A fluorescence-activatable reporter of flavivirus NS2B–NS3 protease activity enables live imaging of infection in single cells and viral plaques

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus. The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus–host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence-activatable sensors to detect the activities of flavivirus NS2B–NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro. A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco's plaque assay, leading to the construction of a multireporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and yellow fever virus. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus–host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

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Activation and Kinetics of Circulating T Follicular Helper Cells, Specific Plasmablast Response, and Development of Neutralizing Antibodies following Yellow Fever Virus Vaccination

The Journal of Immunology ◽

10.4049/jimmunol.2001381 ◽

2021 ◽

pp. ji2001381

Author(s):

John Tyler Sandberg ◽

Sebastian Ols ◽

Marie Löfling ◽

Renata Varnaitė ◽

Gustaf Lindgren ◽

...

Keyword(s):

Yellow Fever ◽

Neutralizing Antibodies ◽

Yellow Fever Virus ◽

Fever Virus ◽

T Follicular Helper Cells ◽

Virus Vaccination ◽

Helper Cells ◽

Follicular Helper ◽

Kinetics Of

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Machine learning models exploring characteristic single-nucleotide signatures in Yellow Fever Virus

10.1101/2021.03.23.436688 ◽

2021 ◽

Author(s):

Álvaro Salgado ◽

Raquel Minardi ◽

Marta Giovanetti ◽

Adriano Veloso ◽

Francielly Morais-Rodrigues ◽

...

Keyword(s):

Machine Learning ◽

Yellow Fever ◽

Protein Function ◽

Urban Areas ◽

Yellow Fever Virus ◽

Fever Virus ◽

Single Nucleotide ◽

Host Interactions ◽

Human Infections ◽

The Tropics

Yellow fever virus (YFV) is the agent of the most severe mosquito-borne disease in the tropics. Recently, Brazil suffered major YFV outbreaks with a high fatality rate affecting areas where the virus has not been reported for decades, consisting of urban areas where a large number of unvaccinated people live. We developed a machine learning framework combining three different algorithms (XGBoost, random forest and regularized logistic regression). This method was applied to 56 YFV sequences from human infections and 27 from non-human primate (NHPs) infections to investigate the presence of genetic signatures possibly related to disease severity (in human related sequences) and differences in the PCR cycle threshold (Ct) values (in NHP related sequences). Our analyses reveal four non-synonymous single nucleotide variations (SNVs) on sequences from human infections, in proteins NS3 (E614D), NS4a (I69V), NS5 (R727G, V643A) and six non-synonymous SNVs on NHP sequences, in proteins E (L385F), NS1 (A171V), NS3 (I184V) and NS5 (N11S, I374V, E641D). We performed comparative protein structural analysis on these SNVs, describing possible impacts on protein function. Despite the fact that the dataset is limited in size and that this study does not consider virus-host interactions, our work highlights the use of machine learning as a versatile and fast initial approach to genomic data exploration.

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Yellow fever virus outbreak in Brazil under current and future climate

Infectious Disease Modelling ◽

10.1016/j.idm.2021.04.002 ◽

2021 ◽

Vol 6 ◽

pp. 664-677

Author(s):

Tara Sadeghieh ◽

Jan M. Sargeant ◽

Amy L. Greer ◽

Olaf Berke ◽

Guillaume Dueymes ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Fever Virus ◽

Future Climate

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Process intensification for the production of yellow fever virus‐like particles (VLPs) as potential recombinant vaccine antigen

Biotechnology and Bioengineering ◽

10.1002/bit.27864 ◽

2021 ◽

Author(s):

Renata G. F. Alvim ◽

Túlio M. Lima ◽

Jerson L. Silva ◽

Guilherme A. P. Oliveira ◽

Leda R. Castilho

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Process Intensification ◽

Recombinant Vaccine ◽

Fever Virus ◽

Vaccine Antigen ◽

Virus Like Particles

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Yellow Fever Outbreak in Eastern Senegal, 2020–2021

Viruses ◽

10.3390/v13081475 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1475

Author(s):

Moussa Moïse Diagne ◽

Marie Henriette Dior Ndione ◽

Alioune Gaye ◽

Mamadou Aliou Barry ◽

Diawo Diallo ◽

...

Keyword(s):

Yellow Fever ◽

Yellow Fever Virus ◽

Mosquito Species ◽

Virus Transmission ◽

Genomic Analysis ◽

Hemorrhagic Fever ◽

Fever Virus ◽

World Health ◽

Effective Vaccine ◽

Ministry Of Health

Yellow fever virus remains a major threat in low resource countries in South America and Africa despite the existence of an effective vaccine. In Senegal and particularly in the eastern part of the country, periodic sylvatic circulation has been demonstrated with varying degrees of impact on populations in perpetual renewal. We report an outbreak that occurred from October 2020 to February 2021 in eastern Senegal, notified and managed through the synergistic effort yellow fever national surveillance implemented by the Senegalese Ministry of Health in collaboration with the World Health Organization, the countrywide 4S network set up by the Ministry of Health, the Institut Pasteur de Dakar, and the surveillance of arboviruses and hemorrhagic fever viruses in human and vector populations implemented since mid 2020 in eastern Senegal. Virological analyses highlighted the implication of sylvatic mosquito species in virus transmission. Genomic analysis showed a close relationship between the circulating strain in eastern Senegal, 2020, and another one from the West African lineage previously detected and sequenced two years ago from an unvaccinated Dutch traveler who visited the Gambia and Senegal before developing signs after returning to Europe. Moreover, genome analysis identified a 6-nucleotide deletion in the variable domain of the 3′UTR with potential impact on the biology of the viral strain that merits further investigations. Integrated surveillance of yellow fever virus but also of other arboviruses of public health interest is crucial in an ecosystem such as eastern Senegal.

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