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Jun 2019

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Production, quantification, and infection of Amazonian Phlebovirus (Bunyaviridae)
亚马孙流域白蛉病毒属(布尼亚病毒科)的产生、定量和感染   

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Abstract

Phlebotomine vectors, sand flies of the order Diptera, are known to transmit Leishmania parasites as well as RNA viruses (arboviruses) to humans. The arbovirus, Icoaraci Phlebovirus (BeAN 24262 - ICOV), used in this study was isolated from Nectomys rodents, a mammalian species that is the same natural sylvatic reservoir of Leishmania (Leishmania) amazonensis. This Leishmania species is distributed in primary and secondary forests in Brazil and other countries in America and causes localized and diffuse anergic skin lesions. In our recent studies, we observed an aggravation of the protozoan infection by ICOV through the modulation of cytokine expression, such as IL-10 and IFN-β, enhancing the parasite load and possibly the pathogenesis. Efficient viral production and quantitation had to be developed and standardized to ensure that immuno-molecular assays provide consistent and reproducible viral infection results. The standardization of these procedures becomes a particularly useful tool in research, with several applications in understanding the interaction between the host cell and Phlebovirus, as well as co-infections, allowing the study of intracellular signaling pathways. Here, we detail a protocol that allows the production and quantitation of the Icoaraci Phlebovirus using BHK-21 cells (baby hamster kidney cells) and subsequent infection of peritoneal macrophages from C57BL/6 mice.

Keywords: Phlebovirus (白蛉病毒属), Bunyaviridae (布尼亚病毒科), Arbovirus (虫媒病毒), Macrophage (巨噬细胞)

Background

In Brazil, approximately 210 different types of arboviruses have been isolated, of which 196 were found in the Brazilian Amazon (Rosa et al., 1998; Azevedo et al., 2007), one of the largest reserves of arboviruses in the world. The Bunyaviridae family is composed of more than 350 viruses divided into five genera, among which Orthobunyavirus, Phlebovirus, Nairovirus, and Hantavirus contain species pathogenic to humans and animals, while the genus Tospovirus is pathogenic only to plants (Walter and Barr, 2011; Ly and Ikegami, 2016).


In most human cases, Phlebovirus cause a wide spectrum of symptoms from mild febrile illness to hemorrhagic fever and death (Bird et al., 2009; Ikegami and Makino, 2011; Brisbarre et al., 2015). A specific member of Phlebovirus, the Rift Valley Fever virus, is potentially dangerous to pregnant domesticated animals such as cattle, goat, and sheep, with infection resulting in fetal malformation and abortion (Swanepoel and Coetzer, 2004). In this work, we used the Icoaraci Phlebovirus (BeAN 24262), which is prevalent in small forest animals (Causey and Shope, 1965). All Phlebovirus are morphologically similar: 80-120 nm in diameter, icosahedral symmetry, and enveloped with two surface glycoproteins (Gn and Gc) (Freiberg et al., 2008). The single-stranded RNA (ssRNA) is segmented and packaged within ribonucleoprotein particles (RNPs) by protein N and associated with an RNA-dependent RNA polymerase (RdRp). The small (S) segment uses the ambisense coding strategy, coding for a nucleocapsid protein (N) and non-structural protein (NSs) (Simons et al., 1990; Giorgi et al., 1991).


Studies in animal models suggest a protective effect of type I interferon (IFN) in infection caused by Phlebovirus (Mendenhall et al., 2009). Most viruses have developed the ability to express proteins capable of preventing the immune response (Habjan et al., 2009). This antagonistic activity has been observed for several non-structural viral proteins (Van Knippenberg et al., 2010) by mechanisms that explore different targets in IFN signaling pathways. Although Phlebovirus exhibit different levels of pathogenicity, the NSs protein is considered an important virulence factor for all members of the genus, as well as other members of the Bunyaviridae family (Habjan et al., 2009; Ikegami et al., 2009).


Several studies have shown the importance of the relationship between viruses and Leishmania parasites in the establishment of reciprocal adaptive advantages that promote survival and persistent infection (Ives et al., 2011; Hartley et al., 2012; Vivarini et al., 2015; Eren et al., 2016; Rath et al., 2018). Our group previously demonstrated that the induction of antiviral pathways by L. amazonensis increases macrophage infection (Pereira et al., 2010; Vivarini et al., 2015). Different Leishmania species associated with host immunological factors determine distinct clinical manifestations of leishmaniasis (Alvar et al., 2012). The production of leishmanicidal mediators, such as IL-12, IL-6, and reactive oxygen species (ROS), by innate immune cells contributes to infection control (Dayakar et al., 2019), but Leishmania parasites are able to subvert the signaling pathways that trigger these mechanisms and instead induce pathways favoring the infectious process. Using Icoaraci Phlebovirus as a model, we demonstrated that this virus potentiates infection by L. amazonensis in macrophages. Increased macrophage infection requires toll-like receptor 3 (TLR3) and protein kinase R (PKR) pathways due to the production of type I IFNs induced by the Leishmania and amplified during co-infection with the virus (Rath et al., 2019). Our results highlight the importance of complex and multifactorial analysis of infectious processes, especially in endemic areas where overlap occurs, as observed between L. amazonensis and Icoaraci Phlebovirus.


