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Assessing the Efficacy of Small Molecule Inhibitors in a Mouse Model of Persistent Norovirus Infection
小分子抑制剂在持续性诺如病毒感染小鼠模型中的疗效评估   

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Antimicrobial Agents and Chemotherapy
Mar 2016

Abstract

Human norovirus is the most common cause of acute gastroenteritis worldwide, resulting in estimated mortality of ~210,000 each year, of whom most are children under the age of five. However, norovirus can infect people of all age groups. There is a risk of prolonged infection in children, the elderly and patients who are immunocompromised. To study the inhibition of persistent norovirus replication by small molecule antivirals in vivo, we used a murine norovirus CR6 strain (MNV.CR6). We demonstrated earlier that efficient small molecules can reduce viral shedding in the stool of infected mice. Here we present how to generate the MNV.CR6 virus stock, infect type I and II interferon receptor knockout AG129 mice via oral gavage, administer antivirals to mice, and quantify viral genome copies in the stool of these mice.

Keywords: Murine norovirus (鼠诺如病毒), Persistent infection (持续感染), Oral gavage (口服强饲), RT-qPCR (RT-qPCR), Antiviral (抗病毒)

Background

Human noroviruses are an important cause of gastroenteritis. Although most norovirus infections are acute and self-limiting, the infection can become chronic in patients with an immunodeficient status, particularly in solid organ and hematopoietic stem cell transplant recipients, patients undergoing chemotherapy and patients with AIDS (Westhoff et al., 2009; Green, 2014; Angarone et al., 2016). Prolonged norovirus infection is also observed in young children and the elderly resulting in an increased duration of illness, increased defecations and virus shedding for up to 47 days (Murata et al., 2007; Aoki et al., 2010). Reduction of immunosuppressive therapy is, when feasible, the strategy of choice to control the infection in transplant recipients. Specific antiviral therapies to treat (chronic) norovirus gastroenteritis are not available. To assess the inhibitory effect of small molecules on persistent norovirus infections, we used a mouse-adapted persistent murine norovirus (MNV.CR6) in type I and II interferon receptor knockout AG129 mice (Strong et al., 2012). MNV is a genogroup V norovirus that is widely used as a surrogate for human noroviruses, which comprises around 30 strains (Karst et al., 2003; Wobus et al., 2004). The MNV.CR6 is avirulent in AG129 and STAT1-/- mice, but replicates for weeks to months in wild type mice and to higher titers in innate immune-deficient mice (Thackray et al., 2007). The MNV.CR6 strain has a tropism for the proximal colon and the cecum, where it persists and replicates more efficiently than the MNV.CW3 strain, which causes acute infection (Arias et al., 2012; Nice et al., 2013).

Materials and Reagents

  1. Overall
    1. Appropriate personal protection to work in a biosafety level 2 (BSL-2) laboratory or A-2 animal facility (gloves, lab coat, hairnet, shoe covers, safety glasses)
    2. Disinfectant: bleach (5,000 ppm) or Virkon S.

  2. In vitro work
    1. Culture flasks (150 cm2, TPP, catalog number: 90856 )
    2. Cell scrapers (Corning, Falcon®, catalog number: 353086 )
    3. Cryotubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 377224 )
    4. Pipet tips (10 µl, 100 µl, 1,000 µl)
    5. Disposable serological pipets (5 ml, 10 ml, 25 ml)
    6. Murine macrophage cells (RAW 264.7, ATCC, catalog number: TIB-71 )
    7. Dulbecco’s phosphate buffered saline (DPBS) (Thermo Fisher Scientific, catalog number: 14190094 )
    8. Dulbecco’s modified eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 41965039 )
    9. Fetal calf serum (FCS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
    10. Sodium bicarbonate (Thermo Fisher Scientific, GibcoTM, catalog number: 25080060 )
    11. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030024 )
    12. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630056 )
    13. Penicillin/streptomycin (P/S) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
    14. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360039 )
    15. Culture medium (see Recipes)

  3. In vivo work
    1. Eppendorf safe-lock tubes, 1.5 ml (Eppendorf, catalog number: 0030120086 )
    2. Syringe + needle for subcutaneous injection (VWR, catalog number: BDAM303176 )
    3. Needle container (Sharpsafe, catalog number: 41602432 )
    4. Bench surface protector (VWR, catalog number: 115-9220 )
    5. Earmarks (Bioseb, catalog number: EP-1005-1 )
    6. Plastic feeding tubes, 20 G x 30 mm, sterile (Instech Laboratories, catalog number: FTP-20-30 )
    7. Sterile 1 ml syringe that fits on the feeding tube (VWR, catalog number: 612-0106 )
    8. Plastic container to temporarily restrain a mouse
    9. Ear tags (Bioseb, catalog number: EP-1005-1 ) + applicator (Style 1005s1)
    10. Type I and II interferon receptor knockout AG129 mice (129/Sv mice), from BK Universal, UKCR6 strain
    11. 2’-C-methylcytidine (2CMC, Carbosynth, catalog number: NM07918 )
    12. Favipiravir (T-705, BOC Sciences, catalog number: B0084-463609 )
    13. Carboxymethylcellulose (CMC, Sigma–Aldrich, catalog number: C9481 )
    14. Sterile saline (NaCl 0.9%, B. Braun)

