Using barcoded Zika virus to assess virus population structure in vitro and in Aedes aegypti mosquitoes

Weger-Lucarelli, James; García, Selene M.; Rückert, Claudia; Byas, Alex D.; O’Connor, Shelby L.; Aliota, Matthew T.; Friedrich, Thomas C.; O’Connor, David H.; Ebel, Gregory D.

doi:10.1016/j.virol.2018.06.004

Cited by 50 publications

(66 citation statements)

References 49 publications

(54 reference statements)

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“…However, we have successfully used RCA to amplify and rescue infectious virus from several infectious cDNA clones. These include emerging zoonotic viruses (Usutu virus; Genus Flavivirus , Family Flaviviridae ) ( Bates et al, 2020 ), and other medically relevant viruses including, Zika virus (Genus Flavivirus , Family Flaviviridae ) ( Weger-Lucarelli et al, 2018 ), West Nile virus (Genus Flavivirus , Family Flaviviridae ) ( Grubaugh et al, 2016 , 2017 ), chikungunya virus (unpublished data; Genus Alphavirus , Family Togaviridae ), and SARS-CoV-2 (unpublished data; Genus Betacoronavirus , Family Coronaviridae ). We anticipate that this system can be used for other positive-sense RNA virus cDNA clones and likely negative-strand viruses as well.…”

Section: Discussionmentioning

confidence: 99%

“…Importantly, the enzyme replicates DNA with high fidelity due to its 3′—5’exonuclease—or proofreading—activity ( Garmendia et al, 1992 ). We have previously used RCA to rescue infectious cDNA clones; however, in these studies, the RCA product was linearized with endonucleases, column purified, and then transfected ( Weger-Lucarelli et al, 2018 ; Aliota et al, 2018 ). In this study, using a clone driven by a cytomegalovirus (CMV) promoter, we show that peak virus yields were similar following the direct transfection of RCA- and plasmid-derived cDNA clones in several cell lines and that small amounts of the RCA product could be used to rescue virus successfully.…”

Section: Introductionmentioning

confidence: 99%

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Rolling circle amplification: A high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

2020

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Alphaviruses (genus Alphavirus ; family Togaviridae ) are a medically relevant family of viruses that include chikungunya virus and Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology. Traditionally, rescuing virus from an infectious cDNA clone requires propagating plasmids in bacteria, which can result in mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative-rolling circle amplification (RCA), an in vitro technology. We demonstrate that the viral yield of transfected RCA product is comparable to midiprepped plasmid, albeit with a slight delay in kinetics. RCA, however, is cheaper and less time-consuming. Further, sequential RCA did not introduce mutations into the viral genome, subverting the need for glycerol stocks and retransformation. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Rolling circle amplification: A high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

2020

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“…Several of these studies revealed founder effects, an extreme reduction in viral population diversity in which a small sub-population of viruses invade a new tissue or host. This has been described for viruses of plants (8) and animals, including HIV (9), HCV (10), Zika (11,12), influenza (3,13), and poliovirus (6). The gastrointestinal (GI) tract restricts dissemination of lumenal contents, including enteric viruses.…”

Section: Introductionmentioning

confidence: 94%

Rapid dissemination and monopolization of viral populations in mice revealed using a panel of barcoded viruses

McCune

Lanahan

tenOever

et al. 2019

Preprint

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17The gastrointestinal tract presents a formidable barrier for pathogens to initiate infection. 18Despite this barrier, enteroviruses, including coxsackievirus B3 (CVB3), successfully penetrate 19 the intestine to initiate infection and spread systemically prior to shedding in stool. However, the 20 effect of the gastrointestinal barrier on CVB3 population dynamics is relatively unexplored, nor 21 are the selective pressures acting on CVB3 in the intestine well-characterized. To examine viral 22 population dynamics in orally infected mice, we produced over one hundred CVB3 viruses 23 harboring unique nine nucleotide "barcodes." Using this collection of barcoded viruses, we 24 found diverse viral populations throughout each mouse within the first day post-infection, but by 25 48 hours the viral populations were dominated by less than three barcoded viruses in intestinal 26 and extra-intestinal tissues. Using light-sensitive viruses to track replication status, we found 27 diverse viruses had replicated prior to loss of diversity. Sequencing whole viral genomes from 28 samples later in infection did not reveal detectable viral adaptations. Surprisingly, orally 29 inoculated CVB3 was detectable in pancreas and liver as soon as 20 minutes post inoculation, 30indicating rapid systemic dissemination. These results suggest rapid dissemination of diverse 31 viral populations, followed by a major restriction in population diversity and monopolization in all 32 examined tissues. These results underscore a complex dynamic between dissemination and 33 clearance for an enteric virus. 35 Importance 36Enteric viruses initiate infection in the gastrointestinal tract but can disseminate to systemic 37 sites. However, the dynamics of viral dissemination are unclear. In this study, we created a 38 library of 135 barcoded coxsackieviruses to examine viral population diversity across time and 39 space following oral inoculation of mice. Overall, we found that the broad population of viruses 40 disseminates early, followed by monopolization of mouse tissues with three or fewer pool 41 members at later time points. Interestingly, we detected virus in systemic tissues such as 42 pancreas and liver just 20 minutes post-oral inoculation. These results suggest rapid 43 dissemination of diverse viral populations, followed by a major restriction in population diversity 44 and monopolization in all examined tissues. 45 46 Introduction 47 RNA virus populations are dynamic. Population diversity arises through mutations introduced by 48 the viral RNA-dependent RNA polymerases, which lack proofreading. Although many mutations 49 are detrimental, viruses can benefit from diversity within viral populations since diversity may 50 facilitate replication and dissemination within hosts (1,2). Viral diversity can be reduced through 51 selective pressures or stochastic events. Because viral diversity is a fundamental parameter in 52 viral evolution and virulence, understanding population dynamics and the factors that influence 53 viral populations is a c...

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“…Bacteria-Free Cloning. Site-directed mutagenesis (SDM) was performed using a bacteria-free cloning method essentially as previously described (27). Mutated fragments of the USUV TMN and UG clones were amplified by PCR using Platinum SuperFi 2X PCR Master Mix with mutagenic primers (Supp.…”

Section: Construction Of Usuv Infectious Clonesmentioning

confidence: 99%

Development and characterization of infectious clones of two strains of Usutu virus

Bates

Chuong

Hawks

et al. 2020

Preprint

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Usutu virus (USUV; genus Flavivirus; family Flaviviridae) is a mosquito-borne, positive-sense RNA virus that is currently causing significant die-offs in numerous bird species throughout Europe and has caused infections in humans. Currently, there are no molecular clones for USUV, hence, hindering studies on the pathogenesis and transmission of USUV. In this report, we demonstrate the development and characterization of infectious clones for two modern strains of USUV isolated from Europe and Africa. We show that the infectious clone-derived viruses replicated similarly to the parental strains in both mammalian and insect cells. Additionally, we observed similar levels of replication and pathogenesis in two mouse models. This reverse genetics system will aid the scientific community in studying and developing USUV infection, transmission, diagnostics, and vaccines.

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Using barcoded Zika virus to assess virus population structure in vitro and in Aedes aegypti mosquitoes

Cited by 50 publications

References 49 publications

Rolling circle amplification: A high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

Rolling circle amplification: A high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

Rapid dissemination and monopolization of viral populations in mice revealed using a panel of barcoded viruses

Development and characterization of infectious clones of two strains of Usutu virus

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