Powerful virus

Kazakhstan Kyrgyzstan Tajikistan Turkmenistan Uzbekistan. Armenia Azerbaijan Georgia. Belarus Ukraine. Follow Us. Previous Next. May 29, GMT. By Charles Recknagel. See comments Print. Someone is infecting Iran's computers with what experts call "the most powerful virus to date.

What is Flame and what does it do? Flame is a computer virus that Tehran says is infecting its computers and which independent experts say is the most powerful virus yet seen.

The virus appears to be a major escalation in the cyberwar that some governments concerned by Iran's nuclear program are suspected of waging against Tehran to sabotage its progress.

The virus infects computers in order to spy on users, steal classified information, and cause the mass deletion of data. It does this by sniffing network traffic, taking screenshots, recording audio conversations, and intercepting keyboard activity. Through a classic denial of service attack, Slammer had a quite real effect on key services. But when it swept across computers worldwide in , it caught security experts off guard by exploiting a flaw in Microsoft Internet Information Server.

That allowed the worm to deface and take down some websites. Perhaps most memorably, Code Red successfully brought down the whitehouse. It only took hours for Love Letter to become a global pandemic, in part because it played on a fundamental human emotion: the desire to be loved.

In that sense, Love Letter could be considered the first socially engineered computer virus. Rather than amateurs working out of their parents' basement, malware creators are often part of an underworld of criminal gang, or working directly for a foreign government or intelligence agency. Sharon Weinberger is a national security reporter based in Washington, D. Lung tissues were collected from B6 mice intranasally inoculated with a mixture of the four strains 2.

Analysis of lung sections obtained at days 2 and 5 p. At day 2 p. At this time point, a limited number of alveolar cells were infected. Interestingly, we also detected epithelial cells simultaneously expressing two or three fluorescent proteins, albeit at a low frequency, suggesting co-infection of these cells Fig.

To quantitatively analyse the co-infection in the bronchial epithelium, we utilized the inForm multispectral imaging software for automated user-trained tissue and cell segmentation, together with Nuance. Supplementary Fig. The ability to visualize cells co-infected with different influenza viruses in vivo is a major advance in technology and will allow us to provide novel insights into influenza co-infection and reassortment processes.

We next tested the utility of Color-flu viruses for the analysis of host responses to infection. As macrophages are involved in innate immunity and acute inflammation in influenza virus-infected lungs, we examined lung sections stained with an antibody to macrophages PE-Mac3 by using confocal microscopy. Macrophages infiltrated regions containing Venus-positive bronchial epithelial cells at day 2 p.

On the basis of this finding, we next employed live imaging to further study the interaction between influenza virus-infected epithelial cells and macrophages in mouse lungs. We also analysed the kinetics of the lung macrophages by tracking individual cells Fig.

Our system can thus be used to monitor the in vivo interactions between virus-infected and immune cells. The Venus fluorescent signal is shown in green. The tracks of individual macrophages are indicated as coloured lines.

Sequential images in the lower panel 1—4 show an enlarged view of the box in the upper right panel. Arrowheads indicate the blebbing of eGFP-positive cells. A heat map of the clustered transcripts for each condition is displayed, with enriched annotations and the enrichment scores for each cluster. A number of studies have assessed the transcriptomics and proteomics profiles of influenza virus-infected mice 19 , As these studies used whole lung samples, the results are the sum of virus-infected and uninfected cells, leading to the dilution of host responses and not allowing one to distinguish the profiles of infected cells from those of uninfected, bystander cells.

As a first step to overcome this shortcoming, we sorted macrophages known to be infected by influenza viruses Fig. Macrophages isolated from the lungs of mice inoculated with PBS naive macrophages served as controls. In fluorescent-positive macrophages, 6, transcripts were differently expressed relative to naive macrophages. By contrast, in fluorescent-negative macrophages obtained from infected mice, only 4, transcripts were differentially expressed relative to the naive macrophages.

This difference likely reflects differences in gene transcription induced by active influenza virus infection. However, it should be noted that the fluorescent-negative cell populations obtained from infected animals may have included infected cells in which the fluorescent signal had not yet been detected as would be expected at an early stage of virus infection.

Hierarchical clustering of differentially expressed transcripts, followed by functional enrichment analysis of each cluster, indicated that both fluorescent-positive and fluorescent-negative macrophages obtained from infected animals exhibit activation of pathways associated with the immune response, cytokine production and inflammation Fig. In addition, we observed that type I IFN genes were among the most upregulated transcripts in the fluorescent-positive macrophages Fig.

