Droplet digital PCR: A novel method for detection of influenza virus defective interfering particles

Schwartz, Samantha L.

doi:10.1016/j.jviromet.2016.08.023

Cited by 27 publications

(24 citation statements)

References 30 publications

(48 reference statements)

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“…For NL09-based viruses, 293T cells transfected with reverse genetics plasmids 16–24 h prior were co-cultured with MDCK cells at 37°C for 40–48 h. Supernatants were then propagated in MDCK cells from low MOI to generate NL09 working stocks. Defective interfering segment content of PB2, PB1, and PA segments was confirmed to be minimal for each virus stock, as described previously 68 ( Supplementary Figure 7 ). All plaque assays were performed in MDCK cells; viruses were also titered by flow cytometry and/or RT qPCR-based methods where indicated.…”

Section: Methodssupporting

confidence: 80%

Collective interactions augment influenza A virus replication in a host-dependent manner

Phipps

Ganti

Jacobs

et al. 2019

Preprint

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22implicated the PA gene segment as a major driver of this phenotype and quantification of viral 33RNA synthesis indicated that both replication and transcription were affected. These findings 34 indicate that multiple distinct mechanisms underlie IAV reliance on multiple infection and 35 underscore the importance of virus-virus interactions in IAV infection, evolution and emergence. 36Importantly, multiple infection with identical viral genomes can also alter infection 58 outcomes. Such cooperation was documented for VSV and HIV, where rates of transcription and 59 replication were enhanced with increasing multiplicity of infection (MOI) 23,24 . Similarly, faster 60 kinetics of virus production were seen at high MOI for poliovirus and IAV 19,25 . In these instances, 61 it is thought that increased copy number of infecting viral genomes provides a kinetic benefit 62 important in the race to establish infection before innate antiviral responses take hold. Indeed, it 63 has been suggested that multiple infection may be particularly relevant for facilitating viral growth 64 under adverse conditions, such as antiviral drug treatment 3,26 . 65For IAV, an important adverse condition to consider is that of a novel host environment. 66IAVs occupy a broad host range, including multiple species of wild waterfowl, poultry, swine, 67 humans and other mammals 27,28 . Host barriers to infection typically confine a given lineage to 68 circulation in one species or a small number of related species 29,30 . Spillovers occur occasionally, 69 however, and can seed novel lineages. When a novel IAV lineage is established in humans, the 70 result is a pandemic of major public health consequence 31,32 . The likelihood of successful cross-71 species transfer of IAV is determined largely by the presence, absence, and compatibility of host 72 factors on which the virus relies to complete its life cycle, and on the viruses' ability to overcome 73 antiviral defenses in the novel host [33][34][35] . 74Our objective herein was to assess the degree to which IAV relies on the delivery of 75 multiple viral genomes to a cell to ensure production of progeny. In particular, we sought to 76 determine whether this phenotype varies with host species. We therefore examined the 77 multiplicity dependence of one human and a panel of avian-origin viruses in multiple host systems. 78Results from all virus/cell combinations tested confirm prior reports that cells multiply-infected with 79 IAV produce more viral progeny than singly-infected cells. Importantly, however, the extent to 80 which viral progeny production is concentrated within the multiply-infected fraction of a cell 81 population varies greatly with virus-host context. Two poultry-adapted H9N2 viruses (A/guinea 82 fowl/HK/WF10/99 (GFHK99) and A/quail/HK/A28945/88 (QaHK88)) exhibit an acute dependence 83 on multiple infection in mammalian systems that is greatly diminished in natural host systems. 84This strong dependence on multiple infection is not seen for the human strain, influenza 85 ...

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Section: Methodssupporting

confidence: 80%

Collective interactions augment influenza A virus replication in a host-dependent manner

Phipps

Ganti

Jacobs

et al. 2019

Preprint

Self Cite

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“…Three biological replicates were tested for each virus stock. Based on the Ct values obtained with terminal and internal assays, relative differences in the amount of defective RNAs between 95/95(DVG-high) and 95/95(DVG-low) were evaluated as previously described [32]. Briefly, a ratio 2 (-CtT) :2 (-CtI) was calculated and compared for both virus stocks.…”

Section: Analysis Of Amount Of Defective Segmentsmentioning

confidence: 99%

Low pathogenic avian influenza virus isolates with different levels of defective genome segments vary in pathogenicity and transmission efficiency

Świętoń

Tarasiuk

Śmietanka

2020

Vet Res

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Defective interfering particles (DIPs) of influenza virus are generated through incorporation of highly truncated forms of genome segments, mostly those coding polymerase complex proteins (PB2, PB1, PA). Such particles are able to replicate only in the presence of a virus with the complete genome, thus DIPs may alter the infection outcome by suppressing production of standard virus particles, but also by stimulating the immune response. In the present study we compared the clinical outcome, mortality and transmission in chickens and turkeys infected with the same infectious doses of H7N7 low pathogenic avian influenza virus containing different levels of defective gene segments (95/95(DVG-high) and 95/95(DVG-low)). No clinical signs, mortality or transmission were noted in SPF chickens inoculated with neither virus stock. Turkeys infected with 95/95(DVG-high) showed only slight clinical signs with no mortality, and the virus was transmitted only to birds in direct contact. In contrast, more severe disease, mortality and transmission to direct and indirect contact birds was observed in turkeys infected with 95/95(DVG-low). Apathy, lower water and food intake, respiratory system disorders and a total mortality of 60% were noted. Shedding patterns in contact turkeys indicated more efficient within-and between-host spread of the virus than in 95/95(DVG-high) group. Sequencing of virus genomes showed no mutations that could account for the observed differences in pathogenicity. The results suggest that the abundance of DIPs in the inoculum was the factor responsible for the mild course of infection and disrupted virus transmission.

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“…Several studies have demonstrated the efficient dPCR platform searching different viruses [20][21][22][23][24][25]. Moreover, rapid, accurate and affordable molecular technology can be predictable with particular emphasis on emerging techniques (next generation sequencing, digital PCR, point of care testing and syndromic diagnosis) to simplify viral diagnosis in the next future [24,25]. Finally, the present findings show that dPCR enables direct and accurate detection of CCoV genome with efficiency and quantitative linearity both from fecal samples and infected cell suspensions.…”

Section: Resultsmentioning

confidence: 99%

Digital PCR Platform as a Tool to Determine the Canine Coronavirus (CCoV) Genome in Clinical Samples

2018

Arch Vet Sci Tecnol

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Canine coronavirus (CCoV) is an important pathogen that affect dogs. Here we assessed a digital real-time based PCR (dPCR) for the detection of CCoV from infected AF-72 cells and directly from faeces from 100 symptomatic dogs. The results obtained from dPCR were compared to real-time TaqMan® based PCR assay (qPCR) and positive samples were submitted to phylogenetic analysis. Thus, dPCR had an equal sensitivity, 101 copies/μl of partial M CCoV gene, when compared to qPCR results with a good agreement in all analysis. These results indicated that the dPCR could be an alternative technique for the diagnosis of CCoV from clinical samples with advantage of simplicity and sensitivity.

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Droplet digital PCR: A novel method for detection of influenza virus defective interfering particles

Cited by 27 publications

References 30 publications

Collective interactions augment influenza A virus replication in a host-dependent manner

Collective interactions augment influenza A virus replication in a host-dependent manner

Low pathogenic avian influenza virus isolates with different levels of defective genome segments vary in pathogenicity and transmission efficiency

Digital PCR Platform as a Tool to Determine the Canine Coronavirus (CCoV) Genome in Clinical Samples

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