Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.
Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.
DENV, an arthropod-borne virus of the Flaviviridae family, contains a positive-sense RNA genome that encodes three viral structural proteins (capsid, premembrane, and envelope) and seven viral nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The four serotypes of DENV (DENV1-4), which infect approximately 390 million people annually, cause a global burden even though governments have directed substantial efforts toward mosquito vector and disease control1. Currently, protective vaccines and therapeutic antiviral drugs are under development and require further long-term validation2. In clinical practice, although a dengue patient with CNS infection is rare, it needs to be further explored to understand the diversity of dengue disease development3. Further investigation and validation are needed; notably, the World Health Organization (WHO) has included the involvement of CNS impairment, such as cognitive impairment, convulsions, encephalopathy, and encephalitis, in the classification of severe dengue3,4. Constructing animal models of DENV infection is indispensable for exploring the neuropathogenesis of DENV infection.
For generating CNS infection by DENV, several studies have executed different routes of DENV infection, including (1) intracerebral inoculation of C57BL/6 mice who received 4 x 103 plaque-forming units (PFU) of nonadapted DENV35,6, (2) intraperitoneal inoculation of BALB/c mice who received 7 x 104 PFU of in vitro neuroadapted DENV47, (3) intracerebral inoculation of Swiss mice who received 1 x 105 PFU of in vivo neuroadapted DENV18, and (4) intracerebral and intraperitoneal co-inoculation of ICR suckling mice who received 1 x 106 PFU of nonadapted DENV29. According to the findings of these studies5,6,7,8,9, DENV infection in mice result in viral replication in the brain, leading to acute viral encephalitis-like syndromes, behavioral changes accompanied by limb paralysis and postural instability, CNS neurotoxicity and inflammation, general and localized plasma leakage through the blood-brain barrier (BBB), and lethality. All the results from these studies5,6,7,8,9 have shown the ability of DENV to infect the CNS and the induction of acute viral encephalitis-like disease following infection.
Based on our current findings9,10,11,12,13,14,15, we have created a murine model of DENV infection as an in vivo platform to examine the therapeutic efficacy of targeted agents/factors against viral replication, as well as neurotoxicity. Here, we report the protocol utilized to create an immunocompetent outbred ICR mouse to study CNS infection and to monitor the development of neuropathies with different severities caused by DENV. The results show the significant progression of encephalitis-like disease in DENV-infected mice in a time-dependent manner.
Experimental protocols of animal study were approved by the Institutional Animal Care and User Committee of the National Defense Medical Center (IACUC number: 16-261), according to guidelines established by the Ministry of Science and Technology, Taiwan.
1. Infection Procedure
- Prepare nonadapted DENV2 (strain PL046) stocks9 (originally obtained from the Centers for Disease Control in Taiwan, ranging from 2.5 x 107 to 1 x 109 PFU/mL).
- Dilute the virus stock to 1 x 106 PFU with Roswell Park Memorial Institute (RPMI) 1640 medium to a total volume of 40 μL.
- Fill one 0.3 mL syringe fitted with a 30 G needle with 10 μL (2.5 x 105 PFU) of diluted virus, and fill another 0.3 mL syringe fitted with a 30 G needle with 30 μL (7.5 x 105 PFU) of diluted virus.
- Hold the 7 day-old ICR suckling mouse and conduct the following steps.
- For the intracerebral injection, hold the mouse in a prone position by pressing the auricle between the index finger and thumb, and intracerebrally inject 10 μL of diluted virus into the lambda area, the point at the intersection of the sagittal and lambdoid suture16.
- After the intracerebral injection, hold the mouse in a supine position by using the index finger and thumb and intraperitoneally inject 30 μL of diluted virus gently into the murine abdomenl.
NOTE: To avoid cannibalism, 75% alcohol is used to create a temporary loss of the mother’s olfactory sensation. Additionally, covering the litters with their mother’s stool and urine is suggested.
- Lay the suckling mice back in their cages and wait for 5 min to check their safety poststimulation with the avidity of mice, including walking and mother milk sucking.
NOTE: Most often, mice demonstrate normal activity poststimulation and there does not seem to be any harmful effects following the technical challenge. During this experiment, the animals were maintained by the Institutional Animal Care and User Committee of the National Defense Medical Center, Taiwan.
