)and malondial-dehyde(MDA)were measured with the kit;Western blot was applied to detect the expression of AMPK/SIRT1/PGC-1α pathway pro-tein in myocardial tissue of rats in each group.Results:Compared with the sham operation group,the myocardial tissue in the model group was seriously damaged,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocar-dial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myocardial tissue were obviously increased(P<0.05),the levels of LVEF,FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were obviously decreased(P<0.05);compared with the model group,the myocardial tissue damage of rats in the low-dose and high-dose minocycline groups were reduced,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myocardial tissue were decreased(P<0.05),the LVEF,the levels of FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were increased(P<0.05);compared with the low-dose minocycline group,the myocardial tissue damage in the high-dose minocycline group was further reduced,the levels of FBG,TC and TG,the cross-sectional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tissue,and the level of MDA in myo-cardial tissue were further decreased(P<0.05),the levels of LVEF,FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were further increased(P<0.05);compared with the high-dose minocycline group,the myocar-dial tissue damage of rats in the high-dose minocycline+Dorsomorphin group increased,the levels of FBG,TC and TG,the cross-sec-tional area of myocardial cells and the proportion of myocardial fiber area,the levels of IL-6 and IL-18 in serum and myocardial tis-sue,and the level of MDA in myocardial tissue were increased(P<0.05),the LVEF,the levels of FS and SOD in myocardial tissue,and the protein expression of p-AMPK/AMPK,SIRT1 and PGC-1α were decreased(P<0.05).Conclusion:Minocycline can reduce oxidative stress and inflammation in DCM rats by activating AMPK/SIRT1/PGC-1α signal,and improve glycolipid metabolism,thereby reducing myocardial injury and repairing cardiac function in rats.

Objective:To study the genotype and molecular characteristics of Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) in Shenzhen so as to provide basis for the prevention and control.Methods:The serum samples of HFRS patients at acute stage were collected from hospitals, and viral RNA from the sera was extracted as a template for typing detection by real-time fluorescent PCR. The nucleotide sequences of M fragment (G2 segment) and S fragment were amplified by reverse transcription-nested polymerase chain reaction (RT-nested PCR). The PCR products were then sequenced and homology and phylogenetic tree analysis were conducted.Results:In a total of 123 HV IgM antibody positive serum samples were collected in Shenzhen from 2015 to 2023, including 97 males and 26 females. Most of patients were young and middle-aged men. Cases were found throughout the year, with high incidence in spring and early summer. The result of fluorescence PCR showed that among 92 clinical specimens, 63 cases (68.5%) were tested positive for hantavirus genotypes, 59 cases (93.7% of positives) were identified as SEOV and 4 cases (6.3% of positives) as HTNV. Notably, all HTNV-positive patients were male. Analysis on nucleotide homology and phylogenetic tree showed that the difference in nucleotide sequences among SEO viruses in Shenzhen was small, and most of them were S2 subtype except for two cases of S3 subtype. The mutation rate of HTN viruses was high, with two cases of H5 subtype and two cases of H8 subtype.Conclusions:The major genotype of hantavirus causing HFRS in Shenzhen is SEO genotype, especially S2 subtype, which is closely related to some strains in Guangzhou, and has high homology with L99, Z37 and other vaccine strains. The existence of S3 subtype cases was detected for the first time in Shenzhen, while some cases of HTNV were still imported.