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.

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
<ol>
	<li>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).</l...

<ol>
	<li>Guzman, M. G., Gubler, D. J., Izquierdo, A., Martinez, E., Halstead, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue+infection.">Dengue infection.</a> Nature Reviews Disease Primers. 2, 16055 (2016).</li><li>Katzelnick, L. C., Coloma, J., Harris, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue:+knowledge+gaps,+unmet+needs,+and+research+priorities.">Dengue: knowledge gaps, unmet needs, and research priorities.</a> Lancet Infectious Diseases. 17 (3), e88-e100 (2017).</li><li>Carod-Artal, F. J., Wichmann, O., Farrar, J., Gascon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+complications+of+dengue+virus+infection.">Neurological complications of dengue virus infection.</a> Lancet Neurology. 12 (9), 906-919 (2013).</li><li>Geneva: World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control: New Edition. , (2009).</li><li>Amaral, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebral+infection+with+dengue-3+virus+induces+meningoencephalitis+and+behavioral+changes+that+precede+lethality+in+mice.">Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice.</a> Journal of Neuroinflammation. 8, 23 (2011).</li><li>de Miranda, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue-3+encephalitis+promotes+anxiety-like+behavior+in+mice.">Dengue-3 encephalitis promotes anxiety-like behavior in mice.</a> Behavioural Brain Research. 230 (1), 237-242 (2012).</li><li>Velandia-Romero, M. L., Acosta-Losada, O., Castellanos, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+infection+by+a+neuroinvasive+neurovirulent+dengue+virus.">In vivo infection by a neuroinvasive neurovirulent dengue virus.</a> Journal of Neurovirology. 18 (5), 374-387 (2012).</li><li>Despres, P., Frenkiel, M. P., Ceccaldi, P. E., Duarte Dos Santos, C., Deubel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+the+mouse+central+nervous+system+in+response+to+infection+with+mouse-neurovirulent+dengue+viruses.">Apoptosis in the mouse central nervous system in response to infection with mouse-neurovirulent dengue viruses.</a> Journal of Virology. 72 (1), 823-829 (1998).</li><li>Tsai, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+retard+dengue+virus-induced+acute+viral+encephalitis.">Microglia retard dengue virus-induced acute viral encephalitis.</a> Scientific Reports. 6, 27670 (2016).</li><li>Cheng, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Nrf2+by+the+dengue+virus+causes+an+increase+in+CLEC5A,+which+enhances+TNF-alpha+production+by+mononuclear+phagocytes.">Activation of Nrf2 by the dengue virus causes an increase in CLEC5A, which enhances TNF-alpha production by mononuclear phagocytes.</a> Scientific Reports. 6, 32000 (2016).</li><li>Ho, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blockade+of+dengue+virus+infection+and+viral+cytotoxicity+in+neuronal+cells+in+vitro+and+in+vivo+by+targeting+endocytic+pathways.">Blockade of dengue virus infection and viral cytotoxicity in neuronal cells in vitro and in vivo by targeting endocytic pathways.</a> Scientific Reports. 7 (1), 6910 (2017).</li><li>Jhan, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-TNF-alpha+restricts+dengue+virus-induced+neuropathy.">Anti-TNF-alpha restricts dengue virus-induced neuropathy.</a> Journal of Leukocyte Biology. 104 (5), 961-968 (2018).</li><li>Kao, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antiparasitic+drug+niclosamide+inhibits+dengue+virus+infection+by+interfering+with+endosomal+acidification+independent+of+mTOR.">The antiparasitic drug niclosamide inhibits dengue virus infection by interfering with endosomal acidification independent of mTOR.