Mouse model of respiratory syncytial virus infection mimicking acute human pathology
Introduction. Human respiratory syncytial virus (RSV) is one of the common causes of lower respiratory tract inflammation in children and elderly persons. RSV infection being a serious public health problem is characterized by high morbidity and mortality, especially in children. There are no licensed vaccines and inexpensive medications for RSV prevention and treatment. The lack of universal animal model which mimics all aspects of human pathology is one of the main obstacles to develop new therapy approaches.
The aim of the study was to develop the model of RSV infection using BALB/c mice and purified and concentrated virus, which allows reproducing the main features of human pathology.
Material and methods. Mice were divided into 4 groups (n = 10). Animals of the 1st group (RSV hd) were intranasally infected with purified RSV strain A2 at a high dose of 5 - 106 pfu/mouse in a volume of 50 mcl of phosphate buffered saline. The 2nd group (RSV ld) received non-purified virus at low dose 105 pfu/mouse. Мice of the 3rd group (RSV-UV) were treated with the same dose (5 - 106 pfu/mouse) and volume (50 mcl) of the UV-inactivated RSV. Animals of the 4th group (Intact) were left untreated. The following indicators were measured: the airway hyperreactivity, cell composition in broncho-alveolar lavage (BAL) samples, the severity of inflammation and levels of pro-inflammatory cytokines in lungs. The body weight of the animals was monitored daily throughout the experiment. In separate experiments mice infected with high dose (5 - 106 pfu/mouse) of purified RSV received ribavirin orally twice a day in dose 85 mg/kg for 5 days.
Results. The virus was detected in the respiratory tract of animals for 5 days after the infection. Additionaly, a 12% body weight loss was observed on day 3, indicating successful infection. Presented protocol allows to induce many manifestations of the human pathology such as: increased airway hyperreactivity (AHR), mucus secreting goblet cell metaplasia in the bronchial epithelium and lung inflammation associated with the proinflammatory cell infiltration. Proinflammatory cytokine genes (Ifng and Tbet) expression was increased compared to intact mice. At the same time, the expression of Th2 cytokines gens (Il4, Il13 and Gata3) didn’t change significantly after infection with RSV. These data indicate the ability of RSV productively replicate in the respiratory tract, induce pulmonary inflammation and shift the immune response toward Th1-type. We also investigated the effect of ribavirin treatment on the replication of RSV in the current model and how it correlated with lung inflammation and airway hyperreactivity. In the experiment, oral daily administration of ribavirin significantly reduced viral load in lungs and lymphocytes number in the BAL. In addition, improvement in airway hyperreactivity was observed after ribavirin-mediated suppression of RSV replication. Thus, this model is sensitive to known antiviral drugs and may be useful for testing new drugs against RSV.
Conclusion. We described the experimental model of RSV infection in mice. This model mimics the main features of human pathology. The described model can be useful for the testing of novel anti-RSV drugs and further understanding of RSV infection immunopathogenesis.
Keywords:human respiratory syncytial virus; murine models; human pathology modeling
For citation: Shilovskiy I.P., Barvinskaia E.D., Nikolskii A.A., Nikonova A.A., Smirnov V.V., Kovchina V.I., Vishnyakova L.I., Yumashev K.V., Kaganova M.M., Rusak T.E., Mitin A.N., Komogorova V.V., Litvina M.M., Sharova N.I., Kudlay D.A., Khaitov M.R. Mouse model of respiratory syncytial virus infection mimicking acute human pathology. Immunologiya. 2022; 2022; 43 (4): 423–39. DOI: https://doi.org/10.33029/0206-4952-2022-43-4-423–439
Funding. The study was supported by Russian Science Foundation grant No. 22-25-00182.
Conflict of interests. The authors declare no conflict of interests.
Authors contribution. Study conception and design – Shilovskiy I.P., Smirnov V.V., Khaitov M.R., Mitin A.N.; material collection and processing – Barvinskaia E.D., Nikolskii A.A., Kovchina V.I., Vishnyakova L.I., Yumashev K.V., Kaganova M.M., Rusak T.E., Komogorova V.V., Litvina M.M., Sharova N.I.; statistical processing – Barvinskaia E.D., Yumashev K.V.; manuscript preparation – Barvinskaia E.D., Shilovskiy I.P.; editing – Kaganova M.M., Kudlay D.A., Nikonova A.A.