Given the socioeconomic impact of Phlebovirus infection and the potential co-infection in cutaneous leishmaniasis, the present protocol for the production, quantitation, and infection of Icoaraci Phlebovirus aims to address the molecular mechanisms associated with the interaction between sand fly-transmitted arboviruses and Leishmania parasite infection in vitro and in vivo in the cell host. Subsequent research using this methodology may reveal important aspects related to the host immune response, helping to outline the pathophysiological phenomena and clinical conditions and clarifying scenarios that may indicate how an antiviral response to double-stranded RNA (dsRNA) can mediate immune-inflammatory and immunopathogenic processes in Phlebovirus infection and co-infections.


Materials and Reagents

  1. Cell culture vessels, plates, and tubes

    1. 25-cm2 cell culture flask (Corning, catalog number: CLS430639)

    2. 100-mm plates (SARSTEDT, catalog number: 82.1472.001)

    3. 6-well cell culture plates (Corning, catalog number: 83.3920)

    4. 24-well cell culture plates (Corning, catalog number: 83.3922)

    5. 15-ml and 50-ml Falcon tubes (BD Falcon)

    6. Cell counting slides with grids

    7. 1.5-ml Eppendorf tubes

    8. Eco Nitrile Gloves (SuperMax)

    9. Filter tips, low-retention: 1,000 µl, 200 µl, 20 µl, 10 µl (Axygen, catalog numbers: 31000LRS, 3200LRS, 320LRS, 310LRS, respectively)

    10. 5-ml serological pipettes (SARSTEDT, catalog number: 86.1253.001)

    11. 10-ml serological pipettes (SARSTEDT, catalog number: 86.1254.001)

    12. 0.22-µm cell culture medium filtration unit (Corning, catalog number: CLS430513-12EA)


  2. Cells

    BHK-21 cells (baby hamster kidney cells, ATCC: CCL-10)

    Cells are maintained in 25-cm2 cell culture flasks with complete DMEM (DMEM – high glucose and supplemented with 10% heat-inactivated FBS: see Recipe 1) at 37°C in a humidified atmosphere containing 5% CO2. After reaching confluence, cells are subcultured to maintain cell viability. For this, remove the medium and rinse the cells with 3 ml warm PBS without calcium and magnesium. Dissociate the cells using 1 ml trypsin-EDTA solution for 1 min. Add 4 ml complete DMEM, transfer to a new 15-ml conical tube, and centrifuge the cells for 5 min at 300 × g. Resuspend the cell pellet in fresh complete medium and split 1:20 in new flasks.


  3. Phlebovirus

    The Icoaraci Phlebovirus (BeAN 24262-ICOV) was obtained from Dr. Pedro Vasconcelos (Instituto Evandro Chagas/SVS/MS).

    The virus was isolated from the liver, spleen, kidney, and heart of Nectomys squamipes in Belém, Pará, Brazil, and subsequently inoculated intracerebrally in 2-day-old Swiss mice. The samples were lyophilized and stored at -80°C. Before use, reconstitute the samples in 0.5 ml Puck’s medium.


  4. Cell culture reagents

    1. 0.25% trypsin-EDTA solution (Sigma-Aldrich, catalog number: T4049), stored at -20°C

    2. Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose and L-glutamine (Lonza, catalog number: 12-604Q)

    3. 100× penicillin-streptomycin solution (HyClone-GE, catalog number: SV30010)

    4. Puck’s medium (PAN Biotech, catalog number: P04-51100)

    5. Phosphate-buffered saline (PBS) (Lonza, catalog number: 17-516Q)

    6. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106)

    7. UltraPureTM DNase/RNase-free distilled water (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977-035)

    8. Methylcellulose (Sigma-Aldrich, catalog number: M7027)

    9. 100-bp DNA ladder, 500 µg/ml (Promega, catalog number: G201A)

    10. 6× gel loading dye (Promega, catalog number: G190A)

    11. Standard agarose for routine gel electrophoresis (UniScience, catalog number: AGR-LE-100)

    12. Ethidium bromide (Sigma-Aldrich, catalog number: E7637)

    13. Tris-base (Promega, catalog number: H5131)

    14. Glacial acetic acid (Sigma-Aldrich, catalog number: A6283)

    15. UltraPureTM 0.5 M EDTA (Ethylenediaminetetraacetic acid, disodium salt dihydrate), pH 8.0 (Thermo Fisher Scientific, catalog number: 15575020)

    16. Crystal violet (Sigma-Aldrich, catalog number: C0775)

    17. Sodium thioglycolate (Sigma-Aldrich, catalog number: T0632), stored at room temperature

    18. Direct-zolTM RNA MiniPrep Plus kit (Zymo Research, catalog number: R2070)

    19. ImProm-IITM Reverse Transcription System (Promega, catalog number: A3800)

    20. Random primers (Promega, catalog number: C1181)

    21. GoTaq® DNA polymerase (Promega, catalog number: M3001)

    22. Complete DMEM (see Recipes)

    23. Semi-solid medium (see Recipes)

    24. Crystal violet solution (see Recipes)

    25. 10× TAE Buffer (1 L) (see Recipes)

    26. 1× TAE Buffer (1 L) (see Recipes)

    27. 1.5% agarose gel (see Recipes)

    28. 100-bp DNA ladder (50 µg/ml) (see Recipes)

    29. 3% sodium thioglycolate (see Recipes)

