Zike病毒感染腎周管狀上皮細(xì)胞后的持續(xù)性細(xì)胞反應(yīng)相關(guān)研究

Zika病毒是一種蟲媒病毒，通過蚊子傳播。該病毒和登革熱病毒相似，感染后有類似的癥狀：發(fā)熱、頭痛、瞼結(jié)膜充血、皮疹、肌肉疼痛和關(guān)節(jié)疼痛，臨床表現(xiàn)相對溫和，病程持續(xù)約4-7天。Zike病毒感染成人以后，能夠引起胎兒畸形以及吉蘭-巴利綜合征，雖然對Zike病毒感染與小頭畸形之間的關(guān)系已經(jīng)有較多的研究，并且取得了不錯的進(jìn)展，但是針對該病毒病理機(jī)制方面的研究，尤其是寄生在人體內(nèi)的Zika病毒，仍然知之甚少。既往的一些研究表明，被感染的患者尿液中的病毒存在時間比血液中存在的時間要長，這說明在人類的泌尿系統(tǒng)中很有可能存在著獨(dú)立的病毒復(fù)制庫。而且在臨床實驗中，人的泌尿系統(tǒng)中該病毒的寄生細(xì)胞很少被發(fā)現(xiàn)。上海復(fù)旦大學(xué)公共衛(wèi)生臨床中心的Chen Jian等研究證明Zika病毒不但能夠感染免疫缺陷小鼠的腎周管狀上皮細(xì)胞（RPTEpiCs），而且可以感染人的永生化及幼稚腎周管狀上皮細(xì)胞（hRPTEpiCs）。此外，已有實驗證明了Zika病毒感染小鼠腎臟以后會引起caspase-3介導(dǎo)的腎臟細(xì)胞凋亡。Zika病毒感染永生化及幼稚hRPTEpiCs也會導(dǎo)致類似的細(xì)胞反應(yīng)。結(jié)合臨床觀察，研究者發(fā)現(xiàn)Zika病毒感染hRPTEpiCs后持續(xù)存在。RNA-Seq測序結(jié)果也顯示被感染的hRPTEpiCs中存在較長的一段轉(zhuǎn)錄基因的變化，包括I型干擾素信號基因和抗病毒反應(yīng)基因。這些都表明在人類的泌尿系統(tǒng)中，hRPTEpiCs是Zika病毒的潛在病毒復(fù)制庫，為Zika病毒在患者尿液中長期存在提供了一種可能性的解釋。

   本實驗主要通過免疫熒光、免疫組化、RNA-Seq測序分析等方法作為實驗手段，主要實驗結(jié)果如下：

Figure 1   Renal infection of Zika virus (ZIKV) in AG6 mice. (A) Components of the nephron. (B) Detection of ZIKV RNA in mouse kidneys. Left panel, in situ hybridization (ISH) using a ZIKV-specific probe. Blue and red arrows indicate renal glomerular and tubular cells, respectively. Hybridization signals of ZIKV RNA were developed by a chromogenic reaction and are observed as black particles. Right panel, hematoxylin and eosin (H&E) staining. Scale bar, 50 μm. (C) Immunofluorescence staining of caspase-3 in ZIKV-infected renal tissues of AG6 mice. Left panel, representative images. Scale bar, 200 μm. Right panel, statistical analyses of caspase-3-positive cells. **Significant difference between mock and infected cells (Po0.01; Student’s t-test).

Figure 2   ZIKV infection in the HK2 cell line. (A) Immunofluorescence staining of HK2 cells infected with SZ01 or MR766 ZIKV using an anti-ZIKV envelope protein antibody. Upper panel, representative images. Scale bar, 20 μm. Lower panel, quantification of infected cells. (B) Light microscopy images showing SZ01 ZIKV-induced CPEs. (C) Production of infectious SZ01 and MR766 ZIKV in HK2 cells. Cytopathogenic effects, CPEs.

Figure 3   ZIKV infection in primary hRPTEpiCs. (A) Immunofluorescence staining of primary hRPTEpiCs infected with SZ01 ZIKV at a MOI of 2.5 for 96 h. Scale bar, 30 μm. (B) Long-term persistence of SZ01 ZIKV in primary hRPTEpiCs. Viral copies in the supernatant of medium were determined by RT-qPCR using ZIKV standard samples. Values are shown as the mean±standard deviation (sd; n=5). (C) MR766 ZIKV-induced apoptosis in primary hRPTEpiCs. Upper panel, representative immunofluorescence images. Scale bar, 200 μm. Lower panel, quantification of ZIKV-positive and caspase-3-positive cells. ***Significant difference between mock and infected cells (Po0.001; Student’s t-test). (D) Production of infectious SZ01 and MR766 ZIKV particles in primary hRPTEpiCs.

Figure 4   RNA-Seq analysis of mock and ZIKV-infected primary hRPTEpiCs at 48 h post infection. (A) Volcano plot of genes differentially expressed in mock and ZIKV-infected hRPTEpiCs. (B) Genes with differential expression between mock and infected hRPTEpiCs were subjected to gene ontology (GO) analyses. The top 10 most significantly upregulated and downregulated biological processes are shown in red and blue, respectively.

其中，RNA的ISH和HE染色、組織的IF和IHC染色結(jié)果均由奧地利TG公司的Tissue FAXS200系統(tǒng)采圖并分析，通過以上實驗，最終得出以下結(jié)論：1.對I型和II型IFN缺陷的小鼠接種Zika病毒會導(dǎo)致其腎小管及腎小球中病毒大量復(fù)制增殖，且腎小球中的病毒量更高。2.Zika病毒在幼稚hRPTEpiCs中長期存在。3.Zika病毒的2種菌株（SZ01和MR766）均能在永生化及幼稚hRPTEpiCs中復(fù)制增殖。綜上，腎臟的hRPTEpiCs很可能是Zika病毒持續(xù)復(fù)制存在的病毒庫，除此以外，本研究還發(fā)現(xiàn)了美洲和亞洲的Zika病毒菌株具有不同的感染方式。其結(jié)果可以為CDC(疾病防治中心)提供理論實驗數(shù)據(jù)支持。