  4. Viral RNA quantification
    1. Multipette tip of 0.2 ml (Eppendorf, catalog number: 0030089413 )
    2. Lightcycler 480–Multiwell plate 96 (Roche Molecular Systems, catalog number: 04729692001 )
    3. RNeasy Mini kit (QIAGEN, catalog number: 74106 )
    4. Ethanol, absolute (Fisher Scientific, catalog number: 10342652 )
    5. iTaq Universal Probes One-Step Kit (Bio-Rad Laboratories, catalog number: 1725141 )
    6. Primers, probe and standard (Baert et al., 2008)

Equipment

  1. Biosafety hood in an A-2 animal facility
  2. Biosafety hood in a BSL-2 laboratory
  3. Incubator (37 °C, 5% CO2, humidified)
  4. Scale to weigh mice
  5. Forceps suitable for picking up mouse feces (autoclavable)
  6. Pipet set (P10, P100, P1000)
  7. Pipetboy (Integra Biosciences, catalog number: 155016 )
  8. Multipette® M4 (Eppendorf, catalog number: 4982000012 )
  9. Multichannel pipette (Eppendorf, catalog number: 3122000035 )
  10. -80 °C freezer
  11. PCR Workstation
  12. Vortex
  13. Centrifuge with a rotor suitable for 1.5 ml tubes
  14. Centrifuge with a rotor suitable for 50 ml tubes
  15. Autoclave
  16. RT-qPCR machine (Roche Diagnostics, model: Roche LightCycler® 96 )
  17. Inverted light microscope
  18. Cell counter

Software

  1. GraphPad Prism Software, version 7
  2. LightCycler® 96 SW 1.1 Software

Procedure

  1. Expansion and quantification of MNV.CR6 virus stock
    Note: Culture and infect cells under a biosafety hood in a BSL-2 laboratory.
    1. Split the RAW264.7 cells using a cell scraper, once a week at a 1:10 ratio in a T150 flask containing 40 ml of 10% media. Maintain the cells in a CO2 incubator at 5% CO2 and 37 °C.
    2. Infect the RAW264.7 cells with MNV.CR6 after 2 or 3 days of incubation i.e., when the cells are nearly confluent but not overgrown. Do this as follows:
      1. Aspirate the growth medium from cells, wash with DPBS and aspirate the DPBS.
      2. Add 1 ml of MNV.CR6 to the cells and distribute evenly, incubate for 1 h at 37 °C.
      3. Add 20 ml of 2% media and place the flask back in the incubator until the full cytopathic effect is observed (± 2-3 days post-infection).
    3. Repeat two freeze-thaw cycles (-80 °C vs. 37 °C), then collect the supernatants containing the virus after centrifugation (10 min, 1,500 x g) and store in aliquots (500-1,000 µl) in cryotubes at -80 °C.
    4. Determine the viral titer by endpoint titration as previously described in Reed et al. (1938). This procedure is based on the detection of cytopathic effect (CPE) by microscopy.
      Note: Titration can be performed with a higher dilution series or extended over a second 96-well plate when the viral stock is strong.
      1. Thaw MNV.CR6 stock at room temperature.
      2. Add 100 µl of 2% DMEM in all wells of a 96-well plate.
      3. Add 50 µl of pure virus stock in the wells B2-G2 of the second column of the 96-well plate.
      4. Homogenize the virus and medium with a multichannel pipette.
      5. Take 50 µl of the mixture and transfer into the next well.
      6. Repeat Steps A4d-A4e until reaching column 9 of the 96 well-plate.
      7. Homogenize the virus and medium in row 9 and discard 50 µl.
      8. Add 10,000 RAW 264.7 cells/well in a final volume of 100 µl in 2% DMEM, to the inner 60 wells.
      9. Incubate for 72 h in 5% CO2 at 37 °C.
      10. Compare the wells with infected cells (columns 2-9) to the wells with non-infected cells (columns 10-11) and evaluate for cytopathic effect (CPE) microscopically.
      11. Calculate TCID50 using the Reed-Muench formula (Reed et al., 1938).

  2. Oral gavage of MNV.CR6 in mice
    Notes:
    1. Handle infected animals under a biosafety hood in an A-2 animal facility under conditions as close as possible to a specific pathogen free (SPF) facility.
    2. Use a protective bench coat to prevent viral contamination of the biosafety hood.
    3. Replace gloves between different experimental groups.
    4. For all experiments the mice were age- and sex-matched, mice were 8-12 weeks of age.

