INTRODUCTION

The Zika virus (ZIKV) is known since 1947 when it was isolated from a rhesus macaque monkey in a yellow fever research institute in the Zika forest of Uganda[1].ZIKV has reached global attention during the epidemic in Brazil in the years 2015/2016. This mosquito-borne virus (Aedes aegypti and Aedes albopictus) was found to circulate only in East and West Africa[2] until a bigger outbreak occurred on the Yap Islands in Micronesia in the year 2007[3]. Another outbreak took place in French Polynesia in the year 2013. Here for the first time the congenital ZIKV syndrome (CZVS), microcephaly, the Guillain-Barré syndrome (GBS) and non-vectorborne transmission (mother to child, sexual, posttransfusion)was retrospectively documented[4-7]. However, the virus came into public focus in the beginning of 2016,when the WHO declared the Public Health Emergency of International Concern (PHEIC) since in context of the Brazil epidemic (WHO Zika Strategic Response Plan 2016) a clear correlation between ZIKV infection of pregnant women and fetal microcephaly development was observed[8,9]. This changed the attention from a side note to a headline, initiating a variety of research efforts to investigate the virus in more detail with respect to epidemiology, virus-associated pathogenesis and virus cell interaction.

ZIKV belongs to the Flaviviridae family, which is closely related to the Spondweni virus serocomplex. As member of the Flavivirus genus, ZIKV contains a singlestranded, RNA with positive polarity. The viral genome encodes a single polyprotein processed by host and viral proteases into three structural proteins - core (C) that forms the capsid, the precursor of the membrane protein(prM), and the envelope protein (E) - and into seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A,NS4B, and NS5 that are responsible for the replication of the viral RNA[10]. The virus replication and morphogenesis occurs in the extranuclear compartment. In ZIKV-infected cells a massive remodeling of the endoplasmic reticulum(ER) to form membranous replication factories and a drastic reorganization of microtubules and intermediate filaments can be observed[11].

青少年特发性脊柱侧凸（AIS）是最常见的脊柱畸形，其病因未明［1］。椎旁肌的不平衡在AIS的发生和进展中具有重要作用［2］。有文献报道，AIS患者两侧椎旁肌的肌电活动和肌纤维类型存在差异，且与患者的侧凸角度进展相关［3］。本研究组前期通过影像学测量发现，AIS患者两侧椎旁肌的肌容积和脂肪浸润率不对称［4］。另有研究显示，AIS患者两侧椎旁肌褪黑素受体和转化生长因子-β信号通路相关分子表达不对称，但对于上述椎旁肌的复杂改变的分子机制仍鲜有报道［5-6］。

There is a variety of reports describing the infection of various primary cells or immortalized cell lines.Moreover, ZIKV was shown to replicate in various human cell types already like skin cells[12] and lung epithelial cells[13]. Not unexpected was the finding that the Aedes C6/36 cells were infectable[12], since this was described for other related viruses already[14]. Furthermore, a lot of animal cell lines were described to be susceptible to ZIKV infection[15]. When mice lacking receptors for IFN-α/β (A129) were infected with the ZIKV, viral RNA could be found in the brain, ovary, spleen and liver[16].

In order to further characterize the virus in human cell lines and to identify cell culture systems that allow the robust production of high amounts of infectious viral particles, ten cell lines were comparatively analyzed for their susceptibility to the ZIKV. Keratinocytes (HaCaT)were included in the following experiments, since the skin is the first tissue the virus comes in contact with via mosquito bite. Moreover neuronal cells (N29.1 and SHSY5Y) were of special interest due to the neurological disorders ZIKV infections may cause. Furthermore the infectivity of the well-established standard cell lines 293T cells, CHO cells, Vero cells, A549 cells, HepG2C3A cells, Huh7.5 cells and COS7 cells was studied.

MATERIALS AND METHODS

Cell culture

16 Dowall SD, Graham VA, Rayner E, Atkinson B, Hall G, Watson RJ, Bosworth A, Bonney LC, Kitchen S, Hewson R. A Susceptible Mouse Model for Zika Virus Infection. PLoS Negl Trop Dis 2016; 10: e0004658 [PMID: 27149521 DOI: 10.1371/journal.pntd.0004658]

ZIKV strains

The cells were infected with the ZIKV strain French Polynesia (PF13/251013-18) (this clinical low passage strain was kindly provided by Professor Musso, Institute Louis Marlade in Papeete, Tahiti).

Infection procedure

The inoculum for the infection experiments was derived from Vero cells that were infected for 72 h with ZIKV Polynesia. The obtained cell culture supernatant was filtrated and characterized by titration using plaque assays. Defined aliquots were stored at -80 ℃. The cell lines were infected with ZIKV at a MOI = 0.1 for 16 h.The inoculum was removed, cells were washed with prewarmed PBS, cultivated with medium for 32 h and harvested after 48 h if not stated differently.

The obtained cell culture supernatant was filtratedand characterized by titration using plaque assays.Defined aliquots were stored at -80 ℃. The cell lines were infected with ZIKV at a MOI = 0.1 for 16 h. At the present stage of knowledge no detailed information about the velocity of the infection process in the different cell culture systems is available. To avoid effects that reflect potential differences in the velocity of the infection process cells were infected for 16 h (overnight) to ensure a high infection level. The inoculum was removed, cells were washed with prewarmed PBS, cultivated with medium for 32 h and harvested after 48 h if not stated differently.

