Name: using zebrafish models of human influenza a virus infections to screen antiviral drugs and characterize host immune cell responses
Uploaded: 2017-01-20T15:00:00
Description: Watch this Scientific Journal Video about Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses at JoVE.com

The authors wish to thank Mark Nilan for zebrafish care and maintenance and Meghan Breitbach and Deborah Bouchard for propagating NS1-GFP and determining IAV titers. This research was supported by NIGMS grant NIH P20GM103534 and the Maine Agricultural and Forest Experiment Station (Publication Number 3493).

All work should be performed using biosafety level 2 (or BSL2) standards described by the U.S. Centers for Disease Control (CDC) and in accordance with directives established by Institutional Animal Care and Use Committees (IACUC). Please confer with the appropriate officials to ensure safety and compliance.
1. Zebrafish Care and Maintenance
<ol>
	<li>Spawn zebrafish and collect the required number of embryos for the experiments. When necessary, mass breeding tanks, like those described by A...

To maximize the benefits gained from using a small animal to model human host-pathogen interactions, it is important to frame research questions and test hypotheses that capitalize on the inherent advantages of the model system. As a model for human IAV infection, the zebrafish has several strengths, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines that label immune cells like neutrophils. The zebrafish has been developed as an increasingly powerful alternativ...

According to the World Health Organization (WHO), influenza viruses infect 5-10% of adults and 20-30% of children annually and cause 3-5 million cases of severe illness and up to 500,000 deaths worldwide1. Yearly vaccinations against influenza remain the best option to prevent disease. Efforts like the WHO Global Action Plan have increased seasonal vaccine use, vaccine production capacity, and research and development into more potent vaccine strategies in order to reduce morbidity and mortality associated with seasonal influenza outbreaks2. Antiviral drugs like neuraminidase inhibitors (e.g. Zanamivir and Oseltamivir) are available in some countries and have proven effective in mitigating symptoms, when administered within the first 48 hr of onset3,4,5. Despite global efforts, containment of seasonal influenza outbreaks remains a formidable challenge at this time, as influenza virus antigenic drift often exceeds current abilities to adapt to the changing genome of the virus6. Vaccine strategies targeting new strains of virus must be developed in advance and are sometimes rendered less than optimally effective due to unforeseen changes in the types of strains that eventually predominate in an influenza season. For these reasons, there is a clear need to develop alternative therapeutic strategies for containing infections and reducing mortalities. By achieving a better understanding of the host-virus interaction, it may be possible to develop new anti-influenza medicines and adjuvant therapies7,8.
The human host-influenza A virus (IAV) interaction is complex. Several animal models of human IAV infection have been developed in order to gain insight into the host-virus interaction, including mice, guinea pigs, cotton rats, hamsters, ferrets, and macaques9. While providing important data that have enhanced the understanding of host-IAV dynamics, each model organism possesses significant drawbacks that must be considered when attempting to translate the findings into human medicine. For example, mice, which are the most widely used model, do not readily develop IAV-induced infection symptoms when infected with human influenza isolates9. This is because mice lack the natural tropism for human influenza isolates since mouse epithelial cells express &#945;-2,3 sialic acid linkages instead of the &#945;-2,6 sialic acid linkages expressed on human epithelial cells10. The hemagglutinin proteins present in human IAV isolates favorably bind and enter host cells bearing &#945;-2,6 sialic acid linkages through receptor-mediated endocytosis9,11,12,13. As a consequence, it is now accepted that in developing mouse models for human influenza, care must be taken to pair the appropriate strain of mouse with the appropriate strain of influenza in order to achieve disease phenotypes that recapitulate aspects of the human illness. In contrast, epithelial cells in the upper respiratory tract of ferrets possess &#945;-2,6 sialic acid linkages that resemble human cells14. Infected ferrets share many of the pathological and clinical features observed in the human disease, including the pathogenicity and transmissibility of human and avian influenza viruses14,15. They are also highly amenable to vaccine efficacy trials. Nevertheless, the ferret model for human influenza has several disadvantages principally related to their size and cost of husbandry that make acquisition of statistically significant data challenging. In addition, ferrets have previously displayed differences in drug pharmacokinetics, bioavailability, and toxicity that make testing efficacy difficult. For example, ferrets exhibit toxicity to the M2 ion channel inhibitor amantadine16. Thus, it is clear that in choosing an animal model to study questions about human IAV infections, it is important to consider its inherent advantages and limitations, and the aspect of the host-virus interaction that is under investigation.