    1. Tag an ear of each mouse with a unique number.
    2. Thaw virus and dilute in 2% media to 106 CCID50 (50% cell culture infectious dose) of MNV.CR6. Give each mouse 200 μl of virus via oral gavage (see below).
    3. Fill the syringe and feeding tube with virus and remove all air bubbles.
    4. Carefully pick up the mouse by the base of its tail, place onto a wire cage.
    5. With the other hand, restrain the mouse by holding the scruff between thumb and forefinger. Place the tail between the little finger and ring finger to stretch the mouse (Figure 1). 


      Figure 1. Correct hand position to hold a mouse for oral gavage

    6. Now gently insert the feeding tube vertically down into the esophagus and administer virus in a steady motion (Figure 2). Any resistance felt indicates improper placement of the feeding tube, in which case take out the feeding tube and re-position.


      Figure 2. Vertical insertion of the feeding tube in the esophagus of the mouse

  3. Administration of small molecule inhibitors
    Notes:
    1. Handle infected animals under a biosafety hood in an A-2 animal facility under conditions as closely as possible to an SPF facility.
    2. Use a protective bench coat to prevent viral contamination of the biosafety hood.
    3. Replace gloves between different experimental groups.

    Subcutaneous injection of 2’-C-Methylcytidine (100 mg/kg/day)
    1. Dissolve 2CMC in sterile saline.
    2. Fill the syringe with virus and remove all air bubbles.
    3. Hold the mouse by the base of its tail and place it onto a wire cage.
    4. Insert the needle parallel to the skin on the back of the mouse. Make sure you are just under the skin (Figure 3). 


      Figure 3. Subcutaneous injection in the back of the mouse

    5. Inject the mouse with 2CMC via the subcutaneous route in a steady motion with a volume (~200 µl) that has a final concentration of 100 mg/kg/day.

    Oral gavage of favipiravir (T-705) (200 mg/kg/day)

    1. Dissolve T-705 in 0.4% CMC.
    2. Administer the appropriate volume of T-705 via oral gavage with a volume (~200 µl) that has a final concentration of 200 mg/kg/day.

  4. Follow up and stool collection 
    Notes:
    1. Handle infected animals under a biosafety hood in an A-2 animal facility under conditions as close as possible to an SPF facility.
    2. Use a protective bench coat to prevent viral contamination of the biosafety hood.
    3. Decontaminate boxes, forceps and scale between different experimental groups.

    1. Evaluate the general condition of the animal on a daily basis and check the overall health (weight, behavior, fur). If an animal reaches the defined humane endpoints (loses more than 15% of body weight in 1-2 days or an overall of > 20% in body weight or displays obvious signs of suffering [lethargy, squinted eyes, dehydration, hunched back]), it has to be humanely euthanized according to the European guidelines (Dir. 2010/63/EU).
    2. Place each mouse individually in a clean plastic container awaiting the defecation.
    3. Use a forceps to collect one piece of feces and place it in a labeled 1.5 ml tube (Figure 4). Repeat for all mice.


      Figure 4. Collection of a stool sample in a labeled tube

    4. Score the feces samples for consistency according to the table below.

    5. Store stool samples in a -20 °C or -80 °C freezer.

  5. Viral RNA extraction from stool samples
    Note: Handle infectious samples under a biosafety hood in a BSL-2 laboratory.
    1. Extract the viral RNA using the RNeasy mini kit of QIAGEN:
      1. Add 400 µl of RLT buffer to each feces sample.
      2. Vortex the samples very well until the feces sample is completely homogenized.
      3. Spin the tubes for 3 min at 10,000 x g.
      4. Transfer 350 µl of the supernatants to a new 1.5 ml tube and add 350 µl of 70% ethanol.
      5. Continue according to manufacturer’s protocol.
    2. Elute the viral RNA in 50 µl RNase free water.
    3. Store the viral RNA in a -20 °C freezer.

  6. Quantification of viral RNA by RT-qPCR
    Note: Prepare the mixtures for the RT-qPCR in a PCR workstation.
    1. Prepare the correct concentrations of primers, probe and the standard dilution (108 to 100 RNA copies).
    2. Thaw viral RNA samples and components of the one-step iTaq Universal Probes kit.
    3. Prepare for each sample a one-step RT-qPCR mix, a total of 16 µl containing: 10 µl iTaq Universal Probes reaction mix, 0.5 µl of iScript advanced reverse transcriptase, RNase free water, 900 nM of MNV primers and 200 nM of MNV probe.
    4. Gently vortex and briefly centrifuge the mix.
    5. In each well of the 96 RT-qPCR well plate, pipet 16 µl of the mix (using a multipipette) and add 4 µl of viral RNA.
    6. Close the RT-qPCR plate properly with a plastic seal.
    7. Place the RT-qPCR plate in a LightCycler and run with the following cycling conditions: reverse transcription at 50 °C for 10 min, initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing and extension at 60 °C for 30 sec.
    8. For the absolute quantification, use a 10-fold dilution series of the MNV DNA standard with known concentration.c

Data analysis

Viral RNA copy numbers of RNA extracts are determined using the standard curve and the LightCycler® 96 SW 1.1 Software. The amount of viral RNA copy numbers per gram of feces can be calculated by weighing the stool samples before extraction. Statistical analysis was performed using Prism 7 software, P-values were determined with the nonparametric Mann-Whitney test. For representative details see our original publication (Rocha-Pereira et al., 2015).