10 Kuno G, Chang GJ. Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses. Arch Virol 2007; 152: 687-696 [PMID: 17195954 DOI: 10.1007/s00705-006-0903-z]

Virus titration assay (plaque assay)

Vero cells were seeded at a density of 3 × 105 cells per well in standard six well plates and infected with cleared, serial dilutions of either cell culture supernatant or cleared cellular lysate 6 h later. Another 2 h later,the inoculum was removed and the cells were washed twice with PBS. Then the cellular monolayers were overlaid with DMEM complete containing 0.4%seaplaque agarose. Four days later, the agarose overlay was removed and the wells were washed with PBS.Afterwards, the cells were fixed with 4% formaldehyde for 10 min and stained with 0.1% crystal violet for plaque visualization.

RNA isolation and cDNA synthesis

RNA from total lysate was isolated using peqGOLD TriFast (PEQLAB Biotechnologie GmbH, Germany)according to the manufacturer’s protocol. cDNA synthesis was performed after DNA digest with DNaseI(Promega, Mannheim, Germany), using 4 μg total RNA,RevertAid H Minus Reverse Transcriptase and random primer (Thermo Scientific, Dreieich, Germany) as suggested by the manufacturer.

RNA from cell culture supernatant was isolated using QIAamp viral RNA Mini Kit (Qiagen, Hilden Germany)as described by the manufacturer. However, the elution volume was decreased to 40 μL per sample.

Quantitative real-time PCR from total lysate and cell culture supernatant

Quantitative real-time PCR (qPCR) from total RNA was performed as described[17]. All relative quantifications were normalized to the amount of RPL27 transcripts.The following primers were used: Zika fwd (5’agatcccggctgaaacactg 3’-bp 1924-1943), Zika rev (5’ttgcaaggtccatctgtccc 3’-bp 1996-1977), ribosomal protein L27 - RPL27 fwd (5’ aaagctgtcatcgtgaagaac 3’)and RPL27 rev (5’ gctgctactttgcgggggtag 3’).

qPCR using RNA isolated from cell culture supernatant was analyzed using Zika LightMix Kit (TIB MOLBIOL,Berlin, Germany) in combination with LightCycler®Multiplex RNA Virus Master (Roche, Mannheim Germany)as described by the companies protocols. In brief,2.7 μL PCR grade water, 0.25 μL Zika Light Mix, 2 μL Roche Master, 0.05 μL RT Enzyme were mixed with 5 μL purified RNA and measured in the LightCycler 480 or Light cycler 1.2 (Roche, Mannheim Germany) with the following program: (1) RT-Step: 55 ℃/5 min; (2)Denaturation: 95 ℃/5 min; and (3) Cycling (45 times):95 ℃/5 s, 60 ℃/15 s, 72 ℃/15 s; Cooling: 40 ℃/30 s.

Western blot analysis

The samples were resolved by sodium dodecyl sulfatepolyacrylamid electrophoresis (SDS-PAGE) at 10% and transferred by semi-dry blotting onto a polyvinylidine difluoride membrane (PVDF) (0.45 μm; Carl Roth,Germany). The membrane was blocked with 5% skim milk solution and then incubated with anti NS1 specific antibody at a 1:1000 dilution (Biofront, United States)overnight. Then the membrane was incubated with a mouse specific secondary antibody coupled with horseradish peroxidase at a 1:2000 dilution (HRP) (GE Healthcare, United Kingdom) and signals were detected with X-ray films (GE Healthcare, United Kingdom).Signals were quantified using ImageJ software.

Immunofluorescence analysis

Immunofluorescence staining was analyzed with a confocal laser scanning microscope (CLSM 510 Meta; Carl Zeiss) and ZEN 2009 software. Cells were fixed with absolute ice-cold ethanol for 10 min. ZIKV envelope protein was stained using anti Flavivirus Group antigen Antibody (clone D1-4G2-4-15 from Merck-Millipore,Darmstadt Germany) and a polyclonal rabbit antiserum was used to detect STAT1 (Merck AB16951). As secondary antibodies served Alexa 488 and Alexa 546(Thermo, Darmstadt Germany). Nuclei were stained with DAPI.

百年青啤在他的掌舵下进行自我颠覆和脱胎换骨，在创新转型中实现质量、效率双提升，成为引领行业转型升级、高质量发展的样本。是他让一个有着百年历史的优秀企业快速转型，也是他，让中国元素十足的青岛啤酒融入世界潮流。

CPE detection

34 Wu Y, Liu Q, Zhou J, Xie W, Chen C, Wang Z, Yang H, Cui J.Zika virus evades interferon-mediated antiviral response through the co-operation of multiple nonstructural proteins in vitro.Cell Discov 2017; 3: 17006 [PMID: 28373913 DOI: 10.1038/celldisc.2017.6]

Transfection and luciferase reporter assay

The used cell lines were transfected with the pISREluc construct (Agilent, United States), using polyethylenimine(PEI) directly after infection. In brief, 3 × 105 cells per six well were infected as described and transfected directly after the addition of virus, using a transfection mix containing 1 μg plasmid DNA, 12 μL PEI (1 mg/mL) in a total volume of 150 μL PBS (1/10 total volume of media).The media was changed the next day and cells were analyzed 48 h post transfection. Here for cells were lysed in a passive lysis buffer (25 mmol/L Tris, 2 mmol/L DTT,2 mmol/L EGTA, 10% glycerol (v/v), 1% TX-100 (v/v),pH7.5) for 10 min on ice. Afterwards, lysate was cleared by centrifugation at 4 ℃ and 5000 × g for 10 min and the luciferase activity of the supernatant was measured in 96 well Orion II plate reader (Berthold) for 10 s after the addition of luciferase buffer (20 mmol/L Tris-HCl pH7.8, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, 33.3 mmol/L DTT, 470 μmol/L Luciferin, 530 μmol/L ATP). Relative light units were normalized to the total protein amount by Bradford protein assay.

