SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although SARS-CoV-2 was reported to alter several cellular pathways, its impact on DNA integrity and the mechanisms involved remain unknown. Here we show that SARS-CoV-2 causes DNA damage and elicits an altered DNA damage response. Mechanistically, SARS-CoV-2 proteins ORF6 and NSP13 cause degradation of the DNA damage response kinase CHK1 through proteasome and autophagy, respectively. CHK1 loss leads to deoxynucleoside triphosphate (dNTP) shortage, causing impaired S-phase progression, DNA damage, pro-inflammatory pathways activation and cellular senescence. Supplementation of deoxynucleosides reduces that. Furthermore, SARS-CoV-2 N-protein impairs 53BP1 focal recruitment by interfering with damage-induced long non-coding RNAs, thus reducing DNA repair. Key observations are recapitulated in SARS-CoV-2-infected mice and patients with COVID-19. We propose that SARS-CoV-2, by boosting ribonucleoside triphosphate levels to promote its replication at the expense of dNTPs and by hijacking damage-induced long non-coding RNAs' biology, threatens genome integrity and causes altered DNA damage response activation, induction of inflammation and cellular senescence.

A new laser device for ultra-rapid and sustainable aerosol sterilization.

The current COVID-19 pandemic has highlighted the importance of aerosol-based transmission of human pathogens; this therefore calls for novel medical devices which are able to sterilize contaminated aerosols. Here we describe a new laser device able to sterilize droplets containing either viruses or bacteria. Using engineered viral particles, we determined the 10,600 nm wavelength as the most efficient and exploitable laser source to be manufactured in a commercial device. Given the lack of existing working models to reproduce a human aerosol containing living microbial particles, we developed a new system mimicking human droplet formation and preserving bacterial and viral viability. This evidenced the efficacy of 10,600 nm laser light to kill two aerosol transmitted human pathogens, Legionella pneumophila and SARS-CoV-2. The minimal exposure time of <15 ms was required for the inactivation of over 99% pathogens in the aerosol; this is a key element in the design of a device that is safe and can be used in preventing inter-individual transmission. This represents a major advantage over existing devices, which mainly aim at either purifying incoming air by filters or sterilizing solid surfaces, which are not the major transmission routes for airborne communicable diseases.

Prevalence of locally undetected acute infections of Flaviviruses in North-Eastern Nigeria.

The burden of Arboviral infections is largely underestimated in Africa, particularly in North-Eastern Nigeria. A total of 200 serum samples were collected from patients exhibiting febrile illness who visited the State Specialist Hospital in Maiduguri for medical attention between March and April 2018. Sera were tested for Flavivirus RNA by a pan-flaviviral primer set using hemi-nested RT PCR. Twenty-six samples were positive for flaviviral RNA and sequence analysis indicated a high number of West Nile virus infections and one case of Zika virus. In-house recombinant NS1-based IgM ELISA indicated 47 % of WNV and 22 % of ZIKV infections. These data were also compared to commercially available assays for West Nile and Zika viruses. Finally, NS1 IgG ELISA was conducted for Dengue, Zika, West Nile and Usutu viruses. For serum samples detected by at least one flavivirus, 945% tested positive by NS1 IgG antibodies, while only 5.5 % of the patients were negative for all. To conclude, there is a high prevalence rate of arbovirus infections in the region, including Zika and Usutu viruses that were not previously detected. Interestingly, the analysis was conducted using in-house tools to allow the implementation of a sustainable surveillance protocol locally.

Comprehensive response to Usutu virus following first isolation in blood donors in the Friuli Venezia Giulia region of Italy: Development of recombinant NS1-based serology and sensitivity to antiviral drugs.

Surveillance of Usutu virus is crucial to prevent future outbreaks both in Europe and in other countries currently naïve to the infection, such as the Americas. This goal remains difficult to achieve, notably because of the lack of large-scale cohort studies and the absence of commercially available diagnostic reagents for USUV. This work started with the first identification of USUV in a blood donor in the Friuli Venezia Giulia (FVG) Region in Northern-Eastern Italy, which is endemic for West Nile virus. Considering that only one IgG ELISA is commercially available, but none for IgM, a novel NS1 antigen based IgG/M ELISA has been developed. This assay tested successfully for the detection of Usutu virus in blood donors with the identification of a second case of transmission and high levels of exposure. Furthermore, two pan-flavivirus antiviral drugs, that we previously characterized to be inhibitors of other flavivirus infectivity, were successfully tested for inhibition of Usutu virus with inhibitory concentrations in the low micromolar range. To conclude, this work identifies North-Eastern Italy as endemic for Usutu virus with implications for the screening of transfusion blood. A novel NS1-based ELISA test has been implemented for the detection of IgM/G that will be of importance as a tool for the diagnosis and surveillance of Usutu virus infection. Finally, Usutu virus is shown to be sensitive to a class of promising pan-flavivirus drugs.