Notes

  1. AG129 mice (129/Sv mice) were originally obtained from BK Universal, UK, and under an annual license agreement bred and housed at our institute under SPF conditions.
  2. All experiments using mice strictly followed the guidelines of and were performed with the approval of the Ethical Committee of the KU Leuven, Belgium (P101/2012).

Recipes

  1. Culture media for RAW264.7 cells

Acknowledgments

The study in which the protocol described here, has been published previously (Rocha-Pereira et al., 2015). The development and use of this protocol were supported by the EU FP7 project SILVER (260644), KU Leuven IOF project HB/14/031 and GOA grant (GOA/10/014). J. Rocha-Pereira and the research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no 608765. J. Van Dycke is an SB Ph.D. fellow of the Scientific Fund for Research of Flanders (FWO). We gratefully acknowledge Prof. Herbert W. Virgin (Washington University, St. Louis, USA) for kindly providing the MNV.CR6 virus. We thank Carolien De Keyzer, Jasper Rymenants and Charlotte Vanderheydt for excellent technical assistance. The authors declare no conflicts of interest or competing interests.

References

  1. Angarone, M. P., Sheahan, A. and Kamboj, M. (2016). Norovirus in transplantation. Curr Infect Dis Rep 18(6): 17.
  2. Aoki, Y., Suto, A., Mizuta, K., Ahiko, T., Osaka, K. and Matsuzaki, Y. (2010). Duration of norovirus excretion and the longitudinal course of viral load in norovirus-infected elderly patients. J Hosp Infect 75(1): 42-46.
  3. Arias, A., Bailey, D., Chaudhry, Y. and Goodfellow, I. (2012). Development of a reverse-genetics system for murine norovirus 3: long-term persistence occurs in the caecum and colon. J Gen Virol 93(Pt 7): 1432-1441.
  4. Baert, L., Wobus, C. E., Van Coillie, E., Thackray, L. B., Debevere, J. and Uyttendaele, M. (2008). Detection of murine norovirus 1 by using plaque assay, transfection assay, and real-time reverse transcription-PCR before and after heat exposure. Appl Environ Microbiol 74(2): 543-546.
  5. Green, K. Y. (2014). Norovirus infection in immunocompromised hosts. Clin Microbiol Infect 20(8): 717-723.
  6. Karst, S. M., Wobus, C. E., Lay, M., Davidson, J. and Virgin, H. W. t. (2003). STAT1-dependent innate immunity to a Norwalk-like virus. Science 299(5612): 1575-1578.
  7. Murata, T., Katsushima, N., Mizuta, K., Muraki, Y., Hongo, S. and Matsuzaki, Y. (2007). Prolonged norovirus shedding in infants <or=6 months of age with gastroenteritis. Pediatr Infect Dis J 26(1): 46-49.
  8. Nice, T. J., Strong, D. W., McCune, B. T., Pohl, C. S. and Virgin, H. W. (2013). A single-amino-acid change in murine norovirus NS1/2 is sufficient for colonic tropism and persistence. J Virol 87(1): 327-334.
  9. Reed, L. J., and Muench, H. (1938). A simple method of estimating fifty per cent endpoints. Am J Epidemiol 27(3): 493-7.
  10. Rocha-Pereira, J., Van Dycke, J. and Neyts, J. (2015). Treatment with a nucleoside polymerase inhibitor reduces shedding of murine norovirus in stool to undetectable levels without emergence of drug-resistant variants. Antimicrob Agents Chemother 60(3): 1907-1911.
  11. Strong, D. W., Thackray, L. B., Smith, T. J. and Virgin, H. W. (2012). Protruding domain of capsid protein is necessary and sufficient to determine murine norovirus replication and pathogenesis in vivo. J Virol 86(6): 2950-2958.
  12. Thackray, L. B., Wobus, C. E., Chachu, K. A., Liu, B., Alegre, E. R., Henderson, K. S., Kelley, S. T. and Virgin, H. W. t. (2007). Murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence. J Virol 81(19): 10460-10473.
  13. Westhoff, T. H., Vergoulidou, M., Loddenkemper, C., Schwartz, S., Hofmann, J., Schneider, T., Zidek, W. and van der Giet, M. (2009). Chronic norovirus infection in renal transplant recipients. Nephrol Dial Transplant 24(3): 1051-1053.
  14. Wobus, C. E., Karst, S. M., Thackray, L. B., Chang, K. O., Sosnovtsev, S. V., Belliot, G., Krug, A., Mackenzie, J. M., Green, K. Y. and Virgin, H. W. (2004). Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol 2(12): e432.

