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Reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients.

ABSTRACT: Influenza A virus (IAV) infection can be severe or even lethal in toddlers, the elderly and patients with certain medical conditions. Infection of apparently healthy individuals nonetheless accounts for many severe disease cases and deaths, suggesting that viruses with increased pathogenicity co-circulate with pandemic or epidemic viruses. Looking for potential virulence factors, we have identified a polymerase PA D529N mutation detected in a fatal IAV case, whose introduction into two different recombinant virus backbones, led to reduced defective viral genomes (DVGs) production. This mutation conferred low induction of antiviral response in infected cells and increased pathogenesis in mice. To analyze the association between low DVGs production and pathogenesis in humans, we performed a genomic analysis of viruses isolated from a cohort of previously healthy individuals who suffered highly severe IAV infection requiring admission to Intensive Care Unit and patients with fatal outcome who additionally showed underlying medical conditions. These viruses were compared with those isolated from a cohort of mild IAV patients. Viruses with fewer DVGs accumulation were observed in patients with highly severe/fatal outcome than in those with mild disease, suggesting that low DVGs abundance constitutes a new virulence pathogenic marker in humans.

SUBMITTER: Vasilijevic J

PROVIDER: S-EPMC5638565 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients.

Vasilijevic Jasmina J Zamarreño Noelia N Oliveros Juan Carlos JC Rodriguez-Frandsen Ariel A Gómez Guillermo G Rodriguez Guadalupe G Pérez-Ruiz Mercedes M Rey Sonia S Barba Isabel I Pozo Francisco F Casas Inmaculada I Nieto Amelia A Falcón Ana A

PLoS pathogens 20171012 10

Influenza A virus (IAV) infection can be severe or even lethal in toddlers, the elderly and patients with certain medical conditions. Infection of apparently healthy individuals nonetheless accounts for many severe disease cases and deaths, suggesting that viruses with increased pathogenicity co-circulate with pandemic or epidemic viruses. Looking for potential virulence factors, we have identified a polymerase PA D529N mutation detected in a fatal IAV case, whose introduction into two different ...[more]

PMID: 29023600

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Project description:Ebola virus (EBOV), of the family Filoviridae, is an RNA virus that can cause a hemorrhagic fever with a high mortality rate. Defective viral genomes (DVGs) are truncated genomes that have been observed during multiple RNA virus infections, including in vitro EBOV infection, and have previously been associated with viral persistence and immunostimulatory activity. As DVGs have been detected in cells persistently infected with EBOV, we hypothesized that DVGs may also accumulate during viral replication in filovirus-infected hosts. Therefore, we interrogated sequence data from serum and tissue samples using a bioinformatics tool in order to identify the presence of DVGs in nonhuman primates (NHPs) infected with EBOV, Sudan virus (SUDV), or Marburg virus (MARV). Multiple 5' copy-back DVGs (cbDVGs) were detected in NHP serum during the acute phase of filovirus infection. While the relative abundance of total DVGs in most animals was low, serum collected during acute EBOV and SUDV infections, but not MARV infections, contained a higher proportion of short trailer sequence cbDVGs than the challenge stock. This indicated an accumulation of these DVGs throughout infection, potentially due to the preferential replication of short DVGs over the longer viral genome. Using reverse transcriptase PCR (RT-PCR) and deep sequencing, we also confirmed the presence of 5' cbDVGs in EBOV-infected NHP testes, which is of interest due to EBOV persistence in semen of male survivors of infection. This work suggests that DVGs play a role in EBOV infection in vivo and that further study will lead to a better understanding of EBOV pathogenesis. IMPORTANCE The study of filovirus pathogenesis is critical for understanding the consequences of infection and for the development of strategies to ameliorate future outbreaks. Defective viral genomes (DVGs) have been detected during EBOV infections in vitro; however, their presence in in vivo infections remains unknown. In this study, DVGs were detected in samples collected from EBOV- and SUDV-infected nonhuman primates (NHPs). The accumulation of these DVGs in the trailer region of the genome during infection indicates a potential role in EBOV and SUDV pathogenesis. In particular, the presence of DVGs in the testes of infected NHPs requires further investigation as it may be linked to the establishment of persistence.

