Directed Evolution of an Influenza Reporter Virus To Restore Replication and Virulence and Enhance Noninvasive Bioluminescence Imaging in Mice

ABSTRACTReporter viruses provide a powerful tool to study infection, yet incorporating a nonessential gene often results in virus attenuation and genetic instability. Here, we used directed evolution of a luciferase-expressing pandemic H1N1 (pH1N1) 2009 influenza A virus in mice to restore replication kinetics and virulence, increase the bioluminescence signal, and maintain reporter gene expression. An unadapted pH1N1 virus withNanoLuc luciferaseinserted into the 5′ end of the PA gene segment grew to titers 10-fold less than those of the wild type in MDCK cells and in DBA/2 mice and was less virulent. For 12 rounds, we propagated DBA/2 lung samples with the highest bioluminescence-to-titer ratios. Every three rounds, we comparedin vivoreplication, weight loss, mortality, and bioluminescence. Mouse-adapted virus after 9 rounds (MA-9) had the highest relative bioluminescence signal and had wild-type-like fitness and virulence in DBA/2 mice. Using reverse genetics, we discovered fitness was restored in virus rPB2-MA9/PA-D479N by a combination of PA-D479N and PB2-E158G amino acid mutations andPB2noncoding mutations C1161T and C1977T. rPB2-MA9/PA-D479N has increased mRNA transcription, which helps restore wild-type-like phenotypes in DBA/2 and BALB/c mice. Overall, the results demonstrate that directed evolution that maximizes foreign-gene expression while maintaining genetic stability is an effective method to restore wild-type-likein vivofitness of a reporter virus. Virus rPB2-MA9/PA-D479N is expected to be a useful tool for noninvasive imaging of pH1N1 influenza virus infection and clearance while analyzing virus-host interactions and developing new therapeutics and vaccines.IMPORTANCEInfluenza viruses contribute to 290,000 to 650,000 deaths globally each year. Infection is studied in mice to learn how the virus causes sickness and to develop new drugs and vaccines. During experiments, scientists have needed to euthanize groups of mice at different times to measure the amount of infectious virus in mouse tissues. By inserting a foreign gene that causes infected cells to light up, scientists could see infection spread in living mice. Unfortunately, adding an extra gene not needed by the virus slowed it down and made it weaker. Here, we used a new strategy to restore the fitness and lethality of an influenza reporter virus; we adapted it to mouse lungs and selected for variants that had the greatest light signal. The adapted virus can be used to study influenza virus infection, immunology, and disease in living mice. The strategy can also be used to adapt other viruses.

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Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0914994107 ◽

2010 ◽

Vol 107 (25) ◽

pp. 11531-11536 ◽

Cited By ~ 268

Author(s):

B. Manicassamy ◽

S. Manicassamy ◽

A. Belicha-Villanueva ◽

G. Pisanelli ◽

B. Pulendran ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza Virus Infection ◽

Reporter Virus ◽

Gfp Reporter

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Next Generation of Computationally Optimized Broadly Reactive HA Vaccines Elicited Cross-Reactive Immune Responses and Provided Protection against H1N1 Virus Infection

Vaccines ◽

10.3390/vaccines9070793 ◽

2021 ◽

Vol 9 (7) ◽

pp. 793

Author(s):

Ying Huang ◽

Monique S. França ◽

James D. Allen ◽

Hua Shi ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Immune Responses ◽

H1n1 Virus ◽

Influenza Virus Infection ◽

H1n1 Influenza ◽

Influenza Viruses ◽

Next Generation ◽

Wild Type ◽

Influenza A H1n1

Vaccination is the best way to prevent influenza virus infections, but the diversity of antigenically distinct isolates is a persistent challenge for vaccine development. In order to conquer the antigenic variability and improve influenza virus vaccine efficacy, our research group has developed computationally optimized broadly reactive antigens (COBRAs) in the form of recombinant hemagglutinins (rHAs) to elicit broader immune responses. However, previous COBRA H1N1 vaccines do not elicit immune responses that neutralize H1N1 virus strains in circulation during the recent years. In order to update our COBRA vaccine, two new candidate COBRA HA vaccines, Y2 and Y4, were generated using a new seasonal-based COBRA methodology derived from H1N1 isolates that circulated during 2013–2019. In this study, the effectiveness of COBRA Y2 and Y4 vaccines were evaluated in mice, and the elicited immune responses were compared to those generated by historical H1 COBRA HA and wild-type H1N1 HA vaccines. Mice vaccinated with the next generation COBRA HA vaccines effectively protected against morbidity and mortality after infection with H1N1 influenza viruses. The antibodies elicited by the COBRA HA vaccines were highly cross-reactive with influenza A (H1N1) pdm09-like viruses isolated from 2009 to 2021, especially with the most recent circulating viruses from 2019 to 2021. Furthermore, viral loads in lungs of mice vaccinated with Y2 and Y4 were dramatically reduced to low or undetectable levels, resulting in minimal lung injury compared to wild-type HA vaccines following H1N1 influenza virus infection.

