摘要：用表达MRFP或GFP的MDV重组剂进行补充荧光生物成像,证实脚部皮肤感染。无论动物的年龄和感染途径如何,在足部皮肤的中间表皮层都能检测到感染,也能产生感染性病毒。

第一次证明马立克病毒（MDV）不仅被传送到羽毛囊,而且被鳞片覆盖的喙和脚的皮肤。

总而言之，我们的研究揭示了 MDV 迅速传播到意想不到的解剖部位，而这些部位是五十多年来标准采样所遗漏的，这为了解这种鸡的致命疾病提供了重要的见解。

体内的生物成像方法揭示了两个鸡马立克病毒复制的新部位,鸡嘴和被鳞片覆盖的脚的皮肤。

用表达MRFP或GFP的MDV重组剂进行补充荧光生物成像,证实脚部皮肤感染。无论动物的年龄和感染途径如何,在足部皮肤的中间表皮层都能检测到感染,也能产生感染性病毒。

综合起来,禽类全身生物成像这项研究，强调了体内的通过识别以前被忽视的鸡寄主中MDV复制和脱落的地点。

马立克病毒生物学和发病机制

MDV是一种与细胞高度相关的病毒,因为感染在宿主体内的传播是通过细胞到细胞的接触发生的。根据目前的MDV发病模式,感染是由吸入受污染环境的感染尘埃或皮屑而引起。

在上呼吸道,感染性尘埃被吞噬细胞,如巨噬细胞、树突状细胞或B细胞吸收,随后将病毒转移至淋巴器官:法氏囊、胸腺囊和脾脏。在这些器官中,MDV可在主要是B细胞和T细胞中有效复制,直到病毒在感染后10-14天左右确定潜伏期。

MDV主要确定CD4+T细胞的潜伏期感染,也可转化为导致致命淋巴瘤的发展。最后,以溶菌方式和/或潜伏感染的T细胞将病毒运送到羽状卵泡上皮,在那里产生传染性的"无细胞"病毒,并从感染后两周开始向环境中传播。

迄今为止,羽毛,特别是毛囊上皮,是已知在环境中脱落传染性病毒的唯一组织,也是MDV传播的唯一来源。

vTK-fLuc感染鸡的体内生物发光成像

One day old-chickens were inoculated intramuscularly with 2000 pfu of vTK-fLuc. Chickens were imaged in vivo with an IVIS spectrum: (A) 6 chickens at 7 dpi, (B) 7 chickens at 10 dpi and 8 chickens at 14 dpi. Chickens indicated by an asterisk were kept alive after in vivo imaging. An image of each chicken imaged is shown. At each timepoint, a naive control bird of matched age (Control, CTL) was imaged for comparison (CTL A, B and C). Note that all chickens imaged at 14 dpi were imaged earlier, either at 7 or 10 dpi. Each chicken image is shown with its own radiance scale (p/s/cm2/sr) due to variations between individuals.

(A) Viral genome loads measured at 10 dpi in PBMCs and feather tips material. (B) Wings and feet of euthanized chicks were imaged at 14 dpi with an IVIS Spectrum. The red Fluorescence was acquired using the spectral unmixing mode. Wings and feet from a control chicken matched for age (CTL C) were imaged for comparison. C. Quantification of the RFP signal on the feet. The quantification was performed using the IVIS software on each foot taken as one ROI. Each dot corresponds to one measure, the horizontal long bar to the median and vertical bar the interquartile range shown per group. Though some animals present a relatively high signal, the difference of fluorescence between the VP22-RFP infected chickens and the control was not significant (Mann-Whitney test, exact p-value = 0.2583, ns. D. Viral genome loads measured at 14 dpi in feather tips material and skin of the feet. For each group, the median is shown as a black line (value indicated above) with the interquartile range.

(A) Viral genome loads measured at 10 dpi in PBMCs. The median is shown as a black line with the interquartile range. (B) Quantification of the GFP signal on the wings and the feet. The quantification was performed using the IVIS Spectrum software on each wing and foot taken as one ROI. Each dot corresponds to one measure, the horizontal long bar corresponds to the median and the vertical bar to the interquartile range per group. (C) Viral loads measured at 14 dpi in the skin of the feet. The median is shown as a black line with the interquartile range. Infectivity assessed by a plaque assay from four infected-chickens (14 dpi) and a control (D, E, F). Primary keratinocytes isolated from the skin of the feet was co-cultivated with CESCs for 4 days. (D) Pictures of an infection plaque (chick #82) and of a non-infected layer (control chick). The GFP was directly detected in the green channel. (E) Number of plaques obtained by coculture for each chicken at 14dpi. (F) Viral loads measured in the coculture of the infectivity test. Each dot corresponds to one chicken, the horizontal long bar to the median and the vertical bar to the interquartile range per group. (G) Primary keratinocytes isolated from the skin feet of infected-chicken, 14 days post-injection. Keratinocytes were stained with mouse monoclonal antibodies to MDV proteins (gB and VP22), revealed with a secondary antibody coupled to Alexa Fluor 594 (red). The nuclei were stained with Hoechst 33342 (blue). The cell autofluorescence and the GFP are visible in the green channel. (H) Infectivity assessed by a plaque assay from a chicken infected by natural route; a control chicken is also shown. The cocultures were stained with mouse monoclonal antibodies to MDV proteins (gB and VP22), revealed with a secondary antibody couplet to Alexa Fluor 594. The nuclei were stained with Hoechst 33342 (blue). The GFP was directly detected in the green channel.

来源：鸡保姆