The zebrafish, Danio rerio, is an animal model that provides unique opportunities for investigating microbial infection, host immune response, and potential drug therapies17,18,19,20,21,22,23,24,25,26,27,28. The presence of &#945;-2,6-linked sialic acids on the surface of cells in the zebrafish suggested its susceptibility to IAV, which was borne out in infection studies and imaged in vivo using a fluorescent reporter strain of IAV19. In IAV-infected zebrafish, increased expression of the antiviral ifnphi1 and mxa transcripts indicated that an innate immune response had been stimulated, and the pathology displayed by IAV-infected zebrafish, including edema and tissue destruction, was similar to that observed in human influenza infections. Furthermore, the IAV antiviral neuraminidase inhibitor Zanamivir limited mortality and reduced viral replication in zebrafish19.
In this report, a protocol for initiating systemic IAV infections in zebrafish embryos is described. Using Zanamivir at clinically relevant doses as a proof-of-principle, the utility of this zebrafish IAV infection model for screening compounds for antiviral activity is demonstrated. In addition, a protocol for generating a localized, epithelial IAV infection in the zebrafish swim bladder, an organ that is considered to be anatomically and functionally analogous to the mammalian lung21,29,30,31, is described. Using this localized IAV infection model, neutrophil recruitment to the site of infection can be tracked, enabling investigations into the role of neutrophil biology in IAV infection and inflammation. These zebrafish models complement existing animal models of human IAV infections and are particularly useful for testing small molecules and immune cell responses because of the possibility of enhanced statistical power, capacity for moderate- to high-throughput assays, and the abilities to track immune cell behavior and function with light-microscopy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>Instant Ocean</td>
			<td>Spectrum Brands</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr>
			<td>100 x 25 mm sterile disposable Petri dishes&#160;</td>
			<td>VWR</td>
			<td>&#160;89107-632</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes&#160;</td>
			<td>Fisherbrand</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine- S (MS-222)</td>
			<td>Western Chemical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary with filament&#160;</td>
			<td>Sutter Instrument&#160;</td>
			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/Brown micropipette puller&#160;</td>
			<td>Sutter Instrument</td>
			<td>&#160;P-97</td>
			<td></td>
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		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Zanamivir</td>
			<td>AK Scientific</td>
			<td>G939</td>
			<td></td>
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		<tr>
			<td>Dumont #5 forceps&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>72700-D</td>
			<td></td>
		</tr>
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			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope immersion oil</td>
			<td>Olympus</td>
			<td>IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope stage calibration slide&#160;</td>
			<td>AmScope</td>
			<td>MR095</td>
			<td></td>
		</tr>
		<tr>
			<td>MPPI-3 pressure injector&#160;</td>
			<td>Applied Scientific Instrumentation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Back pressure unit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>BPU</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder kit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPIP</td>
			<td></td>
		</tr>
		<tr>
			<td>Foot switch</td>
			<td>Applied Scientific Instrumentation</td>
			<td>FSW</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic base</td>
			<td>Applied Scientific Instrumentation</td>
			<td>Magnetic Base</td>
			<td></td>
		</tr>
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			<td>Phenol red&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P-4758</td>
			<td></td>
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			<td>Low temperature incubator</td>
			<td>VWR</td>
			<td>2020</td>
			<td></td>
		</tr>
		<tr>
			<td>SteREO Discovery.V12</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Zeiss</td>
			<td>HXP 200C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cold light source</td>
			<td>Zeiss&#160;</td>
			<td>CL6000 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass-bottom multiwell plate, 24 well</td>
			<td>Mattek</td>
			<td>P24G-0-13-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td colspan="2">IX-81 with FV-1000 laser scanning confocal system</td>
		</tr>
		<tr>
			<td>Fluoview software</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism v6</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A/PR/8/34 (H1N1) virus&#160;</td>
			<td>Charles River&#160;</td>
			<td>490710</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza A X-31, A/Aichi/68 (H3N2)&#160;</td>
			<td>Charles River&#160;</td>
			<td>490715</td>
			<td></td>
		</tr>
		<tr>
			<td>Influenza NS1-GFP</td>
			<td></td>
			<td></td>
			<td>Referenced in Manicassamy et al. 2010</td>
		</tr>
		<tr>
			<td>Tg(mpx:mCherry)</td>
			<td></td>
			<td></td>
			<td>Referenced in Lam et al. 2013</td>
		</tr>
	</tbody>
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using zebrafish models of human influenza a virus infections to screen antiviral drugs and characterize host immune cell responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Here, data showing how systemic IAV infection in zebrafish can be used to test drug efficacy (Figure 1A) are provided. Embryos at 48 hr post-fertilization are injected with APR8 (Figures 1C, 1F), X-31 (Figures 1D, 1G), or NS1-GFP (Figures 1H-1I) via the duct of Cuvier to initiate a viral infection. Another cohort of embryos at 48 hr post-fertilization were injected to serve as controls for viral infection (Figures...