1. Shi T., Denouel A., Tietjen A.K., Campbell I., Moran E., Li X., Campbell H., et al. Global disease burden estimates of respiratory syncytial virus-associated acute respiratory infection in older adults in 2015: a systematic review and meta-analysis. J. Infect. Dis. 2019; 222 (7): 577–83. DOI: https://doi.org/10.1093/infdis/jiz059
2. Allakhverdiyeva L.I., Humbatova U.M. Immune disturbances in children with virus-induced bronchial asthma. Immunologiya. 2013; 34 (4): 217–20. (in Russian)
3. Troeger C., Blacker B., Ibrahim A. Khalil, Puja C. Rao, Cao J., Zimsen R.M.S., et al. Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in 195 countries, 1990–2016: a systematic analysis for the Global Burden of Di- sease Study 2016. Lancet Infect. Dis. 2018; 18 (11): 1191–210. DOI: https://doi.org/10.1016/S1473-3099(18)30310-4
4. Geoghegan S., Erviti A., Caballero M.T., Vallone F., Zanone S.M., Losada J.V., et al. Mortality due to respiratory syncytial virus burden and risk factors. Am. J. Respir. Crit. Care Med. 2017; 195 (1): 96–103. DOI: https://doi.org/10.1164/rccm.201603-0658OC
5. Halasa N.B., Williams J.V., Wilson G.J., Walsh W.F., Schaffner W., Wright P.F. Medical and economic impact of a respiratory syncytial virus outbreak in a neonatal intensive care unit. Pediatr. Infect. Dis. J. 2005; 24 (12): 1040–4. DOI: https://doi.org/10.1097/01.inf.0000190027.59795.ac
6. Muralidharan A., Li C., Wang L., Li X. Immunopathogenesis associated with formaldehyde-inactivated RSV vaccine in preclinical and clinical studies. Expert Rev. Vaccines. 2017; 16 (4): 351–60. DOI: https://doi.org/10.1080/14760584.2017.1260452
7. Janai H.K., Marks M.I., Zaleska M., Stutman H.R. Ribavirin: adverse drug reactions, 1986 to 1988. Pediatr. Infect. Dis. J. 1990; 9 (3): 209–211
8. Wang D., Cummins C., Bayliss S., Sandercock J., Burls A. Immunoprophylaxis against respiratory syncytial virus (RSV) with palivizumab in children: a systematic review and economic evaluation. Health Technol. Assess. (Rockv.). 2008; 12 (36): 1–86. DOI: https://doi.org/10.3310/hta12360
9. Khaitov M.R., Litvin L.S., Shilovsky I.P., Bashkatova Yu.N., Faizuloev E.B., Zverev V.V. RNA interference. New approaches to the development of antiviral agents. Immunologiya. 2010; 31: 69–76. (in Russian)
10. Shilovskiy I.P., Andreev S.M., Kozhikhova K.V., Nikolskii A.A., Khaitov M.R. prospects for the use of peptides against respiratory syncytial virus. Mol. Biol. 2019; 53, (4): 484–500. DOI: https://doi.org/10.1134/S002689841904013X
11. Pinegin B.V., Pashchenkov M.V. Immunostimulators of muramylpeptide nature in the treatment and prevention of infectious-inflammatory processes. Immunologiya. 2019; 40 (3): 65–71. DOI: https://doi.org/10.24411/0206-4952-2019-13007 (in Russian)
12. Budikhina A.S. The role of antimicrobial peptides in the pathology of diseases of the upper respiratory tract. Immunologiya. 2017; 38 (4): 234–8. DOI: https://doi.org/10.18821/0206-4952-2017-38-4-234-238. (in Russian)
13. Ralston S., Hill V. Incidence of apnea in infants hospitalized with respiratory syncytial virus bronchiolitis: a systematic review. J. Pediatr. 2009; 155 (5): 728–33. DOI: https://doi.org/10.1016/j.jpeds.2009.04.063
14. Johnson J.E., Gonzales R.A., Olson S.J., Wright P.F., Graham B.S. The histopathology of fatal untreated human respiratory syncytial virus infection. Mod. Pathol. 2007; 20 (1): 108–19. DOI: https://doi.org/10.1038/modpathol.3800725
15. Welliver T.P., Garofalo R.P., Hosakote Y., Hintz K.H., Avendano L., Sanchez K., et al. Severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses. J. Infect. Dis. 2007; 195 (8): 1126–36. DOI: https://doi.org/10.1086/512615
16. Plekhova N.G. Kodrashova N.M., Geltser B.I., Kotelnikov V.N. Cellular and molecular factors of innate defense and its role in the pathogenesis of pneumonia. Immunologiya. 2017; 38 (2): 124–9. DOI: https://doi.org/10.18821/0206-4952-2017-38-2 (in Russian)
