Single-cell analysis uncovers a novel influenza A virus-derived defective interfering particle for antiviral therapy

Single-cell analysis of virus-infected cells (Heldt and Kupke et al., 2015) enables the characterization of individual highly productive cells, which may support strategies to improve cell culture-based vaccine production. However, the definition of poor producer single cells can also yield valuable information. Here we show that low-productive single Madin-Darby canine kidney (MDCK) cells, infected with influenza A virus (IAV) of strain A/PR/8/34 (PR8), were affected by a yet unrecognized form of defective-interfering particle (DIP). Conventional DIPs (cDIPs) typically contain a deleted form of the viral genome and are therefore unable to reproduce in an infection. However, upon complementation by the co-infection with fully infectious standard virus (STV), interference with the normal viral life cycle can be observed. Interestingly, considering their ability to suppress STV replication, cDIPs are of growing interest for clinical application, i.e. for their use as antivirals (Dimmock and Easton, 2014). Single-cell infection experiments revealed a surprisingly high variability in IAV replication with progeny virus yields that ranged from 0 to roughly 1000 plaque-forming units (PFU) per cell. Intriguingly, low-productive cells (0-10 PFU) displayed an abnormal phenotype, which was caused by the co-infection of a subpopulation of virus, in the following termed OP7 virus. Sequences of the genomic viral RNA (vRNA) of OP7 virions showed a significant amount of nucleotide substitutions in one of the eight vRNA segments, affecting its promotor, encoded proteins and virus packaging signals. We showed that these alterations were all directed towards the predominant genomic replication and packaging of the mutated vRNA over other genome segments. Concurrently, OP7 virions lacked a large fraction of other vRNAs, which constitute its defect in virus replication. Finally, co-infection experiments showed strong interference of OP7 virus with IAV replication, as indicated by a dramatic reduction in the infectivity of released virions. This interference was directed against relevant homologous and heterologous IAV strains, including strains of the current influenza season. Furthermore, we demonstrated interference in human cell lines. Therefore, OP7 virions are a novel form of IAV-derived DIPs with a non-deleted but mutated genomic RNA segment. First, it seems reasonable to investigate the presence of OP7 virions in seed virus preparations, as they can reduce virus titers in a production process, similar to cDIPs (Frensing et al., 2014). Second, OP7 virus may be used for antiviral therapy. As they are not able to reproduce on their own, they may be administered to organisms with no harm. The presence of OP7 virions can then inhibit the propagation of IAV of a natural infection. In addition, the induction of the innate immune response, observed upon infection with OP7 virus, can even further promote the antiviral effect. In the future, the design of efficient production systems for OP7 virions and the execution of animal trials may facilitate its utilization as a novel antiviral agent. References Heldt and Kupke et al. (2015) Nature Commun 6, 8938 Dimmock and Easton (2014) J Virol 88(10), 5217-5227 Frensing et al. (2014) Appl Microbiol Biotechnol 98, 8999-9008 Patent Patent pending for usage of OP7 virions as an antiviral agen

Effects of two sources of tannins (Quercus L. and Vaccinium vitis idaea L.) on rumen microbial fermentation: an in vitro study

The aim of the experiment was to determine the effect of different sources of tannins on the in vitro rumen fermentation with focus on methane production. In the experiment, a rumen simulation system (RUSITEC) equipped with 4 fermenters (1 L) was used in three replicated runs (6 d of adaptation and 4 d of sampling) to study the effects of Quercus cortex extract (QC), Vaccinium vitis idaea (VVI) dried leaf extract and a mixture of VVI/QC on rumen microbial fermentation. Fermenters were fed 10.9 g/d of dry matter (DM) of a 600:400 forage:concentrate diet. Treatments were control, QC (2.725 mL), VVI leaves 0.080 g) and mixture of QC/VVI (1.362 mL+0.040 g) and were randomly assigned to fermenters within periods. The equivalent of 2.5 g of tannins/kg dietary DM from three sources of tannins was evaluated. All tannin sources decreased CH4 and ammonia concentrations, as well as protozoa and methanogen counts (P<0.001). Vaccinium vitis idaea and QC/VVI tended (P=0.005) to reduce the acetate to propionate ratio. There were no changes in nutrient digestion. Results suggest that these sources of tannins, especially VVI have the potential to reduce rumen CH4 production and ammonia concentration without negative effects on in vitro DM digestibility, total volatile fatty acids and pH