Statistical analysis

All statistical analyses were performed with Prism GraphPad 7.0, using multiple t tests for determination of P-values. Error bars are displayed as value ± SEM.

RESULTS

ZIKV replicates efficiently in various cell lines but not in CHO cells

The capacity of various human- and non-human-derived cell lines to produce high amounts of infectious ZIKV particles was analyzed. For this purpose, ten different(human and non-human) cell lines (Table 1) derived from various tissues (neuronal cells, kidney cells,keratinocytes, hepatoma cells and lung epithelia cells)were tested with respect to their susceptibility to ZIKV infection. The investigated cells were infected with an identical MOI of 0.1, using ZIKV Polynesia strain. The intracellular amount of ZIKV-specific genomes was determined by RT-PCR 48 h after infection, revealing that for all analyzed cell lines with the exception of CHO cells a productive infection could be established. With respect to the number of intracellular genomes detected in the various cell lines (Figure 1A), only moderate differences between the permissive cell lines were found. However, the amount of viral genomes must not necessarily correlate with the amount of infectious viral particles. To address this point, the number of infectious particles in the cell lysates was determined.Quantification of the intracellular amount of infectious viral particles by plaque assay (Figure 1B) revealed strong differences between the investigated cell lines of up to 105-fold. The highest amount of intracellular infectious viral particles was found for Vero containing 3.6 × 107 PFU/mL followed by the Huh7.5, COS7, 293T and A549 cells. Again, N29.1 and SH-SY5Y cells showed significantly lower amounts of intracellular viral particles(8.7 × 103 - 2.3 × 103 PFU/mL). The HaCaT cells showed besides the CHO cells, which did not contain infectious viral particles, the lowest amount amongst the investigated cell lines (3 × 102 PFU/mL). Comparison between the quantification of the intracellular viral genomes and the infectious viral particles reveals that there is a correlation, but the differences between the various cell lines are much more pronounced with respect to the amount of infectious viral particles in comparison to the viral genomes.

ZIKV-infected cells differ significantly with respect to the intracellular amount of NS1

Quantification of intracellular viral genomes does not automatically reflect replication. To further analyze ZIKV replication, the intracellular amount of NS1 was determined by western blot analysis and referred to the amount of actin (Figure 1C). The quantification of the western blots demonstrates that between the different cell lines significant differences with respect to the intracellular amount of NS1 can be observed. Nearly the same pattern for the amount of NS1 can be observed as found for the intracellular genomes by RT-PCR.A549, COS7, HepG2/C3A, Huh7.5, Vero and 293T cells showed strongest signals, while lower amounts of NS1 were detected in N29.1 cells. No NS1 was measurable in SHY5Y and CHO cells. For the HaCaT cells in contrast to the qPCR data only a low amount of NS1 was observed.

Analysis of the amount and subcellular distribution of ZIKV envelope protein by confocal immunofluorescence microscopy

To estimate the intracellular amount of ZIKV envelope protein and to analyze the amount of infected vs noninfected cells, confocal immunofluorescence microscopy was performed (Figure 1D). The staining showed that based on the plaque assays the highest producer cells also showed the best ratio between infected vs non-infected cells. The confocal immunofluorescence microscopy shows for the A549, Vero and 293T cells a susceptibility between 72%-66%, while from the Huh7.5 and HaCaT cells approximately 52% and 42% and only approximately 10% of the HepG2/C3A, N29.1 and SHSY5Y were infected after 48 h. In case of the CHO cells,no specific staining was observed confirming that these cells are not permissive for ZIKV.

Pronounced cytopathogenic effect of ZIKV in A549 and Vero cells

To study the impact of ZIKV on cell viability and integrity in the different analyzed cell lines Presto Blue assays for determination of the cell viability and LDH assays for analysis of the cell integrity were performed.For this purpose the cells were infected for 48 h and stained for Presto Blue assays or the supernatant from ZIKV-infected cells was collected 48 h after infection and the LDH activity was determined (Figure 2). Both assays revealed that ZIKV heavily affects cell integrity/cell viability in A549 and Vero cells. Less cell death was observed for HepG2/C3A, HaCaT and N29.1 cells. Based on the data from these assays and microscopic analysis COS7, 293T and SH-SY5Y cells were found to be most resistant to ZIKV induced cytolysis.

Virus release differs strongly in tested cell lines

6 Driggers RW, Ho CY, Korhonen EM, Kuivanen S, Jääskeläinen AJ, Smura T, Rosenberg A, Hill DA, DeBiasi RL, Vezina G,Timofeev J, Rodriguez FJ, Levanov L, Razak J, Iyengar P,Hennenfent A, Kennedy R, Lanciotti R, du Plessis A, Vapalahti O.Zika Virus Infection with Prolonged Maternal Viremia and Fetal Brain Abnormalities. N Engl J Med 2016; 374: 2142-2151 [PMID:27028667 DOI: 10.1056/NEJMoa1601824]

1.1 对象 选取2011年1月—2013年1月上海市浦东新区金杨社区卫生服务中心糖尿病专科门诊就诊的2型糖尿病患者300例。纳入标准:符合2010年美国ADA推荐的2型糖尿病诊断标准［3］;糖尿病病程至少6个月;使用胰岛素注射笔并且自行注射;胰岛素注射时间≥3个月;具有完全的认知和行为能力;知情同意，自愿参与。排除标准:精神疾病患者;使用胰岛素泵、注射器及无针注射器的患者;视力障碍者;急性并发症患者。

