Influenza A Virus Studies in a Mouse Model of Infection

Summary

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Abstract

Influenza viruses cause over 500,000 deaths worldwide¹ and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism². Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Introduction

IAVs are enveloped viruses classified in the Orthomyxoviridae family³. They contain eight single-stranded RNA molecules with negative polarity³. In humans, IAVs cause seasonal epidemics and occasional pandemics of important consequence when novel viruses are introduced in the human population⁴. Moreover, seasonal IAVs are highly and rapidly transmitted between humans producing an elevated economic loss worldwide every year^2,5. IAV symptoms include cough, nasal congestion, fever, malaise, headache, anorexia and myalgia, but the vir....

Protocol

All animal protocols described here were approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC) at the University of Rochester School of Medicine and Dentistry, and comply with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council 22. The facilities and programs of the Vivarium and Division of Laboratory Animal Medicine of the School of Medicine and Dentistry are accredited by AAALAC International and comply with state law, federal statute and National Institutes of Health (NIH) policy. Similar requirements should be applied at each institution to adhere....

Results

Characterization of viral pathogenesis in mice

The pathogenesis of IAV is related to the morbidity and mortality caused by its infection. These two parameters can be evaluated in mice easily: IAV morbidity is associated with body weight loss in infected mice and the percentage of survival will indicate the mortality rate (Figure 1). The body weight and survival in IAV-infected mice are usually monitore.......

Discussion

The mouse model of IAV is widely used for in vivo studies of IAV pathogenesis, immunogenicity and protection efficacy. The small size of mice makes them easy to manipulate and store as compared to other animal models such as ferrets or guinea pigs. Moreover, the ease in terms of animal cost, housing and reproduction allow their use in pre-clinical vaccination tests in which large numbers of animals are needed. Notably, since mice have been used in multiple research disciplines, several molecular and immunology m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Madin-Darby Canine Kidney (MDCK) epithelial cells	ATCC	CCL-34
Six- to eight-week-old female C57BL/6 mice	National Cancer Institute (NCI)	01XBE
Turckey red blod cells	Biolink Inc		Store at 4 °C
Dulbecco’s modified Eagle’s medium (DMEM)	Corning Cellgro	15-013-CV	Store at 4 °C
Fetal Bovine Serum (FBS)	Seradigm	1500-050	Store at -20 °C
Penicillin/Streptomycin/L-Glutamine (PSG) 100x	Corning	30-009-CI	Store at -20 °C
Penicillin/Streptomycin (PS) 100x	Corning	30-00-CI	Store at -20 °C
Bovin Albumin solution (BA)	Sigma-Aldrich	A7409	Store at 4 °C
Bovin Serum Albumin (BSA)	Sigma-Aldrich	A9647	Store at 4 °C
Tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin	Sigma-Aldrich	T8802	Store at -20 °C
Neutral Buffered Formalin 10%	EMD	65346-85	Store at RT
Triton X-100	J.T.Baker	X198-07	Store at RT
Monoclonal Antibody anti-NP Influenza A Virus HB-65	ATTC	H16-L10-4R5	Store at -20 °C
Polyclonal rabbit anti-mouse immunoglobulins/FITC	Dako	F0261	Store at 4 °C
ECL Anti-mouse IgG, Horseradish Peroxidase linked whole antibody	GE Healthcare	LNA931V/AG	Store at 4 °C
TMB substrate set	BioLegend	421101	Store at 4 °C
Vmax Kinetic plate reader	Molecular Devices
Dounce Tissue Grinders	Thomas Scientific	7722-7
Receptor destroying enzyme, RDE (II)	Denka Seiken Co.	370013	Store at -20 °C
Crystal Violet	Fisher Scienctific	C581-100	Store at RT
96-well Cell Culture Plate	Greiner Bio-one	655-180
Cell Culture dishes 100 mm	Greiner Bio-one	664-160
Nunc MicroWell 96-Well Microplates	Thermo Fisher Scienctific	269620
Nunc 96-Well Polystyrene Conical Bottom MicroWell Plates	Thermo Fisher Scienctific	249570
Puralub Vet Ointment	Dechra	9N-76855
Fluorescent microscope	Olympus	Olympus IX81