Viral priming of cell intrinsic innate antiviral signaling by the unfolded protein response.

The innate response to a pathogen is critical in determining the outcome of the infection. However, the interplay of different cellular responses that are activated following viral infection and their contribution to innate antiviral signalling has not been clearly established. This work shows that flaviviruses, including Dengue, Zika, West Nile and Tick-borne encephalitis viruses, activate the unfolded protein response before transcription of interferon regulatory factor 3 induced genes. Infection in conditions of unfolded protein response priming leads to early activation of innate antiviral responses and cell intrinsic inhibition of viral replication, which is interferon regulatory factor 3 dependent. These results demonstrate that the unfolded protein response is not only a physiological reaction of the cell to viral infection, but also synergizes with pattern recognition sensing to mount a potent antiviral response.

Broad spectrum anti-flavivirus pyridobenzothiazolones leading to less infective virions.

We report the design, synthesis, and biological evaluation of a class of 1H-pyrido[2,1-b][1,3]benzothiazol-1-ones originated from compound 1, previously identified as anti-flavivirus agent. Some of the new compounds showed activity in low µM range with reasonable selectivity against Dengue 2, Yellow fever (Bolivia strain), and West Nile viruses. One of the most interesting molecules, compound 16, showed broad antiviral activity against additional flaviviruses such as Dengue 1, 3 and 4, Zika, Japanese encephalitis, several strains of Yellow fever, and tick-borne encephalitis viruses. Compound 16 did not exert any effect on alphaviruses and phleboviruses and its activity was maintained in YFV infected cells from different species. The activity of 16 appears specific for flavivirus with respect to other virus families, suggesting, but not proving, that it might be targeting a viral factor. We demonstrated that the antiviral effect of 16 is not related to reduced viral RNA synthesis or virion release. On the contrary, viral particles grown in the presence of 16 showed reduced infectivity, being unable to perform a second round of infection. The chemical class herein presented thus emerges as suitable to provide pan-flavivirus inhibitors.

Cellular Targets for the Treatment of Flavivirus Infections.

Classical antiviral therapy targets viral functions, mostly viral enzymes or receptors. Successful examples include precursor herpesvirus drugs, antiretroviral drugs that target reverse transcriptase and protease, influenza virus directed compounds as well as more recent direct antiviral agents (DAA) applied in the treatment of hepatitis C virus (HCV). However, from early times, the possibility of targeting the host cell to contain the infection has frequently re-emerged as an alternative and complementary antiviral strategy. Advantages of this approach include an increased threshold to the emergence of resistance and the possibility to target multiple viruses. Major pitfalls are related to important cellular side effects and cytotoxicity. In this mini-review, the concept of host directed antiviral therapy will be discussed with a focus on the most recent advances in the field of Flaviviruses, a family of important human pathogens for which we do not have antivirals available in the clinics.

A method for the detection of virus infectivity in single cells and real time: Towards an automated fluorescence neutralization test.

Virus neutralizing antibodies are critical correlates of protection in vaccine development and are discriminatory in the plaque reduction neutralization test when used for the diagnosis of viral infections. However, neutralization assays are time consuming, labor intensive and highly variable, thus limiting their use. Advances in automated live imaging of cells opened new possibilities for standard virus diagnostic techniques such as neutralization assays. To this end, a reporter cell line based on the translocation of the transcription factor IRF3 in response to infection is proposed. Image acquisition of signal in a microplate format allowed the setup of a rapid, semi-automated and high-throughput fluorescent neutralization test. The study is extended to the live imaging of IRF3 translocations that could potentially cut the time of analysis to few hours. The fluorescent neutralization test is suitable for high-throughput assays and expandable to other viruses of global importance such as Zika virus.

The host cell response to tick-borne encephalitis virus.

Tick-borne encephalitis virus is the most prevalent autochthonous arbovirus in Europe and an important travel-associated virus. Complications of the infection could lead to lethal encephalitis in susceptible individuals. However, despite its clinical relevance and expanding geographical distribution, most of our knowledge on its pathogenesis is inferred from studies on other flaviviruses. Molecular details of the host cell response to infection are scarce leading to a poor understanding of the antiviral pathways and viral countermeasures that are critical to determine the outcome of the infection. In this work the relevant literature is reviewed and the key elements of tick-borne encephalitis virus infection of human cells are identified, which requires further investigation.