针对我院骨科地佐辛注射液在2017年度开始使用量较前明显增大，特抽查2017年1-6月份骨科出院手术患者病历30份，其中使用地佐辛病历12份，对其用药剂量、溶媒、给药方法、是否联用含地佐辛的镇痛泵、手术时间、用药时间进行列表，根据《地佐辛术后镇痛专家建议》[2]《骨科常见疼痛的处理专家建议》[3]《中国髋、膝关节置换术加速康复-围术期疼痛与睡眠管理专家共识》[4]以及地佐辛说明书等相关文献，对地佐辛使用进行点评。其中有9份病例存在不合理使用，9份病历用药情况见表1，主要不合理情况见表2。

Interferon response does not necessarily correlate with the extent of viral infection

Induction of interferon-stimulated genes (ISGs) was analyzed by luciferase reporter assay using the Interferonstimulated response element (ISRE) as promoter-driving luciferase expression. Cells were infected as described,transfected with pISREluc plasmid and the cellular luciferase activity was analyzed 48 h post infection (Figure 4A). Luciferase assay showed an induction of ISGs only for the N29.1 cells. In the rest of the tested cells ISGs were slightly repressed. However, staining of STAT1 suggests a delocalization by ZIKV (Figure 4B). If ZIKV was present in the cells it occurred like STAT1 is drawn to the replication factories and no longer is evenly distributed as seen in uninfected cells.

Taken together, these data indicate that the analyzed cell lines strongly differ with respect to the amount of released viral particles, although comparable amounts of viral genomes are detectable in the supernatant.With respect to the identification of cell culture systems that are suitable to produce high amounts of infectious viral particles, Vero- and COS7 cells as a non-humanderived cell lines and Huh7.5-A549 and 293T cells as human-derived cell culture systems were identified.

DISCUSSION

ZIKV first isolated from a sentinel monkey in the Zika forest in Uganda in 1947[1] is an emerging virus that has spread over the years from Africa, over Asia,Micronesia, French Polynesia to Brazil[18]. Since the Brazil epidemic in 2015/2016[19] ZIKV research has increased dramatically. In this study a ZIKV isolate from Polynesia that also belongs to the Asian lineage like the ZIKV strain causative for the epidemic in Brazil, was used to characterize its potential to infect various human cell lines with the aim to provide cell culture models for investigating the ZIKV life cycle in more detail and to test the suitability of various cell culture systems to produce high amounts of this virus[20]. The French Polynesia strain was utilized since this was described first to cause GBS and microcephaly, like later for the Brazil outbreak[4,7,20]. The virus targets different cell types such as macrophages, fibroblasts, trophoblasts as well as mesenchymal stem cells[21].

In light of the correlation between ZIKV infection of pregnant women and the development of microcephaly in the fetus it is of major interest that ZIKV can be detected in the maternal decidua, fetal placenta and umbilical cells.

In interferon receptor type I-deficient mice (IFNAR KO) ZIKV causes systemic infections in all tissues providing a useful tool for drug target testing[16,22-24].Moreover, several publications have shown to replicate ZIKV in various primary cells like neuronal cells, dendritic cells or keratinocytes[12,14,25,26]. Immortalized cells are already described for infection with ZIK[11,12,14,27].

一个月后，两人办证结婚了，因为辛娜怀孕了。他们在亲友的祝福声中步入了婚姻的殿堂。半年后，两居室里的鲜花变成了尿片子，儿子小龙降生了。产房里，王树林怀抱着早产的儿子万般柔情心疼不已。出院的时候，王树林抱着儿子去医生办公室告辞，几个护士热心的围拢过来，她们快人快语，一个说脸型是妈妈的，皮肤是爸爸的，一个说，要是遗传妈妈的皮肤就更好了，妈妈多白啊，王树林呵呵应笑。辛娜在门口催他。他赶紧问了最后一个问题：婴儿早产不会对以后的智商有影响吧？

In this comparative study here, ten cells lines were tested for their capacity to support replication and production of infectious virus. After infection of the cells,the ongoing replication was monitored by qPCR, western blot and immunofluorescence microscopy analysis,while the capacity to produce infectious virus was investigated by qPCR and plaque assay. It was found that ZIKV replicates in neuronal cells, keratinocytes,lung carcinoma cells, liver carcinoma cells and kidney cells. Vero cells always served as positive control and standard for quantification. ZIKV showed best replication and virus production in A549, COS7, Huh7.5 and 293T cells, followed by HepG2/C3A and HaCaT cells. Significantly lower support was measured for N29.1 and SH-SY5Y cells. In contrast to these cells,CHO cells could not be infected. The finding that 293T cells are susceptible to ZIKV stands in contrast to a previous publication by Hamel et al[12] (2015). Huh7 cells have also been used in some studies and demonstrated to support viral replication[11], but here we used the Huh7-derived Huh7.5 cells clone[28] that has a defect in the RIG-I gene[29]. However, comparable titers were reached for the Huh7 cells measured by Cortese et al[11] and the Huh7.5 cells in our hands, so that there seems to be no benefit using the Huh7.5 cells at least for the production of high titer viral stock, indicating that ZIKV replication is not significantly affected by the functionality of the RIG-I gene. ZIKV propagation in primary keratinocytes has been shown already[12]. Here we used the immortalized keratinocyte cell line HaCaT[30]which turned out to be susceptible to viral infection and also produced a good viral titer. Moreover, the virus replicates very efficiently in COS7 cells[31], showing release of infectious virus to a titer of over 10E7 viral particles/mL. This was not unexpected since these cells,like Vero cells, derive from the kidney of an African green monkey. By using a MOI of 0.1, which is in the lower range if compared to Dengue virus titers used for infections in cell culture, we assured to only identify cells that support viral infection efficiently. The measured viral genomes in the corresponding supernatants resembled the findings from the intracellular quantification, in which the measured genomes in case of CHO cells represent input RNA from the infection procedure. However,comparable amounts of viral RNA were observed inside and outside of the cells.

