Propagation and Characterization of Influenza Virus Stocks That Lack High Levels of Defective Viral Genomes and Hemagglutinin Mutations

Xue, Jia; Chambers, Benjamin S.; Hensley, Scott E.; López, Carolina B.

doi:10.3389/fmicb.2016.00326

Cited by 60 publications

(78 citation statements)

References 48 publications

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“…The first was a standard stock grown from low dose in embryonated chicken eggs; the second was produced in eggs following triple plaque purification to limit the abundance of DI particles (12, 28); and the third was generated by serial passage in cell culture under high multiplicity conditions, which leads to enrichment of DI particles (6, 12, 28). An example of the raw data obtained by ddPCR is shown in Figure 2, with panel A displaying results acquired with terminal PB2 primers and panel B showing results obtained with internal PB2 primers, both combined with cDNA templates derived from the Pan/99wtHis-MDCK P3 virus.…”

Section: Resultsmentioning

confidence: 99%

“…As a consequence, the impact of DI particles on experimental outcomes and natural IAV infections may be substantial. Xue et al recently reported a rigorous method for preparation of influenza A virus stocks designed to limit the prevalence of DI particles (28). Despite this important advance, a robust assay for the quantification of DI segments is lacking.…”

Section: Introductionmentioning

confidence: 99%

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

Schwartz

2016

Journal of Virological Methods

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Defective interfering (DI) particles are viruses that carry one or more large, internal deletions in the viral genome. These deletions occur commonly in RNA viruses due to polymerase error and yield incomplete genomes that typically lack essential coding regions. The presence of DI particles in a virus population can have a major impact on the efficiency of viral growth and is an important variable to consider in interpreting experimental results. Herein, we sought to develop a robust methodology for the quantification of DI particles within influenza A virus stocks. We took advantage of reverse transcription followed by droplet digital PCR (RT ddPCR), a highly sensitive and precise technology for determination of template concentrations without the use of a standard curve. Results were compared to those generated using standard RT qPCR. Both assays relied on the use of primers binding to terminal regions conserved in DI gene segments described to date, and internal primers targeting regions typically missing from DI particles. As has been reported previously, we observed a lower coefficient of variation among technical replicates for ddPCR compared to qPCR. Results furthermore established RT ddPCR as a sensitive and quantitative method for detecting DI gene segments within influenza A virus stocks.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Schwartz

2016

Journal of Virological Methods

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“…However, the burden of defective viral particles can be reduced by growing virus for a 214 short amount of time and at a low MOI, as defective viral particles increase in frequency 215 when viruses are grown at a high MOI where complementation of deleterious genotypes 216 7/53 can occur [79]. The even read coverage across the polymerase segments in our data 217 ( Fig.…”

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confidence: 81%

Comprehensive profiling of translation initiation in influenza virus infected cells

Machkovech

Bloom

Subramaniam

2018

Preprint

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Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. Host transcripts induced by the anti-viral response are enriched for translation initiation at non-canonical start sites and non-AUG start codons. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.

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“…DVGs are present in live attenuated vaccines against polio, measles and influenza viruses 76,[123][124][125][126] . However, their impact in the development of protective immunity and vaccine efficacy has not been formally assessed.…”

Section: Use As Antivirals and Vaccine Adjuvantsmentioning

confidence: 99%

Defective viral genomes are key drivers of the virus–host interaction

2019

Self Cite

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Viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. We are beginning to appreciate the surprising versatility of viral genomes and how replicationcompetent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. This Review summarizes current knowledge of the types of defective viral genomes generated during the replication of RNA viruses and the functions that they carry out. We highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools. Review ARticleNATuRe MicRoBiology extinction interfere with replication of a wild-type viral genome 29,30 . Hypermutations and mutations leading to frame shifts also result in defective viruses 31 (Fig. 1a). One example is mutations introduced by the cellular protein APOBEC3G into pro-retroviruses 32 . APOBEC3G deaminates deoxycytidine nucleotides in the viral DNA, leading to hypermutations that interfere with the ability of the viral genome to integrate into the host genome and replicate. Interestingly, in opposition to an interfering activity for these mutated pro-viruses, it has been proposed that defective proviruses enhance fitness and that complementation among them drives viral persistence and pathogenesis 33,34 .Deletions. The genomic viral species most commonly referred to as DVGs are truncated viral genomes resulting from large internal deletions occurring during viral replication. Deletion DVGs tend to bear single large truncations that remove several or all essential genes required for self-propagation while retaining 5′ and 3′ ends as well as other RNA structural elements required for polymerase binding, replication and/or packaging [35][36][37] . Multiple variants also exist, including DVGs that can be described as 'mosaic' in that they appear to be the result of multiple recombination and rearrangement events, including deletions, insertions, duplications and even inversions of parts of the genome [38][39][40] (Fig. 1b). Copy-backs.Copy-back and snap-back DVGs are rearranged genomes in which a sequence is duplicated in reverse complement to create theoretical stem-like structures (panhandle structures for copy-back DVGs or hairpin structures for snap-back DVGs) 41-43 . Copy-back DVGs have been reported in many negative-sense (ns) RNA viruses and are generated from the 5′ end of the genome in a process that produces DVGs with complementary 3′ and 5′ termini 44,45 . If the complementary region of the DVG comprises almost the entire sequence, the DVG can be further characterized as a snap-back DVG 43 (Fig. 1c). Copy-back DVGs are predicted to be generated when the viral RNA-dependent RNA polymerase (RdRP) detaches from the template and reattaches to the nascent strand, copying back the end of the genome 42,46 .

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Propagation and Characterization of Influenza Virus Stocks That Lack High Levels of Defective Viral Genomes and Hemagglutinin Mutations

Cited by 60 publications

References 48 publications

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

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

Comprehensive profiling of translation initiation in influenza virus infected cells

Defective viral genomes are key drivers of the virus–host interaction

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