FAIR+E pathogen data for surveillance and research: lessons from COVID-19

The COVID-19 pandemic has exemplified the importance of interoperable and equitable data sharing for global surveillance and to support research. While many challenges could be overcome, at least in some countries, many hurdles within the organizational, scientific, technical and cultural realms still remain to be tackled to be prepared for future threats. We propose to (i) continue supporting global efforts that have proven to be efficient and trustworthy toward addressing challenges in pathogen molecular data sharing; (ii) establish a distributed network of Pathogen Data Platforms to (a) ensure high quality data, metadata standardization and data analysis, (b) perform data brokering on behalf of data providers both for research and surveillance, (c) foster capacity building and continuous improvements, also for pandemic preparedness; (iii) establish an International One Health Pathogens Portal, connecting pathogen data isolated from various sources (human, animal, food, environment), in a truly One Health approach and following FAIR principles. To address these challenging endeavors, we have started an ELIXIR Focus Group where we invite all interested experts to join in a concerted, expert-driven effort toward sustaining and ensuring high-quality data for global surveillance and research

Multiscale modeling of influenza A virus replication in cell cultures predicts infection dynamics for highly different infection conditions.

Influenza A viruses (IAV) are commonly used to infect animal cell cultures for research purposes and vaccine production. Their replication is influenced strongly by the multiplicity of infection (MOI), which ranges over several orders of magnitude depending on the respective application. So far, mathematical models of IAV replication have paid little attention to the impact of the MOI on infection dynamics and virus yields. To address this issue, we extended an existing model of IAV replication in adherent MDCK cells with kinetics that explicitly consider the time point of cell infection. This modification does not only enable the fitting of high MOI measurements, but also the successful prediction of viral release dynamics of low MOI experiments using the same set of parameters. Furthermore, this model allows the investigation of defective interfering particle (DIP) propagation in different MOI regimes. The key difference between high and low MOI conditions is the percentage of infectious virions among the total virus particle release. Simulation studies show that DIP interference at a high MOI is determined exclusively by the DIP content of the seed virus while, in low MOI conditions, it is predominantly controlled by the de novo generation of DIPs. Overall, the extended model provides an ideal framework for the prediction and optimization of cell culture-derived IAV manufacturing and the production of DIPs for therapeutic use

TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells.

The cellular protease TMPRSS2 cleaves and activates the influenza virus hemagglutinin (HA) and TMPRSS2 expression is essential for viral spread and pathogenesis in mice. Moreover, severe acute respiratory syndrome coronavirus (SARS-CoV) and other respiratory viruses are activated by TMPRSS2. However, previous studies on viral activation by TMPRSS2 focused on a 492 amino acids comprising form of the protein (isoform 2) while other TMPRSS2 isoforms, generated upon alternative splicing of the tmprss2 mRNA, have not been characterized. Here, we show that the mRNA encoding a TMPRSS2 isoform with an extended N-terminal cytoplasmic domain (isoform 1) is expressed in lung-derived cell lines and tissues. Moreover, we demonstrate that TMPRSS2 isoform 1 colocalizes with HA and cleaves and activates HA. Finally, we show that isoform 1 activates the SARS-CoV spike protein for cathepsin L-independent entry into target cells. Our results indicate that TMPRSS2 isoform 1 is expressed in viral target cells and might contribute to viral activation in the host

The Hemagglutinin of Bat-Associated Influenza Viruses Is Activated by TMPRSS2 for pH-Dependent Entry into Bat but Not Human Cells.