简介

人类诺瓦克病毒是全球急性胃肠炎最常见的原因,估计每年约有210,000人死亡,其中大部分是5岁以下的儿童。 然而,诺如病毒可以感染所有年龄组的人。 儿童,老年人和免疫功能低下的患者有长期感染的风险。 为研究体内小分子抗病毒剂对持续诺如病毒复制的抑制作用,我们使用鼠诺沃克病毒CR6株(MNV.CR6)。 我们之前表明,高效小分子可以减少感染小鼠粪便中的病毒脱落。 在这里,我们介绍如何产生MNV.CR6病毒,通过口服灌胃感染I型和II型干扰素受体敲除AG129小鼠,给小鼠施用抗病毒药物,并量化这些小鼠粪便中的病毒基因组拷贝。

【背景】人诺瓦克病毒是胃肠炎的重要原因。尽管大多数诺罗病毒感染是急性和自限性的,但是在具有免疫缺陷状态的患者中,尤其是在实体器官和造血干细胞移植受体,接受化疗的患者和患有AIDS的患者中,感染可能变成慢性的(Westhoff等人, / em>,2009; Green,2014; Angarone 等。,2016)。在幼儿和老年人中也观察到延长的诺如病毒感染,导致疾病持续时间增加,排便增加和病毒脱落长达47天(Murata等人,2007; Aoki等人,2010)。在可行的情况下,减少免疫抑制治疗是控制移植受者感染的选择策略。特定的抗病毒治疗(慢性)诺如病毒胃肠炎是不可用的。为了评估小分子对持久性诺如病毒感染的抑制作用,我们在I型和II型干扰素受体敲除AG129小鼠中使用小鼠适应性持续鼠类诺罗病毒(MNV.CR6)(Strong等, , 2012)。 MNV是一种genogroup V诺如病毒,广泛用作人类诺如病毒的替代物,其包含约30种菌株(Karst等人,2003; Wobus等人,2004年)。 MNV.CR6在AG129和STAT1 - / - 小鼠中是无毒的,但在野生型小鼠中复制数周至数月,并且在先天性免疫缺陷型小鼠中复制更高滴度(Thackray等, ,2007)。 MNV.CR6菌株对近端结肠和盲肠具有趋向性,其比MNV.CW3菌株更持久和复制,其导致急性感染(Arias等人,2012; Nice ,2013)。

关键字:鼠诺如病毒, 持续感染, 口服强饲, RT-qPCR, 抗病毒

材料和试剂

  1. 总体
    1. 适用于2级生物安全(BSL-2)实验室或A-2动物设施(手套,实验室外套,发网,鞋套,安全眼镜)的个人防护。
    2. 消毒剂:漂白剂(5000 ppm)或Virkon S.

  2. 体外工作
    1. 培养瓶(150 cm 2,TPP,目录号:90856)
    2. 细胞刮刀(Corning,Falcon ,目录号:353086)
    3. Cryotubes(Thermo Fisher Scientific,Thermo Scientific TM,目录号:377224)
    4. 移液吸头(10μl,100μl,1,000μl)
    5. 一次性血清移液管(5毫升,10毫升,25毫升)
    6. 鼠巨噬细胞(RAW 264.7,ATCC,目录号:TIB-71)
    7. 达尔伯克磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,目录号:14190094)
    8. Dulbecco's改良的Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:41965039)
    9. 胎牛血清(FCS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
    10. 碳酸氢钠(Thermo Fisher Scientific,Gibco TM,产品目录号:25080060)
    11. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030024)
    12. HEPES(Thermo Fisher Scientific,Gibco TM,目录号:15630056)
    13. 青霉素/链霉素(P / S)(Thermo Fisher Scientific,Gibco TM,目录号:15140148)
    14. 丙酮酸钠(Thermo Fisher Scientific,Gibco TM,目录号:11360039)
    15. 培养基(见食谱)

  3. 体内工作
    1. Eppendorf安全锁管1.5 ml(Eppendorf,产品目录号:0030120086)
    2. 用于皮下注射的注射器+针头(VWR,目录号:BDAM303176)
    3. 针头容器(Sharpsafe,目录号:41602432)
    4. 台式表面保护器(VWR,产品目录号:115-9220)
    5. Earmarks(Bioseb,目录号:EP-1005-1)
    6. 塑料饲养管,20 G x 30 mm,无菌(Instech Laboratories,目录号:FTP-20-30)
    7. 无菌1毫升注射器,适合饲管(VWR,目录号:612-0106)
    8. 塑料容器暂时限制鼠标
    9. 耳标(Bioseb,产品目录号:EP-1005-1)+涂药器(Style 1005s1)
    10. 来自BK Universal,UKCR6株的I型和II型干扰素受体敲除AG129小鼠(129 / Sv小鼠)
    11. 2' - 甲基胞苷(2CMC,Carbosynth,目录号:NM07918)
    12. Favipiravir(T-705,BOC Sciences,目录号:B0084-463609)
    13. 羧甲基纤维素(CMC,Sigma-Aldrich,目录号:C9481)
    14. 无菌盐水(NaCl 0.9%,B.Braun)

