Name: a murine model of dengue virus-induced acute viral encephalitis-like disease
Uploaded: 2019-04-28T14:00:00
Description: Watch this Scientific Journal Video about A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease at JoVE.com

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>
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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. 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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. 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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.

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

Research

JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day "0", mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day "4", the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into na&#239;ve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.

Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an ...