Name: influenza a virus studies in a mouse model of infection
Uploaded: 2017-09-07T13:00:00.000+00:00
Duration: 10 min 44 s
Description: Watch this Scientific Journal Video about Influenza A Virus Studies in a Mouse Model of Infection at JoVE.com

Pesquisa sobre o vírus da gripe em laboratório LM-S é parcialmente financiada pelo The New York gripe centro de excelência (NYICE), um membro dos NIAID centros de excelência para a investigação de Influenza e vigilância (CEIRS). Agradecemos seu apoio nas correções do manuscrito Wendy Bates.

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F., Martinez-Sobrido, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-competent+influenza+A+viruses+expressing+a+red+fluorescent+protein.">Replication-competent influenza A viruses expressing a red fluorescent protein.</a> Virology. 476C, 206-216 (2014).</li><li>Margine, I., Krammer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+for+Influenza+Viruses:+Implications+for+Universal+Vaccine+Development.">Animal Models for Influenza Viruses: Implications for Universal Vaccine Development.</a> Pathogens. 3 (4), 845-874 (2014).</li><li>Bouvier, N., Lowen, A. 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O modelo do rato do IAV é amplamente utilizado na vivo estudos de eficácia de proteção, a imunogenicidade e a patogênese do IAV. O pequeno tamanho dos ratos torna fácil de manipular e armazenar em comparação com outros modelos animais, como furões ou cobaias. Além disso, a facilidade em termos de custo de animal, habitação e reprodução permitem sua utilização em testes pré-clínicos vacinação em que um grande número de animais é necessários. Notavelmente, desde que os ratos têm sido usados...

IAVs são vírus envelopadas classificados na família Orthomyxoviridae 3. Eles contêm oito moléculas de RNA único-encalhado com polaridade negativa3. Em humanos, IAVs causam epidemias sazonais e ocasionais pandemias de consequência importante quando novos vírus são introduzidas na população humana4. Além disso, IAVs sazonais são altamente e rapidamente transmitidos entre os seres humanos, produzindo uma elevada perda econômica em todo o mundo a cada ano2,5. IAV os sintomas incluem tosse, congestão nasal, febre, mal-estar, dor de cabeça, anorexia e mialgia, mas o vírus também pode produzir uma doença mais grave em pacientes imunocomprometidos6. Na verdade, a Organização Mundial de saúde (OMS) calcula que os vírus da gripe sazonal com 300.000-500.000 mortes em todo o mundo que cada ano1. Existem apenas duas classes de drogas atualmente aprovadas pela Food and Drug Administration (FDA) para a profilaxia de gripe e tratamento em seres humanos: inibidores de neuraminidase (NA) (por exemplo, oseltamivir) e bloqueadores do canal de íon M2 (por exemplo, Amantadina); no entanto, o surgimento de variantes do vírus resistentes aos medicamentos é uma preocupação crescente. Vacinação, portanto, continua a ser a melhor opção de médica para proteger os humanos contra infecções IAVs. Até à data, três tipos de gripe, as vacinas licenciadas pelo FDA para uso humano estão disponíveis: vacinas de proteína hemaglutinina viral recombinante (HA), inactivadas (IIV) vacinas contra a gripe e viver-atenuado gripe vacinas (LAIV)5, 7. as três vacinas são projetadas para induzir resposta imune adaptativa contra a proteína viral HA, o alvo principal dos anticorpos contra IAVs neutralizantes.
Um modelo de mouse validado para estudar IAV infecção na vivo
Modelos animais têm sido usados para estudar, entre outros, IAV patogênese8,9,10,11, virais fatores que contribuem para doença12 e/ou transmissão viral13 ,14, e testar a eficácia de novas vacinas ou antiviral drogas9,10,15. Camundongos (Mus musculus) são o modelo animal mais extensivamente usado para pesquisa IAV por vários motivos: 1) o sistema imunológico é evolutivamente semelhante ao que se apresentam nos seres humanos; 2) compra animal de baixo custo, incluindo, moradia e reprodução; 3) pequeno tamanho facilmente manipular e armazenar; 4) variabilidade do anfitrião mínima para obter respostas homogêneas e resultados; 5) amplo conhecimento da biologia de ratos, incluindo a sequência do genoma; 6) muitos disponíveis de biologia molecular e/ou reagentes Imunologia; 7) disponível nocaute (KO) ratos para estudar a contribuição de uma proteína do hospedeiro na infecção viral; e, 8) múltiplas variedades do mouse que podem ser exploradas para avaliar as bases genéticas de infecções.
Existem várias cepas de rato atualmente disponíveis para estudar IAV na vivo. Idade, estado imune, sexo, estirpe genética de fundo e mouse, bem como rotas de infecção, dose e estirpes virais influenciam o resultado da infecção do IAV em camundongos. As cepas de rato mais comuns utilizadas na investigação IAV são C57BL/6, BALB/C e, mais recentemente, DBA.2 ratos, desde que eles são mais suscetíveis à doença do IAV que as duas linhagens ex16,17,18, 19 , 20. importante, a resposta imune também pode ser diferente dependendo o rato estirpe18,19,20. Assim, é muito importante recuperar todas as informações disponíveis sobre o mouse e a estirpe do IAV para escolher a melhor opção para o experimento a ser realizado.
Embora o rato é um bom modelo animal de infecção para estudos em vivo com IAV, eles têm várias limitações, que precisam ser considerados no projeto experimental. Por exemplo, uma grande limitação do uso de ratos para estudos em vivo é que IAVs não transmitem entre ratos. Assim, para a transmissão de estudos, mais aceitos modelos animais (por exemplo, furões ou cobaias) são usados16,17,21. Além disso, existem várias diferenças entre as manifestações da IAV em camundongos e humanos. Ao contrário dos humanos, os ratos não desenvolvem febre após infecção IAV; por outro lado, eles apresentam com hipotermia16,17. Em camundongos, replicação IAV está concentrada no trato respiratório inferior (pulmões) em vez de vias aéreas superiores. Assim, virulência do IAV em ratos não é sempre correlacionada ao observado em seres humanos. No total, porque as vantagens superam as desvantagens limitadas, rato representa o primeiro modelo animal usado para avaliar a patogênese viral da gripe, imunogenicidade e eficácia protetora em estudos de vacina e antiviral. Além disso, não seria eticamente aceitável para realizar estudos com IAV usando modelos animais grandes sem provas anteriores em um modelo animal pequeno de infecção IAV. Neste manuscrito, descrevemos como infectar camundongos practicar (óssea) com IAV, como monitorar a gravidade e o progresso da infecção viral e como realizar as experiências necessárias para avaliar humorais respostas imunes e eficácia de proteção.