- Evaluate the daily progress of the mice in terms of body weight (by using a microgram balance), acute viral encephalitis-like disease (by disease scoring as described in section 2), and survival rate9,10,11,12,13,14,15.
2. Disease Scoring
- Monitor the grade of acute viral encephalitis-like illnesses. Assign a score of 0 to healthy mice; 1 to mice with minor illness symptoms, including weight loss, reduced mobility, and a hunchback body orientation; 2 to mice that exhibit limbic seizure; 3 to mice the exhibit limbic weakness, including moving with difficulty and anterior limb or posterior limb weakness; 4 for paralysis; 5 for death.
NOTE: Once the score reaches 3, the disease symptoms are considered to be evolving rapidly.
- Plot the scoring of the disease, according to step 2.1, for each day as a curve-based figure by using the mean ± SD of the daily test scores in each group.
Severe dengue-associated neurological complications have been reported in patients for dengue pathogenesis4. Although these cases are rare in the clinic, creating an immunocompetent murine model of DENV infection can be used not only for studying immunopathogenesis but also for exploring CNS infection, neuroinflammation, neurotoxicity, and acute viral encephalitis-like disease. In this study, according to our current model9,10,11,12,13,14,15, 7 day-old ICR suckling mice were inoculated concurrently with DENV2 through an intracerebral microinjection (2.5 x 105 PFU) and an intraperitoneal injection (7.5 x 105 PFU) (Figure 1). Following infection, we performed numerous analyses to show the presence of dsRNA replication, viral protein expression, and virus release in the DENV-infected brain9,10,11,12,13,14,15. These experiments demonstrated that a successful in vivo model of DENV infection, replication, and release has been established.
To evaluate the induction of acute viral encephalitis-like illnesses9,10,11,12,13,14,15, we monitored the changes in body weight in DENV-infected mice. Compared with the body weight changes in the mock group (where the mice were inoculated with RPMI medium only), Figure 2 shows an increase in the mock group but a significant (P < 0.01) decrease in body weight in the infected mice. According to the symptoms of encephalitis-like illnesses, including hunchback posture, limbic seizure, limbic weakness, paralysis, and death, as manifested in DENV-infected mice, Figure 3 shows a significant (P < 0.05) increase in clinical score in the DENV-infected mice. The survival assay in Figure 4 shows a time-dependent reduction in the survival rate (P < 0.001) of the DENV-infected mice. The data indicate the establishment of an infectious model of DENV in the CNS, which shows the progression of acute viral encephalitis-like illnesses.
Figure 1: Experimental design for dengue virus (DENV) infection in suckling mice. Seven day-old ICR suckling mice were inoculated with DENV2 PL046 by concurrent intracranial and intraperitoneal injections. We measured changes in body weight, disease score, and mortality. Please click here to view a larger version of this figure.
Figure 2: The body weight in suckling mice during dengue virus (DENV) infection. In DENV2-infected ICR suckling mice (n = 12), changes in body weight were measured per day. Following a Wilcoxon signed-rank test, the values are presented as the mean ± SD. **p < 0.01. Please click here to view a larger version of this figure.
Figure 3: The encephalitic disease score in suckling mice during dengue virus (DENV) infection. Following DENV2 infection in ICR suckling mice (n = 12), time-kinetic changes in clinical scores for hunchback posture (score 1), limbic seizure (score 2), limbic weakness (score 3), paralysis (score 4), and death (score 5) were measured. Following a Wilcoxon signed-rank test, the values are presented as the mean ± SD. *p < 0.05. Please click here to view a larger version of this figure.
Figure 4: The survival rate in suckling mice during dengue virus (DENV) infection. Time-kinetic changes in survival rates were measured in DENV2-infected ICR suckling mice (n = 12). Following a log-rank test, the values are presented as the mean ± SD. ***p < 0.001. Please click here to view a larger version of this figure.
