The Number of Target Molecules of the Amplification Step Limits Accuracy and Sensitivity in Ultradeep-Sequencing Viral Population Studies

Gallet, Romain; Fabre, Frédéric; Michalakis, Yannis; Blanc, Stéphane

doi:10.1128/jvi.00561-17

Cited by 32 publications

(34 citation statements)

References 36 publications

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“…Genetic markers (22 nucleotides; sequences available upon request) were inserted between the end of the coding regions and the poly(A) signal sequences of the N and S segments. Markers mys2 and mys7 were introduced in the N segment, while markers mys1 and mys8 were introduced in the S segment (21).…”

Section: Methodsmentioning

confidence: 99%

Small Bottleneck Size in a Highly Multipartite Virus during a Complete Infection Cycle

Gallet

Fabre

Thébaud

et al. 2018

J Virol

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Multipartite viruses package their genomic segments independently and thus incur the risk of being unable to transmit their entire genome during host-to-host transmission if they undergo severe bottlenecks. In this paper, we estimated the bottleneck size during one infection cycle of (FBNSV), an octopartite nanovirus whose segments have been previously shown to converge to particular and unequal relative frequencies within host plants and aphid vectors. Two methods were used to derive this estimate, one based on the probability of transmission of the virus and the other based on the temporal evolution of the relative frequency of markers for two genomic segments, one frequent and one rare (segment N and S, respectively), both in plants and vectors. Our results show that FBNSV undergoes severe bottlenecks during aphid transmission. Further, even though the bottlenecks are always narrow under our experimental conditions, they slightly widen with the number of transmitting aphids. In particular, when several aphids are used for transmission, the bottleneck size of the segments is also affected by within-plant processes and, importantly, significantly differs across segments. These results indicate that genetic drift not only must be an important process affecting the evolution of these viruses but also that these effects vary across genomic segments and, thus, across viral genes, a rather unique and intriguing situation. We further discuss the potential consequences of our findings for the transmission of multipartite viruses. Multipartite viruses package their genomic segments in independent capsids. The most obvious cost of such genomic structure is the risk of losing at least one segment during host-to-host transmission. A theoretical study has shown that for nanoviruses, composed of 6 to 8 segments, hundreds of copies of each segment need to be transmitted to ensure that at least one copy of each segment was present in the host. These estimations seem to be very high compared to the size of the bottlenecks measured with other viruses. Here, we estimated the bottleneck size during one infection cycle of FBNSV, an octopartite nanovirus. We show that these bottlenecks are always narrow (few viral particles) and slightly widen with the number of transmitting aphids. These results contrast with theoretical predictions and illustrate the fact that a new conceptual framework is probably needed to understand the transmission of highly multipartite viruses.

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Section: Methodsmentioning

confidence: 99%

Small Bottleneck Size in a Highly Multipartite Virus during a Complete Infection Cycle

Gallet

Fabre

Thébaud

et al. 2018

J Virol

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“…Post-PCR data analyses were carried out as described [29]. The accumulation of ALCV is expressed as the number of copies of monopartite viral genomes.…”

Section: Quantitative Real-time Pcr (Qpcr) Detectionmentioning

confidence: 99%

Co-Acquired Nanovirus and Geminivirus Exhibit a Contrasted Localization within Their Common Aphid Vector

Mattia

Ryckebusch

Vernerey

et al. 2020

Viruses

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Single-stranded DNA (ssDNA) plant viruses belong to the families Geminiviridae and Nanoviridae. They are transmitted by Hemipteran insects in a circulative, mostly non-propagative, manner. While geminiviruses are transmitted by leafhoppers, treehoppers, whiteflies and aphids, nanoviruses are transmitted exclusively by aphids. Circulative transmission involves complex virus–vector interactions in which epithelial cells have to be crossed and defense mechanisms counteracted. Vector taxa are considered a relevant taxonomic criterion for virus classification, indicating that viruses can evolve specific interactions with their vectors. Thus, we predicted that, although nanoviruses and geminiviruses represent related viral families, they have evolved distinct interactions with their vector. This prediction is also supported by the non-structural Nuclear Shuttle Protein (NSP) that is involved in vector transmission in nanoviruses but has no similar function in geminiviruses. Thanks to the recent discovery of aphid-transmitted geminiviruses, this prediction could be tested for the geminivirus alfalfa leaf curl virus (ALCV) and the nanovirus faba bean necrotic stunt virus (FBNSV) in their common vector, Aphis craccivora. Estimations of viral load in midgut and head of aphids, precise localization of viral DNA in cells of insect vectors and host plants, and virus transmission tests revealed that the pathway of the two viruses across the body of their common vector differs both quantitatively and qualitatively.

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“…We tested the sensitivity of this method to common technical considerations. Estimates of evolutionary rates can be influenced by the frequency threshold used to identify within-host variation (Gallet et al, 2017;Grubaugh et al, 2019;McCrone and Lauring, 2016). We focused on within-host variants present in at least 0.5% of sequencing reads at a site (Figure 2A).…”

Section: Rates Of Influenza Virus Evolution Within Hostsmentioning

confidence: 99%

Linking influenza virus evolution within and between human hosts

Xue

Bloom

2019

Preprint

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Influenza viruses rapidly diversify within individual human infections. Several recent studies have deep-sequenced clinical influenza infections to identify viral variation within hosts, but it remains unclear how within-host mutations fare in the global viral population. Here, we compare viral variation within and between hosts to link influenza's evolutionary dynamics across scales. Synonymous sites evolve at similar rates at both scales, indicating that global evolution at these putatively neutral sites results from the accumulation of within-host variation. However, nonsynonymous mutations are depleted in global viral populations compared to within hosts, suggesting that selection purges many of the protein-altering changes that arise within hosts. The exception is at antigenic sites, where selection detectably favors nonsynonymous mutations at the global scale, but not within hosts. These results suggest that selection against deleterious mutations and selection for antigenic change are the main forces that transform influenza's within-host genetic variation into global evolution.

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The Number of Target Molecules of the Amplification Step Limits Accuracy and Sensitivity in Ultradeep-Sequencing Viral Population Studies

Cited by 32 publications

References 36 publications

Small Bottleneck Size in a Highly Multipartite Virus during a Complete Infection Cycle

Small Bottleneck Size in a Highly Multipartite Virus during a Complete Infection Cycle

Co-Acquired Nanovirus and Geminivirus Exhibit a Contrasted Localization within Their Common Aphid Vector

Linking influenza virus evolution within and between human hosts

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