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Linear polysialoside outperforms dendritic analogs for inhibition of influenza virus infection in vitro and in vivo

Biomaterials ◽

10.1016/j.biomaterials.2017.05.028 ◽

2017 ◽

Vol 138 ◽

pp. 22-34 ◽

Cited By ~ 43

Author(s):

Sumati Bhatia ◽

Daniel Lauster ◽

Markus Bardua ◽

Kai Ludwig ◽

Stefano Angioletti-Uberti ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza Virus Infection

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Role of oxidants in influenza virus-induced gene expression

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.274.1.l134 ◽

1998 ◽

Vol 274 (1) ◽

pp. L134-L142 ◽

Cited By ~ 13

Author(s):

Katharine Knobil ◽

Augustine M. K. Choi ◽

Gordon W. Weigand ◽

David B. Jacoby

Keyword(s):

Gene Expression ◽

Cell Death ◽

Influenza Virus ◽

Virus Infection ◽

A549 Cells ◽

Influenza Virus Infection ◽

Pyrrolidine Dithiocarbamate ◽

Oxygen Intermediates ◽

Mrna Induction

Influenza virus-induced epithelial damage may be mediated, in part, by reactive oxygen intermediates (ROIs). In this study, we investigated the role of ROIs in the influenza virus-induced gene expression of antioxidant enzymes and in the activation of nuclear factor-κB (NF-κB), an oxidant-sensitive transcriptional factor. Influenza virus infection increased production of intracellular ROIs in A549 pulmonary epithelial cells. Induction of manganese superoxide dismutase (MnSOD) mRNA correlated with increased MnSOD protein and enzyme activity. Influenza virus infection also activated NF-κB binding as determined by an electrophoretic mobility shift assay. Pretreatment of A549 cells with N-acetyl-l-cysteine attenuated virus-induced NF-κB activation and interleukin (IL)-8 mRNA induction but did not block induction of MnSOD mRNA. In contrast, pyrrolidine dithiocarbamate blocked activation of NF-κB and induction of MnSOD and IL-8 mRNAs. Treatment with pyrrolidine dithiocarbamate also markedly decreased virus-induced cell death. Thus oxidants are involved in influenza virus-induced activation of NF-κB, in the expression of IL-8 and MnSOD, and in virus-induced cell death.

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Geniposide demonstrates anti-inflammatory and antiviral activity against pandemic A/Jiangsu/1/2009 (H1N1) influenza virus infection in vitro and in vivo

Antiviral Therapy ◽

10.3851/imp3152 ◽

2017 ◽

Vol 22 (7) ◽

pp. 599-611 ◽

Cited By ~ 15

Author(s):

Zhang Yunshi ◽

Yao Jing ◽

Qi Xian ◽

Liu Xing ◽

Lu Xieqin ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Antiviral Activity ◽

Influenza Virus Infection ◽

H1n1 Influenza ◽

H1n1 Influenza Virus ◽

2009 H1n1 ◽

Anti Inflammatory

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Multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon IMPRESS

Nature Protocols ◽

10.1038/s41596-019-0275-y ◽

2020 ◽

Vol 15 (3) ◽

pp. 1041-1065 ◽

Cited By ~ 6

Author(s):

Hiroshi Ueki ◽

I-Hsuan Wang ◽

Dongming Zhao ◽

Matthias Gunzer ◽

Yoshihiro Kawaoka

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza Virus Infection ◽

Two Photon ◽

Photon Imaging ◽

Two Photon Imaging

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Immune-related gene expression in response to H5N1 avian influenza virus infection in chicken and duck embryonic fibroblasts published in MIMM volume 48, issues 6-7, March 2011.

Molecular Immunology ◽

10.1016/j.molimm.2011.10.003 ◽

2011 ◽

Vol 49 (1-2) ◽

pp. 413 ◽

Cited By ~ 1

Keyword(s):

Gene Expression ◽

Influenza Virus ◽

Virus Infection ◽

Avian Influenza ◽

Influenza Virus Infection ◽

Related Gene ◽

Embryonic Fibroblasts ◽

H5n1 Avian Influenza Virus ◽

Immune Related Gene ◽

H5n1 Avian Influenza

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Prevention of Murine Influenza a Virus Pneumonitis by Surfactant Nano-Emulsions

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020001100104 ◽

2000 ◽

Vol 11 (1) ◽

pp. 41-49 ◽

Cited By ~ 28

Author(s):

Brian W Donovan ◽

Jon D Reuter ◽

Zhengyi Cao ◽

Andrzej Myc ◽

Kent J Johnson ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza A ◽

Therapeutic Potential ◽

Clinical Signs ◽

Influenza Virus Infection ◽

Initial Study ◽

Ionic Surfactant ◽

Nano Emulsion

Non-ionic surfactant nano-emulsions have extensive anti-microbial activity and are biocompatible with skin and mucous membranes at effective concentrations. Two nano-emulsion formulations (8N8 and 20N10) made from soybean oil, tributyl phosphate and Triton X-100, were tested for their ability to prevent murine influenza virus pneumonia in vivo. In the initial study, CD-1 mice were administered various dilutions of the nano-emulsions intranasally, and safe dosages and concentrations were determined. Non-toxic concentrations of the nano-emulsions were then mixed with influenza virus and applied to the nares of mice. Animals receiving mixtures of two different emulsions (8N8 or 20N10) and a LD50 of virus survived the challenge without evidence of viral infection. To determine if the nano-emulsions could prevent influenza virus infection in vivo when used as a prophylactic treatment, the nano-emulsions (8N8 at 1.0% and 20N10 at 1.0% or 0.2%) were applied to mouse nares 90 min before exposure to 5×105 p.f.u./ml virus by nebulized aerosol. Animals pretreated with the nano-emulsions had significantly decreased clinical signs of infection. Only 26.0% (8N8 at 1.0%), 31.25% (20N10 at 1.0%) and 37.0% (20N10 at 0.2%) of animals pretreated with nano-emulsion died from pneumonitis, whereas >80.0% of mock pretreated animals succumbed to infection ( P<0.005). These findings suggest that non-ionic surfactant nano-emulsions have therapeutic potential for the prevention of influenza virus infection in vivo.

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