17. Russell C.D., Unger S.A., Walton M., Schwarze J. The human immune response to respiratory syncytial virus infection. Clin. Microbiol. Rev. 2017; 30 (2): 481–502. DOI: https://doi.org/10.1128/CMR.00090-16
18. Taylor G. Animal models of respiratory syncytial virus infection. Vaccine. 2017; 35; (3): 469–80. DOI: https://doi.org/10.1016/j.vaccine.2016.11.054
19. Altamirano-Lagos M.J., Fabián E.D., Miguel A.M., et al. Current animal models for understanding the pathology caused by the respiratory syncytial virus. Front. Microbiol. 2019; 10: 873. DOI: https://doi.org/10.3389/fmicb.2019.00873
20. Green M.G., Huey D., Niewiesk S. The cotton rat (Sigmodon hispidus) as an animal model for respiratory tract infections with human pathogens. Lab. Anim. 2013; 42 (5): 170–6. DOI: https://doi.org/10.1038/laban.188
21. Moore M.L., Chi M.H., Luongo C., Lukacs N.W., Polosukhin V.V., Huckabee M.M., et al. A chimeric A2 strain of respiratory syncytial virus (RSV) with the fusion protein of RSV strain line 19 exhibits enhanced viral load, mucus, and airway dysfunction. J. Virol. 2009; 83 (9): 4185–94. DOI: https://doi.org/10.1128/JVI.01853-08
22. Stokes K.L., Chi M.H., Sakamoto K., Newcomb D.C., Currier M.G., Huckabee M.M., Stokes K.L., et al. Differential pathoge- nesis of respiratory syncytial virus clinical isolates in BALB/c mice. J. Virol. 2011; 85 (12): 5782–93. DOI: https://doi.org/10.1128/JVI.01693-10
23. Hashimoto K., Durbin J.E., Zhou W., Collins R.D., Ho S.B., Kolls J.K., et al. Respiratory syncytial virus infection in the absence of STAT1 results in airway dysfunction, airway mucus, and augmented IL-17 levels. J. Allergy Clin. Immunol. 2005; 116 (3): 550–7. DOI: https://doi.org/10.1016/j.jaci.2005.03.051
24. Jafri H.S., Chavez-Bueno S., Mejias A., Gomez A.M., Rios A.M., Nassi S.S., et al. Respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice. J. Infect. Dis. 2004; 189 (10): 1856–65. DOI: https://doi.org/10.1086/386372
25. van Erp E.A., Lakerveld A.J., Mulder H.L., Luytjes W., Ferwerda G., van Kasteren P.B., et al. Pathogenesis of respiratory syncytial virus infection in BALB/c mice differs between intratracheal and intranasal inoculation. Viruses. 2019; 11 (6): 508. DOI: https://doi.org/10.3390/v11060508
26. Ueba O. Respiratory syncytial virus concentration and purification of the infectious virus. Acta Med. Okayama. 1978; 32 (4): 265–72.
27. Khaitov M.R., Shilovskiy I.P., Nikonova A.A., Shershakova N.N., Kamyshnikov O.Y., Babakhin A.A., et al. Small interfering RNAs targeted to interleukin-4 and respiratory syncytial virus reduce airway inflammation in a mouse model of virus-induced asthma exacerbation. Hum. Gene Ther. 2014; 25 (7): 642–50. DOI: https://doi.org/10.1089/hum.2013.142
28. Marcelin J.R., Wilson J.W., Razonable R.R. Oral ribavirin therapy for respiratory syncytial virus infections in moderately to severely immunocompromised patients. Transpl. Infect. Dis. 2014; 16 (2): 242–50. DOI: https://doi.org/10.1111/tid.12194
29. Lukacs N.W., Moore M.L., Rudd B.D., Berlin A.A., Collins R.D., Olson S.J., et al. Differential immune responses and pulmonary pathophysiology are induced by two different strains of respiratory syncytial virus. Am. J. Pathol. 2006; 69 (3): 977–86. DOI: https://doi.org/10.2353/ajpath.2006.051055
30. Rameix-Welti M.A., Goffic R.L., Hervé P.L., Sourimant J., Rémot A., Riffaultet S., et al. Visualizing the replication of respiratory syncytial virus in cells and in living mice. Nat. Commun. 2014; 5: 5104. DOI: https://doi.org/10.1038/ncomms6104
31. Bitko V., Musiyenko A., Shulyayeva O., Bariket S., et al. Inhibition of respiratory viruses by nasally administered siRNA. Nat. Med. 2005; 11 (1): 50–5. DOI: https://doi.org/10.1038/nm1164