</a> PLoS Neglected Tropical Diseases. 12 (8), e0006715 (2018).</li><li>Tsai, T. T., Chen, C. L., Tsai, C. C., Lin, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+heat+shock+factor+1+as+an+antiviral+strategy+against+dengue+virus+replication+in+vitro+and+in+vivo.">Targeting heat shock factor 1 as an antiviral strategy against dengue virus replication in vitro and in vivo.</a> Antiviral Research. 145, 44-53 (2017).</li><li>Jhan, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue+virus+infection+increases+microglial+cell+migration.">Dengue virus infection increases microglial cell migration.</a> Scientific Reports. 7 (1), 91 (2017).</li><li>Benskey, M. J., Manfredsson, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraparenchymal+Stereotaxic+Delivery+of+rAAV+and+Special+Considerations+in+Vector+Handling.">Intraparenchymal Stereotaxic Delivery of rAAV and Special Considerations in Vector Handling.</a> Methods in Molecular Biology. , 199-215 (2016).</li><li>Fong, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+encephalitis/encephalopathy+with+reversible+splenial+lesion+(MERS)+due+to+dengue+virus.">Mild encephalitis/encephalopathy with reversible splenial lesion (MERS) due to dengue virus.</a> Journal of Clinical Neuroscience. 36, 73-75 (2017).</li><li>Sarathy, V. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lethal+murine+infection+model+for+dengue+virus+3+in+AG129+mice+deficient+in+type+I+and+II+interferon+receptors+leads+to+systemic+disease.">A lethal murine infection model for dengue virus 3 in AG129 mice deficient in type I and II interferon receptors leads to systemic disease.</a> Journal of Virology. 89 (2), 1254-1266 (2015).</li><li>Schul, W., Liu, W., Xu, H. Y., Flamand, M., Vasudevan, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dengue+fever+viremia+model+in+mice+shows+reduction+in+viral+replication+and+suppression+of+the+inflammatory+response+after+treatment+with+antiviral+drugs.">A dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs.</a> The Journal of Infectious Diseases. 195 (5), 665-674 (2007).</li><li>Tyler, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Viral+Encephalitis.">Acute Viral Encephalitis.</a> New England Journal of Medicine. 379 (6), 557-566 (2018).</li><li>Yauch, L. E., Shresta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+dengue+virus+infection+and+disease.">Mouse models of dengue virus infection and disease.</a> Antiviral Research. 80 (2), 87-93 (2008).</li><li>Assir, M. Z., Jawa, A., Ahmed, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+dengue+syndrome:+subacute+thyroiditis+and+intracerebral+hemorrhage.">Expanded dengue syndrome: subacute thyroiditis and intracerebral hemorrhage.</a> BMC Infectious Diseases. 12, 240 (2012).</li><li>Kumar, R., Prakash, O., Sharma, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+hemorrhage+in+dengue+fever:+management+and+outcome:+a+series+of+5+cases+and+review+of+literature.">Intracranial hemorrhage in dengue fever: management and outcome: a series of 5 cases and review of literature.</a> Surgical Neurology. 72 (4), 429-433 (2009).</li><li>Simanjuntak, Y., Liang, J. J., Lee, Y. L., Lin, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repurposing+of+prochlorperazine+for+use+against+dengue+virus+infection.">Repurposing of prochlorperazine for use against dengue virus infection.</a> Journal of Infectious Diseases. 211 (3), 394-404 (2015).</li><li>Rocha, B. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue-specific+serotype+related+to+clinical+severity+during+the+2012/2013+epidemic+in+centre+of+Brazil.">Dengue-specific serotype related to clinical severity during the 2012/2013 epidemic in centre of Brazil.</a> Infectious Disease Poverty. 6 (1), 116 (2017).</li></ol>