Equipment

  1. P1000 (Gilson, catalog number: F123602)

  2. P200 (Gilson, catalog number: F123601)

  3. P20 (Gilson, catalog number: F123600)

  4. P2 (Gilson, catalog number: F144801)

  5. Lab refrigerator set at 4°C (Electrolux, model: IF55)

  6. Lab freezer set at -20°C (Prosdocimo, model: F21)

  7. Lab freezer set at -80°C (Panasonic, model: MDF-U56VC-PA)

  8. Incubator at 37°C with 5% CO2 (Sanyo inCU-Safe, model: MCO-17AC)

  9. Laminar flow hood (Esco Class II BSC- AirStream)

  10. Centrifuge 5418 (Eppendorf) at room temperature; set here at 24°C

  11. Centrifuge (ThermoFisher Scientific, model: D-37520)

  12. NanoDropTM ND-1000 Spectrophotometer (ThermoFisher Scientific)

  13. Balance (BEL Analytical Equipment, model: S622)

  14. Veriti 96-Well thermal cycler (Applied Biosystems PCR instruments, model: 9902)

  15. Mini-gel migration system (Loccus Biotecnology, model: LCH-7X8)

  16. Electrophoresis power supply (GIBCO BRL, Life Technologies, 250) and power cables

  17. U.V. transilluminator (Fisher Scientific, model: 88A)

Procedure

Figure 1 illustrates the steps for virus production, quantitation, and infection. A detailed description of the procedures is found in the following items.



Figure 1. Different steps for the production, quantitation, and infection of Icoaraci Phlebovirus. Figure created in the Mind the Graph platform.


  1. Production of the Icoaraci Phlebovirus

    1. Plate 4 × 106 BHK-21 cells in 100-mm plates (7 ml/plate). In order to obtain a good virus concentration, seed approximately 30 plates. For this step, use BHK cells that have undergone no more than five passages.

    2. After exactly 24 h, wash the cells once with 2 ml warm PBS and proceed with viral absorption (next step).

    3. Dilute the virus in 1 ml DMEM without FBS [multiplicity of infection (MOI) 0.01] and absorb the inoculum for 2 h at room temperature. The inoculum volume is not sufficient to cover the whole plate surface, so please move the plate carefully every 20 min to allow the inoculum to move across the plate.

    4. Remove the inoculum, add 7 ml DMEM (supplemented with 10% FBS) (Recipe 1), and incubate at 37°C for 3 days.

    5. Collect the supernatant and centrifuge at 18,000 × g for 1 h at 4°C to concentrate the virus.

    6. Discard the supernatant and resuspend the pellet in 300 µl PBS. Split the volume between 3 cryovials and store at -80°C until the titration.


  2. Quantitation of the Icoaraci Phlebovirus

    1. Viral titers are determined using a plaque assay. For this, plate 1.5 × 106 BHK-21 cells/well in a 6-well plate. Cells should be used up to a maximum of passage 3.

    2. After 24 h, infect the cells (~80% confluence) with 200 µl 10-fold serial dilution of ICOV stock, as described in the previous section, and incubate for 2 h at room temperature. Again, the inoculum volume is not sufficient to cover the well surface, so please move the plate carefully every 20 min to allow the inoculum to move across the plate.

    3. Using a pipette, remove the inoculum completely and cover the monolayer with 2 ml semi-solid medium composed of complete DMEM plus 1% methylcellulose (Recipe 2). In this step, it is very important to ensure complete removal of the inoculum in order for the semi-solid medium to be evenly distributed throughout the cell monolayer.

    4. Three days post-infection, fix and stain the cells with 2 ml 0.1% crystal violet solution containing 10% formaldehyde (Recipe 3) under slight agitation for 12 h at room temperature.

    5. Wash the wells delicately with running water to remove the fixative/dye and allow the plate to dry upside down at room temperature for 24 h. Viral plaques can be counted on the white-light transilluminator, and titers are expressed as plaque-forming units (PFU)/ml (Figure 2).



      Figure 2. Quantitation of Icoaraci Phlebovirus. The supernatant from 48 h after infection was collected, diluted to three different concentrations (10-2, 10-3, and 10-4), and titrated in a BHK-21 monolayer as described above. After 3 days, the cells were fixed and stained with crystal violet.


  3. Obtaining and cultivating peritoneal macrophages

    Mouse colonies are maintained at the Department of Immunology, UFRJ in accordance with the rules established by the National Council for the Control of Animal Experimentation. The animal experimentation protocols used in this project were approved by the Animal Use Ethics Committee (Protocol No. 046/20).

    1. Peritoneal macrophages from 8-week-old C57 Black-6 (C57BL/6) mice are elicited with 2 ml 3% sodium thioglycolate (intraperitoneal injection, Recipe 8) for 4 days, and cells are obtained after euthanization by injecting 8 ml DMEM into the peritoneal cavity. The mice were inclined at a 45° angle with the head down, positioning the intestines cranially, away from the administration area. The needle was then inserted in the lower right quadrant of the abdomen at a 30-45° angle for either the inoculation of thioglycolate or to obtain macrophages.

    2. Centrifuge the cells at 400 × g for 10 min at 4°C.

    3. Resuspend the cells in 1 ml DMEM and count using a hemocytometer under the microscope (10× dilution in the Neubauer chamber with DMEM).

    4. Plate the macrophages on glass coverslips in 24-well plates (2 × 105 cells per well) or 6-well plates (4 × 106 cells per well).

    5. After a 1-h adherence at 37°C, wash the cells with PBS and add complete DMEM. The macrophages are kept for 1 day at 37°C in a 5% CO2 atmosphere and subsequently subjected to infection.


