日本脑炎病毒感染商品猪软腭扁桃体的检测与鉴定

Detection and characterization of Japanese encephalitis virus-infected soft palate tonsils in commercial pigs

摘要

[目的]本试验旨在检测商品猪软腭扁桃体中的日本脑炎病毒(Japanese encephalitis virus,JEV),鉴定 JEV 感染特性。[方法]从屠宰场采集商品猪软腭扁桃体 20 头份,用 RT-PCR 筛选 JEV 阳性样本。将阳性样本的匀浆上清液过滤后接种 BHK-21 细胞进行病毒分离,采用间接免疫荧光、蛋白免疫印迹和噬斑形成试验鉴定分离病毒。PCR 扩增分离毒株的 E 基因序列并对其进行遗传进化分析。用免疫组化和间接免疫荧光法鉴定 JEV 感染猪软腭扁桃体特征。[结果]20 份软腭扁桃体中检测出 2 例 JEV 阳性,检出率为 10%。成功分离到 2 株 GIb 型 JEV,分离株 E 基因核苷酸相似性为 97.5%,分别命名为 JEV NJ202408 株和 NJ202409 株。在 JEV 阳性软腭扁桃体中,JEV 抗原阳性细胞主要分布在淋巴滤泡边缘附近和周围弥散的淋巴组织中,细胞核较大、形态不规则且颜色淡染。免疫荧光检测结果显示,JEV 阳性软腭扁桃体中 JEV 分别与 CD11b、CD163、iNOS、TNF-α 和 MHC-Ⅱ细胞标志物共定位,说明 JEV 阳性细胞多为巨噬细胞或树突状细胞并引发巨噬细胞 M1 极化。JEV 感染并未明显改变猪软腭扁桃体淋巴滤泡内 MHC-Ⅱ⁺B 细胞和淋巴滤泡间 CD3⁺T 细胞的分布。[结论]在实际生产中 JEV 可在商品猪软腭扁桃体定殖,JEV 阳性细胞主要为巨噬细胞或树突状细胞,为 JEV 感染猪的临床监测和日本脑炎的防控提供了参考。

Abstract

[Objectives]This study aimed to detect the Japanese encephalitis virus (JEV) in the soft palate tonsils of commercial pigs and characterize the nature of JEV infection. [Methods]Soft palate tonsils were collected from 20 commercial pigs at abattoirs, and JEV-positive samples were identified using RT-PCR. The homogenate supernatant of each positive sample was filtered and inoculated into BHK-21 cells for virus isolation. The isolated virus was characterized by indirect immunofluorescence, western blot, and plaque formation assays. The E gene sequence of the isolated strain was amplified via PCR and analyzed for genetic evolution. Immunohistochemistry and immunofluorescence were used to examine the characteristics of JEV-infected soft palate tonsils in pigs. [Results]Two JEV-positive cases were detected among 20 soft palate tonsil samples, yielding a detection rate of 10%. Two GIb-type JEV strains were successfully isolated from these samples and were designated JEV strain NJ202408 and strain NJ202409, with a nucleotide similarity of 97.5% in their E genes. In JEV-positive tonsils, JEV antigen-positive cells were primarily located near the edges of lymphoid follicles and within the surrounding diffuse lymphoid tissue. These cells exhibited large, irregularly shaped, pale-staining nuclei. Immunofluorescence analysis showed that JEV NS1' co-localized with CD11b, CD163, iNOS, TNF-α, and MHC-II cell markers, suggesting that most JEV-positive cells were macrophages or dendritic cells and that the infection induced macrophage M1 polarization. JEV infection did not significantly alter the distribution of MHC-II⁺B cells within lymphoid follicles or CD3⁺T cells between lymphoid follicles in the soft palate tonsils of pigs. [Conclusions]In commercial pig production, JEV can colonize the soft palate tonsils, with most JEV-positive cells identified as macrophages or dendritic cells. This finding serves as a reference for clinical monitoring of JEV-infected pigs and for the prevention and control of Japanese encephalitis.

关键词

日本脑炎病毒 / 猪 / 软腭扁桃体 / 感染 / 监测

Key words

Japanese encephalitis virus / pigs / soft palate tonsils / infect / surveillance

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基金资助

国家自然科学基金项目(32273040)

国家重点研发计划项目(2022YFD1800102)