establishing an active hiv infection in a rat model

Establishing an Active HIV Infection in a Rat Model

For EcoHIV EGFP injection, after confirming a lack of response to pedal reflex, shave the hair from the brain region, and sterilize the exposed skin 2 times with 70% ethanol, and a chlorhexidine-based scrub. Secure the rat in a prone position in a stereotaxic apparatus, and make a 5 to 6-centimeter incision through the skin along the scalp midline.
Mark one drilling position at 0.8 millimeters lateral and one 1.2 millimeters rostral to bregma, and drill a 0.4-millimeter diameter hole at each skull position. Load 1.04 times 10 to the sixth transduction units per milliliter of titered EcoHIV Lentivirus solution into a 10-microliter injection syringe, and secure the syringe to the stereotaxic apparatus.
Move the needle close to the surface of one drilling hole and insert the needle 2.5 millimeters into the hole. Infuse 1 microliter of virus solution at rate of 0.2 microliters of virus per minute. When all of the virus has been injected, leave the needle inside the injection area for five minutes before slowly retracting the needle until it is outside of the rat skull.
Close the skin incision with a 4-0 silk thread suture, and sterilize the solution with 70% ethanol. Then, place the rat in a recovery chamber with a heating pad with monitoring until recumbency.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. EcoHIV-EGFP [Ecotropic HIV Enhanced Green Fluorescent Protein] virus stereotaxic surgeries
<ol>
	<li>Anesthetize rats using 3% sevoflurane. When the rats are not responsive to noxious stimuli and their reflexes are absent, proceed to step 1.2.</li>
	<li>Shave the hairs from the brain region and sterilize the skin twice with 70% ethanol and a chlorhexidine-based scrub. Secure the rat in a prone position in the stereotaxic apparatus.</li>
	<li>Make an incision (5-6 cm) through the skin along the scalp midline.</li>
	<li>Mark two drilling positions at 0.8 mm lateral, 1.2 mm rostral to bregma. Drill a hole (diameter 0.4 mm) in each skull position.</li>
	<li>Fill titrated EcoHIV lentivirus solution (1.04 &#215; 106 TU/mL) in a 10 &#181;L injection syringe. Secure the syringe to the stereotaxic apparatus.</li>
	<li>Move down the needle close to the surface of the drilling hole. Measure and move 2.5 mm in depth.</li>
	<li>Infuse 1 &#181;L of virus solution at a rate of 0.2 &#181;L/min. Keep the needle inside of the injection area for 5 min. Slowly move the needle up until it is outside of the rat skull.</li>
	<li>Suture the skin with a 4-0 silk thread.</li>
	<li>Sterilize the incision with 70% ethanol once. Subcutaneously inject butorphanol (Dorolex, 0.1 mg/kg body weight).</li>
	<li>Transfer the rat to a recovery chamber with a heating pad until it wakes up.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic solution</td>
			<td>Cellgro</td>
			<td>30004CI</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>637-137</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethicon Vicryl Plus Antibacterial, 4-0 Polyglactin 910 Suture, 27in. FS-2</td>
			<td>Med Vet International</td>
			<td>VCP422H</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Forceps</td>
			<td>World Precision Instruments</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>World Precision Instruments</td>
			<td>500216</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>Kopf Instruments</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>World Precision Instruments</td>
			<td>500086</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Li, H., McLaurin, et al. <a href="https://app.jove.com/v/62137">A Rat Model of EcoHIV Brain Infection.</a> J. Vis. Exp. (2021).
This video demonstrates the procedure of injecting chimeric HIV viruses into the cortex of a rat&#39;s brain, where the virus RNA integrates into the host genome, replicates, and spreads to establish an active HIV infection in the experimental model.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with an anesthetized rat, disinfect its shaved head, and secure it in a stereotaxic apparatus.
Make a midline incision to expose the skull.
Mark two positions on the skull and drill small holes.
Attach a syringe with chimeric viruses containing ecotropic HIV RNA within a lentiviral vector to the stereotaxic apparatus.
Insert the needle into the hole, targeting the brain cortex cells, including microglia, the brain&#39;s immune cells.
Slowly inject the viruses for their even distribution in the cortex.
Post-injection, suture the incision, disinfect the area, and allow the rat to recover.
The injected virus interacts with the brain cell receptors, fuses, and releases HIV RNA into the cytoplasm.
The RNA is reverse-transcribed into DNA and integrates into the cell&#39;s DNA.
The infected cell expresses the viral components, producing new RNA and proteins.
These assemble and release as new viruses, which increase the viral load in the brain, establishing an active HIV infection.

21 Views. Ustalenie aktywnego zakażenia wirusem HIV w modelu szczurzym

Video: Ustalenie aktywnego zakażenia wirusem HIV w modelu szczurzym - Experiment

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

JoVE Journal

Medicine

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

Immunology and Infection

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Establishing an Active HIV Infection in a Rat Model

Transkrypcja

Establishing an Active HIV Infection in a Rat Model