DENV infection has been detected in the CNS of severe dengue patients3,17, suggesting the possibility of the manifestation of acute viral encephalitis occurred during dengue pathogenesis. Here, we report an in vivo murine model of DENV infection for studying the involvement of CNS dysfunction in severe dengue, particularly with a focus on DENV-induced acute viral encephalitis-like illnesses. As compared with the previous models, particularly for one-route infection (intravenous only, intraperitoneal only, or intracerebral only), an extremely high titer (1 x 108 PFU) of DENV is utilized in immunocompetent mice. However, in immunocompromised mice, a relatively low but variant titer (1 x 105 to 1 x 108 PFU) of DENV can be performed18,19,20. In this study, we used two-route infection to induce concurrent CNS and systemic infection with a low titer (intracranial: 2.5 x 105 PFU; intraperitoneal: 7.5 x 105 PFU) of DENV in immunocompetent ICR suckling mice. The progression of encephalitis-like disease is completely reproducible9,10,11,12,13,14,15. The possible limitation of this study is, therefore, mitigated by using a concurrent intracranial and intraperitoneal injection. Although it is artificial-however, consistent with previous works by others5,6,7,8 and us9,10,11,12,13,14,15-DENV is actively replicated in the experimental murine brain9,10,11,12,13,14,15, and the possible effects of viral factors and host responses on neuroinflammation, as well as neurotoxicity, are, therefore, of interest.
For neuropathogenesis, the stimulation of neuronal cell apoptosis is triggered by DENV infection as well as CNS inflammation, such as microglial cell activation followed by proinflammatory cytokine tumor necrosis factor-α (TNF-α) production8,12,15. Direct neurotoxicity caused by DENV infection and indirect neuroinflammation initiated by host immune responses are speculated to be involved in dengue encephalitis. Acute viral encephalitis is caused by virus-induced CNS inflammation of the brain3,21. Common clinical symptoms are a high fever, headache, stiff neck and back, vomiting, and confusion, while severe cases also develop seizures, paralysis, and coma21. By monitoring the changes in scores with hunchback posture, limbic seizure, limbic weakness, paralysis, and death, the development of these illnesses shows the progression of acute viral encephalitis-like disease in DENV-infected mice9,10,11,12,13,14,15. Generally, those DENV-infected mice with a score of 3 (according to the disease scoring method presented in the protocol) rapidly progressed to paralysis and death. The pathogenic mechanisms causing these illnesses require further investigation; however, it is speculated that neuronal dysfunction followed by neuropathy induction is involved21. Targeting viral replication, as well as TNF-α-mediated CNS inflammation10,11,12,13,14, at least in part, confers therapeutic effects against the progression of dengue-associated acute viral encephalitis-like disease. The protocol of this study could be used for creating an encephalitis-like pathological model of DENV infection.
Some studies have described the induction of intracerebral hemorrhage induced by DENV infection in the brain22,23. We previously showed the induction of BBB disruption in DENV-infected mice9. Although DENV is not recognized as a neurotropic virus, DENV can produce an effective infection in neuronal cells in vivo and in vitro8,11. Dopamine D2 receptor (D2R) is speculated to be a neuronal receptor for DENV infection, and pharmacologically targeting D2R effectively reduces DENV infection in neuronal cells in vivo and in vitro, as well as DENV-induced encephalitic illnesses11,24. Possible therapeutic strategies against acute viral encephalitis are important to develop so that severe dengue cases with neurological complications can be treated. For its future application, this protocol of a disease model using DENV infection in the brain may become an in vivo platform to screen not only the viral and host factors associated with neuropathogenesis but also possible antiviral and anti-encephalitic drugs. Regarding the incidence of variant neurological disorders caused by DENV infection25 by this established protocol, the difference between the variable types and/or strains of DENV for causing neurological dysfunction is needed for validation.
The authors have nothing to disclose.
This study was supported by grants from the Ministry of Science and Technology (MOST107-2321-B-038-001) and the intramural funding 106TMU-CIT-01-2, Taipei, Taiwan.
|Roswell Park Memorial Institute 1640 medium (RPMI)||Gibco||11875-085||Diluting virus|
|0.3 mL Insulin Syringe||BD Ultra-FineII||328838||Intracerebral and intraperitoneal injection|
|Microbalance||METTLER TOLEDO's LabX||AL104||Weight mouse|
|Non-adapted DENV2 (strain PL046)||The Centers for Disease Control of Taiwan||-||Infect mouse|
|Institute of Cancer Research (ICR) suckling mouse||BioLASCO Taiwan Co., Ltd||-||Our murine model|
- Guzman, M. G., Gubler, D. J., Izquierdo, A., Martinez, E., Halstead, S. B. Dengue infection. Nature Reviews Disease Primers. 2, 16055 (2016).
- Katzelnick, L. C., Coloma, J., Harris, E. Dengue: knowledge gaps, unmet needs, and research priorities. Lancet Infectious Diseases. 17, (3), e88-e100 (2017).