32. Smith P.K. Wang S.Z., Dowling K.D., Forsyth K.D. Leucocyte populations in respiratory syncytial virus-induced bronchiolitis. J. Paediatr. Child Health. 2001; 37 (2): 146–51. DOI: https://doi.org/10.1046/j.1440-1754.2001.00618.x
33. Tripp R.A., Moore D., Barskey A., Jones L., Moscatiello C., Keyserling H., et al. Peripheral blood mononuclear cells from infants hospita- lized because of respiratory syncytial virus infection express T helper-1 and T helper-2 cytokines and CC chemokine messenger RNA. J. Infect. Dis. 2002; 185 (10): 1388–94. DOI: https://doi.org/10.1086/340505
34. Elser B., Lohoff M., Kock S., Giaisi M., Kirchhoff S., Krammer P.H., et al. IFN-γ represses IL-4 expression via IRF-1 and IRF-2. Immunity. 2002; 17 (6): 703–12. DOI: https://doi.org/10.1016/s1074-7613(02)00471-5
35. Geevarghese B., Weinberg A. Cell-mediated immune responses to respiratory syncytial virus infection: magnitude, kinetics, and correlates with morbidity and age. Hum. Vaccines Immunother. 2014; 10 (4): 1047–56. DOI: https://doi.org/10.4161/hv.27908
36. Rosenberg H.F., Domachowske J.B. Inflammatory responses to respiratory syncytial virus (RSV) infection and the development of immunomodulatory pharmacotherapeutics. Curr. Med. Chem. 2012; 19 (10): 1424–31. DOI: https://doi.org/10.2174/092986712799828346
37. Puthothu B., Bierbaum S., Kopp M.V., Forster J., Heinze J., Weckmannet M., et al. Association of TNF-α with severe respiratory syncytial virus infection and bronchial asthma. Pediatr. Allergy Immunol. 2009; 20 (2): 157–63. DOI: https://doi.org/10.1111/j.1399-3038.2008.00751.x
38. Monin L., Gaffen S.L. Interleukin 17 family cytokines: Signaling mechanisms, biological activities, and therapeutic implications. Cold Spring Harb. Perspect. Biol. 2018; 10 (4): a028522. DOI: https://doi.org/10.1101/cshperspect.a028522
39. Lora J.M., Zhang D.M., Liao S.M., Burwell T., King A.M., Barker P.A., et al. Tumor necrosis factor-α triggers mucus production in airway epithelium through an IκB kinase β-dependent mechanism. J. Biol. Chem. 2005; 280 (43): 36 510–7. DOI: https://doi.org/10.1074/jbc.M507977200
40. Homaira N., Rawlinson W., Snelling T.L., Jaffe A. Effectiveness of palivizumab in preventing RSV hospitalization in high risk children: a real-world perspective. Int. J. Pediatr. 2014; 2014; 571609. DOI: https://doi.org/10.1155/2014/571609
41. Wright M., Piedimonte G. Respiratory syncytial virus prevention and therapy: Past, present, and future. Pediatr. Pulmonol. 2011; 46 (4): 324–47. DOI: https://doi.org/10.1002/ppul.21377
42. Du L.N., Xie T., Xu J.Y., Kang A., Di L.Q., Shan J.J., et al. A metabolomics approach to studying the effects of Jinxin oral liquid on RSV-infected mice using UPLC/LTQ-Orbitrap mass spectrometry. J. Ethnopharmacol. 2015; 174: 25–36. DOI: https://doi.org/10.1016/j.jep.2015.07.040
43. Xu J.J., Liu Z., Tang W., Wang G.C., Chung H.Y., Liu Q.Y., et al. Tangeretin from Citrus reticulate inhibits respiratory syncytial virus replication and associated inflammation in vivo. J. Agric. Food Chem. 2015; 63 (43): 9520–7. DOI: https://doi.org/10.1021/acs.jafc.5b03482
44. Mosquera R.A., Stark J.M., Atkins C.L., Colasurdo G.N., Chevalier J., Samuels C.L., et al. Functional and immune response to respiratory syncytial virus infection in aged BALB/c mice: a search for genes determining disease severity. Exp. Lung Res. 2014; 40 (1): 40–9. DOI: https://doi.org/10.3109/01902148.2013.859334
45. Zschaler J., Schlorke D., Arnhold J. Differences in innate immune response between man and mouse. Crit. Rev. Immunol. 2014; 34 (5): 433–54. DOI: https://doi.org/10.1615/CritRevImmunol.2014011600
46. Mejias A., Chavez-Bueno S., Rios A.M., Aten M.F., Raynor B., Peromingo E., et al. Comparative effects of two neutralizing anti-respiratory syncytial virus (RSV) monoclonal antibodies in the RSV murine model: time versus potency. Antimicrob. Agents Chemother. 2005; 49 (11): 4700–7. DOI: https://doi.org/10.1128/AAC.49.11.4700-4707.2005