When comparing the intracellular and the extracellular titers of infectious virus, in general higher titers were measurable in the supernatant of the cells compared to the intracellular amount of virus. But for the high producer cell lines like A549, COS7, Huh7.5, Vero and 293T nearly the titers measured outside the cells were reached already inside the cells. This is also reflected by the detection of NS1 in cellular lysate by western blot and by analyzing the Env-protein by immunofluorescence.Here also more NS1 was detectable for the high producers and more infected cells were visualized. However, for unknown reasons only low titers were detected inside the HepG2/C3A cells, but nearly 10E4 more infectious virus was secreted by these cells. This enhancement of viral release may be based on efficient packaging and export of the viral cargo or a lower turnover of viral proteins.

To investigate if the activation of IFN-signaling is causative for the diverging susceptibility of the analyzed cell lines, the induction of ISGs was monitored by pISRE-luciferase reporter experiments upon viral infection.The luciferase assay showed for all infected cell lines a reduction on ISRE activity, except for N29.1 cells.Inhibition of Type Ⅰ and Ⅲ IFN production by interfering with the STAT signaling has been demonstrated in several recent publications for the ZIKV[32-34]. Particularly NS5 is described to counteract IFN signaling by binding STAT2 and to promote STAT2 degradation by the proteasome[32]. Moreover, NS1, NS4B and NS2B3 were also shown to inhibit IFN signaling[34]. In contrast to wt mice having a functional adaptive immune response,IFNAR KO mice are susceptible to ZIKV infection[16,23].This also emphasizes the importance of the IFN response which has to be trapped by the virus in order to establish infection. Astonishingly, the ISRE promoter element was also reduced in its activation in CHO cells, although they do not support viral infection and replication. Since it is not known if the lack of susceptibility of CHO cells for ZIKV infection is due to impaired attachment, entry or post entry steps there exist a variety of possibilities that could lead to an interference with the interferon signaling.The results from the luciferase assay were strengthened by STAT1 staining in infected A549 cells. The changed distribution in infected vs uninfected cells is obvious,but from these experiments it stays uncertain if this is causative for the reduced ISRE activation. The results of this study support ZIKV research by providing different cell culture models based on various tissues, so that the information at hand enables the investigation of ZIKV life cycle in more detail. Also drug testing and pathogenicity studies can be fostered by the shown cell culture models susceptible to ZIKV.

ARTICLE HIGHLIGHTS

Research background

Zika virus (ZIKV) is an emerging virus transmitted mainly by mosquitos, that has spread during the last decades from Africa, to Asia, over Micronesia to the Americans causing an epidemic in Brazil in the years 2016/2017. In order to propagate the virus in cell culture we investigated various cell lines for their susceptibility to ZIKV infection.

Research motivation

To date ZIKV is mainly propagated in Vero cells derived from kidney epithelial cells from African green monkey. This study aimed to investigate the potential of various cell lines to support the viral life cycle in order to provide researchers with suitable cell culture systems for different issues in the field of ZIKV research.

Research objectives

The objectives of this research were to investigate ten human and nonhuman cell lines from various tissues (e.g., hepatocytes, keratinocytes and neuronal cells) with regard to their intracellular amount of viral genomes and infectious viral particles upon ZIKV-infection. Moreover, the amount of secreted viral genomes and infectious viral particles was analyzed in the cell culture supernatants. Furthermore, the amount of infected cells was analyzed by immunofluorescence using an Envelope-specific antibody and the amount of NS1 was analyzed by western blot. In order to draw a conclusion whether parts of the innate immune response are responsible for the found differences in viral support, STAT1 distribution and expression was analyzed.

前震旦纪末期岩浆活动较强烈，成矿母岩为燕山期闪长岩，岩体富含铁质。当高、中温岩体顺层贯入高角度变质绿泥片岩、绢云母石英片岩与大理岩岩层中，受岩浆中的气水热液作用影响，岩层发生热液蚀变，产生阳起石化、透闪石化、绿泥石化、碳酸盐化、黄铁矿化等，并与围岩发生交代，形成磁铁矿。其成因与岩浆中的气水热液有关。

Research methods

Quantification of viral genomes was performed by qPCR. For the detection of genomes from whole cell lysate a standard PCR protocol with SYBR green was used with cDNA as template transcribed from total RNA that was isolated with a Tri-reagent. Viral genomes released into the cell culture supernatant were isolated with a viral RNA isolation kit and subjected to a Taqman-PCR based on a ZIKV-Lightmix Kit. The detection of infectious virus was performed by virus titration assay using serial dilutions from the supernatant or from cleared cellular lysates. The amount of infected cells was analyzed with immunofluorescence microscopy by using an Env-specific antibody and with western blot using NS1-specific antibody. The effect on the innate immunity was monitored by luciferase-reporter assay and STAT1 analysis distribution in the immunofluorescence microscopy.

Research results

17 Himmelsbach K, Hildt E. The kinase inhibitor Sorafenib impairs the antiviral effect of interferon α on hepatitis C virus replication.Eur J Cell Biol 2013; 92: 12-20 [PMID: 23107224 DOI: 10.1016/j.ejcb.2012.09.001]

Research conclusions

The results presented so far provide a toolbox of cell culture systems for ZIKV research in general. However, the analyzed cells differ strongly with respect to the amount of released viral particles, whereas the amount of genomes amongst the cells in the supernatant and inside of infected cells are more or less equal. This is an important finding, since a lot of research and diagnostic is based on qPCR analysis only.