  4. 病毒RNA量化

    1. 0.2 ml(Eppendorf,目录号:0030089413)的Multipette尖端
    2. Lightcycler 480-多孔板96(Roche Molecular Systems,目录号:04729692001)
    3. RNeasy Mini试剂盒(QIAGEN,产品目录号:74106)
    4. 绝对乙醇(Fisher Scientific,目录号:10342652)
    5. iTaq Universal Probes One-Step Kit(Bio-Rad Laboratories,目录号:1725141)
    6. 引物,探针和标准(Baert et。,2008)

设备

  1. A-2动物设施中的生物安全罩
  2. BSL-2实验室的生物安全罩
  3. 培养箱(37℃,5%CO 2,潮湿)
  4. 秤称重老鼠
  5. 镊子适合捡起鼠标粪便(可高压灭菌)
  6. 移液器组(P10,P100,P1000)
  7. Pipetboy(Integra Biosciences,目录号:155016)
  8. Multipette M4(Eppendorf,产品目录号:4982000012)
  9. 多道移液器(Eppendorf,目录号:3122000035)
  10. -80°C冷冻机
  11. PCR工作站
  12. 涡流
  13. 用一个适合1.5毫升管的转子离心。
  14. 用一个适合50毫升管子的转子离心。
  15. 高压灭菌器
  16. RT-qPCR机器(Roche Diagnostics,型号:Roche LightCycler 96)
  17. 倒置光学显微镜
  18. 细胞计数器

软件

  1. GraphPad Prism软件,第7版
  2. LightCycler 96 SW 1.1软件

程序

  1. 扩增和量化MNV.CR6病毒库存
    注意:培养和感染BSL-2实验室生物安全罩下的细胞
    1. 使用细胞刮刀将RAW264.7细胞分裂,在含有40ml 10%培养基的T150烧瓶中以1:10的比例每周一次。将细胞保持在5%CO 2和37℃的CO 2培养箱中。
    2. 在培养2或3天后用MNV.CR6感染RAW264.7细胞(即,当细胞几乎汇合但不长满时)。这样做如下:
      1. 从细胞中吸出生长培养基,用DPBS洗涤并吸出DPBS。
      2. 向细胞中加入1ml MNV.CR6并均匀分布,在37℃孵育1小时。
      3. 加入20毫升2%培养基,并将培养瓶放回培养箱中,直至观察到完全细胞病变效应(感染后±2-3天)。
    3. 重复两次冻融循环(-80℃对37℃),然后在离心(10分钟,1,500×gg)后收集含有病毒的上清液并以等分试样(500-1,000μl )在-80°C的低温管中。
    4. 如先前Reed等人(1938)所述,通过终点滴定确定病毒滴度。该程序基于通过显微镜检测细胞病变效应(CPE)。
      注意:滴定可以用更高的稀释度系列进行,也可以在病毒储存较强时在第二个96孔板上进行。
      1. 在室温下解冻MNV.CR6库存。

      2. 在96孔板的所有孔中加入100μl2%DMEM

      3. 在96孔板第二柱的B2-G2孔中加入50μl纯病毒原液。

      4. 用多通道移液器匀浆病毒和培养基
      5. 取50μl混合物并转移到下一个孔中。
      6. 重复步骤A4d-A4e,直到达到96孔板的第9栏。
      7. 将第10行的病毒和培养基均质化并丢弃50μl。
      8. 在2%DMEM中以100μl终体积加入10,000个RAW 264.7细胞/孔,至内部60个孔。

      9. 在37℃5%CO 2中孵育72小时。
      10. 将孔与感染细胞(第2-9列)与未感染细胞(第10-11列)比较,并用显微镜评估细胞病变效应(CPE)。
      11. 用Reed-Muench公式计算TCID 50(Reed等人,1938)。

  2. 在小鼠中口服MNV.CR6
    注意:
    1. 在尽可能靠近特定病原体(SPF)设施的条件下,在A-2动物设施的生物安全罩下处理感染动物。
    2. 使用防护工作服来防止生物安全罩受到病毒污染。
    3. 替换不同实验组之间的手套。
    4. 对于所有实验,小鼠年龄和性别匹配,小鼠为8-12周龄。

    1. 使用唯一编号标记每只鼠标的耳朵。
    2. 解冻病毒并在2%培养基中稀释至MNV.CR6的106 CCID50(50%细胞培养感染剂量)。
      通过口服灌胃给每只小鼠200μl病毒(见下文)。

    3. 填充注射器和饲管中的病毒并去除所有气泡。

    4. 小心地将鼠标从尾巴的底部拿起,放在电线笼上。
    5. 另一方面,通过握住拇指和食指之间的颈背来限制鼠标。将小尾巴放在小手指和无名指之间,以拉伸鼠标(图1)。&nbsp;