Watch this Scientific Journal Video about Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses at JoVE.com

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

The overall goals of these procedures are to use zebrafish as an influenza model to study the host immune response to viral infection and to screen antiviral compounds. These methods can help answer key questions in the influenza field, such as the role phagocytes play in immunity and inflammation during an infection and the efficacy of potential antiviral therapies. A zebrafish model of influenza allows for previously unanswered questions to be addressed due to the optical clarity of developing zebrafish and the capacity to perform medium to high through-put assays.After preparing reagants and raising embryos to the desired stage according to the text protocol, on the day of the experiment, use number five forceps to dechorionate the embryos. If using the APR-8 or X31 viruses, in a laminar flow hood, dilute the viruses to three point three times 10 to the six egg infective dose 50 percent in sterile ice cold PBS supplemented with zero point two five percent phenol red. If using the NS1-GFP strain, dilute the virus to approximately one point five times 10 to the second PFUs per nanoliter.Simultaneously, set up a control containing allantoic fluid from uninfected chicken eggs diluted similar to the virus. Then, with microloader tips, pipette virus solution into microinjection needles and insert the microinjection needle into the appropriate holder of the injection apparatus. After clipping the needle according to the text protocol, adjust the pressure and timing settings on the pressure injector and or reclip the needle tip until the desired injection volume is achieved.Following anesthetization of embryos, use a plastic pipette to transfer 10 to 20 embryos to a two percent agarose plate and pipette to remove excess liquid. Under the stereomicroscope, use a fire polished and sealed borosilicate glass capillary to gently align and orient the embryos so that the duct of cuvier or the posterior cardinal vein is in line with the microinjection needle. To carry out microinjection of IAV, gently insert the microinjection needle into the duct of cuvier or the posterior cardinal vein.Then, depress the foot pedal to inject the desired volume of IAV into the circulatory system of the embryo. To achieve a systemic infection, it is critical that the injection bolus is swept up into the circulation and distributed throughout the body. If the bolus collects at the site of infection, remove the embryo from the experiment and euthanize it.Five days post fertilization, prepare needles as described earlier in this video. Align TG MPX mCherry larvae containing inflated swim bladders as just demonstrated, except position the larvae so that the needle can pierce the swim bladder and the virus can be deposited into the posterior of the swim bladder. Gently insert the microinjection needle into the swim bladder and inject the desired volume towards the posterior of the organ.The injection solution should collect towards the posterior and the swim bladder's air bubble should be displaced forward. To achieve a swim bladder infection, it is critical that the injection bolus collects towards the posterior of the swim bladder, causing the air bubble to be displaced forward. GFP expression should begin to be observed as early as three hours post injection.After completing the injections, including with control solutions, transfer the larvae to Petri dishes containing sterile egg water and incubate the fish at 33 degrees Celsius. To carry out antiviral drug treatment, at three hours post infection, replace the egg water of the NS1-GFP and control infected fish with sterile water containing zero, 16.7, or 33.3 nanograms per milliliter of Zanamivir. Beginning at 24 to 48 hours post injection, begin to record observations about infection dynamics related to disease pathology, including signs of lethargy and evidence of edema, differences in pigmentation, ocular and cranial facial deformities, and lordosis.Every 24 hours for five days post infection, record the number of dead, morbid, and healthy larvae. Plot data as a stacked bar graph with percentages of healthy, morbid, or dead fish plotted on the Y axis and the treatment group on the x axis. To mount zebrafish for imaging, transfer an individual transgenic MPX mCherry larvae that has been infected with NS1-GFP to a well of a 24 well glass bottom plate and a small droplet of egg water.Repeat with other IAV infected and control fish. Slowly add one percent agarose embryo mounting medium to each well, taking care not to introduce large bubbles. Gently adjust the position of the larva so that each one is mounted on its side.Once the agarose has gelled, use egg water with 200 micrograms per liter of tricaine to gently fill the individual wells. With a confocal microscope and a 20 times objective focused on the swim bladder, capture a z stack series of brightfield and fluorescence images. Quantify and compare the number of MPX mCherry positive neutrophils present in the swim bladders of IAV infected and control injected zebrafish.Shown here are data representing how systemic IAV infection in zebrafish can be used to test drug efficacy. Embryos at 48 HPF were injected with APR8, X31 or NS1-GFP via the duct of cuvier to initiate a viral infection. Embryos serving as controls for viral infection were injected at 48 HPF.By 48 hours post infection, zebrafish injected with IAV exhibited evidence of pericardial edema and circulatory arrest with erythrocytes present throughout the pericardium. Infected fish not exposed to Zanamivir exhibited features of edema in systemic GFP expression. Reduced gross pathology in GFP florescence were observed in NS1-GFP infected larvae exposed to Zanamivir.In this experiment of a localized zebrafish swim bladder infection model, NS1-GFP virus was injected into the swim bladders of five day post fertilization transgenic MPX mCherry larvae and the recruitment of mCherry labeled neutrophils into the swim bladder was tracked. At 20 hours post infection, considerable migration of neutrophils into the swim bladders of IAV infected fish relative to PBS injected controls was observed, indicating that IAV can recruit neutrophils to the swim bladder, just as it recruits neutrophils to the human lung. The implications of these techniques extend towards antiviral therapies because they have the potential to reveal critical insights into host influenza A virus or IAV interactions that may ultimately translate into the clinic.Don't forget that working with influenza can be extremely hazardous, and precautions including vaccination and wearing the appropriate PPE should always be taken while performing this procedure.

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