Research perspectives

Further research should aim on the differences of released viral genomes vs released infectious virus. Are there differences in the release pathway?Which pathways are used for viral egress? Why are certain cell lines not succeptible?

ACKNOWLEDGEMENTS

We thank Didier Musso (Director of the Research and Diagnosis Laboratory, Institute Louis Marlade in Papeete, Tahiti) for disposing ZIKV Polynesia strain (PF 13/251013-18).

REFERENCES

1 Dick GW. Epidemiological notes on some viruses isolated in Uganda; Yellow fever, Rift Valley fever, Bwamba fever, West Nile, Mengo, Semliki forest, Bunyamwera, Ntaya, Uganda S and Zika viruses. Trans R Soc Trop Med Hyg 1953; 47: 13-48 [PMID:13077697 DOI: 10.1016/0035-9203(53)90021-2]

钱纳里将工业化进程分为三个发展时期，包括六个阶段：第一个时期以农业经济为主导，属于前工业化阶段；第二个时期以工业经济为主导，是工业化实现和经济高速发展的阶段，该阶段又划分为工业化初期、工业化中期和工业化后期三个阶段；第三时期以服务业为主导，是经济稳定增长的阶段，划分为发达经济初级期和发达经济高级期两个阶段。参照钱纳里与库兹涅茨的评价指标体系，本文选取人均GDP、三次产业结构、三次产业就业结构和人口城市化率作为衡量工业化发展水平的指标。

“夕阳”怎样“红”？其“基石”是对生命意义的理解，由此想到了近代几位先贤。1842年上半年，丧权辱国的《南京条约》正酝酿签订，75岁的老臣王鼎认为万万不可签约，面折廷争仍无济于事，无奈之下他毅然选择了“尸谏”，临死前留下了“条约不可轻许，恶例不可轻开，穆（彰阿）不可用，林（则徐）不可弃”的诤言。戊戌变法失败后，谭嗣同本可逃生，但为了唤醒民众却主动赴死。吉鸿昌就义前写下了“恨不抗日死，留作今日羞”的诗句，视为抗日而死为无上荣光。古往今来有无数这样的仁人志士，他们同样知道生命可贵，但还知道有比生命更可贵的东西。“夕阳”也好，“午阳”也罢，他们都“红的像那燃烧的火”，虽死犹生。

2 Olson JG, Ksiazek TG, Suhandiman, Triwibowo. Zika virus, a cause of fever in Central Java, Indonesia. Trans R Soc Trop Med Hyg 1981; 75: 389-393 [PMID: 6275577 DOI: 10.1016/0035-9203(81)90100-0]

3 Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL,Lanciotti RS, Pretrick M, Marfel M, Holzbauer S, Dubray C,Guillaumot L, Griggs A, Bel M, Lambert AJ, Laven J, Kosoy O, Panella A, Biggerstaff BJ, Fischer M, Hayes EB. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med 2009; 360: 2536-2543 [PMID: 19516034 DOI: 10.1056/NEJMoa0805715]

4 Oehler E, Watrin L, Larre P, Leparc-Goffart I, Lastere S, Valour F,Baudouin L, Mallet H, Musso D, Ghawche F. Zika virus infection complicated by Guillain-Barre syndrome--case report, French Polynesia, December 2013. Euro Surveill 2014; 19: pii: 20720[PMID: 24626205 DOI: 10.2807/1560-7917.ES2014.19.9.20720]

中间仿古椽头等距等尺寸布置，在底模设置后，采用模板定型。选择无扭曲、无变形的胶合板（表面光滑，预加工成填充形状）进行椽木之间的加工，即根据椽木之间的距离，采用胶合板模型作为填充物，将整体组装成一个长为3m的模板，置于底模上。

5 Calvet G, Aguiar RS, Melo ASO, Sampaio SA, de Filippis I,Fabri A, Araujo ESM, de Sequeira PC, de Mendonça MCL, de Oliveira L, Tschoeke DA, Schrago CG, Thompson FL, Brasil P, Dos Santos FB, Nogueira RMR, Tanuri A, de Filippis AMB.Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study. Lancet Infect Dis 2016; 16: 653-660 [PMID: 26897108 DOI: 10.1016/S1473-3099(16)00095-5]

To investigate whether the data obtained for the analysis of the intracellular amount of viral genomes and infectious viral particles are reflected by the numbers of genomes and infectious viral particles in the supernatant,media from the infected cell cultures were analyzed 48 h after infection. The RT-PCR (Figure 3A) revealed that in accordance to the results obtained for the quantification of the intracellular genomes, CHO released no viral genomes. As observed for the intracellular amount of viral genomes, there were no major differences in the amount of released viral genomes between the different cell lines. The difference between the highest amount observed for A549, HaCaT and Vero cells on the one side and N29.1 or SH-SY5Y cells on the other side is less than 2 fold. As the amount of viral genomes in the supernatant must not correlate with the amount of infectious viral particles, the number of infectious viral particles in the cell culture supernatants was determined by virus titration assays (Figure 3B). In contrast to the moderate differences that were found analyzing the number of viral genomes, strong differences (more than 102-fold) were revealed with respect to the number of infectious viral particles released by the different cell lines. The highest amounts were detected for supernatants derived from Vero-, A549-, COS7-, HepG2/C3A-, Huh7.5-, HaCaT- and 293T-cells that produced nearly the same quantity of infectious viral particles(about 107/mL). The neuronal cell lines N29.1 and SHSY5Y cells released more than 100 times less infectious viral particles than Vero cells. For CHO cells no significant amount of released viral particles was detectable.