<table><tbody><tr><td>Madin-Darby Canine Kidney (MDCK) epithelial cells</td><td>ATCC</td><td>CCL-34</td><td></td></tr><tr><td>Six- to eight-week-old female C57BL/6 mice</td><td>National Cancer Institute (NCI)</td><td>01XBE</td><td></td></tr><tr><td>Turckey red blod cells</td><td>Biolink Inc</td><td></td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Corning Cellgro</td><td>15-013-CV</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Seradigm</td><td>1500-050</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Penicillin/Streptomycin/L-Glutamine (PSG) 100x</td><td>Corning</td><td>30-009-CI</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Penicillin/Streptomycin (PS) 100x</td><td>Corning</td><td>30-00-CI</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Bovin Albumin solution (BA)</td><td>Sigma-Aldrich</td><td>A7409</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Bovin Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9647</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin</td><td>Sigma-Aldrich</td><td>T8802</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Neutral Buffered Formalin 10%</td><td>EMD</td><td>65346-85</td><td>Store at RT</td></tr><tr><td>Triton X-100</td><td>J.T.Baker</td><td>X198-07</td><td>Store at RT</td></tr><tr><td>Monoclonal Antibody anti-NP Influenza A Virus HB-65</td><td>ATTC</td><td>H16-L10-4R5</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Polyclonal rabbit anti-mouse immunoglobulins/FITC</td><td>Dako</td><td>F0261</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>ECL Anti-mouse IgG, Horseradish Peroxidase linked whole antibody</td><td>GE Healthcare</td><td>LNA931V/AG</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>TMB substrate set</td><td>BioLegend</td><td>421101</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Vmax Kinetic plate reader</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Dounce Tissue Grinders</td><td>Thomas Scientific</td><td>7722-7</td><td></td></tr><tr><td>Receptor destroying enzyme, RDE (II)</td><td>Denka Seiken Co.</td><td>370013</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Crystal Violet</td><td>Fisher Scienctific</td><td>C581-100</td><td>Store at RT</td></tr><tr><td>96-well Cell Culture Plate</td><td>Greiner Bio-one</td><td>655-180</td><td></td></tr><tr><td>Cell Culture dishes 100 mm</td><td>Greiner Bio-one</td><td>664-160</td><td></td></tr><tr><td>Nunc MicroWell 96-Well Microplates</td><td>Thermo Fisher Scienctific</td><td>269620</td><td></td></tr><tr><td>Nunc 96-Well Polystyrene Conical Bottom MicroWell Plates</td><td>Thermo Fisher Scienctific</td><td>249570</td><td></td></tr><tr><td>Puralub Vet Ointment</td><td>Dechra</td><td>9N-76855</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Olympus</td><td>Olympus IX81</td><td></td></tr></tbody></table>