Persistent viremia and urine shedding of tick-borne encephalitis virus in an infected immunosuppressed patient from a new epidemic cluster in North-Eastern Italy.

A persistent tick-borne encephalitis virus infection in an immune-suppressed patient is presented. Such an unusual clinical case offers the unique chance of detecting persistent viremia associated to the erythrocyte fraction and shedding of the virus in the urine for more than six weeks. The infection occurred in a new area of the Friuli Venezia-Giulia region (North Eastern Italy) where two additional cases are also being reported.

The stress granule component TIA-1 binds tick-borne encephalitis virus RNA and is recruited to perinuclear sites of viral replication to inhibit viral translation.

UNLABELLED: Flaviviruses are a major cause of disease in humans and animals worldwide. Tick-borne encephalitis virus (TBEV) is the most important arthropod-borne flavivirus endemic in Europe and is the etiological agent of tick-borne encephalitis, a potentially fatal infection of the central nervous system. However, the contributions of host proteins during TBEV infection are poorly understood. In this work, we investigate the cellular protein TIA-1 and its cognate factor TIAR, which are stress-induced RNA-binding proteins involved in the repression of initiation of translation of cellular mRNAs and in the formation of stress granules. We show that TIA-1 and TIAR interact with viral RNA in TBEV-infected cells. During TBEV infection, cytoplasmic TIA-1 and TIAR are recruited at sites of viral replication with concomitant depletion from stress granules. This effect is specific, since G3BP1, another component of these cytoplasmic structures, remains localized to stress granules. Moreover, heat shock induction of stress granules containing TIA-1, but not G3BP1, is inhibited in TBEV-infected cells. Infection of cells depleted of TIA-1 or TIAR by small interfering RNA (siRNA) or TIA-1(-/-) mouse fibroblasts, leads to a significant increase in TBEV extracellular infectivity. Interestingly, TIAR(-/-) fibroblasts show the opposite effect on TBEV infection, and this phenotype appears to be related to an excess of TIA-1 in these cells. Taking advantage of a TBE-luciferase replicon system, we also observed increased luciferase activity in TIA-1(-/-) mouse fibroblasts at early time points, consistent with TIA-1-mediated inhibition at the level of the first round of viral translation. These results indicate that, in response to TBEV infection, TIA-1 is recruited to sites of virus replication to bind TBEV RNA and modulate viral translation independently of stress granule (SG) formation. IMPORTANCE: This study (i) extends previous work that showed TIA-1/TIAR recruitment at sites of flavivirus replication, (ii) demonstrates that TIAR behaves like TIA-1 as an inhibitor of viral replication using an RNA interference (RNAi) approach in human cells that contradicts the previous hypothesis based on mouse embryonic fibroblast (MEF) knockouts only, (iii) demonstrates that tick-borne encephalitis virus (TBEV) is capable of inducing bona fide G3BP1/eIF3/eIF4B-positive stress granules, (iv) demonstrates a differential phenotype of stress response proteins following viral infection, and (v) implicates TIA-1 in viral translation and as a modulator of TBEV replication.

Regulation of transcription factor Twist expression by the DNA architectural protein high mobility group A2 during epithelial-to-mesenchymal transition.

Deciphering molecular mechanisms that control epithelial-to-mesenchymal transition (EMT) contributes to our understanding of how tumor cells become invasive and competent for intravasation. We have established that transforming growth factor ß activates Smad proteins, which induce expression of the embryonic factor high mobility group A2 (HMGA2), which causes mesenchymal transition. HMGA2 associates with Smad complexes and induces expression of an established regulator of EMT, the zinc finger transcription factor Snail. We now show that HMGA2 can also induce expression of a second regulator of EMT, the basic helix-loop-helix transcription factor Twist. Silencing of endogenous Twist demonstrated that this protein acts in a partially redundant manner together with Snail. Double silencing of Snail and Twist reverts mesenchymal HMGA2-expressing cells to a more epithelial phenotype when compared with single silencing of Snail or Twist. Furthermore, HMGA2 can directly associate with A:T-rich sequences and promote transcription from the Twist promoter. The new evidence proposes a model whereby HMGA2 directly induces multiple transcriptional regulators of the EMT program and, thus, is a potential biomarker for carcinomas displaying EMT during progression to more advanced stages of malignancy.