The authors have nothing to disclose.

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 infecti...

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:745px;" width="746">
	<tbody>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Roswell Park Memorial Institute 1640 medium (RPMI)</td>
			<td style="width:211px;">Gibco</td>
			<td style="width:119px;">11875-085</td>
			<td style="width:200px;">Diluting virus</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">0.3-mL Insulin Syringe&#160;</td>
			<td style="width:211px;">BD Ultra-Fine&#173;II</td>
			<td style="width:119px;">328838</td>
			<td style="width:200px;">Intracerebral and intraperitoneal injection</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Microbalance</td>
			<td style="width:211px;">METTLER TOLEDO&#39;s LabX</td>
			<td style="width:119px;">AL104</td>
			<td style="width:200px;">Weight mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Non-adapted DENV2 (strain PL046)</td>
			<td style="width:211px;">The Centers for Disease Control of Taiwan</td>
			<td style="width:119px;">-</td>
			<td style="width:200px;">Infect mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Institute of Cancer Research (ICR) suckling mouse</td>
			<td style="width:211px;">BioLASCO Taiwan Co., Ltd</td>
			<td style="width:119px;">-</td>
			<td style="width:200px;">Our murine model</td>
		</tr>
	</tbody>
</table>

a murine model of dengue virus-induced acute viral encephalitis-like disease

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.

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,<sup c...

Watch this Scientific Journal Video about A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease at JoVE.com

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

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 ...

This protocol of a disease model you see dengue virus infection in the brain become an in vivo platform for screening viral and host vectors associated with neuropathogenesis. Unlike previous models, our models use tools of the infection with a low viral titer, to infect the immunocompetent Institute of Cancer Research suckling mice for encephalitic-like disease procreation induction. Demonstrating the procedure will be Ting-Jing Shen, a PhD candidate from my laboratory.Before initiating the infection procedure, dilute non-adapted dengue virus two stock, to a one times 10 to the fifth plaque forming units per 40 microliters of RPMI 1640 medium concentration. Next, load one 0.3 milliliter syringe equipped with a 30 gauge needle with 10 microliters of diluted virus. And one 0.3 milliliter syringe equipped with a 30 gauge needle, with 30 microliters of virus per animal.For intercerebral delivery of the virus, press the oracle between the thumb and index finger to gently restrain a seven day old suckling Institute of Cancer Research mouse in a prone position and inject the 10 microliter volume of diluted virus into the lambda area, at the intersection of the sagittal and lambdoid suture. After the intercerebral injection, use the thumb and index finger to hold the mouse in a supine position, and gently intraperitoneally, inject the 30 microliter volume of diluted virus, into the murine abdomen. For scoring of the disease progression, assign a score of zero to five to each inoculated mouse daily, according to the grade of acute viral encephalitis-like illnesses, and plot the scores for each day as a curve based figure, using the mean plus and minus the standard deviation of the daily test scores in each group.Compared with the body weight changes measured in mock, medium only inoculated mice, dengue virus inoculated animals exhibit a decrease in body weight at every time point during the trial. In addition, dengue virus infected animals demonstrate a significant increase in hunch back postures, limbic seizures, limbic weakness, paralysis, and death compared to the symptoms displayed by mock inoculated animals. Indeed, dengue infected mice exhibit a time dependent reduction in survival rates that is not observed in mock inoculated animals, confirming the establishment of an infectious model of dengue virus within the central nervous system that demonstrates the progression of acute viral encephalitis-like illnesses in mice.Puncture of the wrong site may result in needle blockage by the score and the overflow of the virus solution outside the head, resulting in failed injection and a failed infection. Following the procedure, brain samples can be harvested for pathological damage, immune cell infiltration, and viral replication analysis by hematoxylin and eosin staining flow cytometry, and plaque formation assay, respectively. Our immunocompetent murine model, provides a platform for exploring the pathogenic mechanisms of dengue virus infection, for the development of dengue virus vaccines, or anti-dengue virus drugs.Dengue virus is a biosafety level two pathogen, and, as such, all experiment with this virus should be completed in a qualified animal biosafety level two laboratory.

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Immunology and Infection

6.4K 조회수.  Taipei Medical University. 뇌에서 뎅기열 바이러스 감염을 볼 질병 모델의이 프로토콜은 신경 병인과 관련된 바이러스 및 호스트 벡터를 선별하기위한 생체 내 플랫폼이된다. 이전 모델과는 달리, 우리의 모델은 낮은 바이러스 성 titer감염의 도구를 사용, 뇌와 같은 질병 출산 유도에 대한 마우스를 빨아 암 연구의 면역 무능 한 연구소를 감염. 절차를 시연하는 것은 내 실험실에서 박사 후보인 팅징 셴...