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目的 分离一例病程超长的呼吸道感染患儿呼吸道标本中的偏肺病毒（Human metapneumovirus,hMPV），分析其体外生物学特征，验证其对广谱抗病毒药物的敏感性，以评估其进一步传播导致婴儿重症感染的风险。方法 采用气-液两相培养的呼吸道上皮类器官（Air-liquid interface human airway epithelial organoid, HAE）分离患儿咽拭子中的偏肺病毒，绘制病毒复制动力曲线；电镜检测病毒形态，免疫荧光检测偏肺病毒N蛋白在HAE表达；对所获病毒进行全基因组序列测定并进行基因标定，构建系统发育树；将分离的病毒感染肺泡类器官及常规细胞LLCMK2，测定病毒载量并观察病毒感染导致的细胞病变；同时在呼吸道类器官及LLC-MK2细胞验证该偏肺病毒分离株对广谱抗病毒药物利巴韦林的敏感性。结果 从一例病程超长的患儿咽拭子中分离获得1株人偏肺病毒临床分离株hMPV-CTan01/Beijing/2023（以下简称hMPV-CTan01），基因组全长13435 bp，属于偏肺病毒A2C亚型，与时下流行株高度一致；hMPV-CTan01能够在HAE中高效复制并产生有活性的子代病毒；HAE上清中观察到典型的偏肺病毒形态，呼吸道上皮细胞中可检测N蛋白的表达。hMPV-CTan01可以感染肺泡类器官，提示其有引发重症感染的风险；同时hMPV-CTan01在LLC-MK2细胞中良好复制，导致细胞出现典型的细胞病变，其体外特征与既往报道一致；抗病毒药物利巴韦林在细胞系LLC-MK2及HAE中均能抑制该毒株的复制。结论 从这例病程超长的患儿呼吸道分离获得的hMPV-CTan01，其体外生物学特征与既往报道一致，并未在体外展现出更强的致病性，利巴韦林能够抑制其在呼吸道类器官和LLC-MK2中的复制，提示其未出现明显的抗药性。上述结果证明，尽管hMPV-CTan01导致该患儿病程较长，但其进一步导致重症感染传播的风险较低。
Abstract：Objective To isolate human metapneumovirus(hMPV) from a pediatric patient with an unusually prolonged respiratory infection, analyze its in vitro biological characteristics, and evaluate its susceptibility to broad-spectrum antiviral agents, thereby assessing the risk of its further transmission leading to severe infection in infants. Methods Human metapneumovirus was isolated from the patient's throat swab using an air-liquid interface human airway epithelial organoid(HAE) model. Viral replication kinetics were determined. Viral morphology was examined by electron microscopy, and expression of the hMPV N protein in HAE was detected by immunofluorescence. The full-length genome of the isolated virus was sequenced and phylogenetically analyzed. The isolated virus was used to infect alveolar organoids and conventional LLC-MK2 cells to measure viral load and observe virus-induced cytopathic effects. Meanwhile, the susceptibility of this hMPV isolate to the broad-spectrum antiviral drug ribavirin was validated in both airway organoids and LLCMK2 cells. Results A clinical isolate of human metapneumovirus, designated hMPV-CTan01/Beijing/2023(hereinafter referred to as hMPV-CTan01), was obtained from the throat swab of the pediatric patient with a prolonged disease course. Its complete genome is 13,435 bp in length, belonging to the hMPV A2C subgenotype, showing high consistency with currently circulating strains. hMPV-CTan01 efficiently replicated in HAE and produced infectious progeny virus. Typical hMPV morphology was observed in the HAE supernatant, and N protein expression was detected in airway epithelial cells. hMPV-CTan01 was able to infect alveolar organoids, suggesting its potential to cause severe infection. Furthermore, hMPV-CTan01 replicated efficiently in LLC-MK2 cells, inducing typical cytopathic effects, and its in vitro characteristics were consistent with previous reports. The antiviral drug ribavirin suppressed the replication of this isolate in both LLC-MK2 cells and HAE. Conclusion The in vitro biological characteristics of hMPV-CTan01, isolated from the respiratory tract of this pediatric patient with a prolonged illness, are consistent with previous reports and do not demonstrate enhanced pathogenicity in vitro. Ribavirin effectively inhibits its replication in both airway organoids and LLC-MK2 cells, indicating no apparent drug resistance. These findings demonstrate that although hMPV-CTan01 was associated with a prolonged disease course in this patient, its risk of further transmission leading to severe infection is low.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.260132
中图分类号:R725.6
引用信息:
[1]石小丫,陆柔剑,赵扬,等.从一例住院患儿分离人偏肺病毒及其在人呼吸道上皮与肺泡类器官中感染特性研究[J].病毒学报,2026,42(03):750-762.DOI:10.13242/j.cnki.bingduxuebao.260132.
基金信息:
国家自然科学基金面上项目（项目号：82072296）,题目：新型冠状病毒SARS-CoV-2与人冠状病毒NL63、HKU1在呼吸道上皮类器官模型HAE中的生物学特征比较研究; 国家重点研发计划项目（项目号：2022YFC2304100）,题目：新冠与流感等大流行风险病原体多肽疫苗的研制;国家重点研发计划项目（项目号：2022YFC2303401）,题目：潜在高危新病毒风险识别的新技术体系研究~~
2026-05-14
2026-05-14
2026-05-14