7 Mlakar J, Korva M, Tul N, Popović M, Poljšak-Prijatelj M, Mraz J, Kolenc M, Resman Rus K, Vesnaver Vipotnik T, Fabjan Vodušek V, Vizjak A, Pižem J, Petrovec M, Avšič Županc T. Zika Virus Associated with Microcephaly. N Engl J Med 2016; 374: 951-958[PMID: 26862926 DOI: 10.1056/NEJMoa1600651]

8 Fauci AS, Morens DM. Zika Virus in the Americas--Yet Another Arbovirus Threat. N Engl J Med 2016; 374: 601-604 [PMID:26761185 DOI: 10.1056/NEJMp1600297]

9 Zhang Q, Sun K, Chinazzi M, Pastore Y Piontti A, Dean NE,Rojas DP, Merler S, Mistry D, Poletti P, Rossi L, Bray M, Halloran ME, Longini IM Jr, Vespignani A. Spread of Zika virus in the Americas. Proc Natl Acad Sci USA 2017; 114: E4334-E4343[PMID: 28442561 DOI: 10.1073/pnas.1620161114]

依据当地《当地城市轨道交通详细勘察阶段岩土工程勘察总体技术要求》中提供的统一岩土分层标准，根据场地内所揭露地层的地质时代、成因类型、岩性特征、风化程度等工程特性，将场地内岩土层分为填土层、淤泥质黏土、岩石全风化带、岩石强风化带、岩石中风化带共七大类，部分地层根据地质描述需要进一步再细分亚层，例如淤泥质黏土中，黏性好，富水性差，透水性弱，为相对隔水层可视为粉质黏土.地质填充如图1，现场地质比较复杂，分沿开挖方向左、右线地质填充图.

童话故事有夸张、想象等多种表现手法，且多利用学生熟悉的事物作为基础。因此，教师可以利用童话故事，开展童话演出的活动，通过表演的方式，培养学生表演能力，培养学生的想象能力，提升其审美水平与情趣。

11 Cortese M, Goellner S, Acosta EG, Neufeldt CJ, Oleksiuk O,Lampe M, Haselmann U, Funaya C, Schieber N, Ronchi P,Schorb M, Pruunsild P, Schwab Y, Chatel-Chaix L, Ruggieri A,Bartenschlager R. Ultrastructural Characterization of Zika Virus Replication Factories. Cell Rep 2017; 18: 2113-2123 [PMID:28249158 DOI: 10.1016/j.celrep.2017.02.014]

12 Hamel R, Dejarnac O, Wichit S, Ekchariyawat P, Neyret A,Luplertlop N, Perera-Lecoin M, Surasombatpattana P, Talignani L, Thomas F, Cao-Lormeau VM, Choumet V, Briant L, Desprès P, Amara A, Yssel H, Missé D. Biology of Zika Virus Infection in Human Skin Cells. J Virol 2015; 89: 8880-8896 [PMID: 26085147 DOI: 10.1128/JVI.00354-15]

13 Frumence E, Roche M, Krejbich-Trotot P, El-Kalamouni C,Nativel B, Rondeau P, Missé D, Gadea G, Viranaicken W, Desprès P. The South Pacific epidemic strain of Zika virus replicates efficiently in human epithelial A549 cells leading to IFN-β production and apoptosis induction. Virology 2016; 493: 217-226[PMID: 27060565 DOI: 10.1016/j.virol.2016.03.006]

14 White LA. Susceptibility of Aedes albopictus C6/36 cells to viral infection. J Clin Microbiol 1987; 25: 1221-1224 [PMID: 3611315]

15 Barr KL, Anderson BD, Prakoso D, Long MT. Working with Zika and Usutu Viruses In Vitro. PLoS Negl Trop Dis 2016; 10:e0004931 [PMID: 27541001 DOI: 10.1371/journal.pntd.0004931]

古元古界秦岭群郭庄组(Pt1g)：该地层出露最为广泛，北以大河韧性剪切带为界，与二郎坪群刘山岩组(Pz1l)呈断层接触，岩性主要为斜长角闪片麻岩和花岗质片麻岩，局部夹条带状大理岩透镜体，是区内钼多金属矿的赋矿围岩；雁岭沟组(Pt1y)：主要分布在研究区的东南部，岩性主要为方解石大理岩、含石墨方解石大理岩、大理岩等，与下伏郭庄组地层呈断层接触(见图2)。

教学实验选择江苏旅游职业学院经贸分院2016级会计专业中专1班、2班为实验班，两班共有学生60人；以同年级同专业的中专3班、4班为对照班，两班共有学生61人。

A549, CHO, COS7, HepG2/C3A, Huh7.5, HaCaT,N29.1, SH-SY5Y, Vero and 293T cells (Table 1) were grown in Dulbecco’s modified eagle medium (DMEM)supplemented with 2 mmol/L L-Glutamine, nonessential amino acids, 100 U/mL penicillin and 100 μg/mL streptomycin in a humidified incubator at 37 ℃ with 5%CO2. Passaging of the cells was carried out three times a week, reaching a maximum density of 90%.

All investigated cell lines except CHO cells supported infection, replication and release of ZIKV. While in infected A549 and Vero cells a pronounced cytopathic effect was observed COS7, 293T and Huh7.5 cells were most resistant.Although the analyzed cell lines released comparable amounts of viral genomes to the supernatant significant differences were found for the number of infectious viral particles. The neuronal cell lines N29.1 and SH-SY5Y released 100 times less infectious viral particles than Vero-, A549- or 293T-cells.However there is no strict correlation between the amount of produced viral particles and the induction of an interferon response in the analyzed cell lines.

18 Hayes EB. Zika virus outside Africa. Emerg Infect Dis 2009; 15:1347-1350 [PMID: 19788800 DOI: 10.3201/eid1509.090442]

19 Heukelbach J, Alencar CH, Kelvin AA, de Oliveira WK,Pamplona de Góes Cavalcanti L. Zika virus outbreak in Brazil.J Infect Dev Ctries 2016; 10: 116-120 [PMID: 26927450 DOI:10.3855/jidc.8217]

20 Baud D, Gubler DJ, Schaub B, Lanteri MC, Musso D. An update on Zika virus infection. Lancet 2017; 390: 2099-2109 [PMID:28647173 DOI: 10.1016/S0140-6736(17)31450-2]

21 El Costa H, Gouilly J, Mansuy JM, Chen Q, Levy C, Cartron G, Veas F, Al-Daccak R, Izopet J, Jabrane-Ferrat N. ZIKA virus reveals broad tissue and cell tropism during the first trimester of pregnancy. Sci Rep 2016; 6: 35296 [PMID: 27759009 DOI:10.1038/srep35296]

22 Lazear HM, Govero J, Smith AM, Platt DJ, Fernandez E, Miner JJ, Diamond MS. A Mouse Model of Zika Virus Pathogenesis. Cell Host Microbe 2016; 19: 720-730 [PMID: 27066744 DOI: 10.1016/j.chom.2016.03.010]

23 Ma W, Li S, Ma S, Jia L, Zhang F, Zhang Y, Zhang J, Wong G,Zhang S, Lu X, Liu M, Yan J, Li W, Qin C, Han D, Qin C, Wang N, Li X, Gao GF. Zika Virus Causes Testis Damage and Leads to Male Infertility in Mice. Cell 2016; 167: 1511-1524.e10 [PMID:27884405 DOI: 10.1016/j.cell.2016.11.016]

24 Miner JJ, Cao B, Govero J, Smith AM, Fernandez E, Cabrera OH, Garber C, Noll M, Klein RS, Noguchi KK, Mysorekar IU,Diamond MS. Zika Virus Infection during Pregnancy in Mice Causes Placental Damage and Fetal Demise. Cell 2016; 165:1081-1091 [PMID: 27180225 DOI: 10.1016/j.cell.2016.05.008]

25 Retallack H, Di Lullo E, Arias C, Knopp KA, Laurie MT,Sandoval-Espinosa C, Mancia Leon WR, Krencik R, Ullian EM, Spatazza J, Pollen AA, Mandel-Brehm C, Nowakowski TJ,Kriegstein AR, DeRisi JL. Zika virus cell tropism in the developing human brain and inhibition by azithromycin. Proc Natl Acad Sci USA 2016; 113: 14408-14413 [PMID: 27911847 DOI: 10.1073/pnas.1618029113]

26 Tang H, Hammack C, Ogden SC, Wen Z, Qian X, Li Y, Yao B,Shin J, Zhang F, Lee EM, Christian KM, Didier RA, Jin P, Song H,Ming GL. Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth. Cell Stem Cell 2016; 18: 587-590[PMID: 26952870 DOI: 10.1016/j.stem.2016.02.016]

27 Chan JF, Yip CC, Tsang JO, Tee KM, Cai JP, Chik KK, Zhu Z,Chan CC, Choi GK, Sridhar S, Zhang AJ, Lu G, Chiu K, Lo AC,Tsao SW, Kok KH, Jin DY, Chan KH, Yuen KY. Differential cell line susceptibility to the emerging Zika virus: implications for disease pathogenesis, non-vector-borne human transmission and animal reservoirs. Emerg Microbes Infect 2016; 5: e93 [PMID:27553173 DOI: 10.1038/emi.2016.99]

28 Blight KJ, McKeating JA, Rice CM. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.J Virol 2002; 76: 13001-13014 [PMID: 12438626 DOI: 10.1128/JVI.76.24.13001-13014.2002]

29 Sumpter R Jr, Loo YM, Foy E, Li K, Yoneyama M, Fujita T,Lemon SM, Gale M Jr. Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I. J Virol 2005; 79: 2689-2699 [PMID:15708988 DOI: 10.1128/JVI.79.5.2689-2699.2005]

30 Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J,Markham A, Fusenig NE. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 1988; 106: 761-771 [PMID: 2450098 DOI: 10.1083/jcb.106.3.761]

31 Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell 1981; 23: 175-182 [PMID: 6260373 DOI: 10.1016/0092-8674(81)90282-8]

32 Kumar A, Hou S, Airo AM, Limonta D, Mancinelli V, Branton W, Power C, Hobman TC. Zika virus inhibits type-I interferon production and downstream signaling. EMBO Rep 2016; 17:1766-1775 [PMID: 27797853 DOI: 10.15252/embr.201642627]

33 Bowen JR, Quicke KM, Maddur MS, O‘Neal JT, McDonald CE,Fedorova NB, Puri V, Shabman RS, Pulendran B, Suthar MS. Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells. PLoS Pathog 2017; 13: e1006164 [PMID:28152048 DOI: 10.1371/journal.ppat.1006164]

Cytolysis was monitored by LDH release assay (Clontech,Japan) and cell viability was assessed by Presto Blue staining (Thermo, Darmstadt Germany) according to the instructions of the manufacturer. Upon cellular damage lactate dehydrogenase (LDH) is released into the cell culture supernatant. This release is indirectly measured based on a calorimetric assay detecting an enzymatically formed formazan product. Presto Blue is a red compound that is taken up by the cells and due to the reducing interior environment turns into a red color that is detectable at 570 nm.