influenza a virus studies in a mouse model of infection

Vírus da gripe causam mais de 500.000 mortes em todo o mundo1 e estão associados a um custo anual de US $ 12 bilhões nos Estados Unidos sozinho, considerando médica direta e as despesas de hospitalização e de absentismo de trabalho2. Modelos animais são cruciais em estudos de Influenza A virus (IAV) para avaliar a patogênese viral, as interações patógeno-hospedeiro, respostas imunes, e a eficácia da vacina atual e/ou nova abordagens bem como antivirais. Ratos são um modelo animal pequeno vantajoso porque seu sistema imunológico é evolutivamente semelhante ao encontrado em seres humanos, eles estão disponíveis de fornecedores comerciais como indivíduos geneticamente idênticos, há múltiplas variedades que podem ser exploradas para avaliar a base genética das infecções, e são relativamente baratos e fáceis de manipular. Para recapitular infecção IAV nos seres humanos através das vias aéreas, os ratos são primeiro anestesiados antes da inoculação intranasal com infecciosas IAVs sob contenção adequada de biossegurança. Após a infecção, a patogênese da IAVs é determinada pelo monitoramento diário da morbidade (perda de peso de corpo) e a taxa de mortalidade (sobrevivência). Além disso, a patogênese viral também pode ser avaliada por avaliar a replicação do vírus no superior (mucosa nasal) ou inferior (pulmões) do trato respiratório de ratos infectados. Respostas humorais após infecção IAV podem ser rapidamente avaliadas por sangramento não-invasiva e detecção do anticorpo secundário ensaios destinados a detectar a presença de total ou anticorpos neutralizantes. Aqui, descrevemos os métodos comuns usados para infectar camundongos practicar (i.n) com IAV e avaliar a eficácia de proteção, humorais respostas imunes e patogênese.

Caracterização de patogênese viral em camundongos
A patogênese da IAV está relacionada a morbidade e a mortalidade causada por sua infecção. Esses dois parâmetros podem ser avaliados em camundongos facilmente: morbidade IAV está associada a perda de peso corporal em camundongos infectados e a porcentagem de sobrevivência irá indicar a taxa de mortalidade (Figura 1). O peso corporal e sobrevi...

Watch this Scientific Journal Video about Influenza A Virus Studies in a Mouse Model of Infection at JoVE.com

Estudos do vírus influenza A em um modelo de infecção em camundongos | Imunologia e Infecção | Vídeo

Influenza A Virus Studies in a Mouse Model of Infection

Vírus da influenza A (IAVs) são importantes patógenos respiratórios humanos. Para entender a patogenicidade de IAVs e realizar testes pré-clínicos de vacina romance abordagens, modelos animais, imitando a fisiologia humana são necessários. Aqui, descrevemos técnicas para avaliar a patogênese do IAV, respostas humorais e eficácia de vacina usando um modelo do rato da infecção.

Estudos do Vírus Influenza A em um Modelo de Infecção de Camundongos

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 ...

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28.2K visualizações.  University of Rochester School of Medicine and Dentistry. Vírus da influenza A (IAVs) são importantes patógenos respiratórios humanos. Para entender a patogenicidade de IAVs e realizar testes pré-clínicos de vacina romance abordagens, modelos animais, imitando a fisiologia humana são necessários. Aqui, descrevemos técnicas para avaliar a patogênese do IAV, respostas humorais e eficácia de vacina usando um modelo do rato da infecção.

Vídeo: Influenza A Virus Studies in a Mouse Model of 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.

Usando imagens Bioluminescent para Investigar sinergismo entre Streptococcus pneumoniae E Vírus da Influenza A em Ratos infantil

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

Imunologia e Infecção

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Alta capacidade método de detecção de vírus Influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

A administração intranasal de recombinantes Vacinas contra Influenza em quimérico rato Modelos para estudar a imunidade mucosal

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Co-imunoprecipitação da proteína do rato Mx1 com a nucleoproteína do vírus da gripe A

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

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.

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.

Usando modelos de Zebrafish de infecções por vírus gripe humana A para Screen antivirais e Caracterizar imune do hospedeiro respostas de células

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

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

Interação de célula de imagem latente na Mucosa traqueal durante a infecção de vírus de Influenza usando microscopia Intravital dois fotões

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

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.

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.

Um modelo murino da doença da encefalite viral aguda induzida pelo vírus da dengue

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

Um luciferase-vírus da gripe do repórter fluorescente para A imagem latente viva e a quantificação da infecção viral

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

Avaliação de células auxiliares tolas e resposta do centro germinal durante a infecção pelo vírus da influenza A em camundongos

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

Um modelo de camundongo para a transição de Streptococcus pneumoniae de colonizador para patógeno após coinfecção viral recapitula a doença exacerbada pela idade

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

Estudos do Vírus Influenza A em um Modelo de Infecção de Camundongos

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