- Carod-Artal, F. J., Wichmann, O., Farrar, J., Gascon, J. Neurological complications of dengue virus infection. Lancet Neurology. 12, (9), 906-919 (2013).
- Geneva: World Health Organization. Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control: New Edition. WHO Guidelines Approved by the Guidelines Review Committee (2009).
- Amaral, D. C., et al. Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice. Journal of Neuroinflammation. 8, 23 (2011).
- de Miranda, A. S., et al. Dengue-3 encephalitis promotes anxiety-like behavior in mice. Behavioural Brain Research. 230, (1), 237-242 (2012).
- Velandia-Romero, M. L., Acosta-Losada, O., Castellanos, J. E. In vivo infection by a neuroinvasive neurovirulent dengue virus. Journal of Neurovirology. 18, (5), 374-387 (2012).
- Despres, P., Frenkiel, M. P., Ceccaldi, P. E., Duarte Dos Santos, C., Deubel, V. Apoptosis in the mouse central nervous system in response to infection with mouse-neurovirulent dengue viruses. Journal of Virology. 72, (1), 823-829 (1998).
- Tsai, T. T., et al. Microglia retard dengue virus-induced acute viral encephalitis. Scientific Reports. 6, 27670 (2016).
- Cheng, Y. L., et al. Activation of Nrf2 by the dengue virus causes an increase in CLEC5A, which enhances TNF-alpha production by mononuclear phagocytes. Scientific Reports. 6, 32000 (2016).
- Ho, M. R., et al. Blockade of dengue virus infection and viral cytotoxicity in neuronal cells in vitro and in vivo by targeting endocytic pathways. Scientific Reports. 7, (1), 6910 (2017).
- Jhan, M. K., et al. Anti-TNF-alpha restricts dengue virus-induced neuropathy. Journal of Leukocyte Biology. 104, (5), 961-968 (2018).
- Kao, J. C., et al. The antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mTOR. PLoS Neglected Tropical Diseases. 12, (8), e0006715 (2018).
- Tsai, T. T., Chen, C. L., Tsai, C. C., Lin, C. F. Targeting heat shock factor 1 as an antiviral strategy against dengue virus replication in vitro and in vivo. Antiviral Research. 145, 44-53 (2017).
- Jhan, M. K., et al. Dengue virus infection increases microglial cell migration. Scientific Reports. 7, (1), 91 (2017).
- Benskey, M. J., Manfredsson, F. P. Intraparenchymal Stereotaxic Delivery of rAAV and Special Considerations in Vector Handling. Methods in Molecular Biology. 199-215 (2016).
- Fong, C. Y., et al. Mild encephalitis/encephalopathy with reversible splenial lesion (MERS) due to dengue virus. Journal of Clinical Neuroscience. 36, 73-75 (2017).
- Sarathy, V. V., et al. A lethal murine infection model for dengue virus 3 in AG129 mice deficient in type I and II interferon receptors leads to systemic disease. Journal of Virology. 89, (2), 1254-1266 (2015).
- Schul, W., Liu, W., Xu, H. Y., Flamand, M., Vasudevan, S. G. A dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs. The Journal of Infectious Diseases. 195, (5), 665-674 (2007).
- Tyler, K. L. Acute Viral Encephalitis. New England Journal of Medicine. 379, (6), 557-566 (2018).
- Yauch, L. E., Shresta, S. Mouse models of dengue virus infection and disease. Antiviral Research. 80, (2), 87-93 (2008).
- Assir, M. Z., Jawa, A., Ahmed, H. I. Expanded dengue syndrome: subacute thyroiditis and intracerebral hemorrhage. BMC Infectious Diseases. 12, 240 (2012).
- Kumar, R., Prakash, O., Sharma, B. S. Intracranial hemorrhage in dengue fever: management and outcome: a series of 5 cases and review of literature. Surgical Neurology. 72, (4), 429-433 (2009).
- Simanjuntak, Y., Liang, J. J., Lee, Y. L., Lin, Y. L. Repurposing of prochlorperazine for use against dengue virus infection. Journal of Infectious Diseases. 211, (3), 394-404 (2015).
- Rocha, B. A. M., et al. Dengue-specific serotype related to clinical severity during the 2012/2013 epidemic in centre of Brazil. Infectious Disease Poverty. 6, (1), 116 (2017).