      图1.正确的手部位置可容纳一只用于灌胃的小鼠

    6. 现在轻轻地将饲管垂直向下插入食道,并以稳定的动作施用病毒(图2)。任何阻力感觉都表示喂食管放置不当,在这种情况下,取出喂食管并重新定位。


      图2.饲养管垂直插入小鼠的食道

  3. 小分子抑制剂的管理
    备注:
    1. 在尽可能接近SPF设施的条件下,在A-2动物设施的生物安全罩下处理感染动物。
    2. 使用防护工作服来防止生物安全罩受到病毒污染。
    3. 替换不同实验组之间的手套。

    皮下注射2' - 甲基 - 乙基胞苷(100mg / kg /天)

    1. 在无菌生理盐水中溶解2CMC

    2. 注入病毒并清除所有气泡。
    3. 将鼠标放在尾巴的底部,并将其放在电线笼上。
    4. 将针平行于鼠标背面的皮肤插入。确保你只是在皮肤下面(图3)。&nbsp;


      图3.在鼠标背部皮下注射

    5. 通过皮下途径用2CMC注射小鼠并以最终浓度为100mg / kg /天的体积(〜200μl)进行稳定运动。

      口服强力霉素(T-705)(200 mg / kg / day)

    6. 溶解0.4%CMC中的T-705
    7. 通过口服灌胃施用适量的T-705,其体积(〜200μl)终浓度为200mg / kg /天。

  4. 跟进和收集粪便&nbsp;
    备注:
    1. 在尽可能靠近SPF设施的条件下,在A-2动物设施的生物安全罩下处理感染动物。
    2. 使用防护工作服来防止生物安全罩受到病毒污染。
    3. 净化不同实验组之间的盒子,镊子和刻度。

    1. 每天评估动物的一般状况并检查整体健康状况(体重,行为,皮毛)。如果动物达到确定的人道终点(在1-2天内失去超过15%的体重或总体体重> 20%或显示明显的痛苦迹象[嗜睡,眼睛斜视,脱水,驼背] ),必须根据欧洲指导方针(Dir。2010/63 / EU)进行人道安乐死。
    2. 等待排便时,将每只小鼠单独放入干净的塑料容器中。
    3. 使用镊子收集一块粪便,并将其放置在标记为1.5毫升的管中(图4)。对所有老鼠重复。


      图4.标记管中粪便样本的收集


    4. 根据下表对粪便样本的一致性进行评分。

    5. 将粪便样品存放在-20°C或-80°C的冷冻箱中。

  5. 从粪便样品中提取病毒RNA
    注意:在BSL-2实验室的生物安全罩下处理感染样本。
    1. 用QIAGEN的RNeasy mini试剂盒提取病毒RNA:

      1. 加入400μl的RLT缓冲液到每个粪便样本

      2. 旋转样品,直到粪便样品完全均匀。
      3. 以10,000gxg旋转管3分钟。
      4. 转移350μL的上清液到一个新的1.5毫升管,并加入350μL的70%乙醇。
      5. 根据制造商的协议继续。
    2. 用50μl无RNase的水洗脱病毒RNA。
    3. 将病毒RNA储存在-20°C冰箱中。

  6. 通过RT-qPCR定量病毒RNA
    注:在PCR工作站中准备用于RT-qPCR的混合物。
    1. 准备正确浓度的引物,探针和标准稀释液(108到100个RNA拷贝)。

    2. 解冻一步iTaq Universal Probes试剂盒的病毒RNA样本和组件
    3. 为每个样品准备一步RT-qPCR混合物,总共16μl,包含:10μliTaq Universal Probes反应混合物,0.5μliScript高级逆转录酶,无RNase水,900 nM MNV引物和200 nM MNV探测。
    4. 轻轻旋转并短暂离心混合物。
    5. 在96个RT-qPCR孔板的每个孔中,吸取16μl混合物(使用多重吸管)并加入4μl病毒RNA。

    6. 用塑料密封件正确关闭RT-qPCR平板
    7. 将RT-qPCR平板置于LightCycler中,并在以下循环条件下运行:在50℃下逆转录10分钟,在95℃下初始变性3分钟,随后进行40个循环的95℃变性15秒,在60℃退火和延伸30秒。
    8. 对于绝对定量,使用具有已知浓度的MNV DNA标准品的10倍稀释系列。

数据分析

使用标准曲线和LightCycler 96 SW 1.1软件确定RNA提取物的病毒RNA拷贝数。每克粪便的病毒RNA拷贝数量可以通过在提取前称量粪便样品来计算。使用Prism 7软件进行统计分析,用非参数Mann-Whitney检验确定P值。有关代表详情,请参阅我们的原始出版物(Rocha-Pereira等人,2015年)。

笔记

  1. AG129小鼠(129 / Sv小鼠)最初从英国BK环球公司获得,并根据SPF条件下培育和安置在我们研究所的年度许可协议进行。
  2. 所有使用小鼠的实验严格遵循比利时KU Leuven伦理委员会(P101 / 2012)的指导方针并进行。

