A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 dengue^3,4. 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 DENV3^5,6, (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 studies^5,6,7,8,9, 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 studies^5,6,7,8,9 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 findings^{9,10,11,12,13,14,15}, 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.

Protokół

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

1. 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).
2. Dilute the virus stock to 1 x 10⁶ PFU with Roswell Park Memorial Institute (RPMI) 1640 medium to a total volume of 40 μL.
3. 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.
4. Hold the 7 day-old ICR suckling mouse and conduct the following steps.
   1. 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.
   2. 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.
5. 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.
6. 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 rate^{9,10,11,12,13,14,15}.

2. Disease Scoring

1. 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.
2. 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.

Wyniki

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 model^9,

Dyskusje

DENV infection has been detected in the CNS of severe dengue patients^3,17, 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 infecti...

Materiały

Name	Company	Catalog Number	Comments
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