Project description:Deletion-containing viral genomes (DelVGs) are commonly produced during influenza A virus infection and have been implicated in influencing clinical infection outcomes. Despite their ubiquity, the specific molecular mechanisms that govern DelVG formation and their packaging into defective interfering particles (DIPs) remain poorly understood. Here, we utilized next-generation sequencing to analyze DelVGs that form de novo early during infection, prior to packaging. Analysis of these early DelVGs revealed that deletion formation occurs in clearly defined hot spots and is significantly associated with both direct sequence repeats and enrichment of adenosine and uridine bases. By comparing intracellular DelVGs with those packaged into extracellular virions, we discovered that DelVGs face a significant bottleneck during genome packaging relative to wild-type genomic RNAs. Interestingly, packaged DelVGs exhibited signs of enrichment for larger DelVGs suggesting that size is an important determinant of packaging efficiency. Our data provide the first unbiased, high-resolution portrait of the diversity of DelVGs that are generated by the influenza A virus replication machinery and shed light on the mechanisms that underly DelVG formation and packaging. IMPORTANCE Defective interfering particles (DIPs) are commonly produced by RNA viruses and have been implicated in modulating clinical infection outcomes; hence, there is increasing interest in the potential of DIPs as antiviral therapeutics. For influenza viruses, DIPs are formed by the packaging of genomic RNAs harboring internal deletions. Despite decades of study, the mechanisms that drive the formation of these deletion-containing viral genomes (DelVGs) remain elusive. Here, we used a specialized sequencing pipeline to characterize the first wave of DelVGs that form during influenza virus infection. This data set provides an unbiased profile of the deletion-forming preferences of the influenza virus replicase. In addition, by comparing the early intracellular DelVGs to those that get packaged into extracellular virions, we described a significant segment-specific bottleneck that limits DelVG packaging relative to wild-type viral RNAs. Altogether, these findings reveal factors that govern the production of both DelVGs and DIPs during influenza virus infection.

Project description:RNA viruses produce abundant defective viral genomes during replication, setting the stage for interactions between viral genomes that alter the course of pathogenesis. Harnessing these interactions to develop antivirals has become a recent goal of intense research focus. Despite decades of research, the mechanisms that regulate the production and interactions of Influenza A defective viral genomes are still unclear. The role of the host is essentially unexplored; specifically, it remains unknown whether host metabolism can influence the formation of defective viral genomes and the particles that house them. To address this question, we manipulated host cell anabolic signaling activity and monitored the production of defective viral genomes and particles by A/H1N1 and A/H3N2 strains, using a combination of single-cell immunofluorescence quantification, third-generation long-read sequencing, and the cluster-forming assay, a method we developed to titer defective and fully-infectious particles simultaneously. Here we show that alpelisib (Piqray), a highly selective inhibitor of mammalian Class 1a phosphoinositide-3 kinase (PI3K) receptors, significantly changed the proportion of defective particles and viral genomes (specifically deletion-containing viral genomes) in a strain-specific manner, under conditions that minimize multiple cycles of replication. Alpelisib pre-treatment of cells led to an increase in defective particles in the A/H3N2 strain, while the A/H1N1 strain showed a decrease in total viral particles. In the same infections, we found that defective viral genomes of polymerase and antigenic segments increased in the A/H1N1 strain, while the total particles decreased suggesting defective interference. We also found that the average deletion size in polymerase complex viral genomes increased in both the A/H3N2 and A/H1N1 strains. The A/H1N1 strain, additionally showed a dose-dependent increase in total number of defective viral genomes. In sum, we provide evidence that host cell metabolism can increase the production of defective viral genomes and particles at an early stage of infection, shifting the makeup of the infection and potential interactions among virions. Given that Influenza A defective viral genomes can inhibit pathogenesis, our study presents a new line of investigation into metabolic states associated with less severe flu infection and the potential induction of these states with metabolic drugs.

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Reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients.

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Reduced accumulation of defective viral genomes contributes to severe outcome in influenza virus infected patients.

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