食谱

  1. RAW264.7电池的培养基

致谢

这里描述的方案的研究以前已经发表(Rocha-Pereira等人,2015)。欧盟FP7项目SILVER(260644),KU Leuven IOF项目HB / 14/031和GOA授予(GOA / 10/014)支持该协议的开发和使用。 J. Rocha-Pereira是Marie Curie COFUND William Harvey国际转化研究学院(WHRI-ACADEMY#343)博士后研究员。 J. Van Dycke是一位SB博士。法兰德斯研究科学基金(FWO)的研究员。我们非常感谢Herbert W. Virgin教授(美国圣路易斯华盛顿大学)亲切地提供MNV.CR6病毒。我们感谢Carolien De Keyzer,Jasper Rymenants和Charlotte Vanderheydt提供了出色的技术支持。作者声明不存在利益冲突或利益冲突。

参考

  1. Angarone,M. P.,Sheahan,A.和Kamboj,M.(2016年)。 诺如病毒移植 Curr Infect Dis Rep 18( 6):17.
  2. Aoki,Y.,Suto,A.,Mizuta,K.,Ahiko,T.,Osaka,K.和Matsuzaki,Y。(2010)。 诺如病毒排泄的持续时间和诺沃克病毒感染老年病人的病毒载量的纵向过程 J Hosp感染 75(1):42-46。
  3. Arias,A.,Bailey,D.,Chaudhry,Y.和Goodfellow,I.(2012)。 开发用于小鼠诺沃克病毒3的反向遗传学系统:长期持续存在于盲肠和结肠。
  4. Baert,L.,Wobus,C. E.,Van Coillie,E.,Thackray,L. B.,Debevere,J.和Uyttendaele,M.(2008)。 使用空斑试验,转染试验和实时逆转录PCR检测鼠诺如病毒1
    应用环境微生物学 74(2):543-546。
  5. Green,K.Y.(2014)。 免疫功能低下宿主感染诺如病毒 Clin Microbiol感染 20 (8):717-723。
  6. Karst,S.M.,Wobus,C.E.,Lay,M.,Davidson,J。和Virgin,H.W。 (2003年)。 STAT1依赖先天免疫诺瓦克样病毒 科学< / em> 299(5612):1575-1578。
  7. Murata,T.,Katsushima,N.,Mizuta,K.,Muraki,Y.,Hongo,S.and Matsuzaki,Y。(2007)。 婴儿或6个月以下的婴儿因胃肠炎持续感染诺如病毒。 Pediatr Infect Dis J 26(1):46-49。
  8. Nice,T.J.,Strong,D.W.,McCune,B.T.,Pohl,C.S。和Virgin,H.W。(2013)。 小鼠诺沃克病毒NS1 / 2的单氨基酸改变足以促进结肠取向和持久性。
  9. Reed,L.J。和Muench,H。(1938)。 估计百分之五十端点的简单方法。 Am J Epidemiol 27(3):493-7。
  10. Rocha-Pereira,J.,Van Dycke,J.和Neyts,J。(2015)。 用核苷聚合酶抑制剂治疗可减少粪便中诺如病毒的脱落,达到检测不到的水平,耐药变异体 抗菌剂Chemother 60(3):1907-1911。
  11. Strong,D. W.,Thackray,L. B.,Smith,T. J.和Virgin,H. W.(2012)。 衣壳蛋白的突出结构域对于确定鼠类诺沃克病毒复制和发病机制在体内是必要和充分的。 J Virol 86(6):2950-2958。
  12. Thackray,L.B.,Wobus,C.E.,Chachu,K.A.,Liu,B.,Alegre,E.R。,Henderson,K.S.,Kelley,S.T。和Virgin,H.W。 (2007年)。 尽管序列分歧有限,但由单个基因组组成的鼠诺沃克病毒表现出生物多样性。 J Virol 81(19):10460-10473。
  13. Westhoff,T.H.,Vergoulidou,M.,Loddenkemper,C.,Schwartz,S.,Hofmann,J.,Schneider,T.,Zidek,W。和van der Giet,M。(2009)。 肾移植受者的慢性诺如病毒感染 Nephrol Dial Transplant > 24(3):1051-1053。
  14. Wobus,C.E。,Karst,S.M.,Thackray,L.B.,Chang,K.O.,Sosnovtsev,S.V.,Belliot,G.,Krug,A.,Mackenzie,J.M.,Green,K.Y。和Virgin,H.W。(2004)。 诺如病毒在细胞培养中的复制揭示了树突状细胞和巨噬细胞的趋向性。 > PLoS Biol 2(12):e432。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dycke, J. V., Neyts, J. and Rocha-Pereira, J. (2018). Assessing the Efficacy of Small Molecule Inhibitors in a Mouse Model of Persistent Norovirus Infection. Bio-protocol 8(9): e2831. DOI: 10.21769/BioProtoc.2831.
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