New World bats have recently been discovered to harbor influenza A virus (FLUAV)-related viruses, termed bat-associated influenza A-like viruses (batFLUAV). The internal proteins of batFLUAV are functional in mammalian cells. In contrast, no biological functionality could be demonstrated for the surface proteins, hemagglutinin (HA)-like (HAL) and neuraminidase (NA)-like (NAL), and these proteins need to be replaced by their human counterparts to allow spread of batFLUAV in human cells. Here, we employed rhabdoviral vectors to study the role of HAL and NAL in viral entry. Vectors pseudotyped with batFLUAV-HAL and -NAL were able to enter bat cells but not cells from other mammalian species. Host cell entry was mediated by HAL and was dependent on prior proteolytic activation of HAL and endosomal low pH. In contrast, sialic acids were dispensable for HAL-driven entry. Finally, the type II transmembrane serine protease TMPRSS2 was able to activate HAL for cell entry indicating that batFLUAV can utilize human proteases for HAL activation. Collectively, these results identify viral and cellular factors governing host cell entry driven by batFLUAV surface proteins. They suggest that the absence of a functional receptor precludes entry of batFLUAV into human cells while other prerequisites for entry, HAL activation and protonation, are met in target cells of human origin

TMPRSS2 isoforms 1 and 2 colocalize with hemagglutinin.

<p>(A) COS-7 cells were transfected with plasmids encoding TMPRSS2 isoform 1 or isoform 2 or with empty plasmid which served as negative control. Subsequently, the cells were infected with FLUAV A/PR/8/34 (H1N1) at an MOI 0.5. At 24 h post infection, the cells were stained for FLUAV-HA (green) and TMPRSS2 isoforms (red) and images were taken at 63 x magnification. White squares show examples of colocalization of HA and TMPRSS2 (yellow signals) and were digitally magnified 2.5x from the original images. Similar results were obtained in three separate experiments. (B) Images obtained in (A) were analyzed with Fiji software, which allows calculation of the Pearson Correlation Coefficient (PCC), a measure for colocalization. The average PCC measured in three separate experiments is shown. For each experiment, 6–8 cells were analyzed. Error bars indicate standard error of the mean (SEM).</p

TMPRSS2 isoform 1 cleaves and activates the influenza virus hemagglutinin.

<p>(A) Expression plasmids encoding FLUAV HA subtypes H1 (left) and H3 (right) and the indicated proteases or empty plasmid (pCAGGS) were transiently cotransfected into 293T cells. At 48 h post transfection the cells were treated with PBS or trypsin, and HA cleavage was determined by Western blotting. Similar results were obtained in three independent experiments. The HA₀ precursor (upper arrow) and the HA₁ (middle arrow) and HA₂ (lower arrow) subunits are indicated. (B) The indicated proteases were transiently expressed in 293T cells and the cells infected with FLUAV A/PR/8/34 (H1N1) at an MOI 0.01 (left) or FLUAV A/Panama/2007/99 (H3N2) at an MOI of 0.1 (right) and treated with either trypsin or PBS. At 48 h post infection, the virus release was measured by determination of infectious particles (ffu/ml) in the culture supernatant. The results of representative experiments performed with triplicate samples are shown. Error bars indicate standard deviations. Similar results were obtained in three independent experiments. ffu, focus forming units.</p

Expression of tmprss2 transcript variants in human organs and cell lines.

<p>RT-PCR analysis of the expression of tmprss2 transcript variants 1 and 2 in organ samples from adult men (left panel) or from cell lines of human origin (right panel). Expression of GAPDH was assessed in parallel. Plasmids encoding isoform 1 and 2 (left panel) or a mix of both plasmids (right panel) served as positive control. Similar results were obtained in two separate experiments.</p