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">
	<tbody>
		<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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Dumont #5 forceps&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>72700-D</td>
			<td></td>
		</tr>
		<tr>
			<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>
		<tr>
			<td>Phenol red&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P-4758</td>
			<td></td>
		</tr>
		<tr>
			<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>
</table>

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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Research

JoVE Journal

Immunology and Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...