  4. Infection

    1. For peritoneal macrophage ICOV infections, adsorption is performed for 1 h at 37°C using an MOI of 1.

    2. Dilute the virus in DMEM without FBS and use 100 µl for 24-well plates and 1 ml for 6-well plates.

    3. Remove the inoculum with a pipette and add 1 ml (24-well plate) or 2 ml (6-well plate) complete DMEM.

    4. The subsequent treatment time, conditions, or other additional infections for which the experimental model is proposed may vary. For example, in Leishmania infections, we allow the parasite to interact for 4 h after viral adsorption and then lyse the cells for analysis of gene expression by qPCR.


  5. Analysis of murine macrophage infection by semiquantitative PCR for viral detection

    To detect and confirm the viral infection and replication in peritoneal macrophages, a semiquantitative PCR must be performed.

    1. After 4, 24, or 48 h of infection, isolate and purify the total RNA using the Direct-zolTM RNA MiniPrep Plus kit according to the manufacturer's instructions.

    2. Quantitate the RNA using the NanoDrop. The expected concentration should be 100-200 ng/µl, and the 260/280 ratio should be 1.8-2.0, ensuring good RNA quality.

    3. First-strand cDNA synthesis is performed in a reaction containing Improm-II Reverse Transcriptase, a mix of dNTPs, and random primers, as described in the manufacturer's instructions.

    4. RT-PCR is performed using 10 µM primers for the N protein gene of ICOV (sense 5’-AGGTGAGGCTGTAAATCTTG-3’ and antisense 5’-TCACATCATCCTTCCAAGTG-3’), 2.5 U GoTaq DNA polymerase, 1.5 mM MgCl2, 10 mM dNTPs, 2 µl cDNA, and 1× appropriate buffer in a final volume of 50 µl. The PCR cycle is as follows: 95°C for 1 min, 48°C for 1 min, and 72°C for 1 min in 40 amplification cycles.

    5. PCR products are separated on a 1.2% agarose gel containing 0.5 µg/ml ethidium bromide in 1× TAE buffer (Recipe 5) at 80-100 V and photographed in a UV transilluminator. The product has 100 bp (Figure 3).



      Figure 3. Peritoneal macrophages from wild-type C57BL/6 mice were infected with stationary-phase promastigotes of L. (L.) amazonensis at a ratio of 5 parasites/cell, Icoaraci Phlebovirus (ICOV), or co-infected with both for 4 h, 24 h, or 48 h. Total RNA was extracted and analyzed by semiquantitative PCR for the Icoaraci N protein.

Recipes

  1. Complete DMEM

    To make 500 ml complete DMEM:

    445 ml 1× DMEM

    50 ml 10% FBS (heat-Inactivated)

    5 ml 1× penicillin-streptomycin (100×)

  2. Semi-solid medium

    Complete DMEM plus 1% methylcellulose

    1. Add 1 g methylcellulose to 100 ml distilled water in an autoclavable flask.

    2. Before autoclaving, add a stir bar in order to mix the solution and maintain sterility. For complete solubilization, store the solution overnight at 4°C and then leave the flask in a magnetic stirrer.

      Note: If a lumpy solution forms, cool the temperature of the solution and mix again. The procedure should be repeated until the solution is homogeneous.

    3. Add cold methylcellulose to the cold complete DMEM (v/v) and thoroughly mix to ensure a homogeneous mixture. Prior to use, wait until the bubbles dissipate and the temperature raises to room temperature.

  3. Crystal violet solution

    0.1% crystal violet in PBS solution

    10% formaldehyde

  4. 10× TAE Buffer (1 L)

    1. Weigh 48.5 g Tris-base, 11.4 ml glacial acetic acid and 20 ml 0.5 M EDTA pH 8.0

    2. Add distilled water up to 1 L

    3. Store at room temperature

  5. 1× TAE Buffer (1 L)

    1. Add 100 ml 10× TAE (Recipe 5) to 900 ml distilled water

    2. Store at room temperature

  6. 1.5% agarose gel

    1. Weigh agarose powder (1.5 g) in an autoclaved glass bottle

    2. Add 100 ml 1× TAE

    3. Heat in a microwave until the agarose is homogeneously dissolved

    4. Cool down at room temperature for a few minutes

  7. 100-bp DNA Ladder (50 µg/ml)

    1. For 240 µl DNA Ladder in a 1.5-ml tube on ice, add 176 µl UltraPureTM DNase/RNase-free distilled water

    2. Add 40 µl 6× gel loading dye and 24 μl 100-bp DNA ladder at 500 µg/ml

    3. Store at 4°C

  8. 3% sodium thioglycolate

    1. Weigh sodium thioglycolate powder (3.0 g) in an autoclaved glass bottle

    2. Add water up to 100 ml

    3. Filter the solution with a 0.22-µm cell culture medium filtration unit

    4. Store at room temperature

Acknowledgments

We thank Professor Pedro Fernando da Costa Vasconcellos for providing the Phleboviruses, which are fundamental for carrying out this work. We thank Professor Clarissa Damaso for the support in the virology methodologies. We also thank all members of the Molecular Parasitology laboratory for helpful discussion. This research was supported by the National Council for Scientific and Technological Development (CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments were performed in accordance with protocols approved by the National Council for Animal Experiment Control and Animal Use Ethics Committee (Protocol No. 046/20 – ID: 01200.001568/2013-87 Validity: 08/30/2022).