Finally, we tested whether the concept of mouse-adapted fluorescent influenza viruses could be applied to other influenza virus strains, such as highly pathogenic avian influenza A H5N1 HPAI viruses, which are a research priority due to the threat they pose to humans. This type of three-dimensional imaging analysis improves our understanding of the spatial distribution of influenza virus-infected bronchi.

Taken together, these findings demonstrate the utility of Color-flu viruses for comparative studies of influenza pathogenesis. Mouse body weight and survival were monitored for 14 days. Virus titres of tissue homogenates were determined by use of plaque assays in MDCK cells. Images of transparent lung tissues with Venus-positive cells in the bronchus red and alveolar area green were obtained by using a two-photon microscope. In this study, we established Color-flu viruses to study influenza virus infections at the cellular level.

Color-flu viruses combine several improvements over existing systems, including robust viral replication, virulence, stable fluorescent protein expression and a set of four different colours that can be visualized simultaneously. We also demonstrated that Color-flu viruses are applicable to a different influenza virus strain. These improvements allowed global transcriptomics analyses of infected and bystander cells and, for the first time, live-imaging of influenza virus-infected cells in the mouse lung.

Previous versions of fluorescent influenza viruses 12 , 13 including our original construct that is, WT-Venus-PR8 were appreciably attenuated in mice. These attenuated fluorescent viruses may still be useful for identifying initial target cells.

However, the immune responses elicited by these highly attenuated, non-lethal viruses most likely differ considerably from those of the mouse-lethal parent virus, making their use for pathogenesis studies problematic.

Here, we solved this problem by passaging viruses in mice. This strategy proved to be successful for two different influenza virus strains, suggesting its broad applicability. A second drawback of previously tested fluorescent influenza viruses is the genetic instability of the added reporter protein We are currently studying the mechanism by which our mouse-adapted viruses stably express fluorescent proteins.

Several groups generated a luciferase reporter-expressing influenza virus that can be used to monitor virus replication in live animals 25 , 26 , 27 ; however, this system needs systemic inoculation of substrate into the animals at every observation point.

In addition, the resolution of their imaging system based on the IVIS system is not adequate for the analysis of cellular immune mechanisms in vivo , which we are able to achieve with our system. Novel technologies for imaging analysis 28 have enabled us to develop a set of four different influenza colour variants that can be distinguished from one another by using Nuance, hence allowing their simultaneous detection. In fact, our pilot study identified lung epithelial cells expressing two or three different fluorescent proteins Fig.

To our knowledge, this is the first visualization of mouse lung cells infected with more than one influenza virus strain. In future studies, these colour variants could be used to address long-standing questions in influenza virus research, such as the frequency of viral co-infections in vivo , which may be critical to better understand influenza virus reassortment and, hence, the generation of novel influenza viruses such as the pandemic viruses of refs 29 , 30 , refs 29 , 30 and refs 31 , By employing our novel tool sets, we were able to detect influenza virus-infected cells in whole-lung tissues of mice, allowing us to observe the location and distribution of influenza viruses in the lung.

Moreover, we were able to observe interactions of virus-infected epithelial cells with immune cells. Such studies will allow us to directly monitor influenza disease progression from acute bronchitis to severe viral pneumonia, which causes considerable morbidity and mortality in highly pathogenic influenza virus infections 33 , In conclusion, Color-flu viruses in combination with advanced imaging technologies are a powerful and versatile tool to elucidate the mechanisms of influenza virus pathogenicity at the cellular level in animals.

In brief, the open reading frame ORF of the NS1 gene without the stop codon was fused with the N terminus of fluorescent reporter genes via a sequence encoding the amino-acid linker GSGG. The fluorescent reporter ORFs were followed by a sequence encoding the GSG linker, a foot-and-mouth virus protease 2A autoproteolytic site with 57 nucleotides from porcine teschovirus-1 ref. In addition, silent mutations were introduced into the endogenous splice acceptor site of the NS1 gene to abrogate splicing The plasmid encoding the Venus reporter protein was a kind gift from Dr A.

WT-Venus-PR8 was generated by using the reverse genetics system as described previously As serial passage in animals typically results in virus populations composed of genetic variants, we recreated MA-Venus-PR8 by using reverse genetics.

Lungs were harvested from PBS-inoculated or Color-flu virus-infected mice for virus titration, flow cytometric analysis and histological experiments at the times indicated in the figure panels. Mice were killed and intracardially perfused with PBS to remove blood cells from the lung. Thirty minutes after the antibody injection, the lungs of the mice were harvested. The lung tissues were collected from the mice at day 1 and 2 p. Cells were stained with appropriate combinations of fluorescent antibodies to analyse the population of each immune cell subset.

Agilent Technologies. Probe intensities were background-corrected and normalized using the normal-exponential and quantile methods, respectively.