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简介

[摘要] Phlebotomine 载体,双翅目白蛉,已知将利什曼原虫和 RNA 病毒(虫媒病毒)传播给人类。该虫媒病毒,Icoaraci白蛉(豆24262 - ICOV ),在这项研究中使用的是从分离泳鼠属啮齿动物,我ammal伊恩物种是同一自然sylvatic水库利什曼原虫(利什曼原虫)亚马孙。这利什曼原虫的物种分布在巴西原始森林和次生林和其他国家在美国和CAU的ES局限型和弥漫无能皮损。在我们最近的研究中,我们观察到的通过细胞因子表达的调节,如IL-10和IFN-原生动物感染的恶化通过ICOV β ,增强了寄生虫负载和可能的发病机制。有效的病毒生产和孔定量牛逼通货膨胀必须要开发和标准化,以确保免疫分子检测提供一致的和可重复的病毒感染的结果。这些程序的标准化成为研究中特别有用的工具,在理解宿主细胞和静脉病毒之间的相互作用以及共同感染方面有多种应用,从而可以研究细胞内信号通路。在这里,我们详细的协议日在允许生产和孔定量吨通货膨胀的的Icoaraci白蛉使用BHK-21细胞(b ABY仓鼠肾CEL LS )和来自C57B腹腔巨噬细胞的随后感染大号/ 6小鼠。

[背景]在巴西,已分离出大约 210 种不同类型的虫媒病毒,其中 196 种发现于巴西亚马逊(Rosa等人,1998 年;Azevedo 等人,2007 年),这是巴西最大的虫媒病毒储备之一。世界。该布尼亚病毒家族由350种多名病毒分为五个属,其中Orthobunyavirus ,白蛉,内罗毕病毒属,和汉坦病毒包含种致病的人类和动物,而属Tospovirus是致病只对植物(沃尔特和Barr,2011 ; Ly的和池上,2016 年)。

在大多数人类病例中,静脉病毒引起从轻度发热性疾病到出血热和死亡的广泛症状(Bird等人,2009 年;Ikegami和Makino,2011 年;Brisbarre等人,2015 年)。的特定成员白蛉,裂谷˚F以往的病毒,是有潜在危险的怀孕家养动物如牛,山羊和绵羊,感染导致胎儿畸形和流产(Swanepoel和科泽尔,2004 )。在这项工作中,我们使用了在小型森林动物中普遍存在的Icoaraci Phlebovirus ( BeAN 24262 ) (Causey和Shope ,1965)。所有静脉病毒在形态上都相似:直径为80 - 120 nm,呈二十面体对称,并被两种表面糖蛋白(Gn和Gc )包裹(Freiberg等,2008)。单链 RNA ( ssRNA ) 被蛋白质 N 分割并包装在核糖核蛋白颗粒 (RNP) 内,并与依赖于 RNA 的 RNA 聚合酶 ( RdRp )相关联。小 (S) 片段使用双义编码策略,编码核衣壳蛋白 (N) 和非结构蛋白 (NS)(Simons等,1990;Giorgi等,1991)。

动物模型研究表明 I 型干扰素 (IFN) 对静脉病毒引起的感染具有保护作用(Mendenhall等,2009)。中号OST病毒已经开发表达的能力能够防止免疫应答的蛋白质(Habjan等人。,2009)。此拮抗活性ħ作为被观察到几种非结构病毒蛋白的(van Knippenberg等人通过在IFN信号传导途径探索不同的目标的机制。,2010)。尽管静脉病毒表现出不同程度的致病性,但 NSs 蛋白被认为是该属所有成员以及布尼亚病毒科其他成员的重要毒力因子(Habjan等,2009 ;Ikegami等,2009)。

多项研究表明,病毒和利什曼原虫寄生虫之间的关系在建立促进存活和持续感染的互惠适应性优势方面的重要性(Ives等,2011;Hartley等,2012;Vivarini等,2015;Eren等人,2016 年;Rath等人,2018 年)。我们的小组先前证明,亚马逊乳杆菌对抗病毒途径的诱导会增加巨噬细胞感染(Pereira等人,2010 年;Vivarini等人,2015 年)。与宿主免疫因素相关的不同利什曼原虫物种决定了利什曼病的不同临床表现(Alvar等,2012)。生产leishmanicidal介质,如IL-12,IL-6 ,和反应性氧物质(ROS) ,由先天免疫细胞有助于小号感染控制(Dayakar等人。,2019),但利什曼原虫寄生虫能够颠覆触发这些机制的信号通路,而不是诱导有利于感染过程的通路。使用Icoaraci Phlebovirus作为模型,我们证明了这种病毒会增强巨噬细胞中L. amazonensis 的感染。由于利什曼原虫诱导产生 I 型干扰素并在与病毒共感染期间扩增,巨噬细胞感染增加需要 Toll 样受体 3 (TLR3) 和蛋白激酶 R (PKR) 通路(Rath等人,2019 年)。我们的结果突出感染过程的复杂和多因素分析的重要性,特别是在发生重叠流行地区,作为之间观察到L.亚马孙和Icoaraci白蛉。

给定的社会经济影响白蛉感染以及潜在的共感染在皮肤利什曼病,为生产本协议,孔定量吨通货膨胀,和感染的Icoaraci白蛉旨在解决所述相关联的分子机制与白蛉传播虫媒病毒和之间的相互作用细胞宿主体内和体外的利什曼原虫寄生虫感染。子序贯研究使用日是方法可以揭示与宿主的免疫反应的重要方面,有助于勾勒出病理生理现象和临床情况和澄清的情况可能表明对于抗病毒反应如何双链RNA (dsRNA的)可以介导免疫炎症和免疫病理的过程白蛉感染和混合感染。

关键字:白蛉病毒属, 布尼亚病毒科, 虫媒病毒, 巨噬细胞


材料和试剂

细胞培养容器、板和管
1. 25 - cm 2细胞培养瓶(Corning,目录号:CLS430639)     
2. 100 - mm 板(SARSTEDT,目录号:82.1472.001)     
3. 6 孔细胞培养板(Corning,目录号:83.3920)     
4. 24 孔细胞培养板(Corning,目录号:83.3922)     
5. 15 - ml 和 50 - ml Falcon 管(BD Falcon)     
6.带网格的细胞计数幻灯片     
7. 1.5 - ml Eppendorf 管     
8.环保丁腈手套(SuperMax )     
9.过滤器尖端,升流-保留:1000微升,200微升,20微升,10微升(爱思进,产品目录号:31000LRS,3200LRS,320LRS,310LRS ,分别地)     
10. 5 - ml 血清移液管(SARSTEDT,目录号:86.1253.001) 
11. 10 - ml 血清移液管(SARSTEDT,目录号:86.1254.001) 
12. 0.22 - µm 细胞培养基过滤装置(康宁,目录号:CLS430513-12EA) 

细胞
BHK-21细胞(b ABY仓鼠肾细胞小号,ATCC:CCL-10)
细胞保持在 25 - cm 2细胞培养瓶中,并带有完整的 DMEM (DMEM–高葡萄糖并辅以 10% 热灭活 FBS –配方 1 ) 在 37 °C 下,在含有 5% CO 2的加湿气氛中。达到汇合后,将细胞传代培养以保持细胞活力。对于第是,除去培养基并用冲洗3细胞毫升温PBS无钙和镁。使用 1 ml 胰蛋白酶-EDTA 溶液分离细胞 1 分钟。4毫升完全DMEM,转移添加到一个新的15 -毫升锥形管,并离心将细胞在300 5分钟×克。重悬细胞沉淀在新鲜的完全培养基和分裂1 :在新的培养瓶中20。

静脉病毒
所述Icoaraci白蛉(豆24262-ICOV)瓦特作为距离Pedro博士获得洛斯(研究所伊万德罗恰加斯/ SVS / MS)。
该病毒从肝,脾,肾,心脏的分离泳鼠属鼩在贝伦,帕拉州,巴西,并随后在2日龄瑞士小鼠脑内接种。将样品冻干并储存在-80 °C 。使用前,在0.5 ml Puck's 培养基中重新配制样品。

细胞培养试剂
1. 0.25% 胰蛋白酶-EDTA 溶液(Sigma-Aldrich,目录号:T4049),储存在 -20°C     
2.含有 4.5 g/L 葡萄糖和 L-谷氨酰胺的 Dulbecco 改良 Eagle 培养基(DMEM)(Lonza,目录号:12-604Q)     
3. 100 ×青霉素-链霉素溶液(HyClone - GE,目录号:SV30010)     
4. Puck 培养基(PAN Biotech,目录号:P04-51100)     
5.磷酸盐缓冲盐水(PBS)(Lonza,目录号:17-516Q)     
6.胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10270106)     
7. UltraPure TM DNase/RNase-free蒸馏水(Thermo Fisher Scientific,Invitrogen TM ,目录号:10977-035)     
8.甲基纤维素(Sigma-Aldrich,目录号:M7027)     
9. 100 - bp DNA 阶梯,500 µg/ml(Promega,目录号:G201A)     
10. 6 ×克EL样染料(Promega公司,目录号:G190A) 
11.用于常规凝胶电泳的标准琼脂糖(UniScience ,目录号:AGR-LE-100) 
12.溴化乙锭(Sigma-Aldrich,目录号:E7637) 
13. Tris-base(Promega,目录号:H5131) 
14.冰醋酸(Sigma-Aldrich,目录号:A6283) 
15. UltraPure TM 0.5 M EDTA(乙二胺四乙酸,二钠盐二水合物),pH 8.0(Thermo Fisher Scientific,目录号:15575020) 
16.结晶紫(Sigma-Aldrich,目录号:C0775) 
17.巯基乙酸钠(Sigma-Aldrich,目录号:T0632),室温储存 
18.指示按ZOL TM RNA MiniPrep试剂Plus试剂盒(ZYMO研究,目录号:R2070) 
19. ImProm -II TM逆转录系统(Promega,目录号:A3800) 
20.随机引物(Promega,目录号:C1181) 
21. GoTaq ® DNA聚合酶(Promega公司,目录号:M3001) 
22.完成 DMEM(见食谱) 
23.半固体培养基(见食谱) 
24.结晶紫溶液(见配方) 
25. 10 × TAE 缓冲液(1 L)(见配方) 
26. 1 × TAE 缓冲液(1 L)(见配方) 
27. 1.5%一garose凝胶(见配方) 
28. 100 - bp DNA 阶梯(50 µg/ml)(见配方) 
29. 3%小号裂果巯基乙(见配方) 

设备

1. P1000(吉尔森,目录号:F123602)     
2. P200(吉尔森,目录号:F123601)     
3. P20(吉尔森,目录号:F123600)     
4. P2(吉尔森,目录号:F144801)     
5. 4°C 实验室冰箱(伊莱克斯,型号:IF55)     
6.实验室冷冻室设置在-20°C(Prosdocimo ,型号:F21)     
7.实验室冷冻室设置在-80°C(松下,型号:MDF-U56VC-PA)     
8. 37°C 培养箱,含5% CO 2 (Sanyo inCU -Safe ,型号:MCO-17AC)     
9.层流罩(Esco Class II BSC-AirStream )     
10. 5418(Eppendorf)室温离心;此处设置为 24°C 
11.离心机(ThermoFisher Scientific,型号:D-37520) 
12. NanoDrop TM ND-1000 分光光度计(ThermoFisher Scientific) 
13.天平(BEL分析设备,型号:S622) 
14. Veriti 96孔吨有源冰箱Ç ycler仪(Applied Biosystems PCR仪器,型号:9902) 
15.迷你凝胶迁移系统(Loccus Biotecnology ,型号:LCH-7X8) 
16.电泳p奥尔š upply(GIBCO BRL,Life Technologies公司,250)和电力电缆 
17.紫外透照仪(Fisher Scientific,型号:88A) 

程序

图1示出了用于步骤病毒生产,孔定量吨通货膨胀和感染。一的程序的详细说明以下项目中找到。


图1不同的步骤的生产,孔定量吨通货膨胀,和感染Icoaraci白蛉。在 Mind the Graph 平台中创建的图。

Icoaraci 静脉病毒的生产
板4×10 6 BHK-21在100细胞中-毫米板(7毫升/板)。为了获得良好的病毒浓度,SE编大约30块板。在这一步中,使用不超过五次传代的 BHK 细胞。
恰好 24 小时后,用 2 ml 温热的 PBS 清洗细胞一次,然后进行病毒吸收(下一步)。
d ilute在1米病毒升不含FBS的DMEM [感染复数(MOI)0.01]和吸收接种物在室温下2小时。接种量不足以覆盖整个板表面,因此请每 20 分钟小心移动一次板,让接种物穿过板。
去除接种物,加入 7 ml L DMEM(补充有 10% FBS)(配方 1),并在 37°C下孵育3 天。
收集上清液并在 4°C 下以18,000 × g离心1 小时以浓缩病毒。
弃去上清液,用 300 µl PBS 重悬沉淀。分割卷之间3个冷冻管并储存于-80℃直至滴定。

孔定量牛逼的通货膨胀的Icoaraci白蛉
病毒滴度使用噬斑测定法测定。为此,在6孔板中加入 1.5 × 10 6 BHK-21 细胞/孔。细胞应最多使用到第3 代。
24小时后,感染细胞(〜80%confluenc ë )用200μ升,在室温下2小时10倍ICOV股票的系列稀释,如前一节中所描述的,并孵育。同样,接种量不足以覆盖孔表面,因此请每 20 分钟小心移动一次板,让接种物穿过板。
使用移液器完全去除接种物,并用 2 ml 由完全 DMEM 加 1% 甲基纤维素组成的半固体培养基覆盖单层(配方 2)。在这一步中,确保完全去除接种物非常重要,以便半固体培养基均匀分布在整个细胞单层中。
三天感染后,修复和染色用2M细胞升含有10%甲醛的0.1%结晶紫溶液(配方3)下轻微搅拌12 ħ在室温下。
洗涤所述孔微妙用流水,除去固定剂/染料和允许所述板以干燥在室温下颠倒24小时。病毒噬斑可以在白色进行计数-光透射仪,和效价表示为空斑形成单位小号(PFU)/米升(图2)。
图2.孔定量吨的通货膨胀Icoaraci白蛉。从48小时感染后,收集上清液,稀释至三种不同浓度(10 - 2 ,10 - 3 ,和10 - 4 ),并且如上所述在一个BHK-21单层滴定。3天后,将细胞固定并用结晶紫染色。

腹腔巨噬细胞的获得和培养
根据国家动物实验控制委员会制定的规则,小鼠菌落维持在UFRJ免疫学系。本项目中使用的动物实验方案经动物使用伦理委员会批准(方案编号 046/20)。
从8凌晨腹膜巨噬细胞ķ -old C57黑6(C57BL / 6)小鼠中引发与2米升3%氢氧化钠巯基乙(腹膜内注射,配方8)4天,并且被细胞获得后安乐死通过注入8米升在DMEM到腹膜腔。将小鼠头部向下倾斜45 °角,将肠道置于头侧,远离给药区域。然后将针以 30 – 45 °角插入腹部右下象限,用于接种巯基乙酸盐或获得巨噬细胞。
将细胞在4°C 下以 400 × g离心10 分钟。
将细胞重悬在 1 ml DMEM 中,并在显微镜下使用血细胞计数器计数(在 Neubauer 室中用 DMEM 稀释10倍)。
将巨噬细胞铺在 24 孔板(每孔2 × 10 5 个细胞)或 6 孔板(每孔4 × 10 6 个细胞)的玻璃盖玻片上。
后一个1 -在37℃。1 H附着,洗涤细胞,用PBS,并添加完全DMEM。巨噬细胞在 37°C、5% CO 2气氛中保持 1 天,然后进行感染。

感染
对于腹腔巨噬细胞 ICOV 感染,在 37°C 下使用 MOI 为 1 进行吸附 1 小时。
稀病毒在不含FBS的DMEM,并使用100μ升用于24孔板和1m升6孔板中。
用移液管取出接种物,加入 1 ml(24 孔板)或 2 ml(6 孔板)完全 DMEM。
随后的处理时间,条件,或用于该实验模型,提出可能会发生变化的其他额外的感染。例如,在利什曼原虫感染,我们允许寄生虫到用于病毒吸附后4小时进行交互,然后裂解细胞用于通过qPCR分析基因表达。

病毒检测半定量 PCR 分析小鼠巨噬细胞感染
为了检测和确认腹腔巨噬细胞中的病毒感染和复制,必须进行半定量 PCR。
后4,24 ,感染,分离物或48小时,并使用指示按纯化的总RNA ZOL TM RNA MiniPrep试剂Plus试剂盒符合荷兰国际集团到制造商的说明。
孔定量泰特使用的RNA纳米滴。预期浓度应为 100 - 200 ng/µl,260/280 比率应为1.8 - 2.0 ,确保良好的 RNA 质量。
第一链 cDNA 合成是在包含Improm -II 逆转录酶、dNTP 混合物和随机引物的反应中进行的,如制造商的说明中所述。
使用 ICOV N 蛋白基因的 10 µM 引物(有义 5'-AGGTGAGGCTGTAAATCTTG -3' 和反义 5'-TCACATCATCCTTCCAAGTG-3')、2.5 U GoTaq DNA 聚合酶、1.5 mM MgCl 2 、NTPs 进行 RT-PCR 、2 µl cDNA和 1 ×合适的缓冲液,终体积为 50 µl。在PCR循环是如下:95℃1分钟,48℃1分钟,和72℃下,在40个扩增循环1分钟。
PCR 产物在 1.2% 琼脂糖凝胶上分离,该凝胶含有0.5 µg/ml 溴化乙锭和 1 × TAE 缓冲液(配方 5),电压为 80 - 100 V,并在紫外透射仪中拍照。该产品有 100 bp (图 3)。
图 3.来自野生型 C57BL/6 小鼠的腹膜巨噬细胞以 5 个寄生虫/细胞、Icoaraci Phlebovirus (ICOV)的比例感染了L. (L.) amazonensis 的静止期前鞭毛体,或同时感染了两者4个小时,24小时,或48小时。提取总 RNA 并通过半定量 PCR 分析Icoaraci N 蛋白。

食谱

完整的 DMEM
制作 500 ml 完整 DMEM:
445 毫升 1 × DMEM
50 毫升 10% FBS(热灭活)
5 ml 1 ×青霉素-链霉素 (100 × )
半固体培养基
完整的 DMEM 加 1% 甲基纤维素
添加1克甲基纤维素至100μm升蒸馏水中的耐高压加热的烧瓶中。
在高压灭菌之前,添加搅拌棒以混合溶液并保持无菌。为了完全溶解,将溶液在 4 ℃下过夜°C,然后将烧瓶留在磁力搅拌器中。
注意:如果形成块状溶液,冷却溶液的温度并再次混合。应重复该过程直至溶液均匀。
将冷甲基纤维素添加到冷的完整 DMEM (v/v) 中并彻底混合以确保均匀的混合物。使用前,请等待气泡消散,温度升至室温。
结晶紫溶液
0.1% 结晶紫的 PBS 溶液
10% 甲醛
10 × TAE 缓冲液 (1 L)
称取 48.5 g Tris-base、11.4 ml 冰醋酸和 20 ml 0.5 M EDTA pH 8.0
添加蒸馏水水达到1升
在室温下储存
1 × TAE 缓冲液 (1 L)
将 100 毫升 10 × TAE(配方 5)添加到900 毫升蒸馏水中
在室温下储存
1.5% 琼脂糖凝胶
在高压灭菌的玻璃瓶中称量琼脂糖粉 (1.5 g)
添加 100 毫升 1 × TAE
热在微波直到所述琼脂糖均匀地溶解
在室温下冷却几分钟
100 - bp DNA Ladder (50 µg/ml)
为240微升DNA梯子在1.5 -在冰上毫升管中,加入176微升超纯TM DNA酶/ RNA酶的蒸馏水
加入 40 µl 6 ×凝胶上样染料和 24 µl 100 - bp DNA 阶梯,浓度为 500 µg/ml
4°C 保存
3%巯基乙酸钠
在高压灭菌的玻璃瓶中称量巯基乙酸钠粉末 (3.0 g)
加水高达100米升
过滤与所述溶液一0.22 -微米的细胞培养基过滤单元
在室温下储存

致谢

我们感谢佩德罗费尔南多·达科斯塔Vasconcellos教授为提供白蛉ES ,这对开展这项工作的基础。我们感谢 Clarissa Damaso教授对病毒学方法的支持。我们也感谢分子寄生虫学实验室的所有成员进行有益的讨论。这项研究是由支持的国家委员会科学和技术发展(的CNPq )和Fundação卡洛斯·查加斯·菲略德安帕罗à Pesquisa做埃斯塔做里约热内卢(FAPERJ)。

竞争利益

作者声明没有竞争利益。

伦理

所有的动物实验均按照全国委员会的动物实验控制和使用的动物伦理委员会批准的方案进行(协议ñ Ö 。046/20 - ID:01200.001568 / 2013-87有效期:2022年8月30日)。

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引用:Rath, C. T., Vivarini, A. C., Pereira, R. M. and Lopes, U. G. (2021). Production, quantification, and infection of Amazonian Phlebovirus (Bunyaviridae). Bio-protocol 11(13): e4072. DOI